Oral Infections

Anne Marie Lynge Pedersen
Editor
Oral Infections
and General Health
From Molecule
to Chairside
123
Oral Infections and General Health
Editor
Oral Infections
and General Health
From Molecule to Chairside
Editor
Department of Odontology
University of Copenhagen
Copenhagen
Denmark
ISBN 978-3-319-25089-2 ISBN 978-3-319-25091-5 (eBook)

DOI 10.1007/978-3-319-25091-5
Library of Congress Control Number: 2015957993
Springer Cham Heidelberg New York Dordrecht London

© Springer International Publishing Switzerland 2016
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About the Editor
Anne Marie Lynge Pedersen, DDS, PhD, is Associate Professor and Head of
the Department of Odontology in the Faculty of Health and Medical Sciences,
University of Copenhagen, Denmark. Dr. Pedersen graduated in 1992 from
the Royal Dental College, Copenhagen, Denmark, and gained her PhD from
the University of Copenhagen in 1997 for a thesis entitled Salivary gland
dysfunction in patients with primary Sjögren’s syndrome. She then joined the
Section of Oral Physiology and Oral Pathology & Medicine, Department of
Odontology, University of Copenhagen, as a research assistant, and pro-
gressed to become head of the department in 2013. From 2009 to 2012,
Dr. Pedersen was Vice President and board member of the Scandinavian
Division of the International Association of Dental Research (IADR), and she
became President of the Division in January 2013. She has also served as an
appointed board member of the Danish Dental Association Research
Foundation (2007–2013) and acts as a consultant to the Danish Dental
Association on topics such as xerostomia, salivary gland function, oral muco-
sal diseases, and pharmacology. Dr. Pedersen has been the organizer of
national and international conferences, seminars, and symposia and is a
referee for many leading international scientific journals.
v
Contents
Part I Background Topics
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Part II Oral Infections and General Health
2 Dental Caries and General Health in Children

and Adults . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Svante Twetman
3 Plausible Mechanisms Explaining the Association
of Periodontitis with Cardiovascular Diseases . . . . . . . . . . . . . . . 19
Bruno G. Loos, Wijnand J. Teeuw, and Elena A. Nicu
4 Linkage Between Periodontal Disease
and Diabetes Mellitus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Palle Holmstrup and Allan Flyvbjerg
5 Linkage Between Periodontal Disease
and Rheumatoid Arthritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Palle Holmstrup and Claus H. Nielsen
6 Association Between Dental Infections and Renal
and Liver Diseases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Jukka H. Meurman
7 Association Between Oral Infections and Cancer Risk . . . . . . . . 59
Jukka H. Meurman
8 Oral Candidiasis and the Medically Compromised Patient . . . . 65
Camilla Kragelund, Jesper Reibel,
and Anne Marie Lynge Pedersen
9 Association Between Oral Infections and Salivary
Gland Hypofunction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Siri Beier Jensen and Anne Marie Lynge Pedersen
vii
viii Contents
Part III Future Diagnostic Methods and Techniques
10 The Oral Microbiome in Health and Disease . . . . . . . . . . . . . . . . 97

Ingar Olsen
11 Salivary Microbiota in Oral Health and Disease . . . . . . . . . . . . 115
Daniel Belstrøm
Part IV Future Perspectives in Management of Oral Infections
12 Use of Probiotics in Future Prevention and Treatment

of Oral Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
Mette Rose Jørgensen and Mette Kirstine Keller
13 Management of Patients with Oral Candidiasis . . . . . . . . . . . . 137
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
Contributors
Daniel Belstrøm, DDS, PhD Section 1, Periodontology and Oral

Microbiology, Department of Odontology, Faculty of Health and Medical
Sciences, University of Copenhagen, Copenhagen N, Denmark
Allan Flyvbjerg, MD, DMSc The Medical Research Laboratories,
Department of Clinical Medicine, Faculty of Health, Aarhus University,
Aarhus C, Denmark
Palle Holmstrup, DDS, PhD, Dr. Odont, Odont Dr. (hc) Section 1,
Periodontology and Oral Microbiology, Department of Odontology,
Faculty of Health and Medical Sciences, University of Copenhagen,
Copenhagen N, Denmark
Siri Beier Jensen, DDS, PhD Section 1, Oral Pathology and Oral
Medicine, Department of Odontology, Faculty of Health and Medical
Sciences, University of Copenhagen, Copenhagen N, Denmark
Mette Rose Jørgensen, DDS, PhD student Section 1, Cariology
and Endodontics, Department of Odontology, Faculty of Health and
Medical Sciences, University of Copenhagen, Copenhagen N, Denmark
Mette Kirstine Keller, DDS, PhD Section 1, Cariology and Endodontics,
Department of Odontology, Faculty of Health and Medical Sciences,
University of Copenhagen, Copenhagen N, Denmark
Camilla Kragelund, DDS, PhD Oral Pathology and Oral Medicine,
Department of Odontology, Faculty of Health and Medical Sciences,
University of Copenhagen, Copenhagen N, Denmark
Bruno G. Loos, DDS, MSc, PhD Department of Periodontology,
Academic Center of Dentistry Amsterdam (ACTA), University of
Amsterdam and VU University Amsterdam, Amsterdam, The Netherlands
Jukka H. Meurman, MD, PhD Department of Oral and Maxillofacial
Diseases, University of Helsinki and Helsinki University Hospital,
Helsinki, Finland
Elena Nicu, DDS, MSc, PhD Department of Periodontology,
Academic Center of Dentistry Amsterdam (ACTA), University
of Amsterdam and VU University Amsterdam, Amsterdam,
The Netherlands
ix
x Contributors
Claus H. Nielsen, MD, MSc, PhD Section 1, Periodontology

and Oral Microbiology, Department of Odontology, Faculty of Health
and Medical Sciences, University of Copenhagen, Copenhagen N,
Denmark
Ingar Olsen, DDS, PhD Department of Oral Biology, Faculty
of Dentistry, University of Oslo, Oslo, Norway
Anne Marie Lynge Pedersen, DDS, PhD Oral Pathology
and Oral Medicine, Department of Odontology, Faculty of Health
Denmark
Jesper Reibel, DDS, PhD, Dr. Odont Oral Pathology
and Oral Medicine, Department of Odontology, Faculty of Health
and Medical Sciences, University of Copenhagen, Copenhagen,
Denmark
Wijnand J. Teeuw, DDS, MSc Department of Periodontology,
Academic Center of Dentistry Amsterdam (ACTA), University
of Amsterdam and VU University Amsterdam, Amsterdam,
The Netherlands
Svante Twetman, DDS, PhD, Odont. Dr. Section 2, Cariology
and Pediatric Dentistry, Department of Odontology, Faculty of Health
Denmark
Part I
Background Topics
Introduction
1
Abstract
The idea for this book came about in relation to a symposium organized by
the editor to present a research update that summarizes new findings related
to the oral microbiota and the interaction between oral infections and gen-
eral health. The symposium was held at the Annual Meeting of the IADR
Continental European Division (CED) and Scandinavian Division (NOF) in
Florence, Italy, in 2013. Consequently, the content of this book is largely
based on contributions from European researchers within the field, but none-
theless the science presented in the various chapters embodies the global
aspects of the topic and is therefore of significant relevance for researchers
as well as health-care professionals throughout the whole world.
The healthy oral cavity is normally colonized by suppression and/or salivary gland dysfunction. A
bacteria, fungi, and viruses. It has been estimated disturbance of the balance between the oral
that more than 600 bacterial species colonize the microbiota and the host immune system results in
oral cavity of which some may be pathogenic and a shift from a healthy state to a diseased state
others are symbiotic or commensal. The normal leading to inflammation and infections of the oral
oral microbiota may be disrupted by a large num- hard and/or soft tissues. The most common oral
ber of factors including poor diet, malnutrition, infectious diseases include dental caries, peri-
poor oral hygiene, tobacco smoking and alcohol odontal disease, and oral candidiasis.
consumption, but also by several systemic dis- Dental caries is one of the most prevalent dis-
eases as well as the medications used for treating eases in humans. Even though the incidence of
them, especially those associated with immuno- dental caries has decreased during the last
decades, it is still a major problem in most indus-
trialized countries as it affects 60–90 % of school-
A.M.L. Pedersen, DDS, PhD aged children and the vast majority of adults and
Section 1, Oral Medicine and Pathology, obviously a larger problem in developing coun-
Department of Odontology, Faculty of Health
tries with poor living conditions and limited
and Medical Sciences, University of Copenhagen,
Noerre Allé 20, Copenhagen N, Denmark availability and accessibility of oral health
e-mail: amlp@sund.ku.dk services. In Chap. 2, Professor Twetman reviews
© Springer International Publishing Switzerland 2016 3

A.M.L. Pedersen (ed.), Oral Infections and General Health: From Molecule to Chairside,
DOI 10.1007/978-3-319-25091-5_1
4 A.M.L. Pedersen
a number of systemic diseases that are associated The prevalence of oral cancer is particularly high
with an increased frequency of dental caries. As among men, and it is the eighth most common
stated by the author, there are still obvious gaps cancer worldwide. Recent research suggests that
of knowledge concerning the interaction between oral infections may play a role in the develop-
caries and general diseases which calls for fur- ment of cancer. In Chap. 7, Professor Jukka
ther studies covering risk factors, associations, as H. Meurman reviews current knowledge on the
well as cost-effective interventions. mechanisms by which oral infections may influ-
Periodontitis is a serious oral infection, which ence the process of carcinogenesis.
is estimated to affect 15–20 % of the adult popu- Oral candidiasis is a common opportunistic
lations worldwide. Severe periodontitis may lead infection usually caused by the overgrowth of
to premature tooth loss. Furthermore, most chil- Candida species. There are several predisposing
dren and adolescents worldwide display signs of to oral candidiasis including use of antibiotics,
gingivitis. An increasing number of studies have steroid inhalers or systemic steroids, high-
suggested that oral infection, especially marginal carbohydrate diet, malnutrition, smoking, wear-
and apical periodontitis, may affect the course ing dentures, and impaired salivary gland
and pathogenesis of a variety of clinically impor- function. The prevalence of oral candidiasis is
tant systemic diseases. In Chap. 3, Professor high in immunocompromised patients such as
Bruno Loos reviews current knowledge on the patients with HIV infection, Sjögren’s syn-
inflammatory mechanisms linking periodontal drome, malignancies, and diabetes. Hence fun-
diseases to cardiovascular diseases. In the past gal infections are becoming a serious public
decades a large number of studies have also health problem, particularly for the growing
demonstrated a linkage between periodontitis population of elderly people, immunocompro-
and diabetes mellitus. This linkage appears to mised patients, as well as patients with salivary
be bidirectional. The proposed mechanisms or gland dysfunction. Although the introduction of
pathways by which these two diseases interact highly active antiretroviral therapy (HAART)
are reviewed by Professor Palle Holmstrup and has made oral candidiasis less common, HIV-
Allan Flyvbjerg in Chap. 4. A possible two-way associated oral lesions still remain significant
interrelationship has also been suggested for with oral candidiasis as the most typical lesion.
periodontitis and rheumatoid arthritis which is An update on oral candidiasis in medically com-
described in details by Professor Palle Holmstrup promised patients and the various current meth-
and Claus Nielsen in Chap. 5. Possible mecha- ods used to diagnose oral candidiasis, their
nisms behind the systemic effect include spread- advantages and disadvantages, as well as with
ing of bacteria present in the periodontal pockets new perspectives in using molecular techniques
as a result of transient bacteremia and release of is given by Associate Professor Camilla
circulating oral microbial toxins and pro-inflam- Kragelund and coworkers in Chap. 8.
matory mediators caused by immunological The last chapter in part II deals with the
injury induced by oral microorganisms. Insidious influence of salivary gland dysfunction, i.e.,
dental infections can worsen the condition and affection of the quantity and quality of saliva,
turn out to be life threatening in immunocompro- on the occurrence of oral infections. In Chap. 9,
mised patients like recipients of kidney and liver Associate Professor Siri Beier Jensen and Anne
organs. Consequently, oral infections should be Marie Lynge Pedersen review the most condi-
diagnosed and properly treated before as well as tions associated with severe salivary gland dys-
during and after organ transplantation. Chap. 6 function and their influence on oral health, i.e.,
by Professor Jukka H. Meurman addresses the Sjögren’s syndrome, cancer therapy, and intake
associations between dental infections and liver of medications.
and renal diseases. According to WHO, the num- Emerging knowledge on the oral microbiota
ber of cancers is estimated to increase by 70 % by challenges the current practice of chairside diag-
the year 2030 due to the aging of the populations. nostics. A number of new molecular techniques
1 Introduction 5
are now used to analyze the microbiome in health oral and systemic diseases as reviewed by
and disease including HOMINGS, oligotyping, Assistant Professor Daniel Belström in Chap. 11.
high-throughput sequencing, whole genome The final part of the book addresses recent
shotgun sequencing, single-cell genome sequenc- research in treating or even preventing oral infec-
ing, metatranscriptomics, and community-wide tions. The field of using probiotics is promising
transcriptome analysis. Chap. 10, part III, by and may offer a novel approach of future han-
Professor Ingar Olson deals with the human oral dling oral functions as reviewed by Dr. Mette
microbiome which contains bacteria, bacterio- Rose Jørgensen and Associate Professor Mette
phages/viruses, archaea, fungi, and protozoa, in K. Keller (Chap. 12). Also the management of
health and common oral diseases. The salivary patients with oral candidiasis is dealt with in this
microbiota is a highly complex microbial com- part of the book, in Chap. 13 by Associate
munity, containing oral microorganisms shed Professor Camilla Kragelund and coworkers.
from various oral surfaces. Saliva can be easily As we discover in this book, it seems justified
and noninvasively collected, and compositional to state that good oral health is important not only
changes of the salivary microbiota may poten- to prevent oral diseases but also to maintain good
tially serve as a biomarker used for screening of general health and vice versa.
Part II
Oral Infections and General Health
Dental Caries and General Health
in Children and Adults
2
Svante Twetman
Abstract
Caries is a biofilm-mediated noncommunicable disease fueled by dietary
sugar, neglected oral hygiene, and reduced saliva flow. General diseases
may influence the oral environment through its pathogenesis, medication,
and/or the caring of the condition. Associations between caries and chronic
diseases are mainly derived from case–control studies with various sample
sizes and quality of matching. Few observational studies are available and
the majority of all research is conducted in childhood and among older
adults. There is an increased caries risk for subjects with obesity, severe
asthma, poorly controlled type 1 diabetes mellitus, and congenital heart
diseases. An elevated caries frequency has also been reported for children
with neuropsychiatric disorders and cleft lip palate and long-term cancer
survivors. Frail elderly with cognitive impairments constitute a growing
age group in society with caries risk due to age- and medication-induced
salivary reduction. However, a general disease may not always have a
negative influence on dental health. Therefore, a regular individual caries
risk assessment is of utmost importance for clinical decision-making and
tailoring of recall intervals. There is good evidence that preventive mea-
sures based on fluoride, saliva stimulation, and sugar awareness can pre-
vent, control, and even arrest caries lesions in medically compromised
patients of all ages.
2.1 Introduction
Dental caries is still the major cause of tooth loss,

S. Twetman, DDS, PhD, Odont Dr and the severe forms of the disease are associated
Section 2, Cariology and Pediatric Dentistry, with pain and feeding problems as well as
Department of Odontology, Faculty of Health and
impaired well-being and quality of life (Selwitz
Medical Sciences, University of Copenhagen,
Noerre Allé 20, Copenhagen N 2200, Denmark et al. 2007). In 2010, untreated caries in perma-
e-mail: stwe@sund.ku.dk nent teeth was the most prevalent chronic

DOI 10.1007/978-3-319-25091-5_2
10 S. Twetman
condition worldwide, affecting 2.4 billion people acid-producing and acid-tolerating bacteria (i.e.,
(Kassebaum et al. 2015). In addition, around 621 mutans streptococci, lactobacilli, bifidobacteria,
million children exhibited untreated caries in their Scardovia), causing dysbiosis (Marsh et al.
deciduous teeth (10th most prevalent condition). 2014). Thus, caries is not a classical infection but
With more than 45 % of the global population should be regarded and handled as a noncommu-
affected, it seems obvious that there is an overlap nicable disease. From a chemical point of view,
with general diseases. Interestingly, there was evi- caries is an imbalance between mineral loss and
dence that the current burden of untreated caries mineral gain; when the loss is dominating over
has shifted from children to adults, with three time, a caries lesion eventually becomes visible.
prevalence peaks at ages 6, 25, and 70 years In a simplified way, demineralization occurs at
(Kassebaum et al. 2015). Therefore, caries is a low pH conditions in the oral biofilm (plaque)
concern throughout the entire life course, and pre- and remineralization at pH levels around neutral
vention and oral health promotion are needed for and above. It is therefore important to understand
all age groups. This is especially important in the that any medical condition, medication, or behav-
light of the current demographic trends with an ior that directly or indirectly affects the pH stabil-
increasing life expectancy and fewer non-dentate ity in the oral environment and favors the
elderly persons. In general dental practice, around overgrowth of aciduric species can be linked to
30 % of all patients have medical issues that need caries. One common example is the use of xero-
to be considered, but an obvious concern is that genic drugs which reduce oral clearance and pro-
medical data often are underreported in dental long an acidic environment. A systematic review
records; patients seem to regard such information of literature has concluded that medication-
as irrelevant for the dental care. The purpose of induced salivary gland dysfunction (MISGD)
this chapter was to review the comorbidity constitutes a significant burden in many patients
between caries and the common general chronic and may be associated with important negative
diseases of which many have strong social and implications for oral health (Aliko et al. 2015).
lifestyle components. The focus lies on children Radiation therapy in connection with head and
and elderly simply because there is a gap of stud- neck cancer is also associated with xerostomia
ies and knowledge concerning young and middle- and hyposalivation and rampant caries develop-
aged adults. The ambition was not to cover rare ment at any age (Jensen et al. 2003). Another
syndromes and conditions described in case common example is intellectual or cognitive
reports. Furthermore, diseases with impact on impairment among elderly that negatively affects
dental erosion, the oral mucosa, and periodontal the ability to maintain a proper oral hygiene. On
conditions are described elsewhere. top of the direct influence of the disease and its
medication, the caring involved with the disease
may increase caries risk. It is especially true in
childhood, when the onset of a chronic disease is
2.2 Link between Caries stressful for the whole family. The oral hygiene
and General Diseases can be put aside for more “urgent” problems, and
compensation through sweets and candies are
Caries is a biofilm-mediated disease resulting often used to comfort the child.
from a complex interaction between the com- To summarize the paragraph above, caries can
mensal microbiota, host susceptibility, and envi- be linked to any medical or behavioral condition
ronmental factors such as diet (Wade 2013). The associated with sugar behavior, polypharmacy,
resident oral microbiota is normally diverse and and impaired ability to regular cleaning. The
beneficial to the host but the stability can be dis- most vulnerable groups seem to be young chil-
rupted by stress. The repeated exposure of dren and frail elderly, although the onset of a gen-
healthy biofilms to dietary sugars, and hence to eral chronic disease may affect the caries burden
low pH, favors the growth and metabolism of at any age. It should however be underlined that a
2 Dental Caries and General Health in Children and Adults 11
Fig. 2.1 Example of a computer-based caries risk model avoid new caries in the near future and that the major
(Cariogram). After input, the computer is weighting pre- cause is a sugar-rich diet (blue sector). The program is
selected algorithms on bacteria, oral hygiene, circum- interactive for patient education and motivation. It can be
stances, and susceptibility toward each other. The green downloaded free of charge in many different languages at
sector indicates that this patient only has 17 % chance to www.mah.se
disease as such, with few exceptions like the basis for subsequent and tailored preventive
Sjögren’s disease, will not necessarily ruin the and/or restorative treatment.
dental health within a certain time period.
Therefore, an individual caries risk assessment
should always be performed as an integrated part 2.3 Common Research Designs
of the clinical decision-making. There is evi-
dence to suggest that comprehensive risk models There are two principal study designs utilized to
are more accurate than the use of single factors, investigate the relationship between general dis-
albeit no existing model performs superior to eases and dental caries; case–control and obser-
another (Mejàre et al. 2014). The use of struc- vational studies (Fig. 2.2a, b). The case–control
tured templates or computer-based software approach is most common and starts with the
(Fig. 2.1) is regarded as best clinical practice due selection of subjects with a defined disease
to its consistency and didactic values (Twetman (cases) and subjects without this disease (con-
et al. 2013). The risk assessment should be trols). The prevalence of caries can be compared
repeated periodically throughout life and espe- in a cross-sectional way, and explanatory vari-
cially when life events occur, such as the onset of ables, or factors related to both the general dis-
a chronic disease (Twetman et al. 2013). The out- ease and caries, can be collected retrospectively
come of these assessments should ideally form through a review of medical and dental records.
12 S. Twetman
a b
study start
exposed
disease
disease
exposed
review records
group
un-
exposed no disease
exposed disease
un-
no disease
exposed
review records
group
no disease
un-
exposed
Fig. 2.2 (a) A case-control study begins by selecting subjects with disease (cases) or no disease (controls). (b) An
observational study begins with exposure and the outcome is registered by time
This approach is easy to implement but the qual- defined cases and controls are selected from the
ity is strongly dependent on the matching process full cohort. The main advantage is that not all
and the validity of the “historical” registrations. patients must be sampled or examined over time
In a trustworthy study, the cases and the controls which keeps down the workload and costs for an
must be carefully matched for sex, age, socioeco- otherwise costly and resource-demanding trial.
nomic and educational background, cultural
norms, and lifestyle. The fact that case–control
studies many times are presenting conflicting 2.4 Obesity/Overweight
results is likely due to inadequate matching.
Other problems that may flaw the conclusions are Obesity and overweight is a growing problem
small sample sizes and diverging disease defini- among children and adults worldwide. The etiol-
tions for the “cases.” From an evidence point of ogy is complex but overeating and calorie-rich
view, the prospective observational study design diets are common compartments. A frequent
is more robust and controllable for the researcher intake of sucrose-containing food and beverages
but requires a considerable budget and time; a may be detrimental for the dental health (Arola
large number of patients must be included and et al. 2009). The relationship between sugar and
followed for years with periodical reexamina- caries is however not as strong today as it was
tions. The major threat for reliable conclusions is decades ago which is commonly explained by the
a large attrition rate (lost to follow-up) and/or a widespread access to fluoride in water and tooth-
low incidence rate of the disease under study. paste. From the evidence perspective, it is beyond
A power calculation must therefore be conducted doubt that fluoride exposure is the key element
prior to the study in order to include a proper in caries prevention and caries arrest at all ages
number of subjects. From an ethical point of throughout life (Griffin et al. 2007; Twetman and
view, it is equally problematic to run a project Dhar 2015). The link between caries and over-
with too few persons as it is to enroll too many in weight in case–control trials is somewhat contro-
demanding examinations and samplings. A mix versial and highly dependent on inclusion criteria
of the abovementioned study designs is the nested and matching. Even systematic reviews have come
case–control approach in which only a few to diverging conclusions; one established a significant
relationship (OR = 3.7) between obesity and den- primary dentition, and the corresponding value in
tal caries in children from industrialized, but not the permanent dentition was 2.0 based on 14 stud-
from newly industrialized countries (Hayden ies (Alavaikko et al. 2011). It was concluded that
et al. 2013), while another failed to note any rela- physicians and dentists should reconsider preven-
tionship between overweight and caries burden tive measures against caries for persons with severe
(Silva et al. 2013). A fact that partly can explain asthma and strongly recommend water rinses
the different conclusions is that dental caries may immediately after the use of inhalators.
be associated with both high and low body mass
index (Hooley et al. 2012). Unfortunately, studies
in adults and elderly are largely lacking, but obvi- 2.6 Diabetes
ously diet recommendations and restrictions in
order to reduce weight and prevent caries go hand Diabetes can affect the stability and profile of the
in hand. Obesity is a part of the metabolic syn- oral biofilm through frequent meals and an
drome, and according to the common risk factor increased output and leakage of glucose in saliva
approach, dentists, together with all other health and gingival crevicular fluid. In the past, when the
professionals, should embrace the current WHO management of diabetes mellitus in childhood
guidelines and motivate their patients to reduce basically relied on slow-acting insulin and a highly
the free sugar intake to less than 10 % of the total restricted diet, subjects with diabetes exhibited less
energy intake (Moynihan and Kelly 2014). Free caries than a non-diseased population. With today’s
sugars include monosaccharides and disaccha- continuous monitoring of glucose, rapid-acting
rides added to foods by the manufacturer, cook, or insulin, or insulin pumps, the type 1 diabetic child
consumer, and sugars naturally present in honey, can live a more or less normal life with a less
syrups, fruit juices, and fruit juice concentrates. restricted diet. Furthermore, the oral health aware-
A further reduction to below 5 % of total energy ness among diabetics has increased in recent years
intake, or roughly 25 g (six teaspoons) per day, with its close link to periodontal problems.
would provide additional health benefits. Consequently, the results from case–control studies
with diabetic patients have therefore slightly
changed over time. According to recent systematic
2.5 Asthma reviews, there is no consistent relationship between
type 1 diabetes mellitus (T1DM) and dental caries
Asthma affects 6–8 % of the population and may in childhood (Ismail et al. 2015), although patients
influence the oral ecology through behavioral and with uncontrolled T1DM and poor oral hygiene
medical pathways. Severe asthma is often associ- may present increased prevalence of dental caries
ated with dry mouth, thirst, and frequent wake-up (Sampaio et al. 2011). It is therefore important to
periods at night when sugar-rich beverages and collect information on the patient’s recent HbA1c
fruit juices must be avoided. Furthermore, the ste- status; values above 8 % may indicate a poor com-
roid-containing inhalators, as well as beta-2 ago- pliance and may be associated with active caries
nists, may have low pH values which favor the development in schoolchildren and adolescents
growth of acid-tolerating phenotypes in the oral (Twetman et al. 2002). Concerning type 2 diabetes
biofilm, and increased levels of mutans strepto- mellitus, no impact on the prevalence of dental car-
cocci are commonly unveiled in the saliva of asth- ies has been reported (Sampaio et al. 2011).
matic children (Alaki et al. 2013). Although there
are conflicting reports on the relationship between
asthma and dental caries in the literature (Maupomé 2.7 Congenital Heart Disease
et al. 2010), a meta-analysis has suggested that
asthma doubles the risk of caries in both primary Congenital heart disease affects around 1 % of all
and permanent dentition (Alavaikko et al. 2011). children, and the condition is commonly associ-
Based on 11 studies, the odds ratio was 2.7 in the ated with oral health problems. This has been
14 S. Twetman
explained by an increased meal frequency, use of patients with cancers should be considered at risk
diuretic sucrose-containing medication, and fre- and candidates for saliva-stimulating measures and
quent episodes of antibiotic treatment (Hansson high-fluoride supplements.
et al. 2012). In addition, enamel defects and hypo-
mineralization are prevalent in children with con-
genital heart disease which may predispose to 2.9 Cleft Lip Palate
caries development. Parental anxiety and over-
compensation with sweets are psychological and Children with cleft lip palate are often claimed to be
behavioral factors often involved in the manage- caries prone in their maxillary incisors due to com-
ment of critically ill children. A case–control promised tooth brushing during the infant period
study has shown that children with congenital (Hasslöf and Twetman 2007; Antonarakis et al.
heart disease have three times more caries than 2013). An elevated prevalence of enamel defects
healthy controls in spite of more prevention may also contribute to caries susceptibility (Sundell
(Stecksén-Blicks et al. 2004). In the same study, a et al. 2015). Consequently, it has recently been
positive relationship between caries and the dura- reported that children with cleft lip palate have more
tion of the digoxin medication was established. caries in the primary but not in the young permanent
Thus, a dental home should be established for dentition compared to non-cleft controls (Sundell
children with congenital heart disease at an early et al. 2015). There was however no clear association
age in order to implement individual treatment to the type or localization of the clefts. The increased
plans with frequent checkups during childhood. caries risk must be taken into account by all mem-
bers of the multi-professional team involved in the
management of children with this syndrome.
2.8 Cancers
Long-term survivors of malignant conditions are 2.10 Neuropsychiatric Disorders

subjected to long-term effects on oral health due to
aggressive treatment protocols based on chemo- Neuropsychiatric disorders in childhood may affect
therapy and radiation (Kaste et al. 2009; Effinger the possibilities to conduct a regular and proper
et al. 2014). In particular, the radiation therapy oral hygiene. For example, studies in children with
affects the salivary gland functions in a permanent ADHD (attention deficit hyperactivity disorder)
or transient way, depending on location and radia- have suggested a 12-time increased risk for high
tion exposure. Consequently, patients who were DMFT values compared to controls that were
post-radiotherapy exhibited higher DMFT values matched concerning age, gender, ethnicity, and
(decayed, missing, filled permanent teeth) com- socio-economy (Broadbent et al. 2004, Blomqvist
pared to those who were post-chemotherapy and et al. 2011). The findings were adjusted for fluoride
healthy controls (Hong et al. 2010). There is also exposure, medical problems, diet, and oral hygiene.
data suggesting that children with leukemia dis- The neuropsychiatric disorders are however highly
played more caries than hospitalized children with- diverse and each family/child is unique, so the car-
out cancer (Willershausen et al. 1998). However, ies risk must be assessed individually and followed
among children that were caries-free at the onset of by targeted preventive measures.
leukemia and displayed a low caries risk, the vast
majority (87 %) were still unaffected after 3 years
(Pajari et al. 2001). This picture seems to be less 2.11 Aging and Cognitive
clear-cut among adults; a study has suggested an Impairment
inverse relationship between head and neck squa-
mous cell carcinoma and dental caries in a case– There is no evidence to suggest a link between
control study, although age and social factors may caries and healthy and vital older persons.
have played a role (Tezal et al. 2013). Nevertheless, However, for those with progressive intellectual
disabilities and dementia, oral health can rapidly 2.12 Caries Management
be jeopardized, and root caries development is an
atypical and increasing problem (Fiske et al. Caries prevention and management of the medi-
2006; Anders and Davis 2010). This is also true cally compromised patient span from community
for subjects with mental illnesses (Kisely et al. measures based on the common risk approach to
2011). The main reasons are difficulties to clean tertiary prevention and treatment under general
and polypharmacy. Alzheimer’s and Parkinson’s anesthesia. Although there is a palette of tech-
disease are common examples on non-regular or nologies for intervention, the core must be
sporadic oral cleaning and loss of ability to clean. focused on evidence-based methods for primary
Saliva plays a crucial role in maintaining oral prevention and noninvasive methods for second-
health through its mechanical clearance, buffer- ary prevention. Examples of suitable strategies
ing effect, and being a source of mucins, immu- are presented in Table 2.1 and some practice
noglobulins, enzymes, and antibacterial agents points are listed in Box 2.1. It is important to
(Sreebny 2000). Increasing age means an increas- keep in mind that there is no “one-size-fits-all”
ing number of prescribed drugs that when com- and that no method works better than its compli-
bined in a “cocktail” very well can be xerogenic. ance. The patient’s wish and demands must be
Even a modest reduction of the unstimulated considered in order to find and suggest the opti-
saliva secretion rate can have a strong impact on mal care for each situation. For example, there
root caries development. It is therefore important are alternative ways to bring in fluoride to the
to assess salivary gland function in elderly people tooth–biofilm interspace on a daily basis; the
and especially in frail elderly with diminishing quality of evidence is varying, but a method with
autonomy. A sialometry can be helpful for moti- low evidence and good compliance may defi-
vating the patient to benefit from saliva- nitely be preferred over a measure with strong
stimulating measures. evidence but poor compliance.
Table 2.1 Examples of caries preventive measures suitable for the medically compromised patient
Measure Age Notes Quality of evidencea
Self-applied measures
Fluoride toothpaste All ages at least 1000 ppm F, twice daily ๨๨๨๨
High-fluoride toothpaste >12 years >1500 ppm F, biofilm metabolic inhibitor, ๨๨ΟΟ
high-risk subjects, root caries arrest
Fluoride mouth rinse >6 years Daily 0.05 % NaF, post-brushing ๨๨ΟΟ
Fluoride chewing gum >6 years Saliva stimulation, remineralization ๨ΟΟΟ
Xylitol chewing gum All ages Saliva stimulation, biofilm metabolic ๨๨ΟΟ
inhibitor
Professional measures
Fluoride varnish All ages 2–4 times per year, sustained fluoride release ๨๨๨Ο
Silver diamine fluoride All ages 44,000 ppm, lesion arrest, black-staining ๨๨ΟΟ
Fissure sealants 6–14 years Prevention and noninvasive occlusal ๨๨๨Ο
treatment
Resin infiltration Perm. teeth Interdental non-cavitated lesions ๨๨ΟΟ
Professional tooth cleaning All ages Continuously repeated ๨ΟΟΟ
a
Quality of evidence according to GRADE (Guyatt et al. 2011): high (๨๨๨๨) = based on high or moderate quality
studies containing no factors that weaken the overall judgment; moderate (๨๨๨O) = based on high or moderate
quality studies containing isolated factors that weaken the overall judgment; low (๨๨ OO) = based on high or moderate
quality studies containing factors that weaken the overall judgment; very low (๨OOO) = the evidence base is insuffi-
cient when scientific evidence is lacking, quality of available studies is poor, or studies of similar quality are
contradictory
16 S. Twetman
gaps of knowledge concerning caries and gen-

Box 2.1 Practice Points eral diseases which calls for well-conducted
• At least 3/10 of all adults have medical studies covering risk factors, associations, as
issues to be considered for oral health. well as cost-effective interventions.
• Patient’s health declaration and medi-
cine list must be continuously updated
and reevaluated. References
• Patients/parents are often well informed
and “experts” on their own disease – lis- Alaki SM, Ashiry EA, Bakry NS, Baghlaf KK, Bagher
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• A 2–3-day logbook is practical and
Aliko A, Wolff A, Dawes C, Aframian D, Proctor G,
valid to capture dietary habits, but a Ekström J, Narayana N, Villa A, Sia YW, Joshi RK,
selective underreporting of food with et al. World Workshop on Oral Medicine VI: clinical
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• A powered toothbrush with rotating
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Livingstone B, Palou A, Picó C, Sanders T, Schaafsma
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G, van Baak M, van Loveren C, van Schothorst
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Plausible Mechanisms Explaining
the Association of Periodontitis
3
with Cardiovascular Diseases
Bruno G. Loos, Wijnand J. Teeuw,

and Elena A. Nicu
Abstract
The association between periodontitis and cardiovascular diseases is now
well established. Cardiovascular diseases include atherosclerosis, coronary
heart (artery) disease, cerebrovascular disease, and peripheral artery dis-
ease. Atherosclerosis is the underlying pathology of cardiovascular dis-
eases. In this chapter, we describe plausible mechanisms to explain the link
between periodontitis, atherosclerosis, and the subsequent cardiovascular
diseases. The explanations for the development and exacerbation of athero-
sclerotic plaques in periodontitis patients include: (1) bacteremia, (2) a pro-
inflammatory state, (3) a prothrombotic state, (4) an overactive immunity,
(5) dyslipidemia, and (6) common genetic risk factors. Most likely, these
plausible mechanisms play all simultaneously a role. Obviously, much
more fundamental and clinic research is needed to further study the asso-
ciations between periodontitis and atherosclerotic diseases.
3.1 Introduction has exponentially increased in this period.

Since the landmark “Editorial” by Friedewald
In the last three decades, the association between et al. in 2009 which appeared simultaneously
periodontal diseases and cardiovascular dis- in the American Journal of Cardiology and the
eases has become apparent. The number of sci- Journal of Periodontology (Friedewald et al.
entific publications and reviews on this topic 2009), and which confirmed the existence of
the relationship between these two types of
conditions, only further evidence has emerged.
A joint workshop organized by the European
B.G. Loos, DDS, MSc, PhD (*)
W.J. Teeuw, DDS, MSc • E.A. Nicu, DDS, MSc, PhD Federation of Periodontology (EFP) and the
Department of Periodontology, Academic Center American Academy of Periodontology (AAP)
of Dentistry Amsterdam (ACTA), University on the broad topic “the oral health-systemic
of Amsterdam and VU University Amsterdam,
health connection” further confirmed the epide-
Gustav Mahlerlaan 3004, Amsterdam 1081 LA,
The Netherlands miological associations between periodontitis
e-mail: b.g.loos@acta.nl and cardiovascular diseases (Tonetti et al. 2013).

DOI 10.1007/978-3-319-25091-5_3
20 B.G. Loos et al.
Cardiovascular diseases include atherosclerosis, Traditionally, we differentiate between two

coronary heart (artery) disease, cerebrovascu- types of periodontitis: aggressive periodontitis
lar disease, peripheral artery disease, and some and chronic periodontitis (Armitage 1999).
other less frequent conditions. Atherosclerosis is Severe periodontitis occurs in about 8–15 % of
the underlying pathology of cardiovascular dis- the population (Demmer and Papapanou 2010)
eases. In the framework of this chapter, where we depending on the definitions used for severe peri-
describe plausible mechanisms to explain the link odontitis and depending on the specific study
between cardiovascular diseases and periodontal population subjected to epidemiological studies.
diseases, we therefore will refer mainly to a pos- In countries with a high availability of dental
sible role of periodontitis in atherogenesis and care, with dental and health awareness, and with
atherosclerosis and its (acute) consequences. preventive measures available, the prevalence of
While the epidemiological associations severe periodontitis may be <10 %, while in
between periodontitis and atherosclerosis and countries with no dental care, the prevalence can
acute atherosclerotic events are now beyond any even be >15 %. Recent studies suggest that
doubt, biological explanations on how the rela- almost half of the population suffers from mild to
tionship may exist are not clear. This chapter moderate periodontitis (Albandar 2011; Eke
describes possible and plausible mechanisms on et al. 2012). Nevertheless, severe periodontitis is
how these two diseases might be linked. In this, a disease occurring only in a minority of popula-
we follow the consensus report on potential tions (8–15 %) (Albandar 2011; Eke et al. 2015).
inflammatory mechanisms linking periodontal Periodontitis is considered to be a complex
diseases to atherosclerosis and its consequences disease: multiple factors play a role simultane-
(Tonetti et al. 2013; Schenkein and Loos 2013). ously in the pathophysiology (Loos et al. 2015).
In periodontitis, the pathobiology behaves in a
nonlinear fashion, where the disease progression
3.2 Definitions of the Conditions rate fluctuates. The disease swings between peri-
ods of exacerbation to periods of quiescence (a
3.2.1 Periodontitis disease-resolving state). In the active phase of the
disease, the total of the host-related and lifestyle-
Periodontal diseases include gingivitis, peri- related causal factors results in an aberrant
odontitis, acute necrotizing ulcerative gingivi- immune reaction against bacteria in the gingival
tis or periodontitis, and periodontal abscesses. sulcus or pocket, in particular against gram-
Specifically, periodontitis has been associated negative bacteria (Loos et al. 2015). The ensuing
with atherosclerosis and acute atherosclerotic inflammatory reactions result in the described
events. Periodontitis is a chronic inflammatory inflammatory infiltrate, proliferation, and ulcer-
disease of the supporting tissues around the roots ation of the pocket epithelium at the cost of sound
of the teeth. This disease results in the irrevers- tooth-supporting structures.
ible destruction of periodontal ligament and
alveolar bone. The gingival tissues are highly
inflamed and the periodontal connective tissues 3.2.2 Atherosclerosis and Acute
are infiltrated with leukocytes; the inflammatory Atherosclerotic Events
reactions are actually at the base of loss of col-
lagen and an increase in the number and size of Atherosclerosis is a disease of the whole vascular
blood vessels, giving the gingiva its swollen and system, with obvious predilection places for
reddish appearance. Over a period of years of more severe disease development (Friedewald
chronic inflammation, a gradual loss of alveolar et al. 2009; Andersen and Jess 2014;
bone and periodontal ligament is apparent. If left Garcia-Gomez et al. 2014; Pearson et al. 2003).
untreated, teeth become mobile, may migrate, Atherosclerosis is the gradual stiffening of arter-
and eventually will exfoliate. ies, the increasing thickness of the intima media
3 Plausible Mechanisms Explaining the Association of Periodontitis with Cardiovascular Diseases 21
of vessels, and at special areas severe atheroscle- there is evidence for the association of periodonti-
rotic plaque formation. This results in the nar- tis with coronary artery disease, with cerebrovas-
rowing of the arterial lumen. This is typically cular disease, mortality due to the latter conditions,
observed in the coronary, carotid, intercranial, and links with peripheral artery disease. However,
and femoral arteries. Severe atherosclerosis and to date, no firm evidence for any biological mech-
severe endothelial dysfunction can result in anism linking periodontitis to atherosclerosis and
blockage and local thrombus formation in the its sequelae is available. To what extent is peri-
artery preventing oxygen-rich blood to supply the odontitis a true risk factor for atherosclerosis and
distal tissues. This is regarded as an “ischemic how can it play a causative role?
event,” such as an acute myocardial infarct, or We now further summarize current knowledge
stroke, or transient ischemic event in the brain. on the possible mechanisms on how periodontitis
The consequences of atherosclerosis are there- and atherosclerotic diseases may be linked
fore the following: (Schenkein and Loos 2013). In brief, the explana-
tions for the associations focus on atherogenesis,
1. Coronary artery disease (CAD) (also known that is, the development and exacerbation of athero-
as coronary heart disease (CHD)). This is a sclerotic plaques (see Box 3.1); these include: (1)
diagnosed, severe atherosclerotic plaque for- bacteremia, (2) a pro-inflammatory state, (3) a pro-
mation and narrowing of the lumen occurring thrombotic state, (4) an overactive immunity, (5)
in the coronary arteries, possibly resulting in dyslipidemia, and (6) common genetic risk factors.
angina pectoris and/or an acute myocardial Most likely, these six potential mechanisms do not
infarction (MI) or acute cardiac death. act individually, but may play all simultaneously
2. Cerebrovascular disease. This is confirmed and may act in concert, making a patient with peri-
severe atherosclerosis in carotid arteries and/ odontitis more susceptible for atherogenesis in
or in intracranial vessels. The patient can time, and more prone to suffer from an acute athero-
experience transient ischemic accidents (TIA) sclerotic event.
mostly with reversible brain damage, or can
suffer from a cerebrovascular ischemic acci-
dent (nonhemorrhagic stroke) with irrevers-
ible brain damage or sudden death. Box 3.1. Atherogenesis and the
3. Peripheral arterial disease (PAD). Severe ath- Atherosclerotic Lesion (Atheroma)
erosclerotic disease can also occur in periph- At some point, an initial atherosclerotic
eral arteries, such as the femoral arteries and/ lesion starts (atherogenesis, atheroma for-
or tibial arteries, but also in the brachial artery. mation). This results in activated and dys-
The patient can experience (transient) leg functional endothelial lining (1), and
muscle pain (intermittent claudication); subsequently, partial loss of integrity of the
necrosis and open ulcers can develop and pos- lining of the blood vessel. Now, on and by
sibly thrombus formation, with the potential these endothelial cells, there is an upregu-
that the thrombus gets dislodged to lung tissue lation of adhesion molecules (ICAM-1,
(pulmonary embolism). VCAM-1, E-selectin, P-selectin) and che-
moattractants (e.g., IL-8, thrombin). These
The above cardiovascular conditions have activated endothelial cells provide the
been linked to periodontitis in many epidemio- adhesion triggers and receptors, for
logical studies, in both cross-sectional studies and increased platelet and leukocyte adhesion
in longitudinal studies. The latter type of studies to them. This results in diapedesis of mono-
has been prospective and retrospective. The cytes and dendritic cells (both possibly
review by Dietrich (2013) (Dietrich et al. 2013), with ingested bacteria), and also T cells,
presented and reviewed by the joint EFP/AAP into the underlying inflammatory lesion.
workshop (Tonetti et al. 2013), concluded that
22 B.G. Loos et al.
Activated platelets may aggregate and may progressive fibrosis and loss of a demarca-
form mini-thrombi. tion between the inflammatory lesion and
The inflammatory lesion (2) in the smooth muscle cells; there is development
intima media of the artery may be in part of a compensatory blood supply for the
initiated and/or propagated by bacteria affected artery and an increased outer mus-
originating from the periodontitis lesion. cle layer. In Fig. 3.1., items 2 + 3 + 4 form
However, many more sources of bacterial the intimal layer (media) of the artery, and
remnants in such atherosclerotic lesions are item 5 is the outer muscle layer.
conceivable and extensive bacterial signa- Ultimately, a disintegrated endothe-
tures have been described (Ott et al. 2006). lial lining (6) can rupture, with exposi-
The inflammatory reactions in the athero- tion of the underlying atherosclerotic
sclerotic lesion can also be propagated by plaque. This generates thrombin from
pro-inflammatory mediators (IL-1, IL-6, prothrombin, which in turn enzymati-
CRP, TNF-alpha) and chemotactic factors cally generates fibrin from fibrinogen,
(e.g., MCP-1) both “spilled over” from the initiating the clotting cascade and resulting
periodontal lesions and produced in the in thrombosis and subsequently to a myo-
liver (8) and also systemically. cardial infarction, ischemic stroke, acute
The atherosclerotic lesion increases peripheral artery disease, or cardiac death.
over a period of years. Now it is character- This slow and chronic process over years
ized by the accumulation of degenerative of atherogenesis may be mediated by
material in the tunica intima (inner layer) repeated bacteremia, a pro-inflammatory
of artery walls (3). The material consists state and activated inflammatory cells, from
of (mostly) a cellular infiltrate of macro- infectious and inflammatory processes else-
phages and the latter fused to foam cells where (Andersen and Jess 2014; Koenig
and lymphocytes (CD4+ Th cells charac- 2013). In this respect, Crohn’s disease and
terized by increased IL-12, IL-18, IFN- ulcerative colitis, chronic periodontitis, but
gamma production), further lipid streaks also rheumatoid arthritis have been impli-
(cholesterol and fatty acids), calcifica- cated (Andersen and Jess 2014; Garcia-
tions, and a variable amount of fibrous Gomez et al. 2014; Kholy et al. 2015).
connective tissue. The atherosclerotic Bacteria (transmigrated from the sub-
plaque intrudes into the lumen of the gingival biofilm into the periodontal
artery and causes the arterial lumen to nar- lesion or in the circulation itself) can acti-
row, which will restrict blood flow. During vate platelets (7; out of scale within
atheroma maturation, one sees more and Fig. 3.1) and can trigger platelet–leuko-
more modified low-density lipoproteins cyte complexes. These can form aggre-
(LDL) phagocytosed within the macro- gates on the dysfunctional endothelium,
phages and foam cells, resulting in an in particular due to the adhesion mole-
increase of pro-inflammatory cytokines cules that are abundantly expressed,
(IL-6, IL-1, TNF-alpha), chemoattractants resulting in the initiation of (micro)
(IL-8), and metalloproteineases (MMPs). thrombus formation.
The active atherosclerotic lesion, now in a Simultaneously, the liver (8) is stimu-
more inflammatory state, induces a further lated and upregulated due to inflammatory
dysfunctional endothelial lining. signals (IL-6), to overproduce clotting fac-
In the mature atheroma, a degeneration tors, resulting in a prothrombotic state.
of smooth muscle cells and an increase Also increased production of acute
of fibroblasts are observed (4), with phase reactants, including CRP, which
Direction of bloodflow
1
7
6 2 8
10 9
Fig. 3.1 Schematic cartoon of an atherosclerotic lesion and events in the process of atherogenesis (Adapted and
reprinted from (Schenkein and Loos 2013))
will increase the pro-inflammatory fibrous cap, weakening the strength

state, are produced in the liver (8; of the vessel, leading to fissuring of
again out of scale within Fig. 3.1). the atheroma.
Within the progressive athero- In a severely progressed stage (10),
sclerotic lesion, decreased collagen the atheroma comprises of a large
production and activation of MMPs necrotic core which is exposed to the
(9) is occurring, resulting in reduc- vasculature within the lesion, leading
tion of the smooth muscle cell con- to contact with platelets and initiation
tent and increased degradation of of coagulation and plaque rupture in
remaining collagen that borders the so-called vulnerable lesions.
3.3 Bacteremia subgingival area (Loos 2005; Nesse et al. 2008).

and Consequences Many of the subgingival bacteria are gram nega-
for the Cardiovascular tive, and in particular, the endotoxin lipopolysac-
System charide (LPS) from these species stimulates the
host immune system. This latter biologic event
It has been known for many years that oral bacte- can be at the basis of the pro-inflammatory state
ria can enter the bloodstream. In particular, in seen in both periodontitis and cardiovascular dis-
cases of severe gingivitis and periodontitis, the ease (see below) (Schenkein and Loos 2013;
inner epithelial lining of the periodontal pockets Tonetti and Graziani 2014). Also the daily epi-
is ulcerated and shows loss of epithelial lining sodes of dissemination of bacteria from the peri-
integrity (Schenkein and Loos 2013; Loos 2005; odontal lesions into the bloodstream can favor
Tonetti and Graziani 2014). This ulcerated pocket atheroma formation and can induce activated
epithelium may add up to 8–20 cm2 (Loos 2005; platelets and platelet–leukocyte complexes, lead-
Nesse et al. 2008). It is called periodontal ing to the prothrombotic state (see below).
inflamed surface area and forms an easy porte It is now well documented that bacteria and
d’entrée for the bacteria, bacterial toxins, and endotoxins from periodontal lesions enter the
other antigenic components residing in the bloodstream (Schenkein and Loos 2013; Tonetti
24 B.G. Loos et al.
Table 3.1 Lines of evidence for the role of bacteria and LPS, may easily gain access to the vascular sys-
specifically periodontal pathogens and atherosclerosis
tem in cases of periodontitis. And biological
(Tonetti and Graziani 2014)
experiments have demonstrated that this phe-
Evidence for the role of periodontal pathogens in
nomenon plays a role in atherogenesis and exac-
atherosclerosis
erbation of atherosclerotic lesions.
DNA of periodontopathogens has been localized within
atherosclerotic plaques
Periopathogens are able to invade endothelial cells, in
particular P. gingivalis
Periopathogens and other oral bacteria may trigger 3.4 Immunologic Reactions
platelet aggregation
P. gingivalis can accelerate the transition of
The increased bacteremias and dissemination of
macrophages to foam cells
Periopathogens can activate the host immune system
endotoxins (LPS) and many other microbiologi-
and cause a pro-inflammatory state cal antigens stimulate the immune response of the
host (Schenkein and Loos 2013). Consequently,
an activated innate immunity may result in hyper-
and Graziani 2014; Reyes et al. 2013). This has active neutrophils, higher levels of complement,
been shown when patients chew, brush their increased levels of acute phase reactants (see
teeth, but also after dental procedures. However, below), and a general exacerbation of reactivity.
the long-standing periodontal lesions and the Also the adaptive immunity is activated. The
chronic daily short-lived bacteremias are mostly latter is an intriguing finding and the conse-
responsible for the negative effects. The possi- quences are not well understood. It is found that
bilities for oral and periodontal bacteria to cause there is an increased titer of antibodies to oral and
harm for the cardiovascular system and to be periodontal bacteria in the circulation in patients
involved in atherogenesis and atherosclerosis are with periodontitis. These antibodies first of all can
summarized in Table 3.1. enter atherosclerotic lesions where such oral spe-
Interestingly, some indirect evidence for a role cies are nested. This can exacerbate such athero-
of bacteria in atherogenesis or in atherosclerotic sclerotic lesions and could increase the endothelial
lesions in general is the finding of a large variety dysfunction and increase the risk of rupture of the
of bacterial signatures in atherosclerotic biopsies lesion with thrombus formation as a result.
(Ott et al. 2006). Specifically, in addition to a Furthermore, enhanced T- and B-cell activation
high variety of species, also P. gingivalis, A. acti- may give rise to autoimmunity. Autoantibodies
nomycetemcomitans, and other periodontal may be generated through the molecular mimicry
pathogens have been identified (Table 3.1). In between bacterial and human heat shock proteins
periodontitis, the prevalence of bacteremias and (HSP). These cross-reactive antibodies in the first
endotoxinemias is higher than in gingivitis or place generated against circulating microorgan-
periodontal healthy subjects as evidenced by isms may react, for example, with HSPs on endo-
higher microbial diversities in atherosclerotic thelial cells. These become dysfunctional and again
biopsies from periodontal patients. Also, there this can enhance the chance for rupture of the endo-
are correlations between the presence of bacterial thelial lining giving rise to thrombus formation
antigens and molecular signatures in the athero- with the chance of ischemia. Anti-HSP antibodies
thrombotic lesions and the severity of periodonti- against the periodontal pathogens P. gingivalis, F.
tis. Moreover, there are correlations reported nucleatum, T. forsythia, and A. actinomycetemcom-
between the composition of the subgingival itans have been found. Also the activated T and B
microflora and the bacterial species in vascular cells against other antigens of these bacteria have
biopsies. Even, interestingly, there is suggestion been found. Further, other autoantibodies include
that some bacterial species are still viable. anticardiolipin and anti-low-density lipoproteins
Taken together, there is biological evidence (anti-LDL-type cholesterol) (Schenkein and Loos
that bacteria and their toxic components, such as 2013; Schenkein et al. 2004).
3.5 Pro-inflammatory State Table 3.2 Risk levels for acute cardiovascular events
based on plasma hsCRP levels
It has been widely accepted that low-grade sys- Risk for acute cardiovascular events Levels of hsCRP
temic inflammation contributes to an elevated risk Low <1 mg/L
for CAD and ischemic stroke (Friedewald et al. Intermediate 1–3 mg/L
2009; Andersen and Jess 2014; Garcia-Gomez High >3 mg/L
et al. 2014; Pearson et al. 2003; Koenig 2013;
Emerging Risk Factors Collaboration et al. 2010;
Ridker 2009). In the last two decades, C-reactive therefore often referred to as hsCRP. Levels of
protein (CRP) has been proposed as an important hsCRP are classified in three categories with
inflammatory biomarker related to acute athero- regard to risk for cardiovascular events
sclerotic events. CRP levels exhibit a continu- (Table 3.2) (Pearson et al. 2003).
ous association with the risk of CAD, ischemic In periodontitis, consistently elevated levels of
stroke, and vascular mortality (Emerging Risk hsCRP have been found (Paraskevas et al. 2008).
Factors Collaboration et al. 2010). Interestingly, Meta-analysis of published case-control studies
lowering of CRP levels after statin therapy is have shown that in periodontitis often CRP levels
associated with a decrease in cardiovascular dis- are in the range of intermediate to high CVD risk,
ease event rate, equivalent to what was observed while the values in controls are in the low- or
for patients who achieved lowered LDL choles- intermediate-risk category. The latter authors
terol treatment goals (Ridker et al. 2005). reported that the weighted mean difference
CRP is an “acute-phase reactant,” a molecule between patients and controls was highly signifi-
produced mainly in the liver, in response to cant (1.56 mg/L). Moreover, it has been shown
inflammatory signals, in particular in reaction to that hsCRP behaves in a dose-dependent manner:
elevated levels of interleukin-6 (IL-6). The acute- severe periodontitis in general shows higher
phase reactants such as CRP have pro- blood plasma levels of hsCRP than moderate or
inflammatory properties. The exact function(s) of mild periodontitis, while healthy controls show
CRP, despite its years of discovery as biomarker, even lower values.
is not yet completely uncovered. CRP is a penta- Furthermore, a meta-analysis on periodontitis
trexin molecule; it functions as soluble pattern treatment studies (n = 23) including 1647 patients
recognition molecule, and one of the most impor- have shown that hsCRP levels are significantly
tant roles for this protein is host defense primar- reduced after periodontal therapy (overall a mean
ily against pathogenic bacteria. It can function as reduction of 0.5 mg/L) (Teeuw et al. 2014). The
an opsonin for pathogens through activation of reduction was specifically significant in a sub-
the complement pathway and through binding of group of periodontitis patients having comor-
Fc-gamma receptors. CRP and other pentatrexins bidities such as diabetes, atherosclerosis, or
can also recognize membrane phospholipids and rheumatoid arthritis. The reduction of hsCRP in
nuclear components exposed on or released by periodontitis treatment studies is clinically rel-
damaged endothelial cells. In this way, it is pro- evant since the hsCRP levels exhibit a continu-
posed to be important for clearance of damaged ous association with the risk of CAD, ischemic
cells and tissues. Because CRP is a strong acute- stroke, and vascular mortality (Emerging Risk
phase reactant, it is widely used as a diagnostic Factors Collaboration et al. 2010); the hsCRP
marker for acute inflammation and/or infection, levels after periodontal treatment can often place
with levels ranging 10–200 mg/l or even higher. patients in a low CVD risk category (Table 3.2).
The levels of CRP related to chronic inflam- These treatment studies with favorable results
mation and proposed as cardiovascular risk bio- suggest further that periodontitis is causally
marker are relatively low and not exceeding related to elevated CRP.
10 mg/L. These levels have been found by apply- Next to hsCRP are other markers of inflamma-
ing a high sensitivity test using nephelometry and tion in the circulation of patients with periodontitis.
26 B.G. Loos et al.
Also these can make periodontitis patients more and Loos 2013; Tonetti and Graziani 2014).
susceptible for an atherosclerotic event as they Collectively, we call this a prothrombotic state,
have atherogenic potential (Schenkein and Loos i.e., an individual may form faster than normal a
2013). These include other acute-phase reactants (small) thrombus and/or this is less efficiently
and immune mediators: interleukin (IL)-1, IL-4, removed. It is hypothesized that this state in peri-
IL-6, IL-18, haptoglobin, serum amyloid A, alpha odontitis could also be one of the mechanisms as
1 anti-chymotrypsin, tumor necrosis factor-alpha, to how periodontitis is associated with ischemic
metalloproteinase (MMP)-9, platelet-activating atherosclerotic events. In Box 3.2, we summarize
factor (PAF), and PAF acetylhydrolase. the normal blood clotting events.
The pro-inflammatory state as characterized
by the elevated levels of hsCRP, and other bio-
markers mentioned above, could enter the blood Box 3.2. The Normal Blood Clotting Events
circulation in essentially two ways: In response to vascular injury, a sequence
of coordinated events ensures blood fluid-
1. The spill over from periodontal lesions: There ity while preventing blood loss. Adhesion,
is substantial literature providing indications activation, and aggregation of platelets are
that inflammatory cytokines and other bio- all steps in primary hemostasis. Under
markers are produced in the periodontal static or low shear conditions of flow, plate-
lesion; from here they are “dumped” or lets circulate in the inactivated discocyte
“spilled” into the blood circulation. These form. Upon vascular or tissue injury, col-
molecules could impact organs and tissues, lagen matrix exposure and local adenosine
such as blood vessels with/without atheroscle- diphosphate (ADP) production result in
rotic lesions and the liver. agonist–receptor interactions (GPIb-von
2. The activated liver: The liver in turn initiates Willebrand factor, GPVI-collagen, P2Y12-
an acute-phase response. The liver produces ADP), which conclude with formation of
higher levels of CRP, and other acute-phase platelet aggregates.
reactants, but also higher levels of comple- Formation of the platelet hemostatic plug
ment molecules and prothrombotic molecules, is followed by true blood clot formation
such as fibrinogen, von Willebrand factor, and (activation of coagulation) and, finally, by
plasminogen. clot dissolution (fibrinolysis). Activation of
coagulation is strongly dependent on upregu-
In summary, through the clearly proven pro- lation of tissue factor (TF) and leads to
inflammatory state in periodontitis, there is a thrombin generation. TF is not normally
highly plausible biologic mechanism why peri- present in the circulation. Normal hemosta-
odontitis is linked to atherosclerosis and acute sis arises when the blood vessels are dis-
atherosclerotic events. Excessive local produc- rupted allowing blood to contact extravascular
tion of pro-inflammatory cytokines, as well as cells expressing TF. Thrombin is active in the
higher levels of these and acute-phase reactants conversion of soluble fibrinogen to insoluble
produced in the liver, can induce or exacerbate fibrin (deposited in the formed blood clot)
inflammatory changes in the endothelium and and is a potent platelet activator. There are
atherosclerotic lesions (Fig. 3.1). three natural anticoagulant mechanisms act-
ing together to prevent thrombosis: (I) the
heparin–antithrombin system, (II) the pro-
3.6 Prothrombotic State tein C pathway, and (III) the tissue factor
pathway inhibitor system.
In recent years, there is a growing body of evi- Fibrinolysis is a natural response to coag-
dence that in periodontitis both a hypercoagula- ulation, leading to fibrin clot breakdown.
ble state and hypofibrinolysis exist (Schenkein
et al. 2009; Wu et al. 2000). Studies on the effects

During fibrinolysis, plasmin is generated of periodontal therapy on fibrinogen levels
from plasminogen (an inactive proenzyme) (reviewed in (Teeuw et al. 2014; D’Aiuto et al.
under the influence of tissue plasminogen 2013)) yielded variable results; some studies
activator (tPA) or urokinase plasminogen reported a significant reduction in the fibrinogen
activator (uPA); this reaction is inhibited by levels in the patient groups that received active
plasminogen activator inhibitor 1 and 2 periodontal treatment; others measured no sig-
(PAI-1, PAI-2) and α2-antiplasmin. The nificant changes post-therapy.
formed plasmin cleaves fibrin into soluble Other markers of coagulation or fibrinolysis
degradation products. (prothrombin fragments 1 + 2, D-dimer, von
Willebrand factor, tissue plasminogen activator
and plasminogen activator inhibitor-1) known to
Table 3.3 Changes or abnormalities in hemostasis be modified in prothrombotic states, rendered
parameters observed in periodontitis inconclusive results when explored in periodonti-
Situation in tis. Bizzarro et al. (Bizzarro et al. 2007) reported
Hemostasis parameters periodontitis increased levels of PAI-1 (Bizzarro et al. 2007)
Biomarkers of coagulation and the levels of PAI-1 decreased after full-mouth
Fibrinogen ൹ extractions (Taylor et al. 2006), whereas another
Fragment 1 + 2 ൹ study failed to demonstrate an association
von Willebrand factor ൹
between PAI-1 and periodontitis (Bretz et al.
P-selectin ൹
2005). Interestingly, there is evidence for a short-
Abnormalities of platelets
term increase in the first week after periodontal
Numbers in the circulation
൹
൹
treatment of hemostatic factors such as PAI-1,
Size (mean platelet volume)
Activation state ൹
D-dimer, and von Willebrand factor (D’Aiuto
Reactivity ൹ et al. 2005a, b; Tonetti et al. 2007; Graziani et al.
Abnormalities of fibrinolysis 2010). These changes correspond to an acute-
tPA
ൻ phase reaction in response to the treatment itself
PAI-1 ൹ measurable by an increased CRP, IL-6, possibly
D-dimer ൹ related to the acute bacteremia following full-
Abbreviations: tPA tissue-type plasminogen activator, mouth subgingival debridement.
PAI-1 plasminogen activator inhibitor 1 In response to hemorrhage, circulating plate-
lets adhere to exposed subendothelial tissues
and recruit additional platelets into aggregates
In periodontitis, the normal hemostasis state that function as procoagulant surfaces. Platelets
and events may be disturbed. In Table 3.3, we are released in the bone marrow from precur-
summarized the findings. One of the very first sors, the megakaryocytes. The process is con-
parameters found to be elevated in periodontitis trolled by thrombopoietin, a liver-synthesized
was fibrinogen (Kweider et al. 1993). Fibrinogen hormone, which stimulates multipotent mega-
is a member of the acute-phase protein family. Its karyoblasts toward maturation. In patients with
concentration rises in acute and chronic inflam- reactive high platelet counts secondary to inflam-
mation mainly as result of increased production mation, pro-inflammatory cytokines such as IL-6
by hepatocytes. Increased concentrations of are responsible for enhanced hepatic produc-
fibrinogen are associated with the development tion of thrombopoietin, resulting in increased
of atherothrombotic disease. Several cohort and platelet production. Untreated periodontitis has
population-based studies have documented an been associated with elevated numbers of plate-
increased level of plasma fibrinogen in periodon- lets (Papapanagiotou et al. 2009; Lopez et al.
titis patients compared to periodontally healthy 2012; Wang et al. 2014), and the association of
individuals (Kweider et al. 1993; Papapanagiotou high platelet counts in periodontitis is further
28 B.G. Loos et al.
strengthened by the observations that plate- be the cause for the observed platelet activation
let numbers decrease after periodontal therapy and reactivity (Table 3.3). Interestingly, strains of
(Wang et al. 2014; Christan et al. 2002). the recognized periodontal pathogens A. actino-
Platelets play a crucial role in the pathogen- mycetemcomitans and P. gingivalis, but also other
esis of atherosclerotic complications, contribut- dental plaque bacteria, such as Streptococcus san-
ing to thrombus formation or apposition after guis, induce platelet activation and aggregation
plaque rupture. The mean platelet volume (MPV) in vitro and in animal studies (Nicu et al. 2009;
is universally available with routine blood counts Assinger et al. 2011, 2012). Platelets become acti-
and is a quantity of the average size of platelets vated by periodontopathogens mainly via toll-like
in a sample. Compared to smaller ones, larger receptor 2 (TLR2) and TLR4.
platelets are more reactive, have more granules, P-selectin is a transmembrane protein present
aggregate more rapidly with collagen, have higher in the Weibel–Palade bodies of endothelial cells
thromboxane A2 level, and express more glyco- and in the α-granules of platelets. P-selection is
protein Ib and IIb/IIIa receptors. Elevated MPV expressed on the cell surface upon activation-
levels have been identified as an independent risk dependent granule exocytosis and plays a central
factor for myocardial infarction in patients with role in cardiovascular disease. Upon interaction
coronary heart disease and for death or recurrent with its receptor P-selectin glycoprotein ligand 1
vascular events after myocardial infarction. In (PSGL-1) on the leukocyte surface, P-selectin
chronic periodontitis, there is an ongoing low- (both platelet, as well as endothelial cell derived)
grade inflammation, and cytokines such as IL-6 is rapidly shed to form soluble P-selectin. Elevated
or IL-3 regulate megakaryocyte ploidy, result- levels of plasma P-selectin have been documented
ing in the production of more reactive and larger in patients with periodontitis (Papapanagiotou
platelets. Therefore, we can expect higher MPV et al. 2009; Assinger et al. 2011).
values in untreated periodontitis. Indeed, higher Another cytokine expressed and released from
MPV in periodontitis patients than in healthy activated platelets is the CD40L (cluster of differ-
controls have been reported (Lopez et al. 2012). entiation 40 ligand); in fact, platelets represent the
In contrast, Wang and coworkers found a lower main source of soluble CD40L. Ligation of
MPV in periodontitis patients at intake, and the CD40L to its receptor CD40 induces a pro-
MPV increased without reaching the levels of inflammatory and prothrombotic response in the
healthy controls 1 month post-periodontal ther- vascular endothelium, as evidenced by the release
apy (Wang et al. 2014, 2015). The source of these of inflammatory cytokines, expression of adhesion
conflicting results is as of yet unknown, but there molecules, activation of matrix metalloproteinases
are some plausible explanations. Lower MPV (MMPs), and procoagulant tissue factor. It initi-
in untreated periodontitis might be the effect of ates the formation of reactive oxygen species and
intensive consumption of large platelets at sites of inhibition of nitric oxide production. Also elevated
overt inflammation. Post-therapy, larger platelets levels of soluble CD40L were found in periodonti-
may represent newly released, young platelets tis patients, and these were correlated with
during rebound from platelet clearance. However, P-selectin (Papapanagiotou et al. 2009; Assinger
MPV variations should be interpreted with cau- et al. 2012), highly suggestive of an activated
tion in unmatched cohort studies, as confound- platelet phenotype in periodontitis. The platelets
ing factors, such as body mass index, systolic and were not only activated, but also hyperreactive. In
diastolic blood pressure, smoking status, glucose response to several species of oral bacteria, plate-
and cholesterol levels, or medication use have all lets from periodontitis patients showed an
been associated with MPV variations. increased membrane exposure of P-selectin and
As described above, regularly occurring bacte- increased formation of platelet–monocyte com-
remias in periodontitis patients underlie chronic plexes compared with controls (Nicu et al. 2009).
production and systemic increases of various Formation of platelet–leukocyte complexes is a
pro-inflammatory immune mediators. These could process that facilitates leukocyte transmigration to
perivascular tissues. However, these interactions higher levels of cholesterol are generated. This is
also occur in circulating blood, leading to acti- called elevated biosynthesis of cholesterol in the
vated platelet–leukocyte aggregates, which are liver. Case-control studies have noted elevated total
hallmarks not only of inflammatory disorders and serum cholesterol, elevated LDL and lowered
sepsis but also of acute myocardial infarction. HDL, and elevated (v)LDL and intermediate-
It is clear that in periodontitis small aberra- density lipoproteins in periodontitis (Schenkein
tions from normal can be found regarding the and Loos 2013). Also increased triglycerides (TGs)
hemostasis physiology. Most is based on cross- have been found in periodontitis.
sectional case-control studies or longitudinal LDL cholesterol can bind to circulating
cohort follow-up studies. Since periodontitis is LPS. The LDL–LPS complex in particular is
treatable, future longitudinal research should very atherogenic. The LDP–LPS complex can
address the question of whether periodontal ther- enter easily atherosclerotic plaques and enhance
apy is capable of reducing the levels of hemosta- the inflammatory reactions/responses inside the
sis biomarkers and reducing the platelet activation atheromas. The latter was also observed in
and reactive state. These studies will also help to in vitro experiments.
further confirm a mechanistic role of the pro- Moreover, LDL can be oxidized (oxLDL) and
inflammatory state in periodontitis being causally autoantibodies to oxLDL have been observed in
related to acute ischemic events. periodontitis (Schenkein et al. 2004). Interestingly,
P. gingivalis can induce foam cell formation in the
presence of exogenous LDL. In sum, from cross-
3.7 Dyslipidemia sectional clinical studies, we see that periodontitis
is associated with dyslipidemia, i.e., elevated total
For more than four decades, the role of high cho- cholesterol and elevated LDL and TGs, with con-
lesterol in atherosclerosis and the acute athero- comitant lower levels of HDL. This total makes
sclerotic events has been established. Total periodontitis patients with dyslipidemia more sus-
cholesterol consists mainly of HDL (high-density ceptible for increased atherogenesis, in particular
lipids) and LDL (low-density lipids). Increased in connection with LPS.
serum levels of especially LDL and very low- A recent review and meta-analysis (Teeuw
density lipoproteins (v)LDL and triglycerides are et al. 2014) has demonstrated that dyslipidemia
considered pro-atherogenic. The (v)LDL can in periodontitis patients can be reduced following
actually diffuse freely into the intimal layer of periodontal therapy. In the context of the current
blood vessels. In atherosclerotic lesions, one discussion on possible mechanisms how
finds foam cells, being multinuclear macrophages periodontitis might be linked to atherosclerosis,
which have phagocytized LDL. Regular macro- this is another clear indication that periodontitis
phages, not yet fused into multinuclear cells, are is actually related to dyslipidemia. But also it is
activated when they have phagocytized LDL and important in terms of prevention of further devel-
vLDL and may exuberate inflammation in the opment of atherosclerotic lesions; treatment of
atherosclerotic lesion. Subsequently, the endo- periodontitis can be helpful, or can be part of a
thelial cells overlaying the atherosclerotic lesion set of treatments, to lower an individual’s risk for
become dysfunctional and express many chemo- an acute atherosclerotic event.
kines and surface receptors.
Actually not only diet and “fatty foods,” but in
general, inflammatory processes seem to be associ- 3.8 A Common Genetic
ated with dyslipidemia, i.e., increased levels of (v) Background
LDL and decreased levels of HDL. In fact, choles-
terol is synthesized in the liver; it is not only Recent papers have identified the same genetic
acquired via diet. When the liver is activated by variants (single nucleotide polymorphisms
cytokines directly via pro-inflammatory cytokines, [SNP]) to be associated with both coronary artery
30 B.G. Loos et al.
disease (CAD) and periodontitis. This is a very aberrant inflammatory reactivity, determined in
intriguing finding. A common genetic back- part by genetic variants in ANRIL, CAMTA1 and
ground for CAD and periodontitis could be inter- VAMP3, could also explain the epidemiological
preted that the host acts in similar – aberrant – ways link between periodontitis and atherosclerosis in
to infections and/or inflammatory processes, irre- coronary arteries (CAD).
spectively where they take place. As stated And recently, yet another CAD risk locus was
before, atherosclerotic plaques are essentially also found to be associated with periodontitis;
inflammatory lesions. For example, we could there is now evidence for PLASMINOGEN (PLG)
think about similar host immune reactions and as a shared genetic risk factor of CAD and peri-
similar pathobiologic pathways to bacteria and odontitis (Schaefer et al. 2015). When plasmino-
bacterial antigens that are transmigrated or dis- gen is converted into plasmin, the latter enzyme
lodged or phagocytized in macrophages/foam can dissolve the fibrinogen fibers that entangle the
cells in atherosclerotic plaques, as well as host blood cells in a blood clot; this is called fibrinoly-
reactions to the bacteria and bacterial compo- sis. Thus, the plasminogen–plasmin axis has an
nents which are entered into periodontal lesions important function in tissue degradation and con-
from the subgingival biofilm. trol in the blood coagulation system. Interestingly,
One of the first and best replicated genetic loci bacteria (including P. gingivalis) can aggregate
for CAD is the ANRIL locus. The ANRIL locus is with plasminogen and can convert plasminogen to
a regulatory region and does not contain a protein- plasmin, and this complex is highly proteolytic
encoding gene. It is a long noncoding antisense and can inactivate plasmin inhibitors causing per-
RNA formerly called CDKN2BAS. Importantly, haps uncontrolled plasmin activity. Although we
it appears to be a pleiotropic genetic region, since have no clear picture yet on the precise conse-
it is also associated with diabetes type 2, ischemic quences of genetic variants in PLG, more and
stroke, and Alzheimer disease. Since 2009, it is more pleiotropic genetic regions are identified and
reported that certain genetic variations in ANRIL may form the basis for common diseases such ath-
are also consistently associated with periodontitis erosclerosis and periodontitis (Vaithilingam et al.
(Schaefer et al. 2009; Ernst et al. 2010). Further, 2014); then, periodontitis is not causally related to
a conserved noncoding element within CAMTA1 atherosclerosis, but rather the sequel of common
upstream of VAMP3, also first identified as a aberrant inflammatory pathways.
genetic susceptibility locus for CAD, was found
to be associated with periodontitis (Bochenek
et al. 2013). Experimental work suggests that 3.9 Perspective and Concluding
ANRIL and VAMP3 are part of biological path- Remarks
ways (regulatory networks) that connect glucose
and fatty acid metabolism steps with immune Epidemiological studies have clearly indicated
responses (Bochenek et al. 2013; Schwenk et al. beyond any doubt that atherosclerosis and athero-
2012). Interestingly, a genome-wide association sclerotic events (CAD, cerebrovascular disease,
study suggested that VAMP3 locus was associ- PAD, and death due to ischemic events) are asso-
ated with a higher probability of subgingival col- ciated with periodontitis. However, it is impor-
onization of periodontal pathogens (Divaris et al. tant to note that it is not proven that periodontitis
2012). Collectively, although speculative, these plays a causative role in the pathophysiology of
shared genetic factors suggest a mechanistic link atherogenesis or atherosclerosis. Atherosclerosis,
between CAD, periodontitis, diabetes, meta- actually the presence of atherosclerotic lesions
bolic syndrome, obesity, and inflammation. The that slowly progress and can rupture with acute
impairment of the regulatory pathways by genetic MI or stroke as consequence, is considered a
factors may be a common pathogenic denomina- complex disease. Like for periodontitis, complex-
tor of at least CAD and periodontitis (Schaefer ity of atherosclerosis means that it is a disease
et al. 2015; Loos 2015). We hypothesize that an process involving multiple causal components,
Chronic oral infection

Bacteria get into
bloodstream
Pro-inflammatory
Bacteremia Endotoxinemia
mediators in pocket
Bacteria
invade cells Pro-inflammatory
Host response/ mediators in liver
Bacteria
inflammation
found in atheromas
Pro-thrombotic
mediators
Bacteria
alive in atheromas
Autoimmune
Bacteria mediators
induce atheroma
Atherothrombotic
Bacteria mutants: lesion Dyslipidemia
less atherogenic
Bacteria in atheromas Genetic background

cause disease
Fig. 3.2 A composite of biologically plausible mecha- et al. (2013) and based on references (Schenkein and Loos
nisms how periodontitis might be linked to atherogenesis 2013; Reyes et al. 2013))
and atherosclerosis (Reprinted with permission for Tonetti
which interplay with each other simultaneously. ies from patient to patient. For example, not every
In complex systems, the causes and effects may patient who suffered from an acute MI may have
behave disproportional, so that a small cause may had high cholesterol or was a smoker.
result in a large effect, and vice versa, and that In this chapter, we have outlined possible
the disease progression rate fluctuates or, rather, mechanisms, as to how periodontitis may be
can move from a non-acute or chronic state to an another risk factor for atherosclerosis. These
acute phase without a special “warning signal.” have been discussed before in the EFP/AAP
Complexity of atherogenesis and atherosclero- workshop (Tonetti et al. 2013) and are summa-
sis reveals heterogeneity in its clinical course rized in Fig. 3.2.
and in the various phenotypes between diseased For the link of periodontitis with atherosclero-
patients. sis, researchers suggest that in fact the daily
There are several main causal risk factors for occurrence of multiple short-lived bacterial dis-
atherosclerosis to occur, including: (i) genetic seminations may be at the base of a possible
risk factors and epigenetic modifications of the causal role of periodontitis. But also the fact that
genetic code; (ii) lifestyle-related factors, such as periodontitis is a chronic inflammatory disease,
diet and fat intake (high/low cholesterol- which can cause a spillover of pro-inflammatory
containing foods); (iii) systemic diseases such as cytokines and induces a pro-inflammatory state,
diabetes and its sequelae on the condition of the dyslipidemia and a prothrombotic state, can pres-
blood vessel walls, smoking, high blood present the risk to increased atherogenesis and/or
sure, and obesity; (iv) now also chronic inflam- enhanced pathology of atherosclerotic lesions.
matory processes and/or chronic infections are However, a common genetic susceptibility for
considered as another cause for atherosclerosis. It atherosclerosis and periodontitis (and perhaps
is important to understand that, while these fac- other linked chronic diseases) could dictate the
tors mentioned above play simultaneously a role way the host responds in general to certain types
in the pathobiology of atherosclerosis, the rela- of inflammatory processes. These diseases may
tive contribution of each of the causal factors var- share the same inflammatory pathways in reaction
32 B.G. Loos et al.
to, for example, bacteria, be it in the gingival tis- Christan C, et al. White blood cell count in generalized
aggressive periodontitis after non-surgical therapy.
sues or be it in the circulation or atherosclerotic
J Clin Periodontol. 2002;29(3):201–6.
lesion. Some pleiotropic genes have been identi- D’Aiuto F, et al. Periodontitis: from local infection to sys-
fied, such as ANRIL, CAMTA1, and PLG. temic diseases. Int J Immunopathol Pharmacol.
Independently, whether there is a true causal 2005a;18(3 Suppl):1–11.
D’Aiuto F, et al. Short-term effects of intensive periodon-
contribution of periodontitis to atherosclerosis,
tal therapy on serum inflammatory markers and cho-
periodontitis treatment studies have indicated lesterol. J Dent Res. 2005b;84(3):269–73.
that the clinical condition of the vascular system D’Aiuto F, Orlandi M, Gunsolley JC. Evidence that peri-
can improve, i.e., the degree of atherosclerosis odontal treatment improves biomarkers and CVD
outcomes. J Clin Periodontol. 2013;40 Suppl 14:
can be reduced by periodontal therapy (Tonetti
S85–105.
et al. 2013; Teeuw et al. 2014). For example, Demmer R, Papapanou P. Epidemiologic patterns of
endothelial dysfunction can be decreased after chronic and aggressive periodontitis. Periodontol
periodontal therapy and other studies showed a 2000. 2010;53:28–44.
Dietrich T, et al. The epidemiological evidence behind the
decrease in the intimedia thickness of the carotid
association between periodontitis and incident athero-
arteries (Loos 2015). Also hsCRP is clearly sclerotic cardiovascular disease. J Clin Periodontol.
reduced after periodontal therapy and even some 2013;40 Suppl 14:S70–84.
studies showed a reduction in blood pressure and Divaris K, et al. Genome-wide association study of peri-
odontal pathogen colonization. J Dent Res. 2012;91(7
dyslipidemia. With these clinical findings in
Suppl):21S–8.
mind, the proposed mechanisms as to how peri- Eke P, et al. Prevalence of periodontitis in adults in the
odontitis may be linked to atherosclerosis and its United States: 2009 and 2010. J Dent Res. 2012;91:
sequelae seem plausible. Obviously, much more 914–20.
Eke P, et al. Update on prevalence of periodontitis in
fundamental and clinic research is needed to fur-
adults in the United States: NHANES 2009–2012.
ther study the associations between periodontitis J Periodontol. 2015;17:1–18.
and atherosclerotic diseases. Emerging Risk Factors Collaboration, et al. C-reactive
protein concentration and risk of coronary heart dis-
ease, stroke, and mortality: an individual participant
meta-analysis. Lancet. 2010;375(9709)):132–40.
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Linkage Between Periodontal
Disease and Diabetes Mellitus
4
Palle Holmstrup and Allan Flyvbjerg
Abstract
The past decades have significantly widened the perspectives of the
chronic oral infectious disease known as periodontitis. The disease is
regarded as a bacterial infection resulting in low-grade inflammation of
the periodontal tissues, and both the associated release of pro-inflammatory
mediators and the presence of bacteria in the periodontal pockets, which,
as the result of daily procedures, may spread after penetration of the vas-
culature, are possible mediators of systemic consequences. This chapter
deals with the possible association between periodontitis and diabetes
mellitus which is believed to possess in a two-way interrelationship.
4.1 Diabetes Mellitus and type 2 DM. While type 1 DM is due to an

autoimmune reaction of polygenic origin with
Diabetes mellitus (DM) comprises a hetero- destruction of the insulin-producing β cells of
geneous group of disorders, characterized by the pancreas, resulting in insulin deficiency, type
increased blood glucose level (Bell and Polonsky 2 DM is related to insulin resistance at cellular
2001). The two most common forms are type 1 and organ levels and altered lipid metabolism due
to inactivity, high intake of food, and obesity in
genetically susceptible individuals. As compen-
sation, the β cells in type 2 DM patients are stim-
P. Holmstrup, PhD, Dr.Odont., Odont.Dr. (h.c.) (*) ulated to increase their insulin secretion, but this
Section 1, Periodontology and Oral Microbiology,
compensatory mechanism may over time become
and Medical Sciences, University of Copenhagen, insufficient to maintain the blood glucose level
20 Norre Alle, Copenhagen N DK-2200, Denmark within the normal range, thereby resulting in overt
e-mail: pah@sund.ku.dk type 2 DM (Kahn 2001; Nolan et al. 2011). Type
A. Flyvbjerg, MD, DMSc 2 DM is the most widespread endocrine disorder
The Medical Research Laboratories, and the prevalence of the disease is increasing due
Department of Clinical Medicine, Faculty of Health,
to a worldwide growth in number of people with
Aarhus University, Nørrebrogade 44, Aarhus C
DK-8300, Denmark overweight and obesity. Thus, in 2025–2030, it is
e-mail: dean.health@au.dk estimated that well above 300 million individuals

DOI 10.1007/978-3-319-25091-5_4
36 P. Holmstrup and A. Flyvbjerg
worldwide will suffer from type 2 DM, the preva- 4.2 Association of Periodontitis
lence being above 6 % (Zimmet et al. 2001; Wild and DM
et al. 2004; Kaul et al. 2012). Globally, type 1
DM, which is the predominant DM type among 4.2.1 Population Data
younger individuals, accounts for 5–10 % of total
DM cases (American Diabetes Association 2009; In the recent decades, the relationship between
SEARCH for Diabetes in Youth Study Group DM and periodontitis has been subjected to
and Liese 2006). Type 2 DM, accounting for the increasing scientific interest because the preva-
remaining DM cases, was previously considered lence of type 2 DM is increasing, and because the
a disease of the elderly, but is increasingly seen bidirectional interaction between the two dis-
in younger generations, now also in children and eases has major impact on the affected patients.
young adults (Pinhas-Hamiel and Zeitler 2005). Dentists and medical doctors should be aware of
Prediabetes is part of the natural history of type the interaction between the two diseases.
2 DM and it is defined by American Diabetes A large body of cross-sectional and longi-
Association (ADA) as “a condition, in which tudinal studies have demonstrated that both
blood glucose levels are higher than normal, but type 1 and type 2 DM patients suffer more peri-
not yet diabetic, known as impaired glucose tol- odontitis than do nondiabetic patients (Glavind
erance or impaired fasting glucose” (The Expert et al. 1968; Hugoson et al. 1989; Thorstensson
Committee on the Diagnosis and Classification of and Hugoson 1993; Grossi et al. 1994, 1995;
Diabetes Mellitus 1997). The condition is highly Dolan et al. 1997; Skrepcinski and Niendorff
prevalent, and a significant number of patients 2000; Xavier et al. 2009; Ochoa et al. 2012;
with prediabetes develop type 2 DM within 10 Poplawska-Kita et al. 2014, for recent reviews,
years (Benjamin et al. 2003). see Casanova et al. 2014; Wu et al. 2015).
A significant proportion of up to 50 % of indi- Gingival inflammation in type 1 diabetic chil-
viduals currently suffering from type 2 DM pre- dren and adolescents is more common than in
sumably remains undiagnosed (Glumer et al. 2003, nondiabetic controls (Ryan et al. 2003). Further,
Guariguata et al. 2011), which result in persistent gingivitis is more common in adults with type
poor metabolic control in individuals with unrec- 2 DM than in nondiabetic controls (Orbak et al.
ognized type 2 DM. Poor metabolic control in DM 2008), and good metabolic control appears to
patients is associated with a number of complica- reduce the prevalence of gingivitis (Albandar
tions including low-grade inflammation and mac- and Tinoco 2002). The progression from gin-
rovascular and microvascular changes, including givitis to periodontitis is dependent on the level
cardiovascular disease (CVD), eye and kidney of metabolic control. Thus, poor metabolic con-
problems, impaired wound healing, and increased trol is an important determinant of periodon-
prevalence and severity of periodontitis (Morain tal breakdown in DM patients (Tervonen and
and Colen 1990; Löe 1993; Valensi et al. 1997; Karjalainen 1997; Iughetti et al. 1999; Garcia
Stratton et al. 2000; King 2008). Among the mac- et al. 2015), and since poor metabolic control
rovascular changes, accelerated atherosclerosis is is often associated with poor oral hygiene, it
part of the development of CVD and cerebrovascu- has been suggested that dental health educa-
lar events (Kannel and McGee 1979; Manson et al. tion is particularly important in this population
1991). The microvascular changes may result in (Aggarwal and Panat 2012). A recent European
renal failure and blindness (Anonymous 1996). study confirmed that well-controlled type 2 DM
DM has been known for years to be an important is not associated with periodontitis and neither
risk factor for periodontitis, and periodontitis is does prediabetes associate with periodontitis
increasingly considered a late complication of DM (Kowall et al. 2015). Moreover, the prevalence
(Löe 1993; Preshaw and Bissett 2013). An overall of periodontal sites with moderate to severe
estimate for risk of periodontitis in DM patients is attachment loss depends on the duration of type
increased by 2–3 times (Casanova et al. 2014). 2 DM (Al-Khabbaz 2014).
4 Linkage Between Periodontal Disease and Diabetes Mellitus 37
There is growing evidence that periodontitis periodontal tissue degradation is still to be resolved
may aggravate the course of DM, but the effect of (Kongstad et al. 2009). An obvious similarity of
periodontitis on glycemic control in type 1 DM the two diseases is the increased level of oxidative
patients is controversial. However, evidence shows stress (Bullon et al. 2009).
a direct correlation between periodontal health and The role of antibodies to periodontopathic bac-
glycemic control in type 2 DM patients (Lakschevitz teria for the development of periodontal tissue
et al. 2011). A systematic review of epidemiologic destruction is not clarified due to contradictory
observational studies concluded that periodontal results of available studies. Whether the antibodies
disease adversely affects DM outcomes, i.e., meta- are primarily protective or contribute to tissue deg-
bolic control and development of late complica- radation is unresolved, and the role of antibodies in
tions, but also that further longitudinal studies are type 2 DM is similarly controversial (Zhu and
warranted (Borgnakke et al. 2013). Thus, clinical Nikolajczyk 2014). On the other hand, B cells from
investigations have associated periodontitis with patients with periodontitis have been shown to pro-
increased risk of complications in DM patients duce a pro-inflammatory cytokine profile similar to
(Saremi et al. 2005) and with increased HbA1c in that of B cells from patients with type 2 DM
nondiabetic patients (Demmer et al. 2010). (Nikolajczyk et al. 2012). Finally, hyperlipidemia
The best evidence of the significance of peri- seems to interact with DM and periodontitis by
odontitis for the course of DM probably comes increasing the risk of both diseases. The production
from clinical studies on the effect of periodontal of pro-inflammatory cytokines is increased by
treatment in DM patients (see below). hyperlipidemia, and this may aggravate both insu-
lin resistance and periodontitis (Zhou et al. 2015).
4.2.2 Biological Similarities

4.2.3 Possible Mechanisms
Both DM and periodontitis may be associated of Association
with a state of low-grade inflammation. Thus, dys-
regulation of the cytokine production is essential There are several ways in which DM may influence
for the pathogenesis of DM (Kolb and Mandrup- the periodontal tissues, including cellular activities
Poulsen 2010), and pro-inflammatory cytokines are therein. These involve an impact of DM on the
increased in both diseases, including tumor necro- composition of the periodontal microflora, but
sis factor α (TNF-α) and interleukin 1β (IL-1β), methodological problems in the studies published
IL-6, and IL-18. These increased cytokine levels so far prevent a firm statement. Thus, it remains
may contribute to insulin resistance and to diabetic unclear whether microbiologic differences between
complications, as well as to destruction of pancre- DM patients and controls may be due to the dia-
atic β cells (Johnson et al. 2006; Graves and Kayal betic state, or the result of more severe periodonti-
2008; Nikolajczyk et al. 2011; Cruz et al. 2013). tis. A recent review concluded that DM and the
Likewise, pro-inflammatory cytokines produced level of glycemic control have no significant effect
locally in the inflamed periodontal tissues, where on the periodontal microbiota (Taylor et al. 2013b).
they are involved in tissue-destructive processes, Both the periodontal ligament connective tissue
may spill into the circulation with systemic impact and the tooth supportive bone may be affected by
and contribute to a state of low-grade inflammation processes closely linked to loss of glycemic control.
(Amar et al. 2003; Elter et al. 2006; Garlet 2010). Most important is probably the formation of
Interestingly, adipose tissue is an important source advanced glycation end products (AGEs) associ-
of cytokine production, and obesity may predispose ated with hyperglycemia (Degenhardt et al. 1998).
to both type 2 DM and periodontitis (Hotamisligil Formation of AGEs is due to a non-enzymatic gly-
et al. 1993; Genco et al. 2005; Pischon et al. 2007; cation of proteins and lipids, which results in func-
Saito and Shimazaki 2007; Lontchi-Yimagou et al. tional changes in intra- and extracellular proteins.
2013), although the significance of obesity for The AGEs formed also imply modified functions of
Diabetes
Adiposity
& dyslipidemia
Hyperglycemia
RANKL/OPG axis AGE/RAGE axis Oxidative stress
Ecological Immune dysfunction, cellular stress &

shift in Adipokines
cytokine imbalance (↑ TNFα, IL-6, IL-1)
subgingival
biofilm
Enhanced Impaired
tissue destruction tissue repair
Periodontitls
Fig. 4.1 Network of potential mechanisms involved in the tion. The end result is a loss of equilibrium where
pathogenesis of periodontitis in diabetes. The hypergly- enhanced periodontal tissue destruction and impaired
caemic state that characterizes diabetes has several delete- repair ensue, leading to accelerated and severe periodonti-
rious effects. It drives the formation of irreversible tis. Importantly, as shown, several of the associations
advanced glycation end-products(AGEs) and the expres- between the different elements in the figure are bidirec-
sion of their cheif signalling receptor RAGE. This interac- tional, for example, the pro-inflammatory state further
tion, in turn, leads to immune cell dysfunction, alters feeds the generation of AGEs, ROS, and adipokines,
phenotype and function of other key cells in the periodon- increases the RANKL/OPG ratio and helps pathogenic
tium, and contributes to cytokine imbalance with increased subgingival bacteria thrive. It is also important to note that
generation of certain pro-inflammatory cytokines. (a) the amount and quality of evidence supporting the
Hyperglycaemia also contributes to enhanced levels of various pathways in this figure varies, and (b) although the
reactive oxygen species (ROS) and a state of oxidative goal is to depict the major mechanisms and networks
stress, both directly and indirectly through the AGE/RAGE described in the literature, other pathways and links
axis, promoting quantitative and qualitative shifts in cyto- among the various elements shown do exist, but cannot
kine profiles. Finally, hyperglycaemia modulates the easily be demonstrated in a single schematic. Finally, the
RANKL/OPG ratio, again directly and indirectly via the processes outlined are potentially modified by several
AGE/RAGE axis, tipping the balance towards enhanced other factors, such as genetics, age, smoking, stress, all of
inflammation and destruction. All the above, comple- which may contribute significantly to inter-individual
mented by the effects of ecological shifts in the subgingi- variations in disease experience (From Taylor et al
val biofilm and the circulating adipokines generated due (2013a,b). Reproduced with permission from the
to diabetes-associated adiposity and dyslipidaemia, drive American Academy of Periodontology, European
this vicious cycle of cellular dysfunction and inflamma- Federation of Periodontology and John Wiley and Sons)
cells and their receptors. The binding of AGE to one apoptosis, reduced bone formation, and increased
of its receptors (RAGE), the expression of which is bone resorption, as thoroughly reviewed recently by
increased in DM, may result in synthesis of Taylor, Preshaw and Bissett (2013), Zhu and
inflammation-stimulating cytokines, activation of Nikolajczyk (2014) and Wu, Xiao and Graves
nuclear transcription factor-κB (NF-κB), and pro- (2015). The first mentioned of these reviews pre-
duction of reactive oxygen species (ROS) (Brownlee sented a model of a mechanism of DM-related bone
2001), all of which may result in increased cellular loss in periodontitis (Fig. 4.1).
The significance of the AGE/RAGE binding in DM-related periodontitis, but evidence from
has been demonstrated in experimental studies clinical studies is so far inconclusive (Taylor
using administration of soluble RAGE, which is et al. 2013b).
the extracellular ligand-binding domain of RAGE, The role of neutrophils in the development of
and administration of a RAGE antagonist pre- periodontitis, in general, is considered protective,
vented periodontitis progression in hyperglycemic and changes in neutrophil function may account
diabetic mice (Lalla et al. 2000). Also, decreased for an increased susceptibility to periodontitis.
levels of TNF-α, IL-6, and matrix metalloprotein- Indeed, neutrophil function in DM patients with
ases (MMPs) in the gingival tissue were found. periodontitis has been studied intensively. The
Other studies have demonstrated that RAGE has outcome of studies based on peripheral neutro-
fundamental influence on the increased periodon- phils may conceivably differ from that of neutro-
tal tissue destruction, which is why antagonists of phils located in periodontal tissues. However,
RAGE have been proposed as a therapeutic tool signs of compromised neutrophil function have
for the management of DM-associated periodon- been presented in humans, since neutrophil-
titis (Lalla et al. 2001). Interaction of AGEs with derived β-glucuronidase and IL-8, which has a
toll-like receptors (TLRs) has been described, chemotactic effect on neutrophils, were depressed
as well as increased expression of TLR2, TLR4, in type 2 DM patients with periodontitis
and TLR9 in periodontitis-affected tissues of DM (Engebretson et al. 2006). Experimental animal
patients, as compared with periodontitis-affected studies in rodent models of DM and/or periodon-
tissue from controls without DM (Rojo-Botello titis have also revealed reduced neutrophil func-
et al. 2012). Further investigations of the TLR- tion (Golub et al. 1982; Sima et al. 2010).
mediated pathways in DM and periodontitis are It is well established that hyperglycemia in
obviously needed. DM patients may predispose to periodontal tis-
B cells, which dominate the inflammatory sue destruction, and a large amount of studies
reaction in the established periodontitis lesion, have scrutinized the possible pathologic pathways,
have been described as a major source of receptor by which DM may have impact on the course of
activator of nuclear factor-κB ligand (RANKL) periodontitis. Fewer studies have dealt with the
with a pro-osteoclastogenic effect (Onal et al. pathways by which periodontitis may affect the
2012), and since RANKL expression is increased course of DM. High levels of CRP in patients with
in mice with type 2 DM (Cao et al. 2010), an both diseases have been associated with increased
exaggerated RANKL expression may potentiate HbA1c levels, and since periodontitis itself may
periodontal bone destruction in type 2 DM account for higher levels of CRP, the additional sys-
patients (Zhu and Nikolajczyk 2014). Another temic inflammation associated with periodontitis
source of RANKL is the T cell, but the role of T may be responsible for the increased HbA1c levels
cells, including whether T-cell produced RANKL in DM patients with periodontitis (Demmer et al.
plays a role in DM-associated periodontitis, 2010). Insulin resistance in periodontitis patients
remains to be clarified. with DM may be promoted by hyperreactive neu-
Several studies have investigated the influ- trophils producing reactive oxygen species, which,
ence of DM on the cytokine profile of patients in turn, may stimulate pro-inflammatory pathways
with periodontitis, and the results reported so far (Allen and Matthews 2011). An interesting asso-
are inconsistent; they are cross-sectional, or they ciation of periodontal microbiota with prediabetes
are lacking confirmative support from other prevalence in young adults has been described in a
studies. Elevated levels of IL-1β in serum and recent cross-sectional study (Demmer et al. 2015).
crevicular fluid from DM patients with chronic Although it is up to future longitudinal studies
periodontitis seem to be the most consistent find- to determine whether such interrelationships are
ing (reviewed by Taylor et al. (2013b) and by causal, the finding that levels of potentially peri-
Atieh et al. (2014)). Studies in animal models odontopathic subgingival bacteria are abundant
have also emphasized the role of TNF-α in pro- in and predictive of prevalent prediabetes is new
longing the bacteria-induced immune response knowledge (Demmer et al. 2015).
A recent review has focused on the signifi- criteria: randomized controlled study in humans,
cance of resistin, a biomarker for the levels of intervention study on diabetic patients with peri-
which are increased in chronic inflammation odontal disease, minimum 3 months follow-up
including periodontitis. Since resistin has been observation, including data on HbA1c and/or
shown to induce insulin resistance in mice, it has fasting plasma glucose change after treatment,
been proposed as a possible link between peri- and clear presentation of population demographic
odontitis and DM (Devanoorkar et al. 2014). data (Corbella et al. 2013). The majority of the
Several experimental studies in rodents have patients included in the studies were affected by
provided insight in the possible interactions uncontrolled type 2 DM, and only one study
between periodontitis and DM (for review, see involved patients with type 1 DM. The meta-
Andersen et al. (2007b)). Interestingly, ligature- analyses showed that nonsurgical periodontal
induced periodontitis has been shown to deteriorate treatment significantly reduces the level of
metabolic control in type 2 DM rats with an HbA1c and fasting plasma glucose in patients
increase in oral glucose tolerance test of as much as with DM. The mean decrease of HbA1c was
30 %, and an increase in IL-1β in adipose tissue 0.4 % after 3 months and 0.3 % after 6 months,
compared to diabetic rats without periodontitis and the decrease in fasting plasma glucose was
(Andersen et al. 2006). In prediabetic rats with lig- 9.0 mg/dL after 3 months and 13.6 mg/dL after 6
ature-induced periodontitis, the glucose tolerance months, and there was no positive effect of
was also significantly impaired, which suggests adjunctive antimicrobials. The authors stated that
that periodontitis may facilitate the development it was difficult to quantify the clinical relevance
of manifest type 2 DM (Andersen et al. 2007a). of the findings in terms of improved glycemic
Moreover, the prediabetic rats with periodontitis control. Another meta-analysis of randomized
developed renal alterations including kidney hyper- clinical trials included five studies of patients
trophy and a tendency for increased glomerular with type 2 DM (Sgolastra et al. 2013). The
volume (Andersen et al. 2008). inclusion criteria were almost similar to the
Although a number of interactions of peri- abovementioned, and the primary outcome vari-
odontitis with DM may appear obvious, there is ables were changes in HbA1c and fasting plasma
still little evidence to understand the mechanistic glucose, while secondary outcomes were changes
pathways of periodontitis’ influence on DM, and in total serum cholesterol, serum triglycerides,
most is presently speculative. and high- and low-density lipoprotein choles-
terol. The result of the meta-analysis was that the
periodontal treatment after 3–6 months resulted
4.2.4 Outcome of Periodontal in a significant reduction in HbA1c, and in fast-
Treatment ing plasma glucose, the mean differences being
0.7 % and 9.0 mg/dL, respectively. Periodontal
A large number of studies have examined the role treatment resulted in no significant differences in
of periodontal treatment for the course of DM, the secondary outcomes. This meta-analysis has
but long-term randomized clinical trials are been criticized for the use of too restrictive exclu-
scarce. The studies are characterized by different sion criteria, which may limit the generalizability
inclusion criteria of patients, including various of the meta-analysis to a fraction of the relevant
types of DM and various diagnostic criteria for a population (Janket 2014). Finally, a meta-analysis
case of periodontitis. Moreover, stratification for has been presented of the effect of nonsurgical
confounders such as smoking, overweight, and periodontal treatment on systemic inflammation
medication is difficult. The current evidence has in patients with type 2 DM (Artese et al. 2015).
been critically reviewed and analyzed in several Exclusion of studies due to study design and
papers. A meta-analysis of the outcome of non- missing data resulted in four included studies
surgical periodontal treatment was performed involving associations with CRP and two involv-
based on 15 papers selected on the following ing associations with TNF-α, the primary
outcome measures being high sensitivity CRP some of their patients for diabetes. In favor of
(hsCRP) or CRP, IL-6, and TNF-α. Adjunctive such an arrangement is the fact that the attitude of
antimicrobial therapy was combined with scaling dentists and their patients is positive to these
and root planing in four of the included studies. A medical examinations performed in the dental
significant reduction as the result of treatment setting (Greenberg and Glick 2012; Greenberg
was found for both TNF-α (−1.33 ng/L) and et al. 2012).
hsCRP (−1.28 mg/L). Taken together, the studies
indicate a positive effect on metabolic control Conclusion
and systemic inflammation of nonsurgical peri- The association of periodontitis with diabetes
odontal treatment. This is particularly evident in has been described as bidirectional, and there
type 2 DM patients. The clinical significance of is substantial evidence that poor glycemic con-
the improvements obtained, however, is uncer- trol in type 1 and type 2 DM patients is a risk
tain. Even small reductions in HbA1c may result of periodontitis, resulting in increased exten-
in significant clinical improvements in diabetic sion and severity of periodontitis. Due to the
complications and mortality. Thus, for every per- global increase in the prevalence of diabetes,
centage point decrease in HbA1c, 35 % reduction the influence of diabetes on the development of
in microvascular complications has been periodontitis may be a growing problem.
reported, and an average at 0.2 % point reduction Current evidence also suggests that periodonti-
in HbA1c level associates with 10 % lower mor- tis may aggravate the course of DM, but fur-
tality in type 2 DM patients (UK Prospective ther longitudinal studies are warranted for a
Diabetes Study UKPDS Group 1998). The firm conclusion to be drawn. The mechanisms
abovementioned reductions in HbA1c levels of by which the two diseases interact are uncer-
0.31–0.65 % after periodontal treatment, thereby tain, but presumably chronic low-grade inflam-
constitutes an important public health benefit. mation enhanced by both diseases plays an
Also, it should be remembered that patients with important part in the interaction, which obvi-
poor glycemic control may have more insuffi- ously involves inflammatory cells and their
cient oral hygiene, and they may visit the dentist products, including cytokines and MMPs. The
more infrequently than those with a better blood formation of AGE results in modified cellular
sugar control, as pointed out by Aggarwal and functions. The existing clinical trials indicate a
Panat (2012). This is why special periodontal positive effect on metabolic control and sys-
treatment efforts are recommendable for this temic inflammation of nonsurgical periodontal
group of patients. As mentioned above, a large treatment, which may result in a clinically rel-
proportion of type 2 DM and prediabetes patients evant decrease of HbA1c. However, further
remain undiagnosed (Glumer et al. 2003; studies are needed to robustly confirm this.
Guariguata et al. 2011), which is a general prob-
lem for the prognosis of the patients’ health con-
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Linkage Between Periodontal
Disease and Rheumatoid Arthritis
5
Palle Holmstrup and Claus H. Nielsen
Abstract
The past decades have significantly widened the perspectives of the
chronic oral infectious disease known as periodontitis. The disease is
regarded as a bacterial infection resulting in low-grade inflammation of
the periodontal tissues, and both the associated release of pro-inflammatory
mediators and the presence of bacteria in the periodontal pockets, which,
as the result of daily procedures, may spread after penetration of the vas-
culature, are possible mediators of systemic consequences. The present
chapter deals with the possible association of periodontitis with rheuma-
toid arthritis, which may possess a two-way interrelationship.
5.1 Rheumatoid Arthritis swelling. RA may occur at any age, but it usually
begins after the age of 40, and women are more
Rheumatoid arthritis (RA) is an autoimmune dis- often affected than men. Besides joints, the dis-
ease affecting 0.5–1 % of adults in developed ease may sometimes affect other organs of the
countries. The disorder is characterized by per- body including the skin, lungs, blood vessels, and
sistent synovial inflammation and destruction of eyes. An important environmental risk factor is
joint tissues including the cartilage and bone smoking (Klareskog et al. 2009).
(Scott et al. 2010). As a consequence, joint defor- Although RA is not regarded as a classical
mity occurs, typically affecting the small joints autoantibody-driven autoimmune disease, auto-
of the hands and the feet where it causes painful antibodies have been widely used as diagnostic
tools. These autoantibodies include rheumatoid
factors, which are directed against the constant
P. Holmstrup, DDS, PhD, DrOdont,
region of immunoglobulins of the IgG isotype
OdontDr (hc) (*) • C.H. Nielsen, PhD, MD, MSc and are present in 70–80 % of the patients with
Section 1, Periodontology and Oral Microbiology, RA (Friswell 2004). Rheumatoid factors occur in
Department of Odontology, Faculty of Health many different acute and inflammatory diseases
and are thus a rather nonspecific marker of RA.
20 Norre Alle, Copenhagen N DK-2200, Denmark
e-mail: pah@sund.ku.dk; Anti-citrullinated protein antibodies (ACPAs), on
claus.henrik.nielsen@regionh.dk the other hand, are also found in 70–80 % of

DOI 10.1007/978-3-319-25091-5_5
46 P. Holmstrup and C.H. Nielsen
patients with RA (Schellekens et al. 2000), and, cations in RA patients (Havemose-Poulsen et al.
with a specificity as high as 98 % (Schellekens 2006). However, the outcomes of the many stud-
et al. 2000), they are a more specific marker for ies are seriously hampered by varying diagnostic
RA. Practically all patients with ACPAs have criteria for both diseases. In one of the studies on
HLA-DRB1 molecules containing the so-called periodontitis, cases were identified on the basis
shared epitope capable of binding citrullinated of mean clinical attachment loss being ≥4 mm,
peptides (peptides containing the nonstandard and the odds ratio for simultaneous occurrence of
amino acid residue citrulline), which are thought RA was as much as 6.09 (95 % CI 1.72–21.55),
to induce pathogenic T-cell responses, primarily indicating a strong association although the con-
subtypes 0401, 0404, and 0408 in the white pop- fidence interval was wide. More extensive cross-
ulation and 0405 in Asians (Nepom and Nepom sectional studies have provided weaker evidence
1992; Wordsworth et al. 1992). In this subgroup of an association, the odds ratios being 1.82–1.94,
of patients, posttranscriptional conversion of and one of these had ranges of 95 % confidence
arginine to citrulline, catalyzed by enzymes des- indicating insignificance (Pablo et al. 2008;
ignated peptidylarginine deiminases (PADs), is Demmer et al. 2011). A 20-year prospective fol-
regarded an important part in the pathogenesis of low-up study including 9,564 American adults
the disease (Schellekens et al. 1998). Smoking is defined periodontitis cases by tooth loss of four
thought to mediate release of PADs, which may or more teeth with attachment loss or worse con-
explain why smoking is a particularly strong risk ditions. Baseline and incident cases of RA were
factor in ACPA-positive patients, who also defined on the basis of self-reported physician
develop bone erosions earlier and more widely diagnosis or physical examination data corre-
spread than anti-CCP-negative patients. Many sponding to criteria 1–4 of the American
investigators therefore consider ACPA-positive Rheumatism Association 1987 criteria (Arnett
and ACPA-negative RA as two different disease et al. 1988). Incident RA was also defined on the
entities. basis of death certificate data or health-care facil-
ity discharge diagnosis of rheumatism. The
adjusted odds ratios for incident RA were
5.2 Association of Periodontitis between 1.12 and 1.67, dependent on number of
and RA missing teeth among participants. Most odds
ratios were statistically insignificant, and there
5.2.1 Population Data was a lack of dose responsiveness (Demmer et al.
2011). A similar result was obtained in a compre-
Several studies have indicated a positive associa- hensive 12-year prospective follow-up study in
tion between periodontitis and RA, sometimes American women (Arkema et al. 2010). Overall,
referred to as a bidirectional interaction between the present data indicating an epidemiological
the diseases (Cantley et al. 2011). On the other association between periodontitis and RA are
hand, most of the available population studies are inconsistent. The varying case definitions used
small case–control studies providing limited evi- for both periodontitis and RA may, in part,
dence of an association between the two diseases. explain the differences in results obtained. Also,
Some studies have demonstrated that patients patients with RA are usually receiving intensive
with RA are more likely to acquire advanced anti-inflammatory treatment, which ameliorate
periodontitis than individuals without RA. This periodontal disease progression. The inconsistent
has been shown for young adults (20–35 years) results may also be due to an inhomogeneous
(Havemose-Poulsen et al. 2006) and for midlife nature of the patients with the two disease cate-
to aged people (Käßer et al. 1997; Mercado et al. gories, which may both contain patients with
2000; Mercado et al. 2001). Based on such find- more than one disease.
ings, it has been proposed to develop systematic A recent systematic review showed that
programs for prevention of periodontal compli- seven out of ten case–control studies had found
5 Linkage Between Periodontal Disease and Rheumatoid Arthritis 47
significantly more clinical tooth attachment loss are characterized by their inflammatory nature
in RA patients compared to controls (Kaur et al. with local degradation of collagen-rich soft and
2013). The same review reported that five of hard tissues mediated by cytokines and collagen-
seven studies found significantly increased tooth olytic enzymes. Based on the above findings, it is
loss in RA patients compared to controls. When likely that there is some degree of coexistence,
combining the results of the included studies in a although it is uncertain whether an association is
meta-analysis, the weighed mean differences causal or noncausal, for instance, due to shared
were significant in both clinical attachment level environmental or other predisposing factors, i.e.,
and tooth loss between RA patients and non-RA smoking, and socioeconomic and genetic risk
controls. This finding is further supported by a factors such as MHC class II HLA-DRB1 (Firatli
Dutch cross-sectional study, in which a signifi- et al. 1996; Katz et al. 1987; Marotte et al. 2006;
cantly higher prevalence of severe periodontitis Bonfil et al. 1999).
in RA patients (27 %) than in controls (12 %) was
seen (De Smit et al. 2012). Furthermore, a case–
control study compared the presence of severe 5.2.3 Possible Mechanisms
periodontitis in 287 patients with RA with that in of Association
a noninflammatory arthritis control group of 330
patients with osteoarthritis, believed to be demo- Both periodontitis and RA have cytokine profiles
graphically similar to the RA group (Mikuls et al. thought to be involved in the tissue-destructive
2014). Anti-cyclic citrullinated peptide antibody- inflammatory processes, including high produc-
positive patients were significantly more likely to tion of TNF-α (Cantley et al. 2011). An impor-
have periodontitis (37 %) than the osteoarthritis tant example is the common pathway of
controls (26.4 %). A multivariate analysis upregulated expression of receptor activator of
accounting for confounding factors showed that nuclear factor kB ligand (RANKL) by fibroblasts
the anti-cyclic citrullinated peptide antibody- and lymphocytes, essential for osteoclast forma-
positive patients remained more likely to have tion (Crotti et al. 2003, Bartold et al. 2010a).
periodontitis than controls, the significant odds Obviously, an exaggerated systemic inflamma-
ratio being 1.59. In this study, tooth loss in the tion induced by periodontal infection might
RA patients was also more common than in the worsen the immune-inflammatory reactions in
control group (Mikuls et al. 2014). Taken the joints of RA patients and vice versa (Golub
together, these studies strongly indicate that the et al. 2006, reviewed by Payne et al. 2015).
periodontal status is worse in RA patients than in In an attempt to identify similarities in the
controls (Kaur et al. 2013; Payne et al. 2015). pathology of periodontitis and RA, hematologi-
However, there is currently little evidence that cal characteristics of patients with RA have been
periodontitis represents a risk factor for RA compared with those of aggressive periodontitis
(Linden et al. 2013). patients. Elevated levels of traditional markers of
inflammation could be seen in patients with gen-
eralized aggressive periodontitis similar to
5.2.2 Biological Similarities patients with RA (Havemose-Poulsen et al.
2006). Other case–control studies have compared
RA has several clinical and pathological charac- erythrocyte sedimentation rate, C-reactive pro-
teristics in common with periodontitis. The dis- tein, ACPAs (measured as autoantibodies to
eases, although chronic in nature, show periodical cyclic citrullinated peptides), rheumatoid factor,
flare-ups with increased tissue-destructive activ- TNF-α, and interleukin (IL)-1β in RA patients
ity in some of the involved sites interposed by with and without periodontitis, as systematically
periods of relative quietness, and both diseases reviewed by Kaur et al. (2013). The outcome of
are quality of life hampering because they are the studies indicates that there is no good evi-
associated with loss of function. Both diseases dence for a correlation between increased levels
of the majority of these factors and presence of while the bacterial enzyme targets carboxytermi-
periodontitis and RA. An exception is IL-1 level, nal arginine residues (McGraw et al. 1999) (after
which appears to be increased in patients with cleavage of protein substrates by bacterial gin-
both diseases (Kaur et al. 2013). Moreover, dys- gipains), human PADs efficiently deaminate
regulation of immunoinflammatory responses internal arginine residues (Sugawara et al. 1982).
has been found for both diseases in a number of Experimentally, PPAD is capable of citrullinat-
studies (Mercado et al. 2001; Bartold et al. 2005; ing human fibrinogen and α-enolase, and it has
Havemose-Poulsen et al. 2005) including similar been suggested that immune complexes formed
patterns of elevated IL-10 plasma levels in RA between these citrullinated proteins and ACPAs
patients and in patients with aggressive periodon- play a pathogenic role in RA (Wegner et al.
titis (Havemose-Poulsen et al. 2005). Thus, gene 2010). Of note, P. gingivalis also expresses an
expression of pro- and anti-inflammatory cyto- enolase, against the citrullinated form of which
kines in peripheral blood mononuclear cells ACPAs from patients with RA react (Lundberg
might be a common denominator for the two dis- et al. 2008). Bacterial enolase might also be
eases, but only few similarities between the two citrullinated by human PADs and act as antigens
diseases with respect to these parameters have in RA. Indeed, ACPAs have been revealed in
been found (Sørensen et al. 2009). Rheumatoid inflamed periodontal tissue (Harvey et al. 2013),
factors and ACPAs have been revealed in sera but direct evidence for a role of P. gingivalis in
from periodontitis patients (Gargiulo et al. 1982; RA remains to be established.
Thé and Ebersole 1991; Havemose-Poulsen et al. The role of protein citrullination in periodon-
2006), and levels of IgM- and IgA-rheumatoid titis also needs further elucidation. It is notewor-
factors in patients with RA were found to corre- thy that the same genetic locus associated with
late with percentage of sites with clinical attach- RA and presentation of citrullinated peptides
ment loss≥2 mm, which is why these variables (HLA-DR4) is also associated with severe and
have been proposed as possible predictors of rapidly progressive periodontitis, mainly sub-
periodontal tissue destruction (Havemose- types HLA-DRB1*0401, - 0404, −0405, and
Poulsen et al. 2005) similarly to their use as pre- −0408 (Bonfil et al. 1999). Increased levels of
dictors of joint erosions (Guillemin et al. 2003; ACPAs in RA patients with periodontitis com-
Bukhari et al. 2002). pared with RA patients without periodontitis
One of the most interesting aspects is the pos- have not been encountered (Pischon et al. 2008).
sible involvement of Porphyromonas gingivalis An essential support for a bidirectional inter-
in the pathogenesis of RA (Rosentein et al. action between the two diseases has been estab-
2004). In some studies, the frequency of anti- lished in experimental studies. Adjuvant arthritis
bodies to P. gingivalis has been shown to be sig- was induced in rats, some of which were subse-
nificantly higher in patients with RA than in quently systemically treated with tissue inhibitor
controls (Mikuls et al. 2009; Okada et al. 2011), of matrix metalloproteinases (TIMP-4). At 3
although this was not the case in another study weeks the rats were examined for signs of peri-
based on a higher number of patients (Moen odontitis. Rats untreated with TIMP-4 showed
et al. 2003). Over recent years, there has been significantly increased periodontal bone loss and
much speculation that P. gingivalis may play a tooth mobility, which was improved in rats
role in generation of the citrullinated proteins, treated with TIMP-4 (Ramamurthy et al. 2005).
which are thought to constitute the pathogenic In another experimental study, induction of
autoantigens in ACPA-positive patients. P. gingi- arthritis in mice with preexisting periodontitis
valis has been shown to produce P. gingivalis resulted in exacerbation of arthritis, as compared
peptidylarginine deiminase (PPAD), which, like to mice without periodontitis (Cantley et al.
human PADs, catalyzes citrullination of pro- 2011). Further evidence for a relationship
teins. There is no amino acid sequence similarity between inflammation and a periodontitis-
between PPAD and human PADs, however, and associated pathogen was provided in another
experimental study in rats. These animals had Conclusion

foam pieces loaded with heat-killed P. gingivalis Several studies have demonstrated an associa-
implanted in their backs with subsequent induction of periodontitis and RA. There is ample
tion of adjuvant arthritis. The study showed that evidence of similarity in the pathogenesis of
severe arthritis developed more rapidly in rats the two diseases, and a large body of studies
with preexisting P. gingivalis-induced inflamma- has shown that patients with RA suffer more
tory lesions distant from the joints than in con- periodontal attachment loss. This is why it has
trols (Bartold et al. 2010a, b). been proposed to develop systematic programs
for prevention of periodontal complications
in RA patients. Some short-term interven-
5.2.4 Outcome of Periodontal tion studies have also shown that periodontal
Treatment treatment may reduce the disease activity in
patients with RA, but larger studies with a lon-
Several studies have examined the effect of peri- ger duration are warranted, and currently there
odontal treatment on biomarkers and course of is little evidence that periodontitis represents
RA, as systematically reviewed and meta- a risk factor for RA. A possible involvement
analyzed by Kaur et al. (2014). As an example, of P. gingivalis in the pathogenesis of RA via
full-mouth scaling and root planing resulted in citrullination of proteins remains to be further
reduced erythrocyte sedimentation rate after 3 evidenced.
months in 26 patients with RA, but there was no
significant effect on the degree of disability or
IgM-rheumatoid factor level (Ribeiro et al. 2005). References
Another clinical trial including 40 patients with
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Association Between Dental
Infections and Renal and Liver
6
Diseases
Jukka H. Meurman
Abstract
The number of patients with kidney and liver diseases is increasing due to
global aging of populations, increased obesity leading to metabolic syn-
drome and diabetes, and behavioral factors such as alcohol consumption,
and contagious diseases such as hepatitis virus infections. Oral and dental
infections may have detrimental effects on the course and treatment of
these diseases and should thus be diagnosed and properly treated. The end
stage of both kidney and liver diseases calls for organ transplantation and
hence lifelong immunosuppression. This renders the patient liable for all
kinds of infections. In these patients, insidious dental infections can turn
out to be life threatening.
6.1 Introduction that less than 25 % of the function remains.

Stages of chronic kidney disease are assessed
The kidneys play a major role in body homeosta- using the glomerular filtration rate (GFR) where
sis by filtering metabolic waste products, and values below 15 ml/min/1.73 m2 mean kidney
they are also involved in a number of critical pro- failure. Severe renal diseases are treated with
cesses such as regulation of electrolyte balance, dialysis and finally with kidney transplantation.
blood pressure control, and stimulation of the red The liver, respectively, is the largest internal
blood cell production by erythropoietin. The organ and no human can survive without it. Liver
global prevalence of chronic kidney disease is helps in digestion by producing bile for lipid
estimated to be 8–16 % (Jha et al. 2013). Serious metabolism, but liver also is the principal chemi-
problems arise when the kidney function drops so cal factory of the body. It synthesizes proteins,
such as albumin, hormones, and blood coagula-
tion factors and is responsible for glucose metab-
olism and storage for many vitamins. The liver
J.H. Meurman, PhD, MD also detoxifies a number of harmful substances
Department of Oral and Maxillofacial Diseases,
such as alcohol, bacterial toxins, and many drugs.
University of Helsinki and Helsinki University
Hospital, PB 41, Helsinki 00014, Finland The renin-angiotensin system is an example of
e-mail: jukka.meurman@helsinki.fi the interplay between the liver and kidneys.

DOI 10.1007/978-3-319-25091-5_6
54 J.H. Meurman
Liver failure leads to the death of the patient turn is a marker of mortality, and albumin can
due to multi-organ failure. There are a number of also be measured from saliva samples of the
etiologic causes for liver diseases such as viral patients (Meurman et al. 2002).
hepatitis and extensive alcohol consumption, to
mention just two. Liver function can be assessed
by several clinical chemistry parameters, for 6.3 Streptococcal
example, by serum glutamyltransferase. Liver Glomerulonephritis
cirrhosis is the result of death of functioning
hepatocytes which leads to fibrosis. Its incidence Before the advent of penicillin, streptococcal glo-
is rising in the industrialized countries mainly due merulonephritis was highly prevalent (Nasr et al.
to obesity and alcohol consumption. For example, 2013). Today it is rare in industrialized countries
in the USA, death rate in liver cirrhosis in 2012 with estimates between 9.5 and 28.5 new cases
was 14.9 in men and 7.1 in women per 100,000 per 100,000 individuals per year (Rodriguez-
individuals (WHO Global Information System Iturbe and Musser 2008). Glomerulonephritis
on Alcohol and Health 2014). The ultimate treat- nevertheless is one of the two types of infections
ment of liver diseases is organ transplantation. caused by Viridans streptococci – the other being
endocarditis. These two disease entities often
occur concomitantly, and rheumatic fever should
6.2 Renal Diseases be mentioned in this context (Neugarten and
Baldwin 1984). Hence, infections of the mouth
The global increase in diabetes directly reflects in and teeth have long been known to be important
the increase of renal diseases because of diabetic causative factors of this renal disease. The caries
nephropathy. End-stage renal failure is also bacterium Streptococcus mutans has also been
mainly caused by diabetes (Atkins 2005). In associated with glomerulonephritis (Okada et al.
addition to diabetic nephropathy, there are sev- 1996). A causal link between periodontitis and
eral other etiologic factors for kidney disease. glomerulonephritis has similarly been suggested
Chronic glomerulonephritis and polycystic kid- (Ardalan et al. 2011). It is thus evident that main-
ney disease usually are slow-progressing dis- taining good oral health is important in such
eases, but there are many other heterogenic renal patients. Administering prophylactic antibiotic
diseases, such as nephrosclerosis, arteriosclerotic before dental treatment to patients with history of
nephropathy, urinary tract obstruction, tubuloin- glomerulonephritis is still recommended even
testinal nephritis, renal amyloidosis, and congen- though scientific evidence for this practice is
ital or hereditary kidney diseases. weak (Del Mar et al. 2004).
Periodontal disease is prevalent in patients
with chronic kidney disease (Akar et al. 2011;
Chambrone et al. 2013). Patients with diabetic 6.4 Liver Diseases
nephropathy have shown particularly poor oral
health compared with patients with glomerulone- Apart from metastatic oral infections and case
phritis which finding supports the known two- reports of patients with liver abscesses from dental
way relationship between oral health and diabetes origin, there is hardly any literature on the role of
(Teratani et al. 2013; Preshaw et al. 2012). oral infection in liver diseases (Gendron et al. 2000;
Periodontal pathogens have also been associated Kajiya et al. 2008). However, oral health status of
with chronic kidney disease (Niedzielska et al. patients with chronic liver disease is known to be
2014; Ismail et al. 2015). Oral health is often poor in general, and many patients suffer from
poor among patients with chronic kidney disease xerostomia (Guggenheimer 2009; Helenius-
(Vesterinen et al. 2011). Periodontitis also reflects Hietala et al. 2013a, b). Xerostomia is often associ-
in low serum albumin concentration in end-stage ated with salivary gland hypofunction, and it has
kidney disease (Kshirsagar et al. 2007). This in been shown that liver transplant recipients have
6 Association Between Dental Infections and Renal and Liver Diseases 55
Fig. 6.1 Dental infections 100

associated with the incidence No dental screening
of complications after liver 90
transplantation. Patients who
had no dental examination 80
and treatment because of
emergency operation had more 70
Cumulative incidence,%
posttransplant complications
than those whose dental 60
problems had been treated
(Modified from Helenius-Hietala 50
et al. 2013)
40
30 Dental screening
20
10
0
0 3 6 9 12 15 18 21 24 27
Months to first infection after transplantation
low whole saliva flow rates, which lead to further nonalcoholic fatty liver (Yoneda et al. 2012). In
deterioration of the oral health (Helenius-Hietala addition, periodontitis has been shown to be
et al. 2013a, b). associated with hepatocellular carcinoma
Helenius-Hietala et al. (2013a, b) have inves- (Tamaki et al. 2011). Periodontitis may further
tigated liver transplant patients and observed enhance alcohol-induced liver damage as shown
increased risk for posttransplant infection com- in an animal experiment (Tomofuji et al. 2008).
plications in those patients by whom no dental The importance of lipid and sugar metabolism of
treatment had been given before the operation the liver was further emphasized in a study where
because of lack of time, in comparison to those periodontitis was associated with hepatic steato-
with more elective transplantation: OR 8.17 sis (Saito et al. 2006). In this investigation, the
(95 % CI 2.19–30.6). Similarly, the assessed need severity of periodontitis increased with elevated
for dental extractions was found to associate with serum values measuring liver function.
reduced time from diagnosis of liver disease to
the need of transplantation and the number of Conclusion
tooth extractions correlated significantly with Scientific evidence of the role of oral infections
change in the Model for End-Stage Liver Disease in renal and liver diseases is still weak. However,
(MELD) score (Fig. 6.1) (Aberg et al. 2014). In it is known that chronic oral infections such as
the same study, Streptococcus viridans was periodontitis affect many systemic metabolic
detected in peritonitis cases only among the pathways and by causing endothelial dysfunc-
patients with dental infections. tion, for example, which is detrimental in all
Nagao et al. (2014) reported that periodontal organs (Janket et al. 2008). Studies have shown
disease may worsen the progression of liver dis- that if eradication of dental infection foci has
ease caused hepatitis virus infection by reducing been neglected, the outcome of patients with
platelet count, for example, with OR 5.80 (95 % kidney or liver disease may be compromised.
CI 2.30–14.92). The periodontal pathogen Hence, maintaining good oral health and treat-
Porphyromonas gingivalis has also been linked ing infection foci properly is highly important
to the progression of liver disease in patients with also in these patient groups (Table 6.1).
56 J.H. Meurman
Table 6.1 Aspects of oral infections in patients with kid- pretransplant dental treatment. Oral Dis. 2013b;19:
ney and liver diseases 271–8.
Ismail FB, Ismail G, Dumitriu AS, Baston C, Berbecar V,
Chronic kidney disease Liver disease
Jurubita R, Andronesi A, Dumitriu HT, Sinescu
Streptococcal Dental infections may I. Identification of subgingival periodontal pathogens
glomerulonephritis is the type cause liver abscesses and association with the severity of periodontitis in
of infection often caused by patients with chronic kidney diseases: a cross-sectional
Viridans streptococci study. Biomed Res Int. 2015;2015:370314.
Periodontal disease is Xerostomia affects doi:10.1155/2015/370314.
prevalent among the patients detrimentally oral Janket SJ, Jones JA, Meurman JH, Baird AE, Van Dyke
health TE. Oral infection, hyperglycemia, and endothelial
Periodontal pathogens have Poor dental health dysfunction. Oral Surg Oral Med Oral Pathol Oral
been associated with chronic associates with Radiol Endod. 2008;105:173–9.
kidney disease complications in liver Jha V, Garcia-Garcia G, Iseki K, Li Z, Naicker S, Plattner
transplantation B, Saran R, Wang AY, Yang CW. Chronic kidney dis-
Treating oral and dental Treating oral and ease: global dimension and perspectives. Lancet.
infections necessary before dental infections 2013;382:260–72.
dialysis/kidney transplantation necessary before liver Kajiya T, Uemura T, Kajiya M, Kaname H, Hirano R,
transplantation Uemura N, Tei C. Pyogenic liver abscess related to
dental disease in an immunocompetent host. Intern
Med. 2008;47:675–8.
Kshirsagar AV, Craig RG, Beck JD, Moss K, Offenbacher
S, Kotanko P, Yoshino M, Levin NW, Yip JK, Almas
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Association Between Oral
Infections and Cancer Risk
7
Jukka H. Meurman
Abstract
Highly prevalent dental infections have been shown to statistically associ-
ate with cancer. Periodontal disease in particular links to head and neck
cancer but also to malignancies in other organs. Metabolism of oral micro-
organisms may lead to carcinogenic substance that also affect oral mucosa
locally thus posing a risk for oral cancer. However, scientific evidence is
still weak in these associations.
7.1 Introduction where, for example, local acetaldehyde production

by oral microorganisms has been shown to pose
Cancer is always characterized by infection, but a marked risk (Kurkivuori et al. 2007; Meurman
infection may also be causally linked to the devel- 2010). However, in general there is a long latency
opment of malignancy (zur Hausen and de Villiers between the initial infection and tumor appearance,
2014). Classical examples are certain human pap- and not at all infected person develops cancer.
illomavirus (HPV) infections and cervical can-
cer and Helicobacter pylori infection and gastric
cancer. In general, chronic inflammations induced 7.2 Oral and Dental Infections
by microorganisms are considered important in and Cancer Epidemiology
the carcinogenesis (Kuper et al. 2000). Recently,
also bacterial and yeast infections of the mouth One unique characteristic in oral and dental infec-
have been statistically associated with the develop- tions is their mostly chronic nature and very
ment of cancer in various organs (Meurman and high prevalence in populations. Dental caries is
Bascones-Martinez 2011; Söder et al. 2015). Oral regarded as one of the most widespread infection
microbiome may also play a role in oral cancer of humans in the world. Also the prevalence of
periodontal disease is very high. The World Health
Organization (WHO) has estimated that 60–90 %
J.H. Meurman, PhD, MD of schoolchildren and nearly 100 % of adults
Department of Oral and Maxillofacial Diseases,
worldwide have dental caries, and, respectively,
University of Helsinki and Helsinki University
Hospital, PB 41, Helsinki 00014, Finland 15–20 % of 35–44-year-old adults have severe
e-mail: jukka.meurman@helsinki.fi periodontal disease (Petersen et al. 2005, WHO

DOI 10.1007/978-3-319-25091-5_7
60 J.H. Meurman
Oral Health 2012). Both caries and periodontal play any part in the development of cancer, or
disease are multi-bacterial infections caused by the modify the process of carcinogenesis, the asso-
oral biofilm (dental plaque). Recent studies have ciation here discussed is evidently of high impor-
shown that oral microbiota is far more complex tance. Namely, these easily preventable diseases
and numerous in species than hitherto understood. (in particular caries and periodontitis) should
It may, in fact, comprise thousands of microbial then be better controlled.
species (Keijser et al. 2008). Studies have also
been conducted analyzing the microbiota at the
site of oral cancer compared with normal mucosa; 7.3 Infection-Driven Mechanisms
in this, for example, the number of streptococci in Carcinogenesis
was decreased in cancer specimens (Schmidt et al.
2014). Correspondingly, saliva may also reflect the In carcinogenesis cells accumulate changes
diversity of microbiota in patients with oral can- in the genetic material which modify their
cer (Pushalkar et al. 2011). Dental bacteremia on function. Cell proliferation, differentiation,
the other hand is common, and thus oral microbes senescence, and apoptosis are involved in
easily gain access to blood circulation and may the regulation of the cell cycle, and all these
then cause systemic complications (Lockhart functions may be involved in carcinogenesis
et al. 2008) and possibly link to cancer in general (Lundberg and Weinberg 1999). Infection and
(Meurman 2010). inflammation, in turn, may interfere with cell
Oral yeast infections need to be mentioned metabolism and functions causing upregula-
here. The global prevalence of Candida infections tion of a number of cytokines and inflammatory
is not known. In certain patient groups such as mediators, which trigger cascade-like reac-
human immunodeficiency virus (HIV) infected, tions further leading to DNA damage, impaired
the prevalence can be 90 %, and WHO has esti- DNA repair, mutations, and uncontrolled cell
mated that approximately 9.5 million people proliferation (Chang and Parsonnet 2010).
would be infected with Candida (The Fungal Infection-driven carcinogenesis thus involves
Research Thrust 2011). Candida is more preva- several mechanisms. These include inflam-
lent on dysplastic and carcinoma lesions than on mation caused by microbial infection, lym-
healthy oral mucosa (McCullough et al. 2002). phoproliferation, infection-induced hormonal
The role of Candida in carcinogenesis is particu- changes that affect epithelial cell proliferation,
larly evident in patients with autoimmune polyen- cell transformation directly caused by infec-
docrinopathy-candidiasis-ectodermal dystrophy tion, and toxic and carcinogenic mechanisms of
(APECED), which is a genetic, autosomal reces- the microbes in question (Chang and Parsonnet
sive disorder. These patients frequently present 2010). These pathways are depicted in Fig. 7.1.
oral and esophageal carcinomas (Rautemaa et al.
2007). The mechanisms how Candida links to the
development of cancer may be partly due to its 7.4 Acetaldehyde Production
invasiveness. Degradation of the epithelial basal by Oral Microorganisms
membrane components and disruption of cell-to-
cell contacts have been shown in experiments with Alcohol is not carcinogenic but the first metab-
Candida (Parnanen et al. 2008, 2010). olite of ethanol, acetaldehyde, is highly car-
As regards cancer, this is among the leading cinogenic. Although the liver is the organism
causes of morbidity and mortality in the world. responsible for 75–90 % of ethanol metabo-
The WHO reported approximately 14 million new lism, extrahepatic pathways also exist. Ethanol
cases and 8.2 million cancer-related deaths in the is oxidized also by mucosal and microbial cells
year 2012 (WHO Cancer 2015). Furthermore, the yielding acetaldehyde, formed by alcohol dehy-
number of cases with malignancies is estimated drogenase enzyme. Acetaldehyde is further
to increase by 70 % by the year 2030 because of metabolized by aldehyde dehydrogenase yield-
aging of the populations. Hence if oral infections ing acetone which is less toxic and less harmful
7 Association Between Oral Infections and Cancer Risk 61
Chronic Secretion of
T-cell
inflammation and activation
infection CXCL-8 & CXCL-10
Epithelial cells
TNF-a, IL- 6,
Recruitment of IL-5, INF-g,
lymphocytes, neutrophils
and macrophages IL-1b, IL-2
ROS
TNF-a: Tumor RNOS
necrosis factor alpha
IL: Interleukin Stimulation of B-cells

INF: Interferon DNA
damage
ROS: Reactive
oxygen species !
RNOS: Reactive Increased cell division
nitrogen oxide species Impaired
CXCL: C-X-C motif DNA repair
chemokine
NF-kB: Nuclear Uncontrolled
proliferation
factor kappa-light
Mutations
chain
!
NF-kB
Normal cells Cancer cells
p53 tumor
suppressor gene
Fig. 7.1 Metabolic pathways in infection-driven carcinogenesis (Picture prepared by Bascones-Martinez)
compound. This, in turn, is oxidized to carbon Later, also oral Candida species were shown to
dioxide and then eliminated from the body. produce acetaldehyde from ethanol partly
Homann et al. (1997) were the first to show explaining why Candida infections as such have
high levels of acetaldehyde in saliva after intake been linked to oral cancer (Nieminen et al.
of alcohol. This group further showed that 2009; Uittamo et al. 2011). Moritani et al.
drinking alcohol and smoking concomitantly (2015) reported that indeed considerable num-
increased salivary acetaldehyde concentrations bers of oral bacteria have the capability to pro-
and that poor oral hygiene associated with this duce acetaldehyde from ethanol. Today it seems
risk (Homann et al. 2000, 2001) (see Fig. 7.2). clear that the ethanol metabolism here discussed
62 J.H. Meurman
Salivary acetaldehyde production in correlation with smoking and

drinking, *p<0.05 versus non-smokers, moderate alcohol consumption
Fig. 7.2 Relationship between drinking alcohol and smoking and salivary acetaldehyde concentration (Modified
from Homann et al. 2000)
is an evident pathologic mechanism in the and cancer. Tezal et al. (2013) investigated in a
development of oral and upper gastrointestinal case–control study how cardiological status
tract cancer. parameters link to head and neck cancer. It
appeared that caries lesions showed an OR 0.55
(95 % CI 0.30–101) regarding cancer. The authors
suggest that the lactobacilli prevalent in caries
7.5 Caries, Periodontitis, lesions might exert beneficial effect and enhance
and Cancer the immune system against cancer.
Virtanen et al. (2014), on the other hand,
Chronic periodontitis in particular associates showed in their observational study of 1390 sub-
with oral cancer risk. In a large study from the jects with 24 years of duration that dental infec-
USA comprising more than 13000 subjects, tions in periodontally healthy subjects associated
clinical attachment loss, a proxy for periodonti- with the incidence of any cancer (OR 2.62, 95 %
tis, associated with the presence of tumor (OR CI 1.18–5.78). Gingivitis was also shown to link to
4.57, 95 % CI 2.25–9.30) and premalignant cancer in this same Swedish cohort study (Söder
lesions (OR 1.55, 95 % CI 1.06–2.27), respec- et al. 2015). Furthermore, after 26 years of obser-
tively (Tezal et al. 2005). Periodontitis was vation in the same study, high gingival index score
found to also associate with tongue cancer risk associated with the incidence of any cancer with
(OR 5.23, 95 % CI 2.64–10.35) (Tezal et al. OR 1.29 (95 % CI 1.00–1.65). The statistical asso-
2007). Concomitant HPV infection seems to ciation between oral infections and cancer has also
play a role in this regard too (Tezal et al. 2009). been observed specifically with certain types of
As regards dental caries, there might be an cancer. Söder et al. (2011) found in their cohort
inverse relationship between this dental disease that missing any molar tooth from the mandible
7 Association Between Oral Infections and Cancer Risk 63
associated with incidence of breast cancer with of cytotoxic effects by using the plating efficiency
index. Oncol Rep. 2007;18:1551–6.
OR 2.36 (95 % CI 1.07–5.21). Missing molars
Chang AH, Parsonnet J. Role of bacteria in oncogenesis.
were the proxy for history of dental infections. It is Clin Microbiol Rev. 2010;23:837–57.
evident, however, that more studies are needed for Homann N, Jousimies-Somer H, Jokelainen K, Heine R,
final conclusion regarding the associations Salaspuro M. High acetaldehyde levels in saliva after
ethanol consumption: methodological aspects and patho-
between caries, periodontal disease, and cancer.
genetic implications. Carcinogenesis. 1997;18:1739–43.
Homann N, Tillonen J, Meurman JH, Rintamäki H,
Lindqvist C, Rautio M, Jousimies-Somer H, Salaspuro
M. Increased salivary acetaldehyde levels in heavy
7.6 Role of Saliva in the Oral drinkers and smokers: a microbiological approach to
Infection-Linked oral cavity cancer. Carcinogenesis. 2000;21:663–8.
Carcinogenesis Homann N, Tillonen J, Rintamäki H, Salaspuro M,
Lindqvist C, Meurman JH. Poor dental status increases
acetaldehyde production from ethanol in saliva: a pos-
Little is known about the role of saliva in oral sible link to increased oral cancer risk among heavy
infection-related carcinogenesis. It has been drinkers. Oral Oncol. 2001;37:153–8.
observed that salivary characteristics differ in Keijser BJ, Zaura E, Huse SM, van der Vossen JM,
Schuren FH, Montijn RC, ten Cate JM, Crielaard
patients with and without head and neck tumors
W. Pyrosequencing analysis of the oral microflora of
so that saliva form those with malignancy showed healthy adults. J Dent Res. 2008;87:1016–20.
more cytotoxic effect on fibroblasts than that Kuper H, Adami HO, Trichopoulos D. Infections as a
from healthy controls (Bloching et al. 2007). Oral major preventable cause of human cancer. J Intern
Med. 2000;248:171–83.
microorganisms may also metabolize dietary
Kurkivuori J, Salaspuro V, Kaihovaara P, et al.
components into carcinogenic substances. Saliva Acetaldehyde production from ethanol by oral strepto-
may contain nitrosamines, for example (Bahar cocci. Oral Oncol. 2007;43:181–6.
et al. 2007). However, more studies are called for Lockhart PB, Brennan MT, Sasser HC, Fox PC, Paster BJ,
Bahrani-Mougeot FK. Bacteremia associated with
before any conclusions can be drawn.
toothbrushing and dental extraction. Circulation.
2008;117:3118–25.
Conclusion Lundberg AS, Weinberg RA. Control of the cell cycle and
The highly prevalent oral infections and den- apoptosis. Eur J Cancer. 1999;35:1886–94.
McCullough M, Jaber M, Barrett AW, Bain L, Speight PM,
tal diseases in particular pose threat to sys-
Porter SR. Oral yeast carriage correlates with presence
temic health if not diagnosed in time and of oral epithelial dysplasia. Oral Oncol. 2002;
properly treated. Studies have shown that 38:391–3.
statistical associations exist between chronic Meurman JH. Oral microbiota and cancer. J Oral
Microbiol. 2010;2. doi:10.3402/jom.v2i0.5195
dental infections and development of cancer.
Meurman J, Bascones-Martinez A. Are oral and dental
Head and neck and upper gastrointestinal diseases linked to cancer? Oral Dis. 2011;17:779–84.
tract cancer may be affected by direct oral Moritani K, Takeshita T, Shibata Y, Ninomiya T, Kiyohara
microbial metabolism leading to carcinogenic Y, Yamashita Y. Acetaldehyde production by major
oral microbes. Oral Dis. 2015;21:748–54.
substances thus triggering malignant devel-
Nieminen MT, Uittamo J, Salaspuro M, Rautemaa R.
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organ may in fact be affected by infection. Candida albicans yeasts in vitro. Oral Oncol. 2009;45(12):
e245–8. doi:10.1016/j.oraloncology.2009.08.002.
Parnanen P, Kari K, Virtanen I, Sorsa T, Meurman
JH. Human laminin-332 degradation by Candida pro-
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Oral Candidiasis and the Medically
Compromised Patient
8
Abstract
Oral candidiasis is a common opportunistic oral infection in humans
caused by overgrowth of Candida species, in particular Candida albicans.
Clinically it usually presents as pseudomembranous or erythematous can-
didiasis. It may be asymptomatic or associated with local discomfort, dys-
geusia and xerostomia. The most common risk factors for oral candidiasis
include treatment with antibiotics, poor oral hygiene, tobacco smoking,
denture wearing and salivary gland hypofunction. A large number of dis-
eases as well as their treatment including diabetes, cancer and cancer ther-
apy, HIV infection and treatment with immunosuppressants are associated
with oral candidiasis. In immunocompromised patients, the localized oral
infection can spread through the bloodstream or upper gastrointestinal
tract leading to severe infection with increased morbidity and mortality.
This chapter focuses on Candida as commensal oral microorganism, the
clinicopathological aspects in medically compromised patients and diag-
nostic methods available regarding oral candidiasis.
8.1 Introduction studies due to geographical variations and varia-

tions in subjects examined, sampling methods
Candida species, in particular Candida albicans, and identification techniques. Possibly, 30–50 %
are part of the normal oral microbiota. The per- of healthy individuals harbour Candida species
centage of carriers varies considerably in different as part of their oral microbiota (Odds 1988), and
it is likely that Candida plays a role in maintain-
ing a balance between microorganisms and the
C. Kragelund, DDS, PhD (*) • J. Reibel, DDS, PhD,
Dr. Odont • A.M.L. Pedersen, DDS, PhD host (Krom et al. 2014).
Oral Pathology and Oral Medicine, C. albicans exists in different morphological
Department of Odontology, Faculty of Health forms: blastospores, yeast form and filamentous
forms, pseudohyphae and true hyphae; where
København, Denmark
e-mail: ckra@sund.ku.dk; jrei@sund.ku.dk; pseudohyphae appearing to be an intermediate
amlp@sund.ku.dk between blastospores and true hyphae (Carlisle

DOI 10.1007/978-3-319-25091-5_8
66 C. Kragelund et al.
et al. 2009). The yeast form is occurring in the penetration and furthermore degrades immu-
normal oral microbiota, although hyphae may be noglobulins which help to evade the host
seen in the absence of infection (Arendorf and defence. Interestingly, several studies have
Walker 1980; Rindum et al. 1994). In general, shown that bacteria coexist with C. albicans in
however, the filamentous forms are related to oral biofilms (Budtz-Jørgensen 1990), and it
invasive infection in the oral mucosa (Carlisle seems that this influences the growth and viru-
et al. 2009). Thus, the ability of the fungus to lence of C. albicans (Thein et al. 2006, 2009;
transform from blastospores to hyphae is impor- Diaz et al. 2014; Cavalcanti et al. 2015). Thus,
tant for the virulence of C. albicans. Interestingly, a symbiotic relationship between Streptococcus
a salivary component, statherin, seems to be able mutans and C. albicans has been shown to syn-
to induce the inverse transition (Leiro et al. 2009). ergize virulence of dental plaque biofilms
Another important ability of C. albicans is “phe- in vivo (Falsetta et al. 2014). Furthermore,
notypic switching” (Soll et al. 2014) that may Streptococcus gordonii glucosyltransferase
enable the fungus to evade the immunological promotes biofilm interactions with C. albicans
defence and even adapt to antifungal agents and (Ricker et al. 2014).
thereby increase virulence. In order to maintain in the oral cavity, the
Apart from C. albicans, other Candida species fungi not only need to grow, reproduce them-
such as C. glabrata, C. krusei and C. tropicalis selves and bind to a surface, they also have to
have been isolated from healthy individuals resist the antimicrobial activity of saliva. Saliva
(Zaremba et al. 2006). C. dubliniensis is an emerg- exhibits antimicrobial activity by killing and
ing species initially recovered from patients inhibiting growth but also by preventing adhe-
infected with human immunodeficiency virus sion and colonization to the surfaces in the oral
(HIV) (Sullivan et al. 1995). Recent studies identi- cavity. Moreover, saliva contains numerous
fied Candida/Pichia, Cladosporium/Davidiella, antimicrobial proteins and peptides of which
Alternaria/Lewia, Aspergillus/Emericella/ histatins, especially histatin 5, are the most
Eurotium, Fusarium/Gibberella, Cryptococcus/ important ones regarding antifungal activity.
Filobasidiella and Aureobasidium as consensus Histatins are small molecular weight proteins
genus-level members of the basal human salivary produced by the human salivary glands, which
mycobiome from healthy human mouth using mul- exhibit fungicidal and fungistatic activities
titag pyrosequencing of panfungal internal tran- against C. albicans and other Candida species
scribed spacer (ITS) primers and massive parallel, like C. glabrata, C. guilliermondii, C. krusei,
high-throughput sequencing of ITS1 amplicons C. lambica, C. parapsilosis, C. pseudotropica-
from saliva (Ghannoum et al. 2010; Dupuy et al. lis, C. stellatoidea and C. tropicalis, as well as
2014). Saccharomyces, Epicoccum and Phoma Saccharomyces cerevisiae and Cryptococcus
were weaker candidates for consensus inclusion. neoformans isolated from healthy and immuno-
However, Malassezia species were included in the compromised patients (Oppenheim et al. 1988;
oral core mycobiome, which is interesting since Tsai and Bobek 1998; Xu et al. 1991). Other
they are important commensals/pathogens of important salivary proteins with antifungal
human skin (Dupuy et al. 2014). properties include lactoferrin and lysozyme
Infection requires recognition and adhesion (Samaranayake et al. 2001). The importance of
to epithelial cells, and following subsequent the salivary antifungal activity becomes evident
multiplication and secretion of extracellular in cases of immune deficiency and disease- and/
matrix, a biofilm can form on the mucosal sur- or medication-induced salivary gland hypo-
faces (Cannon et al. 1995). Hyphae formation function being associated with high rates of
is important for formation of a stable biofilm; oral candidal carriage and Candida infections
thus, hyphal growth is important for the viru- (Costa et al. 2006; Lam et al. 2012; Lin et al.
lence of C. albicans. Secretion of enzymes 1999; Pedersen et al. 2015; Shiboski et al.
such as proteases and lipases facilitates tissue 2015; Yan et al. 2011).
8 Oral Candidiasis and the Medically Compromised Patient 67
8.2 Clinicopathological Aspects
In patients carrying Candida as part of the com-

mensal oral microbiota, overgrowth of C. albi-
cans or non-albicans Candida spp. can cause oral
candidiasis (Rindum et al. 1994). The diagnosis
oral candidiasis is based on clinical signs and/or
oral mucosal symptoms together with positive
test results compatible with candidal overgrowth.
Characterization of oral candidiasis according to
sporadic or recurrent infection, duration of infec-
tion (acute/chronic), symptomatic or asymptom-
atic infection, primary or in relation to other oral
or systemic diseases (primary/secondary/tertiary) Fig. 8.1 Pseudomembranous candidiasis in the soft
is important in unravelling and managing oral palate
candidiasis. Most patients experience sporadic
candidal infection, whereas less experiences
multiple recurrent infections. Information from
medical history and clinical evaluation are used
to classify acute or chronic candidiasis as no arbi-
trary time limit makes clinical sense as individual
host and oral environmental factors influence the
course of the infection. Subjective symptoms in
relation to candidiasis are usually associated to
the clinical type. Oral candidiasis presents clini-
cally in various forms such as pseudomembra-
nous, erythematous and hyperplastic candidiasis
(Ellepola and Samaranayake 2000). It is still
largely unknown why oral candidiasis manifests
in these different variants in different individuals
(Reichart et al. 2000). Furthermore, there are
candida-associated lesions which further compli-
cate the diagnosis.
Pseudomembranous candidiasis is character-
ized by thick, white patches covering part of the
mucous membranes often being the soft palate,
tongue and buccal and lip mucosa (Fig. 8.1). The
Fig. 8.2 Chronic erythematous candidiasis on the dorsal
pseudomembranes can be wiped off easily. In surface of the tongue (“median rhomboid glossitis”)
chronic infections the mucous membrane under-
neath often is erythematous with pinpoint haem-
orrhages. Normally no soreness is associated relation to topical exposure to steroids, e.g.
with the pseudomembranous type, but taste dis- asthma inhalators.
turbances as salty or metallic taste are reported. Erythematous candidiasis is characterized by
Acute neonatal thrush is common as a result of unspecific focal or generalized redness of the
favourable candidal growth conditions because mucous membranes (Fig. 8.2) and is often asso-
of immature oral microbiota and oral immunity. ciated with symptoms as burning and stinging
Chronic pseudomembranous candidiasis can be sensation. Antibiotics are the most common
seen in immunocompromised patients and in cause of acute primary erythematous candidiasis.
Fig. 8.4 Chronic erythematous candidiasis presenting as

denture stomatitis
Fig. 8.3 Chronic hyperplastic candidiasis in the anterior
part of the right buccal mucosa
Table 8.1 Different types of oral candidiasis and differ-
ential diagnostic considerations
Chronic secondary erythematous candidiasis is Differential diagnostic
common in oral lichen planus. Oral candidiasis considerations
Hyperplastic candidiasis (Fig. 8.3) is a chronic Pseudomembranous No similar lesion
infection and has two primary manifestations. Erythematous Erythematous oral lichen
planus, erythroplakia, oral
The nodular type presents as small white slightly cancer
elevated papular lesions giving the mucous mem- Hyperplastic
brane a speckled appearance. The plaque-like Nodular Non-homogeneous
type is homogenous white slightly elevated areas leukoplakia, oral cancer
of the mucous membrane. It is often related to Plaque Homogeneous leukoplakia
smoking and seen in the commissural area of the Denture stomatitis Ill-fitting dentures, poor
denture hygiene
buccal mucosa. The hyperplastic lesions cannot
Angular cheilitis Bacterial infection,
be rubbed off and are usually asymptomatic. The malnutrition, vitamin and/
two types may occur simultaneously. The hyper- or mineral deficiency
plastic types are to some extent controversial as Median rhomboid glossitis Geographic tongue,
they may represent secondary candida infections malnutrition, vitamin and/
in leukoplakias (Holmstrup and Bessermann or mineral deficiency
Linear gingival erythema Gingivitis (bacterial)
1983), and the term candidal leukoplakia is
sometimes used (Sitheeque and Samaranayake
2003). A biopsy is generally indicated to exclude plakia, erythroplakia or oral cancer, secondarily
malignancy, and the treatment result after anti- infected with Candida, or a disorder needing other
fungal therapy should always be monitored. management or supplementary treatment, e.g.
Candida-associated lesions including denture lichen planus or lupus erythematosus (Table 8.1).
stomatitis (Fig. 8.4), angular cheilitis, median
rhomboid glossitis (Fig. 8.2) and linear gingival
erythema may all be associated with Candida 8.2.1 Histopathology
infection but may also be related to other causes,
e.g. ill-filling dentures and bacterial or mixed bac- Candidal hyphae are readily identified in biopsies
terial and fungal infection. Thus, a thorough inves- by the use of an appropriate staining method, e.g.
tigation is mandatory before adequate treatment Periodic Acid-Schiff (PAS) method or Grocott-
can be initiated. In particular it is important to rule Gomori methenamine silver (GMS) method. It
out a premalignant or malignant lesion, e.g. leuko- should be mentioned that there is a risk of
8.3 Oral Candidiasis

in the Medically
Compromised Patient
Oral candidiasis is the result of yeast overgrowth

and penetration of the epithelial mucosal protective
barrier and is evident in patients with various con-
ditions that exhibit immunosuppression including
diabetes.
A large number of local and systemic fac-
tors and conditions may predispose individuals
to oral candidiasis. Factors like treatment with
antibiotics, ill-fitting dentures, poor oral hygiene
and tobacco smoking can favour growth of yeast
Fig. 8.5 Photomicrograph of a biopsy from a chronic
hyperplastic candidiasis. Numerous PAS-positive (red) cells by disrupting the ecological balance on the
hyphae are seen in the parakeratin layer of the epithelium mucosal surface (Baboni et al. 2009; Holmstrup
and Bessermann 1983; Semlali et al. 2014).
false-negative results if only few PAS-stained sec- Treatment with immunosuppressives, cancer
tions are examined (Roed-Petersen et al. 1970). therapy, immune deficiencies, salivary gland
Hyphae are seen in the parakeratin layer of the epi- hypofunction induced by medication or disease
thelium as a superficial infection (Fig. 8.5). Only and diabetes can compromise local or general
in severely immunocompromised patients can defence mechanisms and thereby lead to oral
hyphae be seen in the underlying epithelial layers candidiasis. Often a patient will present several
or in the connective tissue. Typically, the epithe- predisposing factors at the same time, and mul-
lium is hyperplastic and hyperparakeratinized with tiple interventions must be initiated including
leukocytes, in particular polymorphonuclear neu- instruction, motivation and follow-up of oral
trophils, penetrating the epithelium often forming hygiene procedures, diet change, stimulation of
microabscesses in the superficial part of the epi- functional salivary glands, substitution of xero-
thelium in relation to the hyphae. Chronic inflam- genic drugs, diagnosis of oral mucosal diseases,
mation is present in the connective tissue tobacco counselling and smoking cessation and
underneath the epithelium; however, this is often multidisciplinary diagnostic workup of systemic
lacking in severely immunocompromised patients, predispositions.
e.g. patients with HIV infections and AIDS.
8.3.1 Oral Candidiasis as Adverse

8.2.2 Immunological Aspects Drug Reaction
A large variety of proinflammatory and immuno- Systemic antibiotics are the most common cause
regulatory cytokines are generated in the oral of acute mucosal candidiasis. Oral exposure to
mucosa during an infection with Candida topical glucocorticoid steroids is another com-
(Dongari-Bagtzoglou and Fidel 2005). It has mon cause of medication-induced oral candidia-
been shown that highly invasive strains of C. sis, probably due to alterations of the local
albicans trigger production of proinflammatory mucosal host immunity. Removal of the topical
cytokines, including IL-1α, IL-6, IL-8 and TNF-α medication with water after exposure tends to
in epithelial cells and IL-6, IL-8, monocyte che- prevent recurrence. General immune suppression
motactic protein (MCP)-1, MCP-2 and granulo- due to systemic glucocorticoids, cancer chemo-
cyte colony-stimulating factor in endothelial therapy and immunomodulators often causes
cells (Villar et al. 2005; Whiley et al. 2012). mucosal candidiasis, which usually can be
prevented by prophylactic antifungal treatment. of glucose in the blood and saliva may promote
Studies using both culture and pyrosequencing growth and enhance adherence of yeasts to epithe-
have shown that the oral mycobiota in immuno- lial cell surfaces (Samaranayake 1990). Also the
suppressed solid organ transplant recipients is impaired functions of polymorphonuclear leuko-
dominated by Candida species (Charlson et al. cytes leading to reduced phagocytosis, intracellu-
2012; Diaz et al. 2013; Dongari-Bagtzoglou et al. lar killing and chemotaxis may contribute to the
2009). Salivary gland dysfunction resulting in increased colonization of Candida and increased
reduced salivary flow rate (salivary hypofunc- susceptibility to oral candidiasis (Ueta et al. 1993;
tion), compositional changes or a combination of Vazques and Sobel 1995). However, other risk
both often has major consequences for the oral factors such as salivary gland hypofunction, low
microbial balance and local immune defence salivary pH and impaired salivary antimicrobial
leading to an increased risk of dental caries and activity, poor oral hygiene, cigarette smoking and
oral candidiasis (Dawes et al. 2015). The main denture wearing also have a substantial influence
causes of salivary gland hypofunction are intake on candidal colonization and various oral mani-
of medications, systemic diseases like Sjögren’s festations and symptoms of Candida infections in
syndrome and head and neck radiotherapy (Villa both type 1 and 2 DM patients (Budtz-Jørgensen
et al. 2015; Jensen et al. 2010; Pedersen 2014). 1990; Banoczy et al. 1987; Guggenheimer et al.
A number of studies have shown that intake of 2000; Jurevic et al. 2003; Kadir et al. 2002;
xerogenic medication is associated with high Pedersen 2004; Samaranayake 1990; Willis et al.
rates of Candida carriage and oral candidiasis 1999). Inadequately controlled diabetics who
(Almståhl and Wikström 2003, 2005; Kaplan wear dentures have a higher oral candida load
et al. 2008; Pedersen et al. 2015) (for further and higher prevalence of denture stomatitis than
details, see Chap. 9). nondiabetic denture wearers (Guggenheimer et al.
2000; Vitkov et al. 1999).
C. albicans is the most common species iso-
8.3.2 Diabetes Mellitus and Oral lated from the oral cavity of diabetics (Dorocka-
Candidiasis Bobkowska et al. 1996; Kadir et al. 2002;
Samaranayake 1990; Willis et al. 1999), but also
The carriage frequency of Candida and the den- C. dubliniensis, C. glabrata and C. tropicalis have
sity of candidal colonization as well as the rates of been isolated from the oral cavity of patients with
oral Candida infections are increased in patients diabetes (Jurevic et al. 2003). The significance of
with both type 1 and type 2 diabetes mellitus (DM) species in relation to pathogenesis of fungal infec-
(Tapper-Jones et al. 1981; Lamey et al. 1988, tions in diabetics remains to be elucidated.
1992; Hill et al. 1989; Vazques and Sobel 1995;
Bai et al. 1995; Guggenheimer et al. 2000; Kadir
et al. 2002; Jurevic et al 2003; Shenoy et al. 2014). 8.3.3 HIV Infection and Oral
Oral candidiasis is not only more common in Candidiasis
patients with DM than in nondiabetics, the infec-
tions are also more severe (Guggenheimer et al. Oral candidiasis is the most common opportunis-
2000). The increased susceptibility to oral candi- tic infection in HIV-infected patients and in
diasis has been related to poor glycaemic control patients with AIDS (Coleman et al. 1993;
and hence high concentrations of glucose in the Shiboski et al. 2015) and one of the earliest
blood and saliva, long disease duration as well indicators of the progression from HIV-
as presence of diabetic complications (retinopa- seropositive status to AIDS. Oral candidiasis is
thy) (Bai et al. 1995; Bartholomew et al. 1987; strongly associated with immune suppression, as
Dorocka-Bobkowska et al. 1996; Guggenheimer measured by CD4+ lymphocyte counts, and also
et al. 2000; Kadir et al. 2002; Ueta et al. 1993; associated with high viral burden and conse-
Vazques and Sobel 1995). High concentrations quently suggested as a clinical marker of plasma
viral load and the progression of HIV disease fungal infections due to immunosuppressive,
(Glick et al. 1994; Patton 2000). immunomodulating therapy as well as treatment
The candida carriage in HIV-infected patients with antibiotics (Trenschel et al. 2000). Despite
is predominated by C. albicans, but C. dublinien- antifungal prophylaxis, the increased risk for sys-
sis and C. glabrata have also commonly been temic and oropharyngeal fungal infection is still
isolated from oral lesions in HIV-infected patients a matter of concern in these patients, and fungal
(Sullivan et al. 1995; Li et al. 2007). The increased infections remain a significant cause of morbidity
carriage of C. krusei has been associated with the and mortality. The prevalence of oral candidiasis
widespread use of fluconazole prophylaxis in renal transplant recipients ranges from 9.4 to
(Samaranayke and Samaranayke 1994). A recent 46.7 % (Al-Mohaya et al. 2002; de la Rosa-
study on the oral mycobiome using pyrosequenc- García et al. 2005; Güleç et al. 2003).
ing showed a shift in the oral mycobiome in C. albicans has been found to be the most
which Epicoccum and Alternaria were abun- prevalent species isolated from the oral cavity of
dantly colonizing HIV-infected patients, but recipients of kidney transplants (da Silva-Rocha
Candida being abundant in both HIV patients et al. 2014). Also in recipients of liver trans-
and healthy subjects (Mukherjee et al. 2014). plants, the Candida carriage and prevalence of
The introduction of highly active antiretrovi- oral candidiasis are high (40–50 %), and many of
ral therapy (HAART) has changed the epidemiol- these patients also suffer from salivary gland
ogy of oral candidiasis. Thus, several studies hypofunction (Helenius-Hietala et al. 2014).
have reported a decrease in the prevalence and The most common forms of oral candidiasis
recurrence of oral candidiasis in HIV-infected reported in patients receiving cancer therapy are
patients receiving HAART (Greenspan et al. pseudomembranous and erythematous candidiasis
2004; Jiang et al. 2014; Ramírez-Amador et al. (Lalla et al. 2010). There appear to be no relation
2007). However, some studies report rare cases to Candida colonization and the presence or sever-
of increased prevalence. There is substantial evi- ity of oral mucositis in haemopoietic progenitor
dence suggesting that onset of oral candidiasis is cell transplant patients (Epstein et al. 2003;
associated with a progressive reduction in CD4+ Westbrook et al. 2013), whereas C. glabrata has
lymphocyte count and an increase in viral load in been associated with oral ulcerations in this patient
HIV-infected patients receiving HAART group (Laheij et al. 2012). A recent study indicates
(Hodgson et al. 2006; Ramírez-Amador et al. that the mycobiome plays a role in the pathogene-
2007). Oral candidiasis has therefore been sug- sis of acute graft-versus-host disease in blood- and
gested as a clinical marker of immune status and marrow-transplanted patients (van der Velden
a predictor of virologic failure during HAART et al. 2013).
and hence an indicator of HIV disease progres- Several studies have shown that the oral
sion and a tool for monitoring HIV infection in microbiota is disturbed in patients, who has
conjunction with CD4+ lymphocyte counts and received radiotherapy in the head and neck
plasma viral load in HIV-infected patients receiv- region, and in this regard found increased coloni-
ing HAART (Ramírez-Amador et al. 2007). zation of Candida species and higher occurrence
of oral candidiasis (Al-Nawas and Grötz 2006;
Almståhl and Wikström 2003; Almståhl et al.
8.3.4 Recipients of Organ 2008; Brown et al. 1975; Grötz et al. 2003). C.
and Haematopoietic Cell albicans is the cause of the majority of
Transplants and Cancer oropharyngeal infections, but C. glabrata and C.
Therapy tropicalis are emerging causes of these infections
in patients with head and neck cancer. It has been
Recipients of solid organ transplants and haema- shown that about 50 % of patients with head and
topoietic cell transplants and patients receiving neck cancer were colonized with Candida spe-
cancer therapy have a high risk of developing cies prior to radiotherapy, and after the therapy
the percentage has increased to about 75 % (Lalla 8.5.1 Culture of Clinical Samples
et al. 2010). The increased colonization also in Order to Quantify and
translated into an increased rate of oral infec- Identify the Candida Load
tions. Predisposing factors in patients with head
and neck cancer include mucosal injury due to Different culture media can be used to grow and
cancer therapy, salivary gland hypofunction, differentiate Candida spp. Some can easily be
smoking and wearing dentures. prepared, e.g. Sabouraud dextrose and Pagano-
Levin agar media, and others are commercially
available, e.g. CHROMagar™ (CHROMagar,
8.4 Chronic Mucocutaneous France), chromID® Candida (BioMérieux, USA)
Candidiasis and BiGGY Agar (Nickerson Agar) (Sigma-
Aldrich®, USA). Up to four different Candida
Chronic mucocutaneous candidiasis (CMC) is spp. can be differentiated by culture of clinical
persistent or recurrent widespread superficial samples. Swaps, imprints, whole saliva and oral
Candida infection of the oral, oesophageal, rinse can be the source for culture techniques.
digestive, genital, nail mucous membranes and/ In immunocompetent patients, there is no uni-
or skin. Most often C. albicans is the infectious versal arbitrary threshold level of colony-form-
Candida species. CMC is caused by immune ing units (CFU) differentiating between carrier
deficiencies involving the mucosal cutaneous state and candidiasis as individual host and oral
immunity, which can have an inherited but most environmental factors influence the candida
often have sporadic origin. T-cells seem to play load (Epstein et al. 1980). However, in immu-
an essential role, as several T-cell immune defi- nocompromised patients an arbitrary value of
ciencies, e.g. impaired T-cell function, activation >400 CFU has been suggested for antimycotic
and cytokine signalling, have been associated intervention (Ship et al. 2007). Culture is time
with CMC (Lanternier et al. 2013). Findings such consuming and 48 h growth at 37 ° C delays the
as reduced proportion of interleukin-17 (IL-17)- diagnosis. The composition of the Candida spp.
secreting T-cells, autoantibodies directed against in lesional infections may vary from the over-
IL-17 and mutations in IL-17-related genes sug- all composition in the oral cavity, which make
gest that impairment of IL-17 immunity plays a the choice of sampling procedure important
significant role in CMC pathogenesis (Puel et al. (Kragelund et al. 2013).
2012). CMC is associated with three syndromes
including autosomal recessive autoimmune poly-
endocrinopathy syndrome type 1, hyper IgE syn- 8.5.2 Smears
drome and CARD9 deficiency (Al-Herz et al.
2011). CMC as the principal symptom or CMC Exfoliative cytologic examination is an easy and
in association to other skeletal, endocrine or skin inexpensive method for detection of candida
abnormalities initiates in early childhood, and organisms. The suspected area is vigorously
immune dysfunction should be suspected in scraped with a wooden spatula and made into a
patients without predisposing risk factors for oral smear on a glass microscope slide, spray fixed
candidiasis. with a commercial spay or fixed in 70 % ethanol
and stained appropriately, e.g. the PAS or GMS
method. An advantage of this method compared
8.5 Diagnostic Methods to simple culture techniques is that the patho-
genic form of the candida organism (hyphae) is
Identification of candidal overgrowth can be easily identified. However, standardized culture
established by a variety of methods including of saliva or oral rinse samples is more suitable
culture, cytosmears, biopsy and molecular tech- for identifying candida presence and load in the
niques (Table 8.2). oral cavity.
8
Table 8.2 Diagnostic methods, detection techniques and their advantages and disadvantages
Traditional Time Molecular Time
Infection Method techniques (hours) Traditional advantages techniques (hours) Molecular advantage Disadvantages
Focal mucosal Swab CFU 48 Quantification PCR/ 24–48 Species No morphological
infection Imprint Some species MALDI-TOF or identification differentiation
identification 10 min. Time
Cytosmear PAS microscopy ~2 Quantification FISH ~2 Species Expensive
Morphological identification
differentiation
Cytobrush DNA PCR 24–48 Species No morphological
identification differentiation
No quantification
Time
Biopsy PAS microscopy 30 Some quantification FISH ~2 Species Expensive
Oral Candidiasis and the Medically Compromised Patient
Morphological identification
differentiation
Generalized oral Whole saliva CFU 48 Quantification PCR/ 24–48 Species No
infection Some species MALDI-TOF 10 min. identification morphological
Time
Oral rinse CFU 48 Quantification PCR/ 24–48 Species No
Some species MALDI-TOF 10 min. identification morphological
Time
CFU colony-forming unit on culture media, PAS Periodic Acid-Schiff, DNA deoxyribonucleic acid, PCR polymerase chain reaction, MALDI-TOF matrix-assisted laser desorp-
tion ionization-time of flight, FISH fluorescence in situ hybridization
73
8.5.3 Biopsy a cheap and quick technique and is part of rou-

tine procedures in many medical microbiological
Biopsy is particularly relevant in the case of the laboratories (Coronado-Castellote and Jimenez-
hyperplastic candida infections (see above). An Soriano 2013).
appropriate staining method will reveal candida
hyphae in the keratin layer of the epithelium. If a
suspected lesion does not respond to antifungal References
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Association Between Oral
Infections and Salivary Gland
9
Hypofunction
Siri Beier Jensen and Anne Marie Lynge Pedersen
Abstract
Saliva plays an important role in the maintenance of oral health and regu-
lation of the oral microbiota. Saliva lubricates the oral hard and soft tis-
sues, dilutes food detritus and bacteria and enhances the clearance of
microorganisms and dietary carbohydrates from the oral cavity. Saliva
also provides antimicrobial activity via numerous proteins and peptides
including lactoferrin, lactoperoxidase, lysozyme, statherin and histatins.
This chapter focuses on the oral microbiota in patients suffering from sali-
vary gland hypofunction due to Sjögren’s syndrome, radiotherapy of
tumours in the head and neck region, cancer chemotherapy and intake of
medications. Despite the different causes of salivary gland hypofunction,
these patient groups show some similarities regarding the composition of
the oral microbiota with increased colonisation of oral pathogens associ-
ated with dental caries (Streptococcus mutans and Lactobacillus species)
and oral mucosal infections, especially Candida albicans.
9.1 Introduction to formation of the dental pellicle, diluting food

detritus and bacteria and mechanical cleansing
Saliva plays an essential role in the maintenance of the oral cavity. Furthermore, salivary proteins
of tooth integrity and protection against dental such as statherin and proline-rich proteins keep
caries by neutralising acids from food and bac- saliva supersaturated with respect to calcium
teria via salivary buffering systems, contributing phosphate salts thereby preventing demineralisa-
tion. Similarly saliva and its components main-
tain mucosal integrity by constantly covering
S.B. Jensen, DDS, PhD (*) • A.M.L. Pedersen, and lubricating the oral soft tissues and thereby
DDS, PhD preventing injuries as well as adhesion and pro-
Section 1, Oral Pathology and Oral Medicine, liferation of microorganisms. In addition, saliva
provides antimicrobial activity via a large vari-
Copenhagen N, Denmark ety of proteins and peptides including mucins,
e-mail: sirib@sund.ku.dk; amlp@sund.ku.dk lysozyme, lactoferrin, histatins, defensins and

DOI 10.1007/978-3-319-25091-5_9
80 S.B. Jensen and A.M.L. Pedersen
antibodies (secretory IgA) and thereby inhibits reviews results from studies of the oral microbiota
bacterial and fungal colonisation and infections both in rinsing samples and in samples collected
(Dawes et al. 2015; Lagerlöf and Oliveby 1994; from specific sites in patients with chronically
Lenander-Lumikari and Loimaranta 2000). or temporary salivary gland hypofunction due to
The composition of saliva is dependent on the Sjögren’s syndrome, cancer therapy (chemo- and/
rate by which the saliva is produced, the type of or radiotherapy) and intake of medications.
gland, from which the saliva is secreted and the
nature and duration of the stimuli applied to acti-
vate the secretion reflexes (Pedersen et al. 2002a, b). 9.2 Oral Microbiota in Sjögren’s
The composition of salivary antimicrobial pro- Syndrome
teins may therefore vary from one oral site to
another in an individual, and different oral sites Sjögren’s syndrome (SS) is a chronic, systemic
can harbour different microbiota depending on autoimmune inflammatory disorder that affects the
the local morphology, growth conditions and the exocrine glands, particularly the salivary and lacri-
local immune defence. Other factors such as oral mal glands. The most prominent disease manifesta-
hygiene, diet, dental restorations, systemic dis- tions include hyposalivation and keratoconjunctivitis
eases, medication intake and various lifestyle fac- sicca resulting in symptoms of oral and ocular dry-
tors also affect the local oral microbiota (for ness. The aetiology remains unknown, but most
further details see Chap. 2). likely includes an interaction between immunologi-
Under normal conditions, the unstimulated cal, genetic, hormonal and environmental factors.
whole saliva flow rate is on average 0.3–0.4 ml/ The median age of presentation is around 50 years
min, while chewing-stimulated whole saliva flow and it mainly affects women. SS is classified into
rate is about 1.5–2.0 ml/min (Humphrey and two forms: primary SS (pSS) and secondary SS
Williamson 2001; Pedersen et al. 2002a, b). An (sSS). The latter defines the disease entity in the
unstimulated whole saliva flow rate ≤0.1 ml/min presence of another chronic inflammatory connec-
and chewing-stimulated whole saliva flow rate tive tissue disease, most commonly rheumatoid
≤0.5–0.7 ml/min are designated hyposalivation arthritis or systemic lupus erythematosus (Pedersen
(Heintze et al. 1983; Pedersen et al. 2002a, b). and Nauntofte 2005). Diagnosis is often delayed
Xerostomia usually occurs when the unstimu- which reflects the fact that the onset is often insidi-
lated secretion rate has decreased to approxi- ous and patients present various and unspecific
mately 50 % of its normal value in any given symptoms like xerostomia, fatigue, myalgia and
individual, indicating that more than one major arthralgia. Hyposalivation may therefore precede
salivary gland must be affected (Dawes 1987). the diagnosis for several years increasing the risk of
The importance of saliva in the maintenance dental caries and recurrent oral candidiasis. Before
of a natural balance between the host and the oral diagnosis and hence awareness of risk of oral dis-
microbiota becomes evident when the saliva flow eases, the patients may have a frequent intake of
rate is reduced. The most common causes of sali- easily fermentable carbohydrates like candies and
vary gland hypofunction include intake of medi- soft drinks in order to alleviate the symptoms of dry
cations, polypharmacy, systemic diseases such as mouth (Brunström 2002; Cermak et al. 2003) and
Sjögren’s syndrome and cancer therapy including an inadequate oral hygiene which further favour the
chemo- and radiotherapy. Regardless of the aetiol- growth of Streptococcus mutans and Lactobacillus
ogy of salivary gland hypofunction, a shift in the and Candida species.
oral ecology appears to occur already at unstimu-
lated whole saliva flow rates below 0.20 ml/min
with a shift towards a more aciduric and acido- 9.2.1 Dental Caries
philic oral microbiota leading to an increased risk
of dental caries and oral candidiasis (Navazesh Both the quantity and quality of saliva are affected
et al. 1995; Bardow et al. 2001). This chapter in patients with pSS (Kalk et al. 2001; Pedersen
9 Association Between Oral Infections and Salivary Gland Hypofunction 81
et al. 2005; Thorn et al. 1989). The number of erythematous candidiasis and angular cheilitis
decayed, missed and filled teeth is high (Baudet- (Hernandez and Daniels 1989; Lundström and
Pommel et al. 1994; Christensen et al. 2001; Lindström 1995; Pedersen et al. 1999b; Soto-
Pedersen et al. 1999a, 2005) and found inversely Rojas et al. 1998; Tapper-Jones et al. 1980).
correlated to salivary flow rates and especially the C. albicans is the most frequently isolated (66–
unstimulated whole saliva flow rate (Pedersen et al. 72 %) species in patients with SS. It may occur
1999b, 2005). The reduced saliva secretion results alone or mixed with other Candida species such
in reduced bicarbonate concentration, pH and buf- as C. tropicalis, C. pseudotropicalis, C. parapsi-
fer capacity (Bardow et al. 2001; Pedersen et al. losis, C. kefyr and C. glabrata (Kindelan et al.
2005). The clearance of microorganisms and dietary 1998; Soto-Rojas et al. 1998). The prevalence of
sugars is also impaired thereby promoting an envi- Candida as well as the numbers of colony-
ronment dominated by aciduric and acidogenic spe- forming units (CFU) per ml (CFU/ml) not only
cies and prolonged exposure of dietary sugars and varies between studies but also between the pSS
acids to the teeth. Kolavic et al. (1997) found higher and sSS patients (Table 9.1) reflecting differ-
counts of Streptococcus mutans and lactobacilli in ences in the patient groups regarding dental sta-
caries-inactive patients with SS having stimulated tus, oral hygiene habits, comorbidity, medication
parotid flow rates <0.25 ml/min than in subjects intake and/or immune response. Results of saliva
with higher parotid flow rates. The counts and num- cultures and oral rinses correspond well to the
bers of S. mutans and lactobacilli counts have been occurrence of signs and symptoms of oral candi-
found inversely correlated to stimulated whole diasis (Abraham et al. 1998; Kindelan et al. 1998;
saliva flow rates (Lundström and Lindström 1995). Soto-Rojas et al. 1998). Candidal colonisation
Almståhl et al. (1999) also showed that patients and oral candidiasis tend to be more prevalent in
with pSS harboured higher numbers of both S. patients with sSS (Soto-Rojas et al. 1998).
mutans and Lactobacillus species and sSS higher Almståhl et al. (2001) showed that patients with
numbers of Lactobacillus species than healthy sub- pSS had significantly higher levels of C. albicans
jects. In patients with pSS, the shift in the oral in rinsing samples than subjects with hyposaliva-
microbiota appears to occur despite a good oral tion of unknown aetiology. SS patients with
hygiene, as they had higher levels of S. mutans than immeasurable unstimulated whole saliva flow
patients who had received radiotherapy in the head rates have the highest levels of C. albicans in oral
and neck region and patients who took neuroleptics rinses (Almståhl et al. 1999).
(Almståhl et al. 1999). The high number of micro- The microbial samples proving the presence
bial retention sites generated by dental restorations of Candida species on the oral mucosa have usu-
such as fillings, crowns and bridges found in patients ally been obtained by smears or culture swabs
with pSS may also contribute to the shift in oral bac- taken from the dorsum of the tongue, the buccal
teria (Almståhl et al. 1999). Leung et al. (2007) or palatal mucosa, the right tonsillar area and/or
found higher levels of lactobacilli in saliva, espe- the fitting surface of the denture (Almståhl and
cially L. acidophilus, L. fermentum and L. minitus, Wikström 1999; Leung et al. 2008; MacFarlane
and in supragingival plaque from patients with SS and Mason 1974; MacFarlane 1984; Pedersen
than in subjects with normal salivary secretion, but et al. 2002a, b; Rhodus et al. 1997; Soto-Rojas
no differences in the numbers of S. mutans or anaer- et al. 1998; Tapper-Jones et al. 1980). Results
obic gram-negative rods. revealed that patients with SS have a significant
higher mucosal colonisation of C. albicans than
healthy subjects (Leung et al. 2008; MacFarlane
9.2.2 Oral Candidiasis 1984; Radfar et al. 2003; Rhodus et al. 1997;
Soto-Rojas et al. 1998; Tapper-Jones et al. 1980;
Recurrent oral candidiasis is prevalent among Yan et al. 2011) and pSS compared with patients
patients with SS, and the most common clinical with oral lichen planus (Pedersen et al. 2002a, b).
presentation of Candida albicans colonisation is Patients with sSS harboured higher numbers of
Table 9.1 Frequency of Candida species determined semiquantitatively and the numbers of colony-forming units per
ml (CFU/ml) in patients with primary Sjögren’s syndrome (pSS) and secondary Sjögren’s syndrome (sSS)
Microbiological tests pSS sSS References
Tongue smear 33 % 76 % Sota-Rojas et al. (1998)
Tongue swab culture 52 % 76 % Sota-Rojas et al. (1998)
Saliva culture/oral rinse 76 % 79 % Sota-Rojas et al. (1998)
81 % 67 % Kindelan et al. (1998)
65 % 60 % Almståhl et al. (1999)
72 % 48.1 % Leung et al. (2007)a
Supragingival plaque 84 % 55.6 % Leung et al. (2007)a
Numbers of CFU/ml
Tongue/palate swab culture 3.1 × 106 (mean) 1.2 × 105 Rhodus et al. (1997)
Saliva culture 419/μl (mean) 739/μl Sota-Rojas et al. (1998)
>104 (35.7 %) >104 (39.1 %) Ergun et al. (2010)
Oral rinse 2100 (median) 1710 Kindelan et al. (1998)a
380 (median) 500 Almståhl et al. (1999)b
1025.5 (mean) 155 Leung et al. (2007)a
Supragingival plaque 1.8 × 106/g (mean) 0.4 × 106 Leung et al. (2007)a
a
The study included denture wearers
b
The study only included dentate subjects
C. albicans (CFU 3.1 × 106) than patients with early candidiasis without clinical apparent lesions
pSS (CFU 1.2 × 105), which was attributed to the or a less virulent strain of Candida. Regarding
presence of an additional inflammatory disease in site specificity, it is noteworthy that C. albicans
sSS as well as to low saliva flow rates (Rhodus was found twice as frequent in the supragingival
et al. 1997). The presence and density of C. albi- plaque than on the tongue in patients with pSS,
cans were also inversely correlated to saliva flow but could not be detected in the gingival crevicu-
rates (Hernandez and Daniels 1989; Radfar et al. lar region using the paper point technique
2003; Rhodus et al. 1997; Tapper-Jones et al. (Almståhl et al. 2001b).
1980). Hernandez and Daniels (1989) found that
patients with SS and with chronic erythematous
candidiasis were older and had long duration of 9.2.3 Other Microorganisms
oral symptoms, more inflammation in their labial
salivary glands and lower stimulated parotid flow A sparse number of studies have examined the
rates than SS patients without this oral lesion. In bacteria in oral mucosal cultures of patients with
contradiction to oral rinses, not all mucosal cul- SS. MacFarlane and Mason (1974) found signifi-
tures correspond to the clinical signs and symp- cantly higher numbers of Staphylococcus aureus
toms. Thus, MacFarlane (1984) found that 73 % and coliform bacilli in SS patients without clini-
of the patients with pSS had clinical signs of oral cal signs of inflammation than in healthy sub-
candidiasis, although cultures obtained from the jects. Veillonella species, Neisseria pharyngis,
dorsal part of the tongue only were positive in Micrococcus mucilaginosus, S. salivarius and S.
52 % of the cases. This difference between clini- aureus have also been isolated in higher num-
cal signs and results of Candida cultures may bers from the tongue, palate, throat and dentures
reflect difficulties in obtaining representative of patients than in healthy subjects (MacFarlane
material from the dry mucosa (Lundström and 1984). However, Almståhl and Wikström (1999)
Lindström 1995; Soto-Rojas et al. 1998). found no differences in the numbers of S. aureus
Furthermore, patients with Candida infection do and enterics between patients with pSS and
not necessarily exhibit oral lesions which can be healthy subjects, and the numbers of S. salivarius,
attributed to an asymptomatic carrier status or Neisseria pharyngis and Veillonella species were
lower in pSS patients. Regarding site specificity, 2010). Salivary secretion may even be lower in
a higher density of streptococci, S. salivarius, individuals with cancer prior to the initiation of
Fusobacterium nucleatum, Prevotella interme- treatment (Harrison et al. 1998; Napeñas et al.
dia, Prevotella nigrescens and S. aureus has been 2013). Chemotherapy can induce compositional
demonstrated on the dorsal part of the tongue changes in saliva. Decreased saliva flow rates
than on the buccal mucosa and in the vestibulum combined with the finding of slightly increased
(Almståhl et al. 2001b). Species usually associ- salivary sodium and chloride concentrations as
ated with gingivitis such as F. nucleatum, P. inter- well as decreased inorganic phosphate concentra-
media and P. nigrescens were found in slightly tion suggest that salivary gland acinar secretion
lower levels in the gingival crevice region of the and duct modification mechanisms are impaired
patients with pSS, but in higher levels of the sSS by cancer chemotherapy (Jensen et al. 2008a).
patients, than in control subjects (Almståhl and The concentration and output of secretory IgA
Wikström 1999). In this regard, it is noteworthy has been found to decrease both during and fol-
that the susceptibility to gingivitis and periodon- lowing chemotherapy (Harrison et al. 1998;
titis has not been found increased in patients with Jensen et al. 2008a; Laine et al. 1992; Main et al.
pSS compared to healthy controls (Boutsi et al. 1984; Meurman et al. 1997a) and the concentra-
2000; Kuru et al. 2002; Pedersen et al. 1999b; tion of lysozyme to decrease after chemotherapy
Schiødt et al. 2001; Tseng et al. 1990), which is (Meurman et al. 1997a). The salivary peroxidase
supported by the rare detection of Porphyromonas system is impaired during chemotherapy due to
gingivalis and Actinobacillus actinomycetemcom- a lower concentration of thiocyanate and its oxi-
itans in the gingival crevice region of patients with dised form hypothiocyanite with antibacterial
pSS (Almståhl et al. 2001b). However, increased properties (Mansson-Rahemtulla et al. 1992).
levels of antibodies to A. actinomycetemcomitans Findings on saliva pH and buffer capacity are
and Porphyromonas gingivalis, but not to P. inter- inconsistent and have been shown to decrease,
media, in addition to an increased incidence of be unchanged or with regard to buffer capacity
periodontal disease have been reported in patients even be increased in response to cancer chemo-
with SS (Çelenligil et al 1998; Ergun et al. 2010), therapy (Avsar et al. 2007; Jensen et al. 2008a;
but they did not discriminate between pSS and Nemeth et al. 2014; Pajari et al. 1989; Schum
sSS, and it is likely that patients with sSS display et al. 1979). Thus, salivary gland hypofunction,
an increased risk of periodontal diseases due to changed composition and reduced output of anti-
its concomitant presence with rheumatoid arthri- microbial substances may impair the oral host
tis (for further details on rheumatoid arthritis and defence against microorganisms, thus individuals
periodontitis, see Chap. 4). in cancer chemotherapy may be more susceptible
to oral infections.
9.3 Oral Microbiota in Patients

Receiving Chemotherapy 9.3.1 Dental Caries
During cancer chemotherapy, there is a signifi- Increased amount of dental bacterial plaque, gin-
cantly increased risk of oral infections due to the gival inflammation and increased salivary counts
immunosuppressive effect and the direct cyto- of caries-related bacteria, mutans streptococci and
toxic effect of the drugs on oral epithelial barrier lactobacilli have been found during and after che-
function. Studies have shown that chemotherapy motherapy (Avsar et al. 2007; Jensen et al. 2008b).
may induce temporary salivary gland hypofunc- However, other studies have shown that the sali-
tion, although there is some controversy whether vary concentrations of S. mutans and lactobacilli
it is caused by the chemotherapy per se or by may decrease during chemotherapy in spite of sali-
other factors, e.g. concomitant intake of xero- vary gland hypofunction. This could be ascribed
genic medication like antiemetics (Jensen et al. to the concomitant use of antibiotics, antifungals
and/or chlorhexidine mouth rinses during chemo- whole saliva flow rate, lower buffer capacity and
therapy as well as cytotoxic effect of the chemo- a change in the oral microbiota towards higher
therapy itself (Meurman et al 1997b; O’Sullivan salivary counts of S. mutans and Lactobacillus
et al. 1993). S. mutans has been found to be sen- have been observed during and after chemo-
sitive to daunorubicin, a cytotoxic antibiotic used therapy and transplantation (Dahllof et al. 1997;
in chemotherapeutic regimens (O’Sullivan et al. Dens et al. 1996). However, stimulated saliva
1993). Along this line, a study showed that salivary flow rates reached normal values 1 year after
S. mutans counts decreased, whereas lactobacilli cancer treatment, and no significant differences
counts increased during chemotherapy (Meurman in caries prevalence were found between bone
et al. 1997b). Other studies have not revealed any marrow-transplanted children receiving chemo-
changes in the composition of the oral microbiota therapy and healthy children 4 years following
(Bergmann 1991; O’Sullivan et al. 1993; Wahlin treatment, but all participants also underwent
and Holm 1988), but an initial doubling of the con- preventive dental care (Dahllof et al. 1997).
centration of microorganisms concomitant with a In other paediatric populations, significantly
transient decrease of stimulated whole saliva flow more caries, poor oral hygiene and significantly
rate during chemotherapy (Bergmann 1991). A lower stimulated whole saliva flow rate have
study of the supra- and subgingival dental plaque been found following childhood chemotherapy
in adult acute leukaemia patients during chemo- (Alberth et al. 2004; Avsar et al. 2007; Nemeth
therapy found that the percentage of total viable et al. 2014; Pajari et al. 1995).
counts of S. mutans in supragingival dental plaque
increased and the percentage in subgingival dental
plaque decreased (Reynolds et al. 1989). However, 9.3.2 Oral Candidiasis during and
the percentage of viridans streptococci (S. mutans After Chemotherapy
not specified) has also been shown to be lower in
the supragingival dental plaque of children with The oral yeast counts and especially the preva-
acute leukaemia during chemotherapy than in lence of Candida species may increase signifi-
healthy individuals (Sixou et al. 1998). cantly from a prevalence of about 50 % in the
As chemotherapy is a time-limited treatment normal population to 73 % in cancer patients dur-
and caries is a process that progresses relatively ing chemotherapy, and the weighted prevalence of
slowly, it may be debatable whether it is possible clinical oral fungal infection has been estimated
to assess an increased progression rate of car- to be 38 % (Lalla et al. 2010). C. albicans is the
ies during chemotherapy. One study found that predominant yeast in the oral microbiota during
5 years after chemotherapy, salivary counts of chemotherapy and accounts for up to 88 % of the
S. mutans and lactobacilli were on the same low salivary yeasts (Samaranayake et al. 1984). Other
levels as baseline values before chemotherapy potential virulent Candida species may colo-
(Meurman et al. 1997b). Another study found no nise the oral cavity during cancer treatment with
significant correlation between salivary immu- weighted prevalence of 16.6 % for Candida trop-
noglobulin levels in stimulated whole saliva and icalis, 5.5 % for Candida glabrata and 3 % for
S. mutans or Lactobacillus counts in long-term Candida krusei (data pooled for chemotherapy
(6 months to 10 years) event-free paediatric and radiation therapy) (Lalla et al. 2010).
patients treated for childhood malignancies by Clinical candidiasis and angular cheilitis
chemotherapy (Dens et al. 1995). The salivary have been found to correlate to higher oral
immunoglobulin level was within normal lim- yeast counts and low saliva flow rates (Wahlin
its, but there was a negative correlation between and Holm 1988; Wahlin 1991). A follow-up
secretory IgA concentration and caries preva- study found that salivary yeast counts remained
lence (DMFT/dmft), although only significant high in spite of normal saliva flow rates 5 years
in some age groups. In bone marrow transplant after chemotherapy for lymphoma (Meurman
patients, a significant decrease in stimulated et al. 1997b). The salivary concentrations of
s-IgA, IgG, IgM and lysozyme in stimulated dose that the tissue has received. Thus, saliva
whole saliva were concomitantly found to be flow rates may remain severely decreased, and
significantly decreased as compared to baseline the compositional changes may persist in
values (Meurman et al. 1997a). These findings response to the decrease in saliva flow rates
suggest that the disease itself or the chemother- (Jensen et al. 2003, 2010). The standard thera-
apy may affect the body defences against peutic radiation dose for head and neck carci-
Candida in the long term. noma amounts to a total dose of 60–70 Gy. The
major salivary glands may have the potential to
gradually recover within 1–2 years if gland-
9.4 Oral Microbiota in Patients sparing radiation regimens have been applied, for
Receiving Radiation Therapy example, intensity-modulated radiation therapy,
and if it has been achievable to keep the radiation
Radiotherapy (RT) of tumours in the head and dose to the gland tissue below thresholds of
neck region often includes the major and minor ~26 Gy to the parotid gland and ~39 Gy to the
salivary glands in the radiation field depending submandibular gland (Murdoch-Kinch et al.
on the anatomical location and the extension of 2008; Vissink et al. 2010). A compensatory
the tumour. RT can cause severe salivary gland increase in saliva flow rate from salivary glands
hypofunction (Jensen et al. 2003, 2010). The not included in the radiation field may be seen
severity of salivary gland hypofunction depends (Eisbruch et al. 2001).
on the volume of salivary gland tissue included in
the radiation field and on the total radiation dose
(Vissink et al. 2010). RT targets cells with a rapid 9.4.1 Dental Caries
mitotic turnover like tumour cells and damages
the DNA thereby leading to cell death. Acinar Irradiation-induced salivary gland hypofunction
salivary gland cells are radiosensitive in spite of is associated with a shift of the normal oral
their slow mitotic turnover (Berthrong 1986), and microbiota increasing the risk to develop rampant
the serous cells appear to be more sensitive to dental caries (Vissink et al. 2003). Higher levels
radiation than the mucous ones (Kashima et al. of S. mutans and Lactobacillus species are often
1965). Radiation damage to the salivary glands observed in the oral cavity during and after RT
may be seen as early as 1 week after initiation of compared to preradiation levels (Brown et al.
RT (Dreizen et al. 1977) and results in both acute 1975, 1978; Keene and Fleming 1987; Llory
and long-term effects characterised by reduced et al. 1972; Schwarz et al. 1999; Vuotila et al.
saliva flow rates, high saliva viscosity and 2002). Oral colonisation with S. mutans has been
changes of saliva composition. During RT, saliva found lower and stimulated saliva flow rates
flow rates decrease and may even reach immea- higher at the end of treatment in patients receiv-
surable levels (Vissink et al. 2010). With decreas- ing unilateral RT as compared to bilaterally irra-
ing flow rates, the salivary pH drops and the diated patients (Beer et al. 2002). In oral rinses, it
buffer capacity decreases both during and after has been demonstrated that the predominant
RT (Jensen et al. 2003; Valdez et al. 1993). RT acid-producing species of the oral microbiota
also affects the salivary antimicrobial compo- may change from S. sanguis, S. mitis and S. sali-
nents. The salivary concentrations of IgA and varius before RT to S. mitis, S. salivarius and lac-
IgG, lactoferrin, lysozyme and peroxidase have tobacilli with a concomitant decrease in saliva
been shown to increase during RT due to acute flow rates after RT (Tong et al. 2003). The acid-
tissue destruction, but after RT they decrease due sensitive S. sanguis appears to be inhibited by the
to reduced functioning of the glands (Brown more acidic oral environment after RT (Tong
et al. 1976; Jensen et al. 2003; Makkonen et al. et al. 2003). The decrease in the presence of S.
1986). In the long term, recovery of salivary sanguis after RT has been shown in other studies
gland function is dependent on the total radiation (Brown et al. 1975, 1978). Vuotila et al. (2002)
found unchanged levels of S. mutans after RT as due to the risk of developing oropharyngeal candi-
compared to preradiation levels. Interestingly, a diasis (Lalla et al. 2010).
study found no difference in the microbial diver-
sity, composition or number of colony-forming
bacterial units (mutans streptococci and lactoba- 9.5 Medication-Induced Salivary
cilli), nor did they find a difference in the stimu- Gland Dysfunction and Oral
lated whole saliva flow rate, saliva pH and Microbiota
buffering capacity when comparing caries-free
and caries-active irradiated nasopharyngeal The most common cause of salivary gland dys-
patients (Zhang et al. 2015). function is the intake of prescribed medications
RT patients suffering from impaired saliva (Handelman et al. 1986; Närhi et al. 1992; Smidt
secretion are to be considered a high-risk group et al. 2010, 2011; Villa et al. 2015; Österberg et al.
regarding dental caries as the oral environment 1984). Xerostomia is a common complaint, espe-
favours acidogenic and acidophilic species. cially in elderly people above the age of 65 years,
and is often reported as an adverse effect of med-
ications. The prevalence is ranging from 11 to
9.4.2 Oral Candidiasis 72 % (Smidt et al. 2011; Desoutter et al. 2012).
Xerostomia is defined as the subjective feeling of
The number of yeasts increases in the oral cavity oral dryness (Fox et al. 1987; Shetty et al. 2012),
of cancer patients with hyposalivation due to RT, and salivary gland dysfunction denotes changes
and oropharyngeal candidiasis is a frequent com- in the quantity and/or quality of saliva (Villa
plication during and after RT for head and neck et al. 2015). More than 75 % of adults aged 65
cancer (Lalla et al. 2010). The weighted preva- and older take at least one prescription medica-
lence of oral candidiasis during head and neck RT tion (Chrischilles et al. 1992; Smidt et al. 2010).
has been estimated to be 37 % (Lalla et al. 2010). Xerostomia has been associated with 80 % of
The increase in the oral yeast colonisation is the most commonly prescribed medications, and
observed during RT, and the colonisation level several of them have adverse effects directly on
remains elevated after RT. The weighted preva- the mechanisms responsible for saliva secretion
lence of oral fungal colonisation during radiation (Smith and Burtner 1994; Sreebny 2010; Smidt
therapy has been estimated to be 75 % (Lalla et al. et al. 2011; Villa et al. 2015; Aliko et al. 2015).
2010). C. albicans is the predominant yeast spe- Regardless of the type of medication, saliva flow
cies associated with oral candidiasis in RT patients rates have been shown to decrease as the number
(Redding et al. 1999; Thaweboon et al. 2008). of medications increases also known as polyphar-
However, other Candida species are frequently macy (Thorselius et al. 1988; Närhi et al. 1992;
isolated from the oral cavity in RT and may cause Smidt et al. 2010). Xerostomia is not only associ-
oral candidiasis, e.g. C. glabrata, C. tropicalis, C. ated with a decrease in the salivary flow rate, it
parapsilosis and C. krusei (Bulacio et al. 2012; de may also be attributed to a change in quality of the
Freitas et al. 2013; Lalla et al. 2010; Schelenz et al. saliva (Crogan 2011; Ekström et al. 2012; Tabak
2011). It has been shown that the increase in C. 1995). The duration of medication intake also
albicans in oral rinses is positively related to the affects the saliva flow rates and the prevalence
radiation dose and the volume of parotid gland tis- of xerostomia (Navazesh et al. 1996; Thomson
sue included in the radiation field (Rossie et al. et al. 2006). Navazesh et al. (1996) found that
1987). A direct correlation between the increase in unstimulated and stimulated whole saliva flow
C. albicans in saliva and reduced saliva flow rates rates were significantly lower in adults who had
during RT has also been shown (Epstein et al. been taking medication for more than 2 years as
1998; Karbach et al. 2012). The increased coloni- compared to those who had been taking medica-
sation of oral yeasts in RT patients in combination tion for less than 2 years.
with salivary gland hypofunction emphasises the The mechanisms by which medications can
importance of optimal oral hygiene in RT patients influence salivary secretion and cause salivary
gland dysfunction are complex, and this is also in the oral microbiota. A cross-sectional study
reflected in the variation and severity of oral demonstrated that root caries was more prevalent
complications. It is believed that medications in those taking antihypertensives than in control
can interact with the salivary secretory reflex at subjects (Streckfus et al. 1990). Also the whole
several sites (Ekström et al. 2012; Proctor 2015; saliva flow rates have been found lower and the
Sreebny 2010; Villa et al. 2015). Thus some levels of mutans streptococci and lactobacilli
medications act at the level of the central nervous higher in patients taking antihypertensives com-
system such as antidepressants (both tricyclic pared to control subjects (Nonzee et al. 2012). A
and non-tricyclic), opioids, sedatives, anxiolyt- study on 848 community-dwelling elderly peo-
ics and decongestants (pseudoephedrine). Also ple in South Australia found only a moderate
antihypertensives acting on central alpha-2 adren- association between medication intake and root
ergic receptors (e.g. clonidine) have been shown caries experience (Thomson et al. 1995).
to reduce salivary secretion in humans (Proctor However, a more detailed analysis on the various
2015; Sreebny 2010). Other medications like drugs revealed that patients who took antide-
anticholinergics for overactive bladder, antiemet- pressants and antiulcer agents had a significantly
ics, tricyclic antidepressants, serotonin reuptake higher root caries index and a 5-year follow-up
inhibitors, certain neuroleptics, antihistamines study including 528 community-dwelling elderly
and antihypertensives (alpha-1 adrenergic and South Australians did not reveal a strong associ-
beta-adrenergic blocking agents) are acting at ation between intake of medication and caries,
the peripheral level of the neuro-glandular junc- apart from intake of antiasthmatics. Along this
tion interfering with cholinergic muscarinic line, Ryberg et al. (1991) found that long-term
(M3), adrenergic, peptidergic and/or histaminer- treatment with β2-adrenoceptor agonists in
gic receptor systems (for reviews Proctor 2015; patients with asthma was associated with an
Sreebny 2010). Antidepressants, i.e. inhibitors of impaired saliva secretion, which was followed
serotonin and noradrenaline transporters respon- by an increased incidence of dental caries and
sible for reuptake, appear to cause salivary gland higher number of DMS (Decayed-Missing-
dysfunction through activation of alpha-2 adren- Surfaces) after 4 years of follow-up. These find-
ergic receptors by elevating endogenous levels of ings were supported by Alaki et al. (2013), who
noradrenaline. Both centrally and peripherally act- also found higher levels of mutans streptococci
ing medications include tricyclic antidepressants, and lactobacilli in asthmatic patients taking anti-
the serotonin reuptake inhibitors, some neurolep- asthmatics more than three times a day com-
tics and antihistamines (Clemmesen 1988; Del pared with other asthmatic patients. A recent
Vigna de Almeida et al. 2008; Hunter and Wilson study showed that patients on diuretics had a
1995; Proctor 2015; Sreebny 2010). Furthermore, higher prevalence of xerostomia, periodontitis,
some medications like diuretics indirectly influ- dental caries and mucosal lesions than control
ence the salivary secretion by affecting the elec- subjects (Prasanthi et al. 2014). A study by
trolytes and water homeostasis (Nederfors et al. Rindal et al. (2005) demonstrated that patients
1989). Finally, a large number of factors influence taking antidepressants had a higher number of
the effect of medications on salivary secretion, dental restorations (a proxy for dental caries)
such as the dose and the absorption and excretion than the non-medicated ones. Bardow et al.
rates of the drug as well as drug interactions (Del (2001) showed that patients with a daily intake
Vigna de Almeida et al. 2008). of xerogenic medications had low unstimulated
and stimulated whole saliva flow rates and
decreased salivary outputs of bicarbonate, cal-
9.5.1 Dental Caries cium, phosphate and protein and higher levels of
Lactobacillus species. These results were sub-
A relatively limited number of studies have stantiated by Almståhl et al. (2003) who found
investigated the association between medication- higher numbers of lactobacilli in oral rinses from
induced salivary gland dysfunction and changes patients with medication-induced hyposalivation
than in control subjects, but lower levels than in showed that intake of xerogenic medication was
patients with pSS and patients with RT-induced significantly associated with a high oral mucosal
hyposalivation which was attributed to much inflammation score. Furthermore, in patients tak-
lower saliva flow rate in the two latter groups. It ing antihypertensives, the mean levels of Candida
has also been shown that patients with medica- species were higher than in the control subjects
tion-induced hyposalivation had a supragingival (Nonzee et al. 2012). Other studies have found
plaque comprising high levels of mutans strepto- higher Candida levels in patients with medication-
cocci and lactobacilli and hence an increased induced hyposalivation and in medicated men
risk of developing caries (Almståhl and (Almståhl and Wikström 2005; Parvinen et al.
Wikström 2005). An additional number of stud- 1984), and Kreher et al. (1991) showed that
ies have reported that the numbers of lactobacilli C. glabrata was the most frequent yeast strain in
are higher in medicated patients compared to the oral cavity of medicated patients.
non-medicated and also associated with low
saliva flow rates (Fure 2003; Närhi et al. 1994;
Parvinen et al. 1984). 9.5.3 Periodontal Disease
Moreover, several liquid formulations of med-
ications have a high sugar content that may influ- One study has found that periodontal disease
ence the oral microbiota and hence increasing the (assessed by Russell’s periodontal index and
risk of medication-induced caries (Donaldson plaque index) was more prevalent in patients tak-
et al. 2015). Beighton et al. (1991) showed that ing diuretics and who also had low whole saliva
the salivary level of mutans streptococci, lactoba- flow rates (Nonzee et al. 2012). The authors sug-
cilli and yeasts in elderly patients treated with gest that the high prevalence of periodontitis
sucrose-containing medication was significantly could be due to decreased cleansing activity and
higher than in patients taking non-sucrose- reduced microbial activity by saliva. However,
containing medication. apparently there is no substantial evidence sug-
gesting that medication-induced salivary gland
dysfunction is associated with an increased risk
9.5.2 Oral Candidiasis of periodontal disease (Aliko et al. 2015).
Periopathogens such as P. gingivalis and A. acti-
The number of studies investigating the associa- nomycetemcomitans are rarely detected in supra-
tion between medication-induced salivary gland gingival plaque samples from patients with
dysfunction and oral candidiasis is sparse. It has medication-induced salivary gland hypofunction
been shown that medicated elderly people have a or in patients with hyposalivation due to other
higher Candida load and also higher frequency of causes (Almståhl and Wikström 2005).
oral candidiasis and lower saliva flow rates than
the non-medicated ones (Pedersen et al. 2015). A Conclusions
higher frequency of Candida isolation and palatal Patients with SS often have a severe and per-
inflammation has also been found in patients manent reduction in their salivary gland func-
treated with psychotropic agents, and who were tion leading to reduction in the salivary pH
wearing complete upper dentures, than in control and decreased clearance of microorganisms,
subjects (Lucas 1993). However, other risk factors dietary sugars and acids in the oral cavity.
were more common among the psychiatric This leads to a shift in the oral microbiota
patients including cigarette smoking, sugar con- including colonisation of more acidophilic
sumption and a poor denture hygiene. A recent species such as S. mutans, lactobacilli and C.
study reported that intake of anxiolytics, and low albicans and consequently an increased risk
salivary flow rates, were associated with higher of dental caries and oral candidiasis.
levels of Candida in patients with oral lichen pla- During cancer chemotherapy, there is a sig-
nus (Bokor-Bratic et al. 2013). Janket et al. (2007) nificantly increased risk of oral infections due
to the immunosuppressive effect and the direct dental caries and salivary characteristics in children.
Oral Health Prev Dent. 2013;11(2):113–20.
cytotoxic effect of the drugs on oral epithelial
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Aliko A, Wolff A, Dawes C, Aframian D, Proctor G,
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Ekström J, Narayana N, Villa A, Sia YW, Joshi RK,
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Part III
Future Diagnostic Methods
and Techniques
The Oral Microbiome in Health
and Disease
10
Ingar Olsen
Abstract
This chapter deals with the human oral microbiome which contains bacte-
ria, bacteriophages/viruses, archaea, fungi, and protozoa. Modern molecu-
lar techniques used to analyze this microbiome are dealt with such as
HOMINGS, oligotyping, high-throughput sequencing, whole-genome
shotgun sequencing, single-cell genome sequencing, metatranscriptomics,
and community-wide transcriptome analysis. The oral microbiota in health
is described as well as that in periodontal disease and dental caries.
Furthermore, the architecture of biofilms in periodontitis and caries is
visualized. Our knowledge on the oral microbiota challenges the current
practice of chairside diagnostics.
10.1 The Human Oral Microbiome and periodontitis through ecological changes.
Another noteworthy feature is that it is personal-
The human oral microbiome is composed of a ized, meaning that each person harbors a unique
variety of different microorganisms such as bac- microbiota. This implies that the human microbi-
teria, bacteriophages/viruses, yeasts, archaea, ome is more different between individuals than
and protozoa. It has been suggested that these within an individual (Fig. 10.1). It has also been
organisms cause diseases by a synergistic or shown that characteristics of an individual’s life
cooperative way and that the interspecies interac- history can be associated with the composition of
tions have a crucial role whether the oral micro- the microbiome (Ding and Schloss 2014) and
biota causes disease or not (He et al. 2014). What that the phylogenetic microbial structure varies
is remarkable for this microbiota is also that its with aging (Xu et al. 2014).
commensals contribute to disease, e.g., to caries
10.1.1 Bacteria
I. Olsen, PhD, DDS
Bacteria have been considered the dominating part
Department of Oral Biology, Faculty of Dentistry,
University of Oslo, Oslo, Norway of the microbiome in man. However, while some
e-mail: ingar.olsen@odont.uio.no six billion bacteria are present in the oral cavity, it

DOI 10.1007/978-3-319-25091-5_10
98 I. Olsen
Fig. 10.1 Humans are far more different from each other in phyla living in and on our bodies, including the oral cavity,
their microbial composition than in their genomic composi- but their relative abundance can be drastically different. On
tion. The colors on the left side of each individual represent the other hand, our genomic composition is nearly identical,
bacterial phyla, while the colors on the right side indicate with only a small fraction (ca 0.1 %) differing across indi-
host genomic similarity. For the most part we contain similar viduals (Adapted from Califf et al. 2014)
contains potentially 35 times that many bacterio- 10.1.2 Bacteriophages/Viruses

phages/viruses (Edlund et al. 2015). When
Dewhirst et al. (2010) established the Human Oral Oral viruses in saliva are dominated by bacterio-
Microbiome Database (HOMD) (http://www. phages (Pride et al. 2012). Also dental plaque is
homd.org/), it comprised over 600 prevalent bacte- inhabited by a community of bacteriophages
rial taxa at the species level with distinct subsets (Naidu et al. 2014). Bacteriophages constitute the
predominating at different sites such as teeth, gin- major part of the oral virome with relatively few
gival sulcus, tongue, cheeks, hard/soft palate, and eukaryotic viruses identified such as herpesvi-
tonsils. The HOMD included 619 taxa from 13 ruses, papillomaviruses, enteroviruses, and circo-
phyla: Actinobacteria, Bacteroidetes, Chlamydiae, viruses (Grinde and Olsen 2010; Naidu et al.
Chloroflexi, Euryarchaeota, Firmicutes, Fuso- 2014). The mouth has been found to have more
bacteria, Proteobacteria, Spirochaetes, SR1, genetic elements than the stool, i.e., viruses, plas-
Synergistetes, Tenericutes, and TM7. The analysis mids, and transposons, although it has fewer bac-
comprised 1179 taxa. Among these 24 % were teria (Zhang et al. 2013). Bacteriophages may
named, 8 % were cultivated but unnamed, and serve as reservoirs for genes functioning in the
68 % were uncultivated phylotypes. Later the oral cavity. Phage members of the oral virome
number of oral phyla has been extended to 15, but can carry genes involved in resistance to comple-
96 % of the sequences are accounted for by only 6 ment degradation of immunoglobulins, adhesion
phyla: Actinobacteria, Bacteroidetes, Firmicutes, to cells lining the oropharynx, and antibiotic
Fusobacteria, Proteobacteria, and Spirochaetes resistance (Pride et al. 2012; Muniesa et al. 2013;
(Wade 2013). Recently, Camanocha and Dewhirst Abeles et al. 2014; Quirós et al. 2014).
(2014) developed primer pairs for making phylum- Oral viruses have gene functions that may be
selective 16S rRNA clone libraries and identified involved in the pathogenic roles of their host bac-
species from the lesser known oral phyla or candi- teria (Pride et al. 2012). The same salivary viruses
date divisions including Synergistetes, TM7, could be identified at all time points over 60 days
Chlorobi, Chloroflexi, GN02, SR1, and WPS-2. despite being present in low numbers (Abeles
10 The Oral Microbiome in Health and Disease 99
et al. 2014), reflecting that the oral viral ecosys- The largest difference in composition was
tem is stable. Most oral viruses are lysogenic and between supra-/subgingival plaque and saliva.
live in harmony with their hosts (Abeles and Differences in virus composition were signifi-
Pride 2014; Ly et al. 2014), and they may be cantly related to the health status of viruses in
important in shaping the microbial diversity of plaque, but not to those in saliva. Noteworthy,
the oral cavity. Another peculiarity is that viral there was a significant increase in myoviruses
communities of the mouth are highly personal- (generally lytic) in subgingival biofilm suggest-
ized (Willner et al. 2011; Pride et al. 2012), even ing that these viruses may have a great impor-
more personalized than bacterial communities tance to local bacterial diversity and that the virus
when analyzed with 16S rDNA sequencing may serve as useful indicators of the oral health
(Abeles et al. 2014). A noteworthy feature is also status. Since viruses have the potential to form
that oral viruses vary according to host sex, rather microbial communities as well as to elicit host
than among individuals (Abeles et al. 2014). The immune response, they probably play an impor-
human oral viral community is probably a result tant role in human health (Edlund et al. 2015).
of the unique viral exposures of each individual Also, the fact that they are personal, persistent,
(Abeles et al. 2014), but considerably more of the and gender specific suggests that they can be
oral virobiota of people living together is shared important in the interplay between host genetics
than could be expected by chance (Robles- and the environment.
Sikisaka et al. 2013). Eukaryotic viruses such as
Torque Teno viruses (TTVs) and SEN viruses
have been found in the bloodstream of healthy 10.1.3 Archaea
people (Pride et al. 2012; Abeles and Pride 2014).
Blood of healthy persons have previously been Archaea were originally considered a primitive
considered sterile. Both these groups of viruses form of life that thrives in extreme environments.
are present in the human oral cavity (Pride et al. However, high numbers of methane-producing
2012). Also herpesviruses, shed in the mouth archaea (methanogens) have now been detected
from healthy individuals, can be found in human in the oral cavity (Belay et al. 1988), the gas-
blood (De Vlaminck et al. 2013). Therefore not trointestinal tract (Karlin et al. 1982), and
only bacteria but also viruses can translocate vagina (Belay et al. 1990) of human beings.
through mucosal surfaces to the bloodstream and The reported oral archaea contain the gen-
possibly be involved in systemic diseases. era Methanobrevibacter, Methanobacterium,
It is well known that the human oral cavity Methanosarcina, and Methanosphaera and the
contains a large and diverse variety of bacteria. order Thermoplasmatales (He et al. 2014). The
What viruses it contains has to a great extent been main species is Methanobrevibacter oralis.
overlooked. This particularly relates to the peri- Archaea have been detected in saliva, periodon-
odontal microbiota, although herpesviruses titis, peri-implantitis, pericoronitis, and infected
including Epstein-Barr virus and cytomegalovi- root canals (Brusa et al. 1987; Belay et al. 1988;
rus can be present in high copy counts in aggres- Kulik et al. 2001; Lepp et al. 2004; Vianna et al.
sive periodontitis and may interact with 2006, 2009; Vickerman et al. 2007; Conway de
periodontopathogenic bacteria to cause the dis- Macario and Macario 2009; Jiang et al. 2009;
ease (Sunde et al. 2008; Slots 2011; Contreras Matarazzo et al. 2011, 2012; Faveri et al. 2011;
et al. 2014). Ly et al. (2014) examined samples Mansfield et al. 2012; Bringuier et al. 2013). These
from saliva of periodontally healthy and diseased studies detected a higher frequency of archaea
patients and found that the communities of in oral infections than in health. Thus the rela-
viruses inhabiting saliva and subgingival and tive abundance of archaea in subgingival plaque
supragingival biofilms were composed mainly of increased with the severity of periodontitis and
bacteriophages. The virome composition was decreased with the reduction of periodontitis after
greatly reflected by the site it was collected from. treatment. Archaea may therefore be associated
100 I. Olsen
with periodontitis but the diversity of archaea skin, were for the first time included in the oral
is limited (Li et al. 2009). Almost all sequenced core mycobiome. The oral fungal community
amplicons fell in the genus Methanobrevibacter showed a consistent intraindividual stability over
of the Euryarchaeota phylum with M. oralis-like time, but there was high interindividual variability
species as the most dominant. In root canal infec- (Monteira-da-Silva et al. 2014).
tions, presence of archaea was associated with Interactions between fungi and bacteria, e.g.,
clinical symptoms (Jiang et al. 2009). Although between Candida and streptococci, may influence
discussion of the clinical role of Euryarchaeota oral health (Diaz et al. 2014). A symbiotic relation-
(including Methanobrevibacter smithii, M. ora- ship between S. mutans and C. albicans has been
lis, and Methanosphaera stadtmanae) continues found to synergize virulence of plaque biofilms
(Horz and Conrads 2010), and archaea are emerg- in vivo (Falsetta et al. 2014). Thus S. gordonii glu-
ing organisms in complex human microbiomes cosyltransferase promotes biofilm interactions
(Dridi et al. 2011), methanogenic archaea do not with C. albicans (Ricker et al. 2014). Fungi proba-
seem to induce oral diseases directly. However, bly have a role in maintaining a balance between
they may promote anaerobic infections through microorganisms and the host (Krom et al. 2014).
syntropic interactions with true pathogenic fer-
menting bacteria, e.g., through interspecies H2
transfer, thereby favoring growth of certain bac- 10.1.5 Protozoa
teria (Matarazzo et al. 2012). Thus, a positive
correlation has been found between methanogens Protozoa are parts of the normal microbiome.
and Synergistes species in oral infections (Vianna The best known are Entamoeba gingivalis and
et al. 2006; Vartoukian et al. 2007). Trichomonas tenax (Vozza et al. 2005). They are
present in subjects who neglect their oral hygiene
and predominantly in subgingival plaque from
10.1.4 Fungi patients with periodontal disease (Lange et al.
1983). Both have been linked to gingivitis and
Dupuy et al. (2014) performed massive parallel, they were once considered pathogens. T. tenax has
high-throughput sequencing of internal transcribed been correlated with xerostomia, burning mouth,
spacer 1 (ITS1) amplicons from saliva after robust and periodontal pockets (Kurnatowska 1993;
extraction methods. Their findings confirmed Kurnatowska and Kurnatowski 1998). Later, it has
nearly every community member from a similar become clear that these organisms increase when
study by Ghannoum et al. (2010) who had detected the oral hygiene deteriorates. Their increase may
74 cultivable and 11 non-cultivable fungal genera be due to nutrients accessible from debris and
in the oral cavity by using multitag pyrosequencing bacteria (Wade 2013). It is interesting though that
of panfungal ITS primers. A consensus on genus- metronidazole, frequently used as an effective sup-
level members of oral fungi (core mycobiome) was plement in the treatment of periodontitis, is active
thereby reached. This study was the first to demon- against both Entamoeba and Trichomonas.
strate not-yet-cultivated fungi in the oral cavity. It
was suggested that such organisms could be the rea-
son for failure in the treatment of oral fungal infec- 10.2 Techniques to Analyze
tions. Consensus members of the saliva microbiome the Oral Microbiota
were Candida/Pichia, Cladosporium/Davidiella,
Alternaria/Lewia, Aspergillus/Emericella/Euroti It should be realized that every technique that has
um, Fusarium/Gibberella, Cryptococcus/Filoba been used to detect oral microorganisms has its
sidiella, and Aureobasidium. Weaker candidates strengths and limitations. Not all of these tech-
for consensus inclusion were Saccharomyces, niques will be dealt with here. Microscopy and
Epicoccum, and Phoma. Interestingly, Malassezia culture were long standard methods for assess-
species, that are important commensals of human ment of the oral microbiota. Later, culture helped
Fig. 10.2 Site specificity of predominant bacterial spe- any subject (clear box), < 15 % of the total number of
cies in the mouth. Bacterial species or phylotypes were clones assayed (yellow box), and ≥ 15 % of the total num-
selected on the basis of their detection in multiple subjects ber of clones assayed (green box). The 15 % cutoff for low
for a given site. Distributions of bacterial species in oral and high abundance was chosen arbitrarily. Marker bar
sites among subjects are indicated by the columns of represents a 10 % difference in nucleotide sequences
boxes to the right of the tree as follows: not detected in (From Aas et al. 2005)
us become more familiar with this microbiota 1998). Since there was reason to believe that also
when methods for recovery of anaerobic bacteria not-yet-cultivated bacteria could be involved in
were developed. However, it soon became clear disease methods, targeting the small subunit
that only half of the oral microbiota could be cul- (16S) ribosomal RNA molecule was used. These
tured. Therefore culture-independent methods efforts have provided a vast amount of knowl-
were exploited, particularly DNA-DNA hybrid- edge and description of the oral microbiota. They
ization and PCR-based assays. DNA-DNA have also shown that the oral microbiota is not
hybridization (checkerboard) relied though on uniform but varies from site to site (Fig. 10.2).
bacteria that could be cultivated for the making of The information has been collected in the first
whole genomic probes (Socransky et al. 1994), curated collection of a human-associated micro-
but reverse-capture checkerboard hybridization biome, HOMD, which provides a description of
did not (Paster et al. 1998). Checkerboard DNA- the organisms and their genomics together with a
DNA hybridization was helpful delineating 16S rRNA identification tool (Dewhirst et al.
bacteria clinically related to periodontitis such as 2010), and later in the CORE database that is a
the red and the orange complex (Socransky et al. phylogenetically curated 16S rDNA database of
102 I. Olsen
the core oral microbiome (Griffen et al. 2011). 10.2.3 High-Throughput Sequencing
Although 16S rRNA gene amplification and (Pyrosequencing)
Sanger sequencing significantly increased our
knowledge of the major components of the oral 16S rRNA sequencing using next-generation
microbiota, they did not provide information of sequencing has provided a wealth of new knowl-
the entire microbiota. Organisms that are present edge on the genetic composition of the oral micro-
in low amounts were first revealed by pyrose- biome in health and disease. The most useful of
quencing (next-generation sequencing methods). these approaches have relied on the 454 (Roche)
pyrosequencing platform. In Table 10.1, the advan-
tages and limitations of different high-throughput
10.2.1 HOMINGS sequencing platforms are summarized.
HOMINGS (http://homings.forsyth.org) apply

the speed and efficiency of the next-generation 10.2.4 Whole-Genome Shotgun
sequencing using the Illumina platform. Almost Sequencing
600 oral bacterial taxa can be identified with this
technique which provides genus-level identifica- Whole-genome shotgun sequencing (WGS) can
tion of the remaining sequences for 129 genera. It provide highly accurate sequences in an economic
is thus more comprehensive than its predecessor way and has a fast turnaround (Hasan et al. 2014).
HOMIM which gave simultaneous microarray WGS metagenomic sequencing has proved to be a
detection of about 270 of the most prevalent, cul- powerful tool for studying the human microbiome.
tivated, and not-yet-cultivated oral bacterial At present, WGS metagenomic data contain mil-
species. lions to billions of short reads and offer an unprec-
edented opportunity to identify species at or near
strain level and their abundance.
10.2.2 Oligotyping Analysis

of the Human Oral 10.2.5 Single-Cell Genome
Microbiome Sequencing
A limited taxonomic resolution has often pre- Remarkable in the identification of bacteria is sin-
vented understanding the census of bacterial pop- gle-cell genome sequencing which enables not
ulations in healthy individuals. By using 16S only identification of microbes but links their func-
rRNA gene sequence data from nine sites in the tions to species, which is not feasible with metage-
oral cavity, Eren et al. (2014) identified 493 oli- nomic techniques. It also analyzes low-abundance
gotypes from their V1-V3 data and 360 oligo- species that can be lost in community-based analy-
types from the V3-V5 data. The oligotypes were ses and can be useful in complementing metage-
associated with species-level taxon names by nomic analyses (Yilmaz and Singh 2012). An
comparing with HOMD. The authors discovered ultimate goal of single-cell sequencing is recovery
closely related oligotypes differing sometimes by of genome sequences from each cell within an
only a single nucleotide that showed widely dif- environment (Clingenpeel et al. 2015).
ferent distributions among oral sites and samples.
Different habitat distributions of closely related
oligotypes indicated a level of ecological and 10.2.6 Metatranscriptomics
functional biodiversity not recognized previ- of the Oral Microbiome
ously. This technique combined with Shannon during Health and Disease
entropy has the capacity to analyze entire micro-
biomes and discriminate between closely related Although new techniques have revealed what
but distinct taxa in different habitats. organisms are present in the oral microbiome,
Table 10.1 Comparison of next-generation sequencing platforms
Current,
Machine Modal read approximate cost
(manufacturer) Chemistry lengtha (bases) Run time Gb per run (US$)b Advantages Disadvantages
High-end instruments
454 GS FLX+ (Roche) Pyrosequencing 700–800 23 h 0.7 500,000 Long read lengths Appreciable hands-on
time
High reagent costs
High error rate in
homopolymers
HiSeq 2000/2500 Reversible terminator 2 × 100 11 days 600 (regular 750,000 Cost-effectiveness Long run time
(Illumina) (regular mode) or 120 Steadily improving Short read lengths
mode) or (rapid run read lengths HiSeq 2500 instrument
2 days (rapid mode)c Massive throughput upgrade not available at
run mode)c Minimal hands-on time of writing
10 The Oral Microbiome in Health and Disease
time (available end 2012)

5500xl SOLiD (Life Ligation 75 + 35 8 days 150 350,000 Low error rate Very short read lengths
Technologies) Massive throughput Long run times
PacBio RS (Pacific Real-time sequencing 3000 (maximum 20 min 3 per day 750,000 Simple sample High error rate
Biosciences) 15,000) preparation Expensive system
Low reagent costs Difficult installation
Very long read
lengths
Bench-top instruments
454 GS Junior (Roche) Pyrosequencing 500 8h 0.035 100,000 Long read lengths Appreciable hands-on
time
High reagent costs
High error rate in
homopolymers
Ion Personal Genome Proton detection 100 or 200 3h 0.01–0.1 (314 80,000 (including Short run times Appreciable hands-on
Machine (Life chip), 0.1–0.5 OneTouch and Appropriate time
Technologies) (316 chip), or server) throughput for High error rate in
up to 1 (318 microbial homopolymers
chip) applications
(continued)
103
Table 10.1 (continued)
104
Current,
Machine Modal read approximate cost
(manufacturer) Chemistry lengtha (bases) Run time Gb per run (US$)b Advantages Disadvantages
Ion Proton (Life Proton detection Up to 200 2h Up to 10 145,000 + 75,000 Short run times Instrument not available
Technologies) (Proton I chip) for compulsory Flexible chip at time of writing
or up to 100 server reagents
(Proton II
chip)
MiSeq (Illumina) Reversible terminator 2 × 150 27 h 1.5 125,000 Cost-effectiveness Read lengths too short
Short run times for efficient assembly
Appropriate
throughput for
microbial
applications
Minimal hands-on
time
Adapted from Loman et al. (2012)
a
Average read length for a fragment-based run
b
Approximate cost per machine plus additional instrumentation and service contract
c
Available only on the HiSeq 2500
I. Olsen
they do not tell anything about the viability of the 10.2.7 Community-Wide
organisms or their functions. Therefore efforts Transcriptome Analysis
have been made recently to use microbiomics, of the Oral Microbiome
metagenomics, and transcriptomics to better in Subjects With and Without
understand the role of the oral microbiome in Periodontitis
health and disease. This may also help us to more
efficiently prevent these diseases and provide a Our knowledge on the in situ activities of the
personalized treatment. organisms and their interaction with each other
Our indigenous microbiota is closely linked to and with the environment is limited. Such knowl-
health. However, when disrupted the same micro- edge may be obtained by characterizing gene
biota can induce disease. Such diseases are charac- expression profiles of the microbiome. In situ
terized by changes in the relative amounts of genome-wide transcriptome variation was stud-
different species. While such changes in the ied in the subgingival microbiome of six peri-
microbiota occur, it is also clear that the members odontally healthy individuals and seven
of the microbial communities can differ markedly individuals with periodontitis (Duran-Pinedo
between individuals (Ge et al. 2013). This applies et al. 2014). The overall metabolic activities
to the microbiota of both healthy and diseased defining disease were related to iron acquisition,
individuals. In a study based on nine patient- lipopolysaccharide synthesis, and flagella syn-
matched healthy and diseased samples, 160,000 thesis. It was both noteworthy and unexpected
genes were compared in healthy and diseased peri- that the majority of virulence factors upregulated
odontal communities (Jorth et al. 2014). Massive in periodontitis came from organisms not consid-
parallel RNA sequencing was used to demonstrate ered as major pathogens. Also remarkable was
changes in the composition and gene expression of that one of the organisms with characterized gene
the microbiota in health and periodontitis. It was expression profile was from the uncultured can-
shown that both communities exhibited defined didate division TM7 exhibiting upregulation of
differences in metabolism that were conserved putative virulence factors in disease. This dem-
between patients. In contrast, the metabolic gene onstrated the importance of in situ metatranscrip-
expression of individual species within the com- tomic studies for studying the possible etiological
munity varied greatly between patients. Disease- role of uncultured organisms. Unexpectedly, no
associated communities also showed conserved viral sequence was detected in either the metage-
changes in metabolic and virulence gene expres- nome or the metatranscriptome.
sion. Thus, by using transcriptional profiling the
authors could determine changes in the composi-
tion and gene expression of the human oral micro- 10.3 Oral Microbiota in Health
biota in health and periodontitis.
By using metatranscriptome analysis of peri- The oral microbiota in health is highly diversified.
odontal biofilm in vitro, it was demonstrated that It consists of approximately 600 predominant spe-
addition of periodontal pathogens to a healthy cies (Dewhirst et al. 2010) that contribute to the
biofilm multispecies model had a drastic effect in health and physiology of the oral cavity. Two main
changing the gene expression profiles of the types of tissues are colonized: soft and hard tis-
organisms of the healthy community (Frias- sues. It is also clear that the oral cavity contains
Lopez and Duran-Pinedo 2012). Chaperones different niches for bacterial growth with different
were highly upregulated, possibly due to stress, bacterial profiles that are site and subject specific
and there was a significant upregulation of ABC (Fig. 10.2). Even close sites such as the dorsal and
transporter systems and putative transposases. lateral sides of the tongue dorsum (Aas et al. 2005)
With pathogens present, proteins related to and the vestibular and lingual surfaces of incisors
growth and division, as well as a large portion of and canines (Simon-Soro et al. 2013) have dif-
transcription factors, were upregulated. ferent microbiotas. The oral microbiota has, due
106 I. Olsen
to its continuum with the external environment, polymicrobial synergy and dysbiosis (PSD) can
developed features to counteract challenges from be mentioned. This variability may partly be con-
foreign bacteria. There is probably a core microbi- sidered results of increased knowledge related to
ome for health which is common to all individuals instrumental analytical improvements. However,
(Zarco et al. 2012). In addition, there is a variable rather than mentioning the microorganisms
microbiome unique to individuals depending on involved under each etiological heading, space will
lifestyle and physiological differences. Supporting be devoted here to the most recent concept, PSD.
the existence of a core microbiome was that iden- In the PSD model, it is recognized that the
tical bacterial sequences were detected in the oral gingival crevice is colonized by a diverse micro-
cavities of unrelated healthy persons (Zaura et al. biota where compatible microorganisms assem-
2009). Transcription profiling defined a functional ble into heterotypic communities. These are in
core microbiota of nearly 60 species in dental equilibrium with the host. The organisms are
plaque (Peterson et al. 2014), and Wang et al. controlled by the host, despite their production of
(2013) described a core disease-associated com- toxic products such as proteases, overgrowth, and
munity in periodontitis by metagenomic sequenc- pathogenicity. Noteworthy, the microbial compo-
ing. A study based on a large set of near full-length nents of these communities vary over time from
sequences in 10 healthy individuals identified 10 person to person and from site to site. The viru-
variables shared by 11 bacterial species (Bik et al. lence of the entire community is increased by
2010). However, there were also significant inter- keystone pathogens such as P. gingivalis which
individual differences. This supported the pres- can have interactive communication with acces-
ence of both a core and a variable microbiome in sory pathogens like the mitis group of strepto-
the oral cavity. Based on several literature reports cocci, thereby orchestrating inflammatory disease
(Zarco et al. 2012) the major genera with the larg- by remodeling a normally benign microbiota into
est representation in the oral cavity were found to a dysbiotic one (Hajishengallis and Lamont
include Streptococcus, Veillonella, Granulicatella, 2012; Hajishengallis et al. 2012). The host
Gemella, Actinomyces, Corynebacterium, Rothia, immune response is not impaired and the abun-
Fusobacterium, Porphyromonas, Prevotella, dance of the dysbiotic community increases,
Capnocytophaga, Neisseria, Haemophilus, Trepo- destroying tissue homeostasis and causing
nema, Lactobacterium, Eikenella, Leptotrichia, destruction of periodontal tissues. PSD is
Peptostreptococcus, Staphylococcus, Eubacteria, probably not the last model of periodontitis that
and Propionibacterium. will be launched, but it is attractive from the point
that it reconciles the joint effects of a synergetic
and a dysbiotic microbial community, rather than
10.3.1 Microbiota in Periodontal select organisms.
Disease In terms of the microorganisms related to
periodontitis, it should be mentioned that it is
Over the years, there have been several mile- now moderate evidence in the literature to sup-
stones and hypotheses on the microbial etiol- port the association of 17 species or phylo-
ogy of periodontitis (Hajishengallis and Lamont types from the phyla Bacteroidetes, Candidatus
2012). Etiologies related to specific organisms Saccharibacteria, Firmicutes, Proteobacteria,
(amoeba, spirochetes, fusiforms, or strepto- Spirochaetes, and Synergistetes with periodon-
cocci), nonspecific plaque hypothesis/mixed titis. Also the archaea domain seems to have an
anaerobic infections, microbial shift in periodon- association with this disease (Pérez-Chaparro
titis, specific plaque hypothesis, red complex et al. 2014). As already mentioned, every human
bacteria (Porphyromonas gingivalis, Tannerella body carries a personalized microbiome that is
forsythia, Treponema denticola), ecological important for maintaining health but also for elic-
catastrophe hypothesis, disruption of periodon- iting disease (Zarco et al. 2012; Califf et al. 2014).
tal tissue homeostasis, keystone pathogens, and According to Schwarzberg et al. (2014) who used
next-generation sequencing, there is not a single consisted of Rothia and Streptococcus and was
microbial composition that represents a healthy related to health.
periodontal state and that recovery from peri- In a study by Ly et al. (2014), the oral bacte-
odontal disease appears to shift from a personal- riophage membership was significantly changed
ized disease state to a personalized healthy state. in persons with periodontitis compared to healthy
Although there may be a consensus that particu- subjects, mainly as a result of abundance of myo-
lar communities will shift according to disease, viruses in subgingival plaque. Myoviruses are
there may not be a healthy part of these bacteria mainly lytic. Their predominance in subjects
that is consistent across individuals. In contrast with periodontitis suggested an active role for
to this Griffen et al. (2012), using 16S multiple viruses in driving bacterial diversity in the peri-
region pyrosequencing, found differences between odontal pocket. They were more abundant than
health- and periodontitis-associated bacterial com- siphoviruses which generally have a lysogenic
munities at all phylogenetic levels and distinct lifestyle. In supragingival plaque, however, there
community profiles. Spirochaetes, Synergistetes, was no difference between myoviruses and
and Bacteroidetes were prominent phyla in dis- siphoviruses. The altered ecology suggested for
ease, while Proteobacteria was detected at higher bacterial involvement in periodontitis could
levels in healthy controls. Their data confirmed the therefore also involve bacteriophages.
association of species such as P. gingivalis, T. den-
ticola, and T. forsythia with disease, but Filifactor
alocis appeared to be at least as prevalent and 10.3.2 Biofilm Architecture
disease associated. Abusleme et al. (2014), using in Periodontitis
454 pyrosequencing of 16S rRNA gene libraries,
found that periodontitis communities were high Sampling of dental plaque will destroy its archi-
in Spirochaetes, Synergistetes, Firmicutes, and tecture making it difficult to conclude firmly on
Chloroflexi among other taxa, while the propor- the relative pathogenic role of taxa. When dif-
tion of Actinobacteria, especially Actinomyces, ferent materials were kept for several days in
was more abundant in health. periodontal pockets of patients with periodon-
A number of bacterial taxa and genes have titis and examined with electron microscopy
been found to differ between health and disease. and fluorescence in situ hybridization (FISH),
Until now data sets across studies have not been those parts of carriers extending into the deep-
compared directly, and we do not know if the est zone of the pocket were mainly colonized
microbial variations observed across studies are by spirochetes and Gram-negative bacteria
consistent. Kirst et al. (2015) used 16S rRNA (Wecke et al. 2000). Those kept in shallower
sequencing to survey the subgingival microbiota regions were colonized by streptococci. The
in 25 subjects with chronic periodontitis and 25 methods allowed detailed analysis of the archi-
controls and compared their data with those of the tecture of biofilms and identification of putative
Human Microbiome Project (HMP) (Turnbaugh periodontal pathogens with single-cell resolu-
et al. 2007; Griffen et al. 2012; Abusleme et al. tion. Previous investigations had revealed pres-
2013). They found a significantly altered micro- ence of novel yet uncultivated organisms at a
biota with decreased heterogeneity in periodon- high frequency in periodontal pockets (Moter
tal disease. Comparison with the other data sets et al. 1998). All patients with rapidly progres-
showed that the subgingival microbiota clus- sive periodontitis (n = 53) harbored oral trepo-
tered by study. However, differences between nemes that were either new species such as T.
periodontal health and disease were greater than maltophilum or uncultivable phylotypes. When
the technical variations between the studies. enamel slices were used to examine the micro-
Two microbial clusters were detected. One was biota development of dental plaque, channels
driven by Fusobacterium and Porphyromonas or pores filled with extracellular polymers were
and was associated with periodontitis; the other seen throughout the biofilm (Wood et al. 2000).
108 I. Olsen
Fig. 10.4 High numbers of group I treponemes (orange)

in a subgingival biofilm, most of which are yet uncultured.
The carrier section was hybridized with probe TRE I
together with FUNU for detection of Fusobacterium
Fig. 10.3 Fluorescence in situ hybridization (FISH) of a
nucleatum⁄canifelinum (light blue), which forms a cluster
subgingival biofilm showing the close spatial relationship
in the lower left corner, and DAPI (dark blue) (From
between facultatively anaerobic Streptococcus spp.
Marsh et al. (2011) with permission)
(orange) and obligately anaerobic Fusobacterium spp.
(magenta). Subgingival biofilms of periodontitis patients
were obtained using a carrier system. Bacteria were visu-
alized in 3 μm cross sections of the biofilms using the fol-
lowing probes simultaneously: probe EUB338, which
10.3.3 Bacteria Associated
detects most bacteria (green); probe Strep1⁄2, which with Caries
shows streptococci; probe FUS664, which detects most
Fusobacterium spp.; and nonspecific nucleic acid stain Recent theories divide the dental caries process
DAPI (blue). Details of oligonucleotide probes are avail-
into three reversible stages: the dynamic stabil-
able at probeBase (http://www.microbial-ecology.net/pro-
bebase/) (From Marsh et al. (2011) with permission) ity stage, the acidogenic stage, and the aciduric
stage (Takahashi and Nyvad 2008, 2011; Nyvad
et al. 2013). The microbiota on clinically sound
Staining and confocal microscopy showed that enamel consists mainly of non-mutans strepto-
the most viable and active areas of the biofilm cocci and Actinomyces. Here acidification is mild
were in the central parts and parts lining the and infrequent which is reflected in a balanced
channels. Plaque biofilms in the gingival crev- demineralization/remineralization or a shift in
ice had a thin densely adherent layer on the sur- the mineral balance toward a net mineral gain
face of the root, while the bulk of the biofilm (dynamic stability stage). Acidification becomes
had a looser structure particularly where there moderate and frequent when sugar is added.
was contact with the epithelial lining of the gin- This may increase the acidogenicity and acidur-
gival crevice or periodontal pocket (Fig. 10.3). ance of non-mutans bacteria. There can also be a
In outer layers structures such as corncob, test- selective increase in more aciduric strains such
tube brush, or rosette formations were detected as low pH non-mutans streptococci. In the end,
together with not-yet-cultivated organisms such this will shift the demineralization/remineraliza-
as spirochetes and members of the TM7 phy- tion balance, so that a net mineral loss occurs,
lum (Fig. 10.4). In the plaque itself interact- leading to initiation/progression of dental caries
ing bacteria exhibited a spatial organization, (acidogenic stage). If the acidogenic conditions
e.g., between streptococci and Fusobacterium become severe and prolonged, aciduric bacte-
nucleatum. ria will predominate by acid-induced selection
(aciduric stage). At this stage, mutans strepto- approaches such as metagenomic, metatranscrip-
cocci, lactobacilli, aciduric strains of non-mutans tomic, metaproteomic, and metabolomic analysis
streptococci, Actinomyces, bifidobacteria, and should be used to provide better information on the
yeasts may become dominant. dynamic caries process. The precise determina-
Different components of the microbiota tion of function requires the analysis of individual
may play different roles in initial enamel cells and cultures. In this context, it is important
lesions compared to caries extension into den- that previously uncultured microorganisms are
tin. The hydroxyapatite-rich enamel likely being brought to culture. Emphasis should also be
requires a more acidic microbiota for demin- made to obtain site-specific sampling of microbial
eralization than dentin. The highly acidogenic communities for studying the molecular ecology
species include S. mutans, acidogenic non- in situ of caries (Dige et al. 2014).
mutans streptococci, Actinomyces species, and The next-generation sequencing technique
Bifidobacterium/Scardovia species (Chalmers was combined with a metagenomic technique
et al. 2015), whereas caries progression into and showed that individuals who had never suf-
dentin may involve proteolysis by Prevotella fered from caries had an overrepresentation of
species of proteins denatured by acidic species functional genetic categories such as genes for
(Hashimoto et al. 2011). It seems likely that the antimicrobial peptides and quorum sensing. They
proteolytic component also will lead to pulp tis- did not carry mutans streptococci (Belda-Ferre
sue necrosis considering the frequent detection of et al. 2012). Interestingly, several isolates belong-
Gram-negative taxa in root canal infections. ing to healthy conditions inhibited the growth of
As for the microorganisms involved in caries, cariogenic bacteria when they were co-cultured.
direct pyrosequencing of samples from dental Thus, the metagenomic approach enabled quanti-
cavities showed that cavities are not dominated tation of the most abundant bacteria and con-
by S. mutans but contain a complex community firmed presence of bacteria with a protective
of bacterial species (Belda-Ferre et al. 2012). effect against cariogenic species.
This supported previous 16S rRNA sequencing
studies (Corby et al. 2005; Aas et al. 2008) and
the idea that dental caries is a polymicrobial dis- 10.3.4 Architecture of Biofilms
ease. Pyrosequencing also supported that oral in Caries
bacteria are specific at different stages of caries
progression (Jiang et al. 2014). In children with In occlusal caries, FISH showed a distinct dif-
severe dental caries, the genera Streptococcus, ference in the bacterial composition between
Granulicatella, and Actinomyces had increased different ecological niches in the caries pro-
significantly (Jiang et al. 2013). cess (Dige et al. 2014). Biofilms located at the
By performing comprehensive 16S DNA pro- entrance of fissures had an inner compact layer
filing of the dental plaque microbiome of both of microorganisms structured in palisades often
caries-free and caries-active microbiomes, the with a columnar pattern (Fig. 10.5). They were
signatures associated with dental health outnum- often identified as Actinomyces and were cov-
bered those associated with dental caries by ered by a loosely structured bacterial layer con-
nearly twofold (Peterson et al. 2013). It was sug- sisting of various genera that were similar to
gested that a shift in the abundance of groups of supragingival biofilm. Within the proper fissure
species, rather than the appearance of new cario- the biofilm appeared less metabolically active as
genic species or the pathogenicity of a single spe- estimated from low fluorescence signal intensity
cies, best describes the distinction between and presence of material of nonbacterial origin.
caries-free and caries-active microbiota. Invasion of bacteria, often Lactobacillus and
Detection of major bacteria present in dental Bifidobacterium spp., into dentinal tubules was
caries needs to be followed by information on seen only at advanced stages of caries with cavity
the metabolic activity of the biofilm. Therefore, formation.
110 I. Olsen
Fig. 10.5 (a–c) Images of in vivo biofilms on dental Fusobacterium spp. (purple/magenta in f). Note that the
occlusal surfaces. (a–c) Toluidine blue-stained sections biofilm could be divided into an inner compact layer of
showing an overview of occlusal surfaces with shallow palisade-like bacteria (d–h) often with a columnar pattern
fissure-like morphology (a), groove-like morphology, (b) (g, h) on top of which a looser structured layer (d, e, f, h,
and cavitated caries lesion (c). Arrows refer to the areas i) with non-stained voids (d, i) was seen. The outermost
illustrated in b, d, i, j, and l, respectively. (d–i) Confocal part of the decalcified enamel showed a thin auto-
laser scanning microscopy images of microbial coloniza- fluorescent layer without bacteria (blue or green in d, g),
tion patterns from above the entrance of shallow fissures and invaginations of developmental origin were often
and groove-like occlusal surfaces. In all confocal laser filled with bacteria (d, g, arrows). All images are oriented
scanning microscopy images, red represents all bacteria with the biofilm surface upward. Scale bars: 500 μm a–c)
that are neither Streptococcus spp. (yellow/green in d–i) and 25 μm (d–i) (Adopted from Dige et al. (2014) with
nor Actinomyces spp. (purple/magenta in e, g–i) nor permission)
10.3.5 Future Chairside Diagnostics Belay N, Johnson R, Rajagopal BS, et al. Methanogenic
bacteria from human dental plaque. Appl Environ
of Dental Plaque Microbiol. 1988;54:600–3.
Belay N, Mukhopadhyay B, Conway de Macario E, et al.
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Belda-Ferre P, Alcaraz LD, Cabrera-Rubio R, et al. The
health and disease, and we have been able to
oral metagenome in health and disease. ISME
study microbial communities on a large scale due J. 2012;6:46–56.
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J. 2010;4:962–74.
organisms are not responsible for disease but
Bringuier A, Khelaifia S, Richet H, et al. Real-time PCR
rather rely on the supplementary action of other quantification of Methanobrevibacter oralis in peri-
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taxa from Chlorobi, Chloroflexi, GN02, Synergistetes,
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SR1, TM7, and WPS-2 phyla/candidate divisions.
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Clingenpeel S, Clum A, Schwientek P, et al. Reconstructing
Acknowledgment The author wants to acknowledge each cell’s genome within complex microbial commu-
funding through a grant from the European Commission nities – dream or reality? Front Microbiol. 2015;5:771.
(FP7-HEALTH-306029 “TRIGGER”). doi:10.3389/fmicb.2014.00771.
Contreras A, Botero JE, Slots J. Biology and pathogenesis
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Salivary Microbiota in Oral Health
and Disease
11
Daniel Belstrøm
Abstract
The salivary microbiota is a highly complex microbial community, pre-
sumably comprised by bacteria shed from various oral surfaces, and since
saliva can be easily and noninvasively collected, compositional changes of
the salivary microbiota may potentially serve as a biomarker used for
screening of oral and systemic diseases. This chapter describes the compo-
sition of the salivary microbiota in oral health and disease, with special
emphasis on recent studies that employed modern molecular methods for
analysis of the salivary microbiota.
11.1 Salivary Microbiota only 0.73 % of total DNA present in saliva

in Oral Health (Lazarevic et al. 2012).
The composition of the salivary microbiota in
The salivary microbiota contains a unique indi- oral health has been addressed in detail. A com-
vidualized bacterial community shaped by con- prehensive study from 2012 performed as part of
tentious interaction with the surroundings, as the Human Microbiome Project analyzed and
bacteria are shed from different oral locations compared bacterial community profiles in sam-
and captured in saliva. Interestingly, 1 ml of ples collected from 10 different sites of the diges-
saliva contains >100 million bacteria, which tive tract in each of more than 200 healthy
means that a person with a normal saliva flow of individuals (Segata et al. 2012). The study
750 ml will excrete >5 g of solid bacteria through reported that the salivary microbiota in oral
saliva in 24 h (Curtis et al. 2011). However, health mostly resembled that of the throat, the
although the salivary microbiota is highly diverse, tonsils, and dorsum of the tongue. Firmicutes,
bacterial DNA has been reported to constitute Bacteroidetes, Proteobacteria, and Fusobacteria
were described as the predominant phyla consti-
tuting 40 %, 25 %, 20 %, and 10 % of the micro-
D. Belstrøm, DDS, PhD
biota, respectively. The predominant genera were
Section 1, Periodontology and Oral Microbiology,
Department of Odontology, Faculty of Health and Streptococcus and Veillonella accounting for 20
Medical Sciences, University of Copenhagen, and 15 % of the microbiota in saliva. Interestingly,
Copenhagen, Denmark major differences between supra- and subgingival
e-mail: dbel@sund.ku.dk

DOI 10.1007/978-3-319-25091-5_11
116 D. Belstrøm
microbiota and salivary microbiota were evident, than samples from the corresponding industrial-
indicating that bacteria shed from these surfaces ized individuals and also reported finding of 40
only accounts for a minor fraction of the salivary bacterial genera in samples from Batwa Pygmies
microbiota in oral health. Recently, next- that had not previously been identified in the oral
generation sequencing was used to investigate cavity. Therefore, the authors suggested that the
saliva from two adults, and five datasets from the differences observed could be a result of a differ-
Human Microbiome Project, and identified more ent diet and lifestyle between the populations
than 175 bacterial species present in saliva in oral investigated (Nasidze et al. 2011). Analogous, in
health. Furthermore, it was estimated that the a recent publication from 2014, data based on
predominant microbiota of saliva comprised as Human Oral Microbe Identification Microarray
much as 900 different bacterial species (Hasan (HOMIM) analysis of saliva samples from 293
et al. 2014), indicating that the salivary microbi- orally healthy adult Danes suggested that saliva
ota is probably far more complex than hitherto microbiota in oral health is influenced by indi-
anticipated. In that respect, an interesting finding vidual smoking habit as well as socioeconomic
was reported in 2014, as an investigation of saliva status. However, in this particular study, no asso-
samples from 97 healthy children aged 6–12 ciation was identified between different dietary
years revealed that 43 % of the samples were habits and composition of the salivary microbiota
culture-positive for Streptococcus aureus, and in (Belstrøm et al. 2014c). Thus, future studies are
6 % of the samples, S. aureus were identified at a warranted to further elucidate the impact of diet
level of >103 CFU/mL, proposing that saliva may and lifestyle on the composition of the salivary
harbor low proportions of pathogens as commen- microbiota. The oral microbiota, including that
sal members of the salivary microbiota in oral of saliva, is developing from the moment of birth,
health (Petti et al. 2014). and it has been hypothesized that during fetal life
It has been suggested that composition of the the fetus may in fact be exposed to the oral micro-
salivary microbiota is influenced by geographic biota, thereby facilitating an ability of the new-
location and individual lifestyle. To elucidate this born to better distinguish microbial friend from
perspective, an extensive study from 2010 ana- foe after birth (Zaura et al. 2014). Interestingly,
lyzed saliva samples collected from 120 individ- the salivary microbiota has been demonstrated to
uals living in each of 12 different locations be relatively simple in a newborn child, and sali-
worldwide, i.e., 10 individuals from each loca- vary microbiota has been reported insignificant
tion, by means of 16S rRNA sequencing. A total from stool microbiota of the same newborn until
of 101 genera were identified, and out of these 39 15 days after birth (Costello et al. 2013).
were not previously identified in the oral cavity. Furthermore, a delayed compositional differenti-
Furthermore, the study reported a major diversity ation of the salivary microbiota in low-birth-
of the salivary microbiota, but it was concluded weight children has been demonstrated (Costello
that this diversity was only minimally influenced et al. 2013). In a recent study, saliva samples
by geographic location, since the diversity among from newborn babies and saliva samples from
individuals from the same location was almost their mothers/primary caregivers were collected
the same as between individuals living at differ- and compared, and based on the data presented,
ent locations (Nasidze et al. 2009). Another high diversity of salivary microbiota in both
report by the same group addressed composition study groups were evident. However, higher
of salivary microbiota in samples collected from diversity was reported in samples from adults, as
two geographically different industrialized 27 genera were observed with greater prevalence
African populations and compared these with in saliva from adults, whereas Streptococcus was
saliva samples from Batwa Pygmies, an isolated the only genus significantly more prevalent in
hunter society from Uganda. This study revealed saliva from newborns (Cephas et al. 2011).
that the salivary microbiota from the Batwa Likewise, in another study from 2011, saliva
Pygmies expressed significantly more diversity samples from 74 children (aged 3–18) were ana-
11 Salivary Microbiota in Oral Health and Disease 117
lyzed by pyro-sequencing, and it was demon- and P. intermedia) were identified in 88 % of

strated that the composition of salivary microbiota samples from this adult population, comprised
was altered through transition from deciduous of both periodontitis subjects and orally healthy
dentition through mixed dentition until perma- individuals (Könönen et al. 2007). Likewise,
nent dentition, as the proportion of Firmicutes another study employed 16S rRNA-based PCR
declined, and proportions of Bacteroidetes and for examining the presence of 6 periodontal
Proteobacteria as well as total bacterial diversity pathogens (Prevotella nigrescens, A. actino-
increased (Crielaard et al. 2011). Interestingly, mycetemcomitans, P. gingivalis, T. forsythia, P.
recent studies have revealed that the salivary intermedia, and T. denticola) in saliva samples
microbiota may be altered in patients with inflam- from 41 orally healthy children (6–13 years) and
matory bowel disease (Said et al. 2014), which in reported high carriage in saliva from healthy chil-
combination with evidence highlighting dynamic dren of P. nigrescens (80 %), T. denticola (32 %),
associations between microbial communities in A. actinomycetemcomitans (24 %), and P. gin-
different anatomic sites (Ding and Schloss 2014) givalis (12 %) (Kulekci et al. 2008). Moreover,
suggest that alteration in relation to local and sys- high carriage of suggested periodontal pathogens
temic diseases may stress the salivary microbiota, was also reported in a group of one-year-old chil-
possibly resulting in detectable compositional dren, regardless of whether they were delivered
alterations of the salivary microbiota. Such alter- premature and with low birth weight or they were
ations could potentially be used for identification full-term infants (Merglova et al. 2014). Finally,
of disease-prone individuals at early stages of another study using a 16S rRNA-based PCR
disease. identification of 4 periodontal pathogens (A. acti-
nomycetemcomitans, P. gingivalis, F. nucleatum,
and T. denticola) in children with and without
11.2 Salivary Microbiota Fanconi’s anemia reported frequent detection of
in Subjects all 4 pathogens in saliva samples, with no sig-
with Periodontitis nificant differences between healthy and diseased
children (Lyko et al. 2013). Collectively these
Since the introduction of the bacterial complex reports demonstrate that suggested periopatho-
theory by Socransky and coworkers (1998), gens may be present as commensal bacteria in
much literature has focused on the etiologi- the salivary microbiota. However, several studies
cal association of specific putative periodon- have reported that the salivary microbiota may
tal pathogens, i.e., Porphyromonas gingivalis, be altered in subjects with periodontitis (Feng
Prevotella intermedia, Tannerella forsythia et al. 2014; Liljestrand et al. 2014; He et al. 2012;
(previously Tannerella forsythensis), Treponema Paju et al. 2009). For example, one study ana-
denticola, Fusobacterium nucleatum, and lyzing saliva samples from 1198 Finnish adults
Campylobacter rectus in chronic periodontitis showed that presence of two or more suggested
and Aggregatibacter actinomycetemcomitans in periodontal pathogens in saliva was associated
aggressive periodontitis. Thus, several papers with periodontitis as determined by number of
have addressed the presence of these specific teeth with deepened periodontal pockets (Paju
bacteria as part of the salivary microbiota, and et al. 2009). In line, an investigation using quan-
high salivary carriage of suggested periodontal titative real-time PCR (qRT-PCR) for detection
pathogens has been reported in subjects with peri- of 4 periodontal pathogens reported significant
odontitis, but also in orally healthy individuals. association between salivary carriage of P. gingi-
For example, a large Finnish survey (n = 1294) valis and P. intermedia and chronic periodontitis
employed a 16S rRNA-based PCR method with compared to healthy controls (He et al. 2012).
species-specific primers and reported that at least This finding was confirmed in a recent report, as
1 of 6 periopathogens (T. forsythia, T. denticola, presence of 8 periopathogens were found to be
P. gingivalis, C. rectus, A. actinomycetemcomitans higher in saliva samples from Chinese adults with
118 D. Belstrøm
chronic and aggressive periodontitis compared to before and after periodontal therapy. This study
healthy controls (Feng et al. 2014). Interestingly, demonstrated that supragingival microbiota dif-
a study from 2014 demonstrated that high sali- fers significantly from salivary microbiota and
vary carriage of P. gingivalis was associated with described that whereas periodontal therapy
increased saliva and serum levels of P. gingivalis- resulted in a major shift in supragingival micro-
specific IgA and IgG, and it was proposed that biota, the salivary microbiota was only modestly
combined information about saliva carriage of P. influenced by periodontal therapy. Therefore, the
gingivalis and markers of pathogen-specific host authors concluded that shifts of the salivary
responses could be used as a biomarker for iden- microbiota might not be a strong predictive mea-
tification of periodontitis patients (Liljestrand surement of periodontal therapy (Yamanaka et al.
et al. 2014). This finding is in line with another 2012). Further large-scale interventional studies
study demonstrating that salivary carriage of P. using advanced molecular methods are war-
gingivalis, in combination with salivary levels of ranted, to elucidate whether local bacterial altera-
matrix metalloproteinase (MMP)-8 and interleu- tions in periodontitis lesions are reflected by
kin 1-β (IL-β), was more strongly associated with detectable compositional changes of the salivary
periodontitis than one of the tree markers alone microbiota.
(Salminen et al. 2014).
Compositional changes of the salivary micro-
biota in relation to periodontitis have been inves- 11.3 Salivary Microbiota
tigated in a few studies recently (Yamanaka et al. in Subjects with Dental
2012; Belibasakis et al. 2013; Belstrøm et al. Caries
2014b). In one study, saliva samples from sub-
jects with aggressive periodontitis (n = 21) and The salivary microbiota in relation to dental car-
chronic periodontitis (n = 20) and saliva samples ies has been addressed in multiple studies pri-
from healthy control subjects (n = 18) were com- marily in children (Nurelhuda et al. 2010; Ling
pared using quantitative fluorescent in situ et al. 2010; Luo et al. 2012; Chaffee et al. 2014;
hybridization (FISH) analysis and demonstrated Relvas et al. 2014) but also in adult populations
significantly higher prevalence of Synergistetes (Yang et al. 2012; Wennerholm and Emilson
cluster A in samples from subjects with aggres- 2013; Belstrøm et al. 2014a). Based on their
sive and chronic periodontitis. This member of prominent position in the specific plaque hypoth-
the salivary microbiota might therefore associate esis and their suggested role of true caries patho-
with periodontitis (Belibasakis et al. 2013). gens (Parisotto et al. 2010), salivary levels of
Additionally, an investigation from 2014, in Streptococcus mutans and Lactobacillus species
which 586 saliva samples from an adult Danish in relation to dental caries have been investigated
population including 139 periodontitis patients using culture-based methods and different molec-
and 447 healthy controls were analyzed by means ular techniques (Guo and Shi 2013). Collectively,
of the HOMIM technology, identified 12 S. mutans and Lactobacillus have been reported
periodontitis-associated phylotypes of saliva. to be present in saliva from children and adoles-
Interestingly, newly suggested periodontal patho- cents with dental caries as well as in orally
gens as Parvimonas micra and Filifactor alocis healthy subjects. For example, one study reported
were associated with saliva samples from peri- that 47 % and 57 % of a population of adolescent
odontitis subjects, indicating that local bacterial had detectable amounts of S. mutans and lactoba-
accumulation during periodontitis may shed to, cilli in saliva, respectively, and that 22 % and
and be identified in, saliva (Belstrøm et al. 34 % of the population had >103 CFU/ml of these
2014b). Finally, a recent investigation employed bacteria in saliva (Relvas et al. 2014). In another
pyro-sequencing of 16S rRNA, for analysis of study, saliva samples were collected from 243
supragingival plaque samples and saliva samples mothers and their newborn infants until 24
collected from 19 subjects with periodontitis, months postpartum, and presence of S. mutans
and lactobacilli in saliva samples from the moth- for analysis of bacterial diversity and reported
ers was compared to caries incidence in the chil- identification of 156 genera belonging to 10
dren at age 3. The study demonstrated that high phyla in saliva samples from this cohort.
levels of salivary carriage of S. mutans and lacto- Furthermore, salivary microbiota was found to
bacilli in mothers were associated with a cumula- significantly differentiate from microbiota of
tive incidence ratio of 1.9 of the children having supragingival plaque. However, no genera identi-
dental caries at age 3. Furthermore, it was dem- fied were found to be significantly associated
onstrated that children of mothers with high sali- with saliva samples from subjects with dental
vary carriage of S. mutans and lactobacilli were caries (Ling et al. 2010). Another study, examin-
more likely to be S. mutans- and Lactobacillus- ing saliva samples from 6- to 8-year-old Chinese
positive themselves (Chaffee et al. 2014). children (caries-active n = 30, healthy controls
Likewise, another report suggested that a com- n = 20) using the HOMIM technique, identified
mercialized test facilitating semiquantitative 94 bacterial phylotypes representing 30 genera
measurements of salivary S. mutans levels could belonging to 6 phyla, out of which 8 and 6 phylo-
be used for caries risk assessment (Wennerholm types were significantly associated with saliva
and Emilson 2013). Furthermore, recent studies samples from subjects with dental caries and
have revealed that the use of probiotics (Cannon healthy controls, respectively (Luo et al. 2012). A
et al. 2013) and meticulous oral hygiene instruc- comprehensive report employing 16S rRNA gene
tion (Liu et al. 2014) may result in lowered pro- amplicon-based and whole-genome-based deep
portions of S. mutans in saliva of children. It has sequencing technologies for analysis of saliva
also been reported that salivary levels of S. microbiota in samples from 19 caries-active and
mutans may be altered during different orthodon- 26 healthy control subjects (aged 18–22), also
tic procedures (Ortu et al. 2014; Jung et al. 2014), reported saliva microbiota in subjects with dental
and it has been reported that children with aller- caries to be significantly more diverse than
gic rhinitis have higher salivary levels of S. observed in healthy control samples. In addition,
mutans than healthy children (Wongkamhaeng 147 caries-associated operational taxonomic
et al. 2014), which is why salivary levels of S. units (OTUs) were identified (Yang et al. 2012).
mutans may be altered as a consequence of other Likewise, a study from 2014 investigating saliva
parameters than dental caries. samples from 621 adult Danes (caries-active
Other papers have addressed community pro- n = 174, healthy controls n = 447) by means of the
files of saliva microbiota using advanced molecu- HOMIM technology found the diversity of sali-
lar methods and demonstrated that dental caries vary microbiota in subjects with dental caries to
in children and adults associates with an altered be reduced compared to healthy controls and
salivary microbiota. Collectively, these studies identified 10 bacterial phylotypes to be present at
suggest that alterations of saliva microbiota may different levels in samples from subjects with and
be more profound than hitherto anticipated based without dental caries (5 caries associated and 5
on data from studies only analyzing S. mutans health associated) (Belstrøm et al. 2014a).
and lactobacilli levels of saliva. Thus, alterations Finally, in a recent investigation, functional gene
in richness and diversity of the salivary microbi- signatures were addressed in saliva samples from
ota in samples from subjects with dental caries 10 subjects with dental caries and 10 healthy con-
have been reported, and other members of the trols (aged 18–23 years) using a functional gene
salivary microbiota have been suggested to be microarray (HuMiChip 1.0), and it was demon-
associated with saliva samples from subjects with strated that disease-associated functional gene
dental caries. In one study, saliva and supragingi- signatures were associated with saliva samples
val plaque samples from 3- to 6-year-old children from subjects with dental caries (Yang et al.
with and without dental caries were investigated 2014). Thus, current evidence suggests that not
using high-throughput barcoded pyro-sequencing only may the composition of salivary microbiota
and PCR-denaturating grading gel electrophoresis be altered in subjects with dental caries but
120 D. Belstrøm
potentially also the functional abilities of the 11.5 Future Perspectives: Using
microbiota may be different in saliva from sub- the Salivary Microbiota
jects with dental caries compared to oral health. as a Biomarker of Health
and Disease
11.4 Methodological Microbial analysis of supra- and subgingival

Considerations for Studying plaque samples can be tenacious and expensive,
Salivary Microbiota since separate sampling and analysis from multiple
diseased sites is often required (Yoshizawa et al.
Some reports describing the composition of the 2013). On the other hand, the use of saliva is con-
salivary microbiota have been rather conflict- siderably more cost-effective since only one sample
ing. One explanation might be that different from each subject may be collected and analyzed,
culture-based techniques and molecular tech- thereby making saliva an ideal fluid for analysis of
niques were employed, making comparison larger populations, which may be suitable for chair-
between investigations difficult. Thus, several side analysis in the dental office (Giannobile et al.
reports have demonstrated that meticulous col- 2011; Schafer et al. 2014). Thus, saliva has been
lection and zealous handing of saliva samples in proposed as “the mirror of the body,” and much
a standardized manner is required for obtaining scientific activity has focused on identification of
solid validated results, which can be compared salivary biomarkers in relation to oral health and
across publications (Lazarevic et al. 2010, disease (Giannobile et al. 2011). However, it is
2012, 2013a; Rasiah et al. 2005). Also, it has important to acknowledge that the most common
been reported that use of systemic antibiotics oral diseases, e.g., periodontitis and dental caries,
in children with acute otitis media (Lazarevic are conditions with multifactorial etiologies, which
et al. 2013b), and chemotherapy/radiation ther- stresses the importance for combined informa-
apy in subjects with nasopharyngeal carcinoma tion from areas of genomics and transcriptomics
(Xu et al. 2014) altered the salivary microbiota. for identification of disease-prone individuals
Thus, careful consideration of inclusion and (Ai et al. 2012). Thus, with further technological
exclusion criteria is prerequisite when ana- developments, it is reasonable to believe that future
lyzing salivary microbiota. Furthermore, pre- chairside analysis of saliva samples measuring
vious reports has revealed that application of compositional changes of the salivary microbiota
various DNA extraction protocols (Lazarevic in combination with inflammatory biomarkers
et al. 2013a) and culture-independent molecu- could provide diagnostic aid for general practic-
lar techniques have profound influence on the ing dentists, enabling identification of periodontitis
data obtained (Lazarevic et al. 2012). Finally, and dental caries-prone individuals at early stages
in a study from 2005 using denaturating gra- of disease, thereby facilitating individualized non-
dient gel electrophoresis (DGGE) for analysis invasive treatment (Yoshizawa et al. 2013).
of saliva samples, it was suggested that saliva
microbiota remains stable for years (Rasiah
et al. 2005). In contrast, a recent study from
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Part IV
Future Perspectives in Management
of Oral Infections
Use of Probiotics in Future
Prevention and Treatment
12
of Oral Infections
Mette Rose Jørgensen and Mette Kirstine Keller
Abstract
The interest for probiotic bacteria to combat biofilm-related diseases in the
oral cavity has arisen during the last decade. The probiotic concept has
successfully been used to control gastrointestinal diseases, such as
antibiotic-associated diarrhoea and acute infectious diarrhoea. The poten-
tial mechanisms of action in the oral cavity are not fully understood, but
there are thoughts to be a local effect in the mouth and systemic effect
through immune regulation. The purpose of this chapter is to summarise
our current knowledge of the potential for probiotics to improve oral and
dental health and to discuss its potential in the future prevention of oral
diseases. Despite some limitations, the currently available clinical studies
appear expectant, and there seem to be evidence of probiotics to improve
oral conditions such as dental caries, periodontitis and the colonisation of
Candida. However, more long-term clinical studies are needed before
evidence-based guidelines can be released.
12.1 Introduction “life”. Probiotic bacteria are defined as “Live

microorganisms, which when administered in
Probiotics in the dental practice is an emerg- adequate amounts, confer a health benefit on the
ing and promising strategy to combat biofilm- host” (WHO) (Meurman and Stamatova 2007).
related diseases in the oral cavity (Laleman and Probiotics used in general health should be non-
Teughels 2015). The term probiotic derives from pathogenic and non-toxic, and they should be
the Greek language meaning pro “for” -biotic able to survive in the gastrointestinal tract and
temporarily colonise in the gut.
The use of microorganisms to promote health
M.R. Jørgensen, PhD student, DDS (*) can be dated back to ancient times, where
M.K. Keller, PhD, DDS Romans fermented food with microorganisms
Section 1, Cariology and Endodontics, Department
and used it therapeutically. In 1906, the French
of Odontology, Faculty of Health and Medical Sciences,
University of Copenhagen, Copenhagen N, Denmark physician Henry Tissier isolated the bacteria
e-mail: mrj@sund.ku.dk; mke@sund.ku.dk Bacillus bifidus communes, later reclassified as

DOI 10.1007/978-3-319-25091-5_12
126 M.R. Jørgensen and M.K. Keller
the genus Bifidobacterium, and some years later, Table 12.1 General definitions within probiotic research
he treated diarrhoea with stool transplants con- Resident Consists of the common microflora
taining bifidobacteria. In 1907, the Nobel Prize– microbiota (microorganisms found in most
humans), the supplemental microflora
winning scientist Elie Metchnikoff postulated
(microorganisms characterising the
that Bulgarians lived longer because of their individual) and the transient
consumption of Bulgarian yoghurt (containing microflora (microorganisms
lactic acid bacteria) promoting good health. The temporarily present in the body)
word “probiotic” was introduced in 1965 by Probiotic Living microorganisms which, when
ingested, provide a health benefit on
Lilly and Stillwell and probiotics were defined the host
as “substances produced by microorganisms that Prebiotic Non-digestible food ingredients, e.g.
stimulate the growth of another” (Lilly and oligosaccharides, inulin and lactulose,
Stillwell 1965). that stimulate the growth and/or
In present time, the probiotic concept has suc- activity of selected beneficial bacteria
cessfully been used to control gastrointestinal Synbiotic Nutritional supplements combining
probiotics and prebiotics
diseases, and probiotics also seem to alleviate synergistically, which beneficially
symptoms of allergy and diseases with immuno- affect the host
logical pathology (Meurman 2005). The most
fully documented probiotic intervention is the
treatment of acute infectious diarrhoea, antibiotic- During the last decade, the interest in probiot-
associated diarrhoea and inflammatory bowel ics as an alternative, preventive, and therapeutic
disease (Crohn’s disease and ulcerative colitis). approach in the oral cavity has arisen. The effi-
Furthermore, probiotics are also commonly used cacy of probiotic bacteria in the oral cavity has
for conditions in which firm evidence is lacking been investigated for conditions including dental
such as vaginal candidiasis, Helicobacter pylori caries, gingivitis, periodontitis, halitosis, coloni-
infection of the stomach and upper respiratory sation of oral Candida, oral mucositis and xero-
infections (Saha et al. 2012). stomia. Within dentistry, L. rhamnosus GG and
There are a number of different microorgan- L. reuteri are the most intensely studied probiotic
isms that can be classified as probiotics, including species, and they have shown their potential in
bacteria and yeasts. The most common probiotic interacting with S. mutans and reducing colonisa-
strains belong to the genera Lactobacillus (e.g. tion of Candida. The intention of this chapter is
L. acidophilus, L. rhamnosus, L. bulgaricus, L. to briefly outline current knowledge on the poten-
reuteri and L. casei) and Bifidobacteria (e.g. B. tial for use of probiotic bacteria to prevent oral
bifidum, B. longum and B. infantis), but also, diseases and to improve oral and dental health.
some Streptococcus spp. (e.g. S. thermophilus
and S. salivarius) and Saccharomyces spp. (e.g.
S. boulardii and S. cerevisiae) have been intro- 12.2 Mechanisms of Action
duced as potential probiotics (Isolauri et al.
2002). Prebiotics are defined as non-digestible The mechanisms of action of probiotics are not
food ingredients that stimulate selected benefi- fully understood but are thought to be locally in
cial bacteria already established in the colon and the mouth by competing for adhesion sites and
thus in effect improve host health (Table 12.1) nutrients with the oral pathogens and by inhibi-
(Dahlén et al. 2012). The most commonly used tion of growth of pathogens by production of
prebiotics are carbohydrate substrates, e.g. oli- bacteriocins or other products (acid or perox-
gosaccharides, with the ability to promote the ide). Thus, probiotics modify the composition
components of the normal intestinal microflora. of the oral biofilm or the metabolic activity.
When the prebiotics and probiotics are applied There also seems to be a systemic regulation of
together, the concept is defined as synbiotics the immune response during intake of probiotics
(Anusha et al. 2015). (Devine and Marsh 2009; Stamatova and
12 Use of Probiotics in Future Prevention and Treatment of Oral Infections 127
Meurman 2009). Alterations in levels of both Taipale et al. 2012). Nonetheless, two recent sys-
salivary IgA (Ericson et al. 2013) and cytokines tematic reviews conclude that probiotic bacteria
in the gingival crevicular fluid (Twetman et al. decrease the mutans streptococci counts as long
2009) have been registered after exposure to as there is a regular intake of the probiotic bacte-
probiotic bacteria. Generally, the effects of pro- ria (Cagetti et al. 2013; Laleman et al. 2014). The
biotic bacteria are strain specific and cannot be strains used in the clinical intervention trials vary
applied directly to other strains. Also, the same among different lactobacilli strains and a few dif-
strains can have different effect in different indi- ferent bifidobacteria and even some streptococci
viduals (Koll-Klais et al. 2005). strains. There does not seem to be any clear-cut
difference between the outcomes of the trials
based on the strains used in the study.
12.3 Probiotics and Oral Diseases Since the lactobacilli used in most studies
have strong acidogenic abilities, a natural reserva-
12.3.1 Dental Caries tion in relation to caries would be that addition
of lactobacilli to the oral cavity would involve an
Dental caries is demineralisation of the tooth increased risk of an acidogenic shift of the oral
induced by microbial production of acid in the microbiota. However, two clinical trials found
dental plaque. Hence, modulation of the dental no increase in biofilm acidurity after exposure to
plaque by probiotic bacteria has naturally been of probiotic lactobacilli strains (L. reuteri SD2112,
interest. L. reuteri DSM 17938 and L. reuteri PTA 5289)
Since the first study on the topic by Näse et al. (Keller and Twetman 2012; Marttinen et al. 2012).
in 2001, there have been an increasing number of There are fewer studies with caries incidence,
clinical trials with caries-related end points. root caries arrest or remineralisation of carious
However, the majority of these studies have other lesions as outcome. The six studies with caries
end points than caries such as microbial counts or incidence as end points are listed in Fig. 12.1,
plaque index. Mutans streptococci are the most which displays the prevented fraction of the stud-
common microbial end point, but some studies ies. The calculated mean of preventive fraction is
also look at lactobacilli. Most of these studies 33 %. The most popular vehicle for the probiotic
show an inhibitory effect of probiotics on sali- bacteria was milk which was supplemented with
vary mutans streptococci, but a few does not find 2.5 ppm fluoride in one study. The first study
any difference after the intervention period from Finland (Näse et al. 2001) also used milk as
(Stecksen-Blicks et al. 2009; Lexner et al. 2010; the vehicle for L. rhamnosus GG during a
Preventive fraction %
Wattanarat (2015)
Stensson (2014)
Hasslöf (2013)
Taipele (2013)
Stecksén-Blicks (2009)
Näse (2001)- 3–4 year
Fig. 12.1 Preventive Näse (2001)- 1–6 year

fractions in clinical studies
with caries as an end point 0 20 40 60 80 100
7-month intervention period. They found a pre- streptococci return to previous levels when the
ventive fraction of 56 % among the children probiotic bacteria are ceased. Hence, the effect
being 3–4 years of age but only 21 % when look- seems to be dependent of a continuous distribution
ing at the entire study population of 1–6 years of of the probiotic bacteria. However, the follow-up
age. Besides the dental health benefits, the study study by Stensson reported on an effect 7 years
also showed a reduction in antibiotic treatments after the original intervention. This could point
for respiratory infections in the test group towards the importance of intervention in early
(Hatakka et al. 2001). The promising results childhood to secure a long-term effect. Despite the
encouraged a similar study in Sweden where pre- promising results from the existing studies, there
schoolers aged 1–5 years were provided with is still insufficient evidence to give any clinical
milk containing L. rhamnosus LB21 and 2.5 ppm recommendations, and more long-term studies
fluoride on a daily basis (Stecksen-Blicks et al. with caries as an end point are needed.
2009). Their results showed a 75 % preventive
fraction but due to the study design, it was not
possible to determine how much of this can be 12.4 Gingivitis and Periodontitis
attributed to the fluoridation or the addition of
probiotic bacteria. Secondly, this study also Gingivitis and periodontitis are diseases related
found an additional beneficial effect in fewer pre- to the gum and surrounding bone caused by
scriptions of antibiotics to the children in the test bacterial dental plaque and the host immune
group. A more recent study from Finland chose response. Exposure of the gingival tissues to
another vehicle based on the young age of the dental plaque results in inflammation within the
participants (0–12 months) and used a pacifier tissues which manifests in clinical signs of gingi-
with room for probiotic tablets to obtain longer vitis, e.g. change in colour, swelling of the tissue,
oral exposure to the probiotic strain L. rhamno- increased gingival exudate and bleeding upon
sus LB21 (Taipale et al. 2013). At age four, how- provocation. The clinical features of chronic
ever, the caries preventive fraction was rather periodontitis are, in addition to the characteris-
modest (5 %) which might be due to uncertain tics of gingivitis, loss of clinical attachment and
compliance. loss of alveolar bone. Today, treatment strate-
Two Swedish studies are follow-up studies on gies include hygiene improvement, mechanical
trials conducted 6 and 7 years ago, respectively. debridement (scaling and root planing), surgery
After having eaten gruel with added L. paracasei and antibiotic treatment. One of the main etio-
F19 during infancy, the children in the test group logical factors in periodontal inflammation is the
had statistically significant less eczema at age 6 shift of the periodontal microbiota towards gram-
but not statistical significant results on caries negative species and the absence of so-called
incidence (Hasslof et al. 2013). The dropout rate beneficial bacteria. Theoretically, restoring the
was rather high and together with generally low reduced number of these beneficial bacteria via
level of caries, it may account for failure to probiotic administration might be of interest in
achieve statistically significant results. In com- the treatment of periodontal disease (Teughels
parison, the results of the other study were more et al. 2008).
promising. Stensson et al. (2014) reports a pre- A Medline search in June 2015 revealed 24 in
ventive fraction of 29 % 7 years after the original vivo human clinical studies concerning the effect
intervention with five drops of L. reuteri ATCC a of probiotic bacteria on periodontally healthy
day during the first 12 months of age. individuals or patients with gingivitis or peri-
It has been discussed whether colonisation of odontitis. Selected studies are shown in
the probiotic strains is necessary to gain an effect. Table 12.2. These studies mostly report end
The studies with microbial end points show a points related to amount of plaque (PI), gingival
change in levels of mutans streptococci as long as condition (GI), bleeding on probing (BOP),
the probiotic strains are administered but mutans probing pocket depth (PPD) and subgingival
12
Table 12.2 Human clinical studies with outcome related to periodontal disease
Infection type at Effect of probiotic
baseline Reference Strain Vehicle, time Assessment criteria treatment
Healthy volunteers Kang et al. (2006) W. cibaria CSM1 Rinse, 1 day OHI-S, PI Yes
Shimauchi et al. (2008) L. salivarius WB21 Tablets, 8 weeks PI, GI, BOP Yes
Iwamoto et al. (2010) L. salivarius WB21 Tablet, 4 weeks BOP, PPD Yes
Sinkiewicz et al. (2010) L. reuteri ATCC 55730/ATCC PTA 5289 Diet, 12 weeks PI, periopath. Yes
Gingivitis Krasse et al. (2006) L. reuteri Chewing gum, 2 weeks GI, PI Yes
Twetman et al. (2009) L. reuteri ATCC 55730/ATCC PTA 5289 Chewing gum, 2 weeks BOP, GCF, cytokines Yes
Exp. Staab et al. (2009) L. casei Shirota Milk drink, 8 weeks PI, GI, GCF Yes
Exp. Slawik et al. (2011) L. casei Shirota Milk drink, 4 weeks PI, BOP Yes
Iniesta et al. (2012) L. reuteri ATCC 55730/ATCC PTA 5289 Tablets, 8 weeks PI, GI No
Exp. Hallström et al. (2013) L. reuteri ATCC 55730/ATCC PTA 5289 Tablets, 3 weeks PI, GI, BOP No
Periodontitis Riccia et al. (2007) L. brevis Lozenges, 4 days GI, PI, BOP, calculus Yes
Tsubura et al. (2009) B. subtilis E-300 Rinse, 30 days PPD, BOP, GI No
SRP Vivekanada et al. (2010) L. reuteri DSM 17938/ATCC PTA 5289 Lozenges, 3 weeks PI, GI, BOP, PPD, CAL Yes
Vicario et al. (2013) L. reuteri ATCC 55730/ATCC PTA 5289 Tablets, 4 weeks PI, BOP, PPD Yes
SRP Teughels et al. (2013) L. reuteri DSM 17938/ATCC PTA 5289 Tablets, 12 weeks PPD, CAL, BOP, Yes
Use of Probiotics in Future Prevention and Treatment of Oral Infections
periopath.
SRP Shah et al. (2013) L. brevis Lozenges, 2 weeks GI, PI, PPD, CAL Yes
Szkaradkiewicz et al. (2014) L. reuteri ATCC PTA 5289 Tablets, 2 weeks GI, PPD, CAL Yes
Tekce et al. (2015) L. reuteri DSM 17938/ATCC PTA 5289 Lozenges, 3 weeks PI, GI, BOP, PPD Yes
Exp. experimental gingivitis, SRP scaling and root planing, OHI-S oral hygiene index, PI plaque index, GI gingival index, BOP bleeding on probing, PPD probing pocket depth,
CAL clinical attachment level, GCF gingival crevicular fluid, periopath. periopathogens
129
Fig. 12.2 Percent Reduction in BOP %

reduction in BOP between
Ince (2015)
probiotic and placebo
groups
Vicario (2013)
Teughels (2013)
Slawik (2011)
Vivekanada (2010)
Twetman (2009)
Tsubura (2009)
0 20 40 60 80 100
microbiota associated with periodontal diseases. plaque scores were significantly lower in the pro-
In the majority of the studies, a significant effect biotic group, but no differences were seen in gin-
of probiotic treatment was obtained in the probi- gival scores. These findings were confirmed by
otic group compared to placebo. However, the Karuppaiah et al. (2013).
studies are heterogeneous and have been subject Krasse et al. (2006) were the first to investi-
to methodological criticism mainly due to a gate the effect of chewing gum containing L.
diverse patient population, lack of descriptions of reuteri on patients with chronic gingivitis. They
the extent and severity of the periodontal disease, found a reduction in PI and GI after 2-week inter-
potential confounding factors, high risk of bias vention. However, Iniesta and co-workers (2012)
and inconsistent end points (Dhingra 2012; were not able to confirm these findings. Three
Laleman and Teughels 2015). clinical trials have looked upon the effect of pro-
Eight clinical studies have looked at clinical biotics on subjects with experimental gingivitis
periodontal parameters in periodontally healthy (Hallström et al. 2013; Slawik et al. 2011; Staab
individuals (Burton et al. 2013; Iwamoto et al. et al. 2009): two with positive and one with a
2010; Kang et al. 2006; Karuppaiah et al. 2013; negative outcome (Table 12.2).
Mayanagi et al. 2009; Shimauchi et al. 2008; Bleeding on probing (BOP) is a widely used
Sinkiewicz et al. 2010; Zahradnik et al. 2009). In criterion to diagnose gingival inflammation.
a randomised, double-blind, placebo-controlled Seven studies found decreased BOP after treat-
trial, Shimauchi et al. (2008) investigated the ment with probiotic bacteria compared with
effect of L. salivarius WB21 on 66 healthy sub- placebo (Fig. 12.2) (Ince et al. 2015; Slawik
jects and found periodontal parameters improved et al. 2011; Teughels et al. 2013; Tsubura et al.
after 8-week intervention. Mayanagi et al. (2009) 2009; Twetman et al. 2009; Vicario et al. 2013;
and Zahradnik et al. (2009) found a reduction in Vivekananda et al. 2010). However, Teughels
selected periopathogens (e.g. P. gingivalis) in et al. (2013) did not find this decrease significant.
subgingival plaque and saliva of healthy individ- Nine recent studies (2007–2015) have inves-
uals after treatment with lactobacilli spp. Two tigated the effect of probiotics on patients with
randomised clinical trials have looked into the chronic periodontitis (Ince et al. 2015; Riccia et al.
efficacy of probiotics on gingival health in chil- 2007; Shah et al. 2013; Szkaradkiewicz et al. 2014;
dren. In the first study by Burton and co-workers Tekce et al. 2015; Teughels et al. 2013; Tsubura
(2013), 100 children (5–10 years) were included et al. 2009; Vicario et al. 2013; Vivekananda
to assess changes in plaque score and gingival et al. 2010) (Table 12.2). Longitudinal studies
score after 3 months treatment with either S. sali- have shown the efficacy of the standard treatment
varius M18 or placebo. At treatment end, the approach consisting of systematic scaling and root
planing (SRP) on root surfaces and optimal oral dentures and smoking (Anil et al. 2014; Pires
hygiene. Three studies combined the SRP with et al. 2002; Shay et al. 1997; Torres et al. 2002).
probiotic treatment. In a randomised placebo- A number of antifungal agents are available for
controlled trial from 2013, Teughels et al. proved the treatment of oral candidiasis, e.g. the polyenes
the effect of L. reuteri (Prodentis)-containing pro- (nystatin) and the azoles (fluconazole). However,
biotic lozenges as an adjunct to SRP in patients since oral candidiasis is caused by an ecologi-
with chronic periodontitis. Significantly larger cal imbalance (dysbiosis) in the oral biofilm that
PPD reductions, especially in moderate and deep favours fungal overgrowth, a certain interest has
pockets, were evident. been addressed to a bioecological approach for
Antibiotic treatment can be an adjunct to prevention and management.
mechanical therapy when these are found to be In the past decade, several clinical studies
insufficient. However, repeated use of antibiotics have investigated the ability of probiotic bacte-
increases the risk of drug-resistant microorgan- ria to hamper the growth of Candida in the oral
isms. Shah et al. (2013) compared the efficacy of cavity. Hatakka and co-workers (2007) were
probiotic tablets (L. brevis CD2) alone, in combi- the first to conduct a randomised double-blind,
nation with doxycycline and doxycycline alone placebo-controlled trial on the effect of probiot-
after SRP in patients with aggressive periodonti- ics on the prevalence of oral Candida. The study
tis. The study showed that all three alternatives included 276 elderly people consuming probiotic
had similar reducing effect on PI, GI and PPD cheese (L. rhamnosus GG, L. rhamnosus LC705,
after 2 months. This finding is encouraging since Propionibacterium freudenreichii spp. shermanii
a reduction in the use of antibiotics in the dental JS) or placebo cheese for 16 weeks. The inves-
practice is desirable. tigators found that the prevalence of high yeast
In summary, within the limitations of the clinical count (≥104 colony-forming units (cfu)/mL saliva)
studies performed to date, the results are encourag- diminished by 32 % in the probiotic cheese group,
ing and display that probiotics might be a valid while it increased by 21 % in the control group.
supplement to the gold standard treatment of gingi- These findings were confirmed in two later stud-
vitis and chronic periodontitis patients. However, ies following consumption of yoghurt containing
further long-term studies with more homogenous lactobacilli and bifidobacteria spp. (Dos Santos
end points are needed before evidence-based treat- et al. 2009; Mendonca et al. 2012). The probiotic
ment recommendations can be released. concept has also been proven effective in reduc-
ing the number of Candida species in patients with
Candida-associated stomatitis (Li et al. 2014) and
12.4.1 Colonisation of Oral Candida in candidiasis-asymptomatic elderly denture wear-
Species ers (Ishikawa et al. 2015).
Our knowledge within this field was just
The yeast Candida can cause a number of recently confirmed by Kraft-Bodi and co-workers
disorders in the oral cavity, known as oral candidi- (2015) who investigated the effect of a twice
asis. Seven Candida species are the clinically most daily intake of probiotic lactobacilli lozenges on
important, of which C. albicans, C. tropicalis and the prevalence and counts of oral Candida spe-
C. glabrata are most frequently isolated (80 %). cies in 215 frail elderly living in nursery homes in
Candida species are commensal microorganisms the southern parts of Sweden. The results revealed
in the oral cavity in 40–60 % of the population and a significant reduction of high Candida counts
only cause disease when disturbances in the oral (>104 cfu/mL) in saliva in the test group com-
microbial balance occur (Teughels et al. 2008). pared to placebo after 12-week treatment with L.
Oral candidiasis is therefore often seen in elderly reuteri (DSM 17938 and ATCC PTA 5289)
people and is frequently associated with antibi- (Fig. 12.3). The same results were seen regarding
otic treatment, hyposalivation, impaired local or Candida prevalence in plaque after probiotic
systemic immune system, neglected oral hygiene, treatment.
Fig. 12.3 Percent

distribution of Candida
scores in saliva at baseline
and at follow-up in the
probiotic and placebo
groups
In conclusion, our knowledge from the find- infections (Delanghe et al. 1997; Quirynen et al.
ings of these studies suggest that probiotic bacte- 2009). The oral malodours can be produced by
ria added to food, tablets or lozenges may reduce anaerobic bacteria by degradation of food resi-
oral Candida counts, and we thereby might be dues, exfoliated epithelial cells and salivary pro-
one step closer to clinical recommendations on teins (Scully and Greenman 2008). It has been
the use of daily probiotic supplementation in proposed that bacteriotherapy in order to replace
patients at risk for oral candidiasis. halitosis-associated species may create a more
long-lasting effect than the current methods reduc-
ing the total bacterial numbers by mechanical
12.5 Oral Mucositis measures such as brushing, flossing, and tongue
scraping, antibacterial agents, and complex bind-
Oral mucositis is a frequent and painful side effect ing of malodorous compounds by zinc (Burton
of cancer chemotherapy and radiotherapy to cancer et al. 2005; Fedorowicz et al. 2008; Outhouse et al.
in the head and neck region. The condition can 2006; Porter and Scully 2006). Several studies
result in suboptimal oral hygiene, problems with have looked at the effect of probiotic bacteria on
adequate nutritional intake and reduced quality of oral malodour (Burton et al. 2006; Iwamoto et al.
life. The immunosuppressed patients have earlier 2010; Kang et al. 2006; Keller et al. 2012; Sutula
been mentioned as a group where probiotic bacteria et al. 2013; Suzuki et al. 2014). A number of them
should be avoided or used only with caution. find a reduction in either VSC (volatile sulphur
However, a randomised, controlled trial with 200 components) or organoleptic score. However, they
patients found no serious adverse effects of the pro- all have fairly small study groups (n = 22–46) and
biotic product (Sharma et al. 2012). Furthermore, some are not placebo controlled.
they found a statistically significant reduction in the
prevalence of mucositis (7 % vs. 28 %) and reduc-
tion of the proportion of patients with severe (grades 12.7 Vehicles, Dose and Safety
III and IV) mucositis (52 % vs. 77 %).
The safety of administration of probiotic bacteria
must also be considered. Overall, the use of pro-
12.6 Halitosis biotics is considered safe. Some strains used as
probiotics, also strains used in oral health, have
Oral malodour is often caused by compounds from been isolated from infections such as endocardi-
the oral cavity but some compounds may also be tis (Cannon et al. 2005). However, in both Finland
produced by nasal, oropharyngeal or pharyngeal and Sweden where use of probiotic products
containing Lactobacillus is common, there have the oral cavity differs from the gut with regard to
been no increase in Lactobacillus-associated oral microbiota, mucosal structure and composi-
bacteremia in a period with increasing use of the tion of fluids like saliva (Meurman and Stamatova
strains (Isolauri et al. 2002; Salminen et al. 2004; 2007). Future studies should focus on defining the
Sullivan and Nord 2006). In another study, the optimal strains for the various dental diseases.
safety of two strains used to promote gastrointes- Moreover, more studies are needed to clarify
tinal health in infants was evaluated, and no side whether a mix of different strains works better than
effects were found (Saavedra et al. 2004). single strains, determine the optimal daily dosage
However, in some high-risk groups, adverse and find a vehicle system allowing a prolonged
effects have been reported (Cilieborg et al. 2011). retention time in the oral cavity.
So far, no serious adverse effects have been
reported in clinical trials regarding oral health- Conclusion
care. Some of the reported adverse effects include The beneficial effect of probiotic bacteria has
flatulence or mild abdominal discomfort. A few been suggested and studied within several
studies have addressed this issue as a primary areas of dentistry. Despite limitations of the
outcome and no adverse effects have been found studies, such as surrogate end points, hetero-
either in animal models (Tanzer et al. 2010) or in geneous design and small sample sizes, the
humans (Burton et al. 2011). The acidogenicity majority of the studies show promising results.
of lactobacilli could lead to increased risk of car- Still, there is not sufficient evidence to make
ies, and hence, acidogenicity (Pham et al. 2009) any clinical recommendations but when for
and sugar fermentation (Haukioja et al. 2008; either prevention or treatment of oral infec-
Hedberg et al. 2008) of probiotic strains have tions, probiotic bacteria should be used as an
been studied in vitro. However, as previously adjunct to the existing options.
mentioned, no increased plaque acidogenicity
has been found in vivo.
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Management of Patients with Oral
Candidiasis
13
Abstract
Oral candidal infections are medically treated with antifungal agents. In
the fungal cell membrane, steroid ergosterol is the target of the antifungals
on the market, but similarity with the human cell membrane may cause
host toxicity and unintended reactions. Management of oral candidiasis
depends on several factors, some are host-sensitive parameters, systemic
diseases and drug exposure, and others are infection-sensitive parameters,
duration of the infection and the virulence of the infecting Candida spe-
cies. Treatment failure might be associated with acquired or native azole
resistance in particular in patients with recurrent oral candidiasis. This risk
can be reduced if different types of antifungal drugs are used over time or
are combined. This chapter focuses on antifungal treatment of the medi-
cally compromised patient with oral candidiasis by highlighting the
advantages and disadvantages of different antifungals.
13.1 Introduction Human and fungal cell membrane steroids differ,

which has been the focus of the antifungals on
Oral candidal infections are medically treated the market. Human cell membranes consist
with antifungal agents with different indications. mainly of cholesterol, whereas fungal cell mem-
Fungi are eukaryotes like humans, which is chal- branes consist of ergosterol, and ergosterol and
lenging for the drug selectivity as similarity its biosynthesis have been the drug target of anti-
between the human host and the pathogen may fungals (Xie et al. 2014).
cause host toxicity and unintended reactions. Antifungal agents for treatment of oral candi-
diasis fall into two main categories: polyenes
(nystatin and amphotericin B) and azoles (micon-
C. Kragelund, DDS, PhD (*) • J. Reibel, DDS, PhD, azole, clotrimazole, fluconazole, ketoconazole,
Dr. Odont • A.M.L. Pedersen, DDS, PhD and itraconazole). Antifungal agents seem to
Oral Pathology and Oral Medicine, Department of
interact with the ability of candidal adherence to
Odontology, Faculty of Health and Medical Sciences,
University of Copenhagen, Copenhagen, Denmark buccal epithelium and some are up-concentrated
e-mail: ckra@sund.ku.dk; jrei@sund.ku.dk; in epithelial cells, e.g. itraconazole. Topical and
amlp@sund.ku.dk systemic formulations are available.
DOI 10.1007/978-3-319-25091-5_13
13.2 Polyenes p450 inhibitors, i.e. CYP2C9 and CYP3A4.

These enzymes are involved in the metabolism of
Nystatin and amphotericin B are ergosterol syn- approximately 40 % of the marketed drugs. Thus,
thesis inhibitors and broad-spectrum antifungal azole-drug interaction must be taken into serious
agents. They interact with ergosterol in the cell consideration in medically compromised patients.
membrane of yeasts and yeastlike fungi, thus, all Clotrimazole cream has a low skin penetration
the Candida spp. The binding to ergosterol causes and has no clinical relevant drug-drug interac-
pore formation and ion leakage. Depending on tions. Miconazole is a potent CYP2C9 and
the drug concentration on the application site, CYP3A4 inhibitor. Only 25 % of the miconazole
polyenes have a fungistatic or fungicidal effect. administered topically is absorbed from the skin
Amphotericin B has not absolute specificity for and mucous membranes of the mouth, ventricle,
fungal cell membranes as it also binds to choles- and intestine, but the first-pass intestinal and liver
terol of the human cell membranes, which causes enzymes are inhibited significantly. Miconazole
significant systemic toxicity. has clinical relevant drug-drug interactions where
Thus, polyenes are only available for topi- dose adjustment of the implicated drug is impor-
cal treatment of oral candidiasis. When admin- tant, e.g. warfarin and cyclosporine. Clotrimazole
istered perorally both are poorly absorbed from and miconazole have a bacteriostatic effect that is
the oral, gastric, and intestinal mucous mem- beneficial in treating angular cheilitis.
branes and mostly eliminated faecally unchanged. Fluconazole is both available as oral suspen-
Accordingly, they have no known severe drug-drug sion for topical/systemic treatment and as capsules
interactions. Nystatin has a bad bitter taste and for systemic use. The oral suspension has both
formulations are often sweetened with sucrose, topical and systemic effects as it is absorbed intes-
which significantly increases the risk of dental tinally and secreted in saliva in high concentration
caries. Special formulations without sucrose can after swallowing. The majority of fluconazole is
be prepared if not available on the market. The excreted unchanged by the kidneys, why knowl-
adverse drug effects of topical polyenes are usu- edge of reduced renal function is relevant. If the
ally mild and of gastrointestinal character. glomerular filtration rate (GFR) is < 50 %, the dos-
age is reduced with 50 % because of 50 % longer
elimination time. Fluconazole is a potent inhibitor
13.3 Azoles of CYP2C9 and a moderate inhibitor of CYP3A4
why there is a risk of drug-drug interaction with a
Azoles are cytochrome p450 enzyme inhibitors long list of drugs (The American Society of
and the antifungal effect is interaction with the Health-System Pharmacists (ASHP) 2015, The
fungal ergosterol synthesis through binding to Danish Health and Medicines Authority 2015).
the cytochrome p450 enzyme, CYP51. A fungi- Itraconazole is available for systemic use and
static effect is achieved by ergosterol depletion, is absorbed intestinally. It is only recommended
changes in cell membrane permeability and to adults. The absorption is dependent on gastric
membrane-bound proteins, and synthesis of cell pH, why it must be administered before a meal,
toxic sterols. Some Candida spp., i.e. C. krusei and proton pump inhibitors reduce the absorp-
and C. glabrata, are native resistant or have tion. Itraconazole is metabolized in the liver and
reduced sensibility to azoles (Arendrup 2013). is a potent CYP3A4 inhibitor. Like fluconazole,
Azoles can enhance the effect of oral antidiabet- itraconazole interacts with a long list of drugs
ics leading to hypoglycaemia. (The American Society of Health-System
Azoles are available for topical and systemic Pharmacists (ASHP) 2015, The Danish Health
use. Azoles for oral candidiasis are classified into and Medicines Authority 2015).
imidazoles with a two-nitrogen azole ring Ketoconazole is recommended for topical
(clotrimazole and miconazole) and triazoles with skin infections only. In 2013 the European
a three-nitrogen azole ring (fluconazole and Medicines Agency (EMA) assessment and the
itraconazole). All these azoles are cytochrome US Food and Drug Administration (FDA) recom-
13 Management of Patients with Oral Candidiasis 139
Table 13.1 Advantages and disadvantages of topical and systemic antifungal treatment of oral candidiasis
Sensitive parameters Topical Systemic
Polyenes Azoles Azoles
Difficult compliance − − +
Complicating systemic disease/condition + + −
Risk of drug-drug interactions + − −
Azole insusceptibility + − −
Treatment expense − − +
+ Advantage, − disadvantage
Table 13.2 Relevant factors for management of patents with oral candidiasis
Host-sensitive parameters
1. Predispositions for oral candidiasis Local
Systemic
2. Health and medical status of the patient affecting drug Chronic diseases
metabolism Drug exposure
Infection-sensitive parameters
1. Classification of infection Duration of infection (acute/chronic)
Primary/secondary/tertiary infection
Sporadic/recurrent
Clinical manifestations
2. Candida species Susceptibility to antifungals
Resistance to antifungals
mended that ketoconazole was no longer used for amphotericin B, echinocandins, and pyrimidines
systemic treatment of candidiasis because of high for oral candidiasis may be indicated in seriously
risk of severe adverse reactions, i.e. liver damage, medically compromised patients and should only
drug interactions, and adrenal gland problems be done in specialist settings.
(European Medicines Agency 2013; US Food
and Drug Administration 2013).
The adverse drug effects of azoles are usually 13.5 Management of Patents
mild and of gastrointestinal character. Topical with Oral Candidiasis
preparations may give rise to local skin
irritation. Management of oral candidiasis depends on sev-
The treatment expense for different antifun- eral factors; some are host-sensitive and others
gals and different formulations may vary consid- are infection-sensitive parameters (Table 13.2).
erably and must also be taken into account in
treatment planning. The general advantages and
disadvantages of topical and systemic treatment 13.5.1 Host-Sensitive Parameters
are listed in Table 13.1.
Host-dependent parameters are very important
factors when managing oral candidiasis.
13.4 Specialist Treatment Identification of predisposing factors is crucial
for successful treatment outcome (Table 13.3). If
Second-generation azoles such as voriconazole the underlying causes are not eliminated or iden-
and posaconazole are spared for specialist set- tified, the chance of relapse is high.
tings and selected patients with life-threatening The drug formulation is relevant in patients
candidiasis or severe immunocompromising con- with special needs, e.g. topical formulation in
ditions. Intravenous antifungal therapy with patients with dysphagia and capsules in patients
Table 13.3 Local and systemic predisposing factors and conditions

Background Intervention
Local Poor oral hygiene Instruction, motivation, and follow-up of oral hygiene
procedures
High-carbohydrate diet Information, motivation, and follow-up
Salivary gland hypofunction caused by Stimulation of functional salivary gland tissue with
medications, head and neck radiotherapy, sugar-free pastilles or chewing gum, substitution
or systemic diseases like Sjögren’s syndrome of drug if possible
Mucosal traumas Identify cause and eliminate
Mucosal diseases Diagnostic workup, information, treatment, and
follow-up
Topical steroid Instruction in appropriate behaviour
Tobacco Tobacco counselling, smoking cessation
Systemic Immune deficiency (acquired or idiopathic) Diagnostic workup and intervention.
Systemic diseases Often need for multidisciplinary cooperation
Nutritional deficiency
with reduced compliance. Patients with hyposali- shorter intervention period. Acute candidiasis in
vation cannot dissolve pastilles and lozenges. general needs topical treatment for 2 weeks and
The health and medical status of the patient systemic treatment for 1 week, whereas the chronic
is important as the choice of antifungals depends infection needs longer intervention (Table 13.4).
on whether the patient can eliminate the drug in The anatomic area of infection influences the
order to avoid toxic or adverse reactions. choice of drug vehicle. Cream is used for perioral
Moreover, knowledge of the patient’s drug infections but is not applicable on wet oral mucous
intake is vital as severe drug-drug interactions membranes. Topical formulations for intraoral
with the antifungals can be relevant (Table 13.1 infections are pastilles, lozenges, oral gel, and oral
and 13.4). suspension. All topical agents need frequent dos-
In treatment of pregnant women, knowledge age as oral clearance results in falling of the con-
of teratogenic risks in relation to drug exposure is centration to a subtherapeutic level after
always important as is unintended drug exposure approximately 6 hours. To achieve maximal reten-
of breastfed children. Generally, well-known and tion time, topical treatment must be applied after
well-documented drugs are recommended during meals and oral hygiene procedures, and the patient
pregnancy and breastfeeding. Topical nystatin must also be instructed not to drink and rinse the
and amphotericin B are well-documented old mouth after application. Thus, topical treatment is
antifungal agents and have a low systemic effect challenging for the compliance of the patient.
because of little absorption. Topical miconazole All lesions in multifocal infections need to be
for vaginal candidiasis in pregnant women is treated; thus, these are most easily treated with
widely used without report of higher incidence of systemic administration of the antifungal agent.
foetal malformations. Foetal malformations have However, this is not always possible because of
been reported in relation to high-dose flucon- other host- or infection-sensitive parameters, e.g.
azole and also itraconazole should be avoided in azole-drug interactions (Table 13.1).
pregnancy and during periods of breastfeeding. Some of the Candida spp. commonly isolated
from the oral cavity are not sensitive to azole
antifungal agents, e.g. C. krusei and C. glabrata,
13.5.2 Infection-Sensitive Parameters and drug resistance can be acquired by other spe-
cies (Tables 13.1 and 13.4) (Arendrup 2013).
Infection-sensitive parameters influence the dura- Acquired resistance to polyenes is very rare
tion of treatment as acute infection usually needs a and accordingly little described. The low
13
Table 13.4 Central host- and infection-sensitive parameters regarding the management of patients with chronic oral candidiasis with antifungals
Host factors Infection factors
Administration Drug Formula Dosage Diseases Drug interaction Classification In-sensitive Comments
Candida species
Topical Clotrimazole Cream 1 % 2–3 daily – – Perioral * – >12 years of old
continues until 10
days after lesions
healed
Miconazole Cream 2 % 2 daily – + Perioral * +
continues until
10 days after
lesions healed
Ketoconazole Cream 2 % 1–2 daily Liver monitoring + Perioral +
continues until
some days
after lesions
Management of Patients with Oral Candidiasis
healed
Oral gel 2.5 ml × 4 – + Intraoral + Blockage of
daily in 4–6 airway in
weeks neonatal children
Nystatin Oral suspension 1 ml × 4 daily – – Intraoral –
100,000 IE/ml in 4–6 weeks
Pastille 100.000 After meals x 4 daily – – Intraoral – Not if
IE/unit 4 weeks hyposalivation
Amphotericin Lozenge 10 mg 4 times daily for – – Intraoral – Not if
B 4 weeks hyposalivation
Fluconazole Oral suspension 50–100 mg daily for Dosis↓ reduced + Multi-focal +
10 mg/ml 4 weeks renal function infection
Systemic Fluconazole Tablet or capsule 1 daily in 2–3 weeks Dosis↓ reduced + Multi-focal + High compliance
50–100 mg renal function infection
Itraconazole Tablet or capsule 1 daily in 2 weeks Not cardiac + Multi-focal + High compliance
100 mg insufficiency infection
Caution liver
disease
–: not relevant
+: relevant
*: some additional antibacterial effect
141
Indications, drug and formula availability may vary in different countries

incidence is believed to be related to the fungi- tribute to the high individual exposure. However,
cidal effect of polyenes. The exact mechanisms the environmental exposure to azoles is even
of resistance are not clear but are suggested to be higher as azoles are widely used as fungicides in
related to mutations in the ergosterol biosynthe- agriculture, industry, and domestically, e.g. seed
sis pathway (Maubon et al. 2014). On the con- and postharvest treatment, wood preservatives,
trary, acquired resistance towards azoles is widely textiles, toiletries, and human and animal excre-
described and believed to be related to their fun- tions when in antifungal treatment (Parker et al.
gistatic effect. 2014).
There are four main azole resistance The risk of iatrogenic selection of azole-
mechanisms: insensitive Candida spp. in patients with recur-
rent oral candidiasis is reduced if the drugs for
1. Reduced affinity to drug target CYP51 the antifungal therapy vary between different
2. Increased amount of drug target CYP51 drugs over time or are combined (Xie et al. 2014).
3. Increased efflux of azoles from the fungal cell Still, caution and restriction of antifungal use are
4. Genomic rearrangements many years behind the professional and public
alertness on unnecessary use of antibiotics.
The mode of azole action is inhibition of the However, in 2013 fluconazole-resistant Candida
fungal ergosterol synthesis through binding to was listed by the US Department of Health and
the CYP51 enzyme leading to ergosterol deple- Human Services as having equivalent threat level
tion, cell membrane disruption, and synthesis of to human health as methicillin-resistant
cell toxic sterols. Azole failure leads to ergosterol Staphylococcus aureus (MRSA) (Centers of
synthesis, unharmed cell membrane integrity, Disease Control and Prevention. Antibiotic resis-
and reduced susceptibility to azole-induced tance threats in the United States 2013).
cellular stress, and the end point is survival of the
fungus.
Point mutation of the ERG11 gene coding for 13.5.3 Prophylaxis of Oral Candidiasis
CYP51 reduces the azole’s affinity to CYP51.
Upregulation of the ERG11 gene increases the The majority of patients experience sporadic oral
amount of CYP51 and enables substrate competi- candidiasis, but some experience recurrent oral
tion towards ergosterol synthesis. Upregulation candidiasis with varying disease-free periods.
of multidrug transporters reduces the intracellu- Recurrent oral candidiasis might affect quality of
lar azole concentration, and thereby the therapeu- life and oral functions because of oral symptoms,
tic dose is not achieved. Genomic alteration as and there is a risk of infection spreading to other
chromosomal rearrangements, aneuploidy, and areas of the body. Identification of predisposing
loss of heterozygosity causes reduced suscepti- factors is the important first step followed by inter-
bility to azole-induced cellular stress (Xie et al. vention for elimination of the predisposing factors
2014). All mechanisms reduce the intended effect (Table 13.3). This may not be possible in patients
of azoles on the fungal cell membrane and may with local predisposing factors like oral mucosal
induce multi-resistance towards azoles. The diseases, e.g. oral lichen planus and leukoplakia,
increased incidence of non-C. albicans infections or systemic predisposing factors, e.g. disease-
in the Western part of the world and the increase related or iatrogenic immunosuppression.
of azole-insensitive C. albicans infections is Depending on the type of predisposing factor,
believed to be a result of increased human expo- other topical treatments with antifungal effect
sure to environmental and medical azoles can be successful, e.g. chlorhexidine mouth rinse
(Arendrup 2013). in patients with oral lichen planus (see below).
Over-the-counter azoles for treatment of Prolonged or repeated treatment with antifungal
‘self-diagnosed’ candidiasis, antifungal prophy- drugs may be necessary, and systemic treatment
laxis, and prolonged antifungal treatment con- with azoles is most widely used. However, there
13 Management of Patients with Oral Candidiasis 143
is a risk of acquired resistance and iatrogenic 0.12 % or above successfully reduced candida
selection of azole-insensitive Candida spp. This yeast in bone marrow-transplanted, mechanically
risk can be reduced if the drugs for the antifungal ventilated, and HIV patients (Lam et al. 2012).
therapy vary between different drugs over time or Local allergic and anaphylactic reactions are
are combined (Xie et al. 2014). reported for chlorhexidine.
Repeated short-term oral treatment may be
indicated in patients with recurrent candidiasis
13.6 Other Topical Treatments and poor oral hygiene, e.g. mouth rinse with
with Antifungal Effect 0.12 % chlorhexidine twice daily for maximum of
two weeks when symptoms begin. Dentures can
In order to minimize the individual and environ- be soaked overnight in 0.2 % chlorhexidine after
mental exposure to antifungals, old and new mechanical cleaning in patients with denture sto-
alternative agents are used or investigated for matitis (Ellepola and Samaranayake 2001).
treatment and prophylaxis of oral candidiasis.
13.6.2 Fluoride
13.6.1 Chlorhexidine
Topical fluoride treatment affects the composition
Chlorhexidine is antiseptic and has a broad- and thickness of the oral biofilm on tooth structures
spectrum antibacterial and antifungal effect. in particular. Candida is part of the oral microfilm
Depending on the concentration it is fungistatic (Williams et al. 2011). Amine fluoride and stannous
or fungicidal. At high concentrations it destroys fluoride have been shown in vitro to have antifungal
the fungus cell membrane resulting in coagula- effect close to that of chlorhexidine (Flisfisch et al.
tion of cellular proteins and cell death. At lower 2008). A combination of potassium fluoride and
doses it inhibits the fungi adhesion to surfaces amphotericin B increases the destabilizing effect of
as it binds to acrylic, epithelial, and tooth struc- amphotericin B on the fungal cell membrane (Li
tures and interacts with the fungus adhesion by and Breaker 2012). Thus high fluoride treatment is
modifying fungal cell surface hydrophobicity indicated in patients with poor oral hygiene.
and hyphae formation. Even at low dose of long
time after chlorhexidine exposure, the fungi are
suppressed, which is referred to as “the post- 13.6.3 Probiotics
antifungal effect of chlorhexidine” (Ellepola and
Samaranayake 2001). Adverse reactions include Probiotics are live microorganisms, which when
chemical-induced epithelial desquamation, tem- administered in adequate amounts confer a health
porary brown discoloration of teeth and dentures, benefit for the host (FAO/WHO 2001). The
and taste disturbances, which are related to treat- probiotic bacteria have been administered to
ment duration and the concentration. The extent patients in dairy products such as cheese, yoghurt,
of epithelial desquamation increases with con- and milk and as pharmaceutical products as pow-
centration at the site. Thus, chlorhexidine is not der, lozenges, tablets, and chewing gums. In order
suitable for long-term treatment (Flotra 1973). to keep an adequate number of probiotic bacteria
In vitro it has been found that chlorhexidine in the mouth, they need to be administered fre-
and nystatin suppress each other’s antifungal quently and after oral procedures with increased
properties as they form chlorhexidine-nystatin oral clearance, e.g. meals and oral hygiene proce-
complexes (Barkvoll and Attramadal 1989). dures. The Bifidobacterium and Lactobacillus are
Chlorhexidine is available as mouth rinse, oral the two genera investigated in relation to oral
gel, and cream and usually in 0.12, 0.2 and 1 % Candida spp., and the oral load of Candida has
concentrations. Randomized clinical trials (RCT) been shown to decrease (Hatakka et al. 2007;
have shown that chlorhexidine concentrations of Mendonca et al. 2012; Dos Santos et al. 2009;
Ishikawa et al. 2015; Kraft-Bodi et al. 2015). One Flotra L. Different modes of chlorhexidine application
and related local side effects. J Periodontal Res.
study investigated probiotic powder for treatment
1973;12:41–4.
of denture stomatitis, but still, the impact of probi- Hatakka K, Ahola AJ, Yli-Knuuttila H, Richardson M,
otics on treatment and prophylaxis of oral candi- Poussa T, Meurman JH, et al. Probiotics reduce the
diasis needs to be investigated in detail (Li et al. prevalence of oral candida in the elderly-a randomized
controlled trial. J Dent Res. 2007;86:125–30.
2014) (see Chap. 12).
Ishikawa KH, Mayer MP, Miyazima TY, Matsubara VH,
Silva EG, Paula CR, et al. A multispecies probiotic
reduces oral Candida colonization in denture wearers.
13.6.4 Essential Oils J Prosthodont. 2015;24:194–9.
Kraft-Bodi E, Jorgensen MR, Keller MK, Kragelund C,
Essential oils from plants have been investigated Twetman S. Effect of probiotic bacteria on oral
Candida in frail elderly. J Dent Res. 2015. 94;9 suppl.:
for antifungal alternative alone or in combination 181S–6S.
with antifungal drugs. There are challenges using Lam OL, Bandara HM, Samaranayake LP, McGrath C, Li
essential oils. One is that genetic identical plants LS. Oral health promotion interventions on oral yeast
produce chemically different essential oils with in hospitalised and medically compromised patients: a
systematic review. Mycoses. 2012;55:123–42.
different antifungal properties depending on grow- Li D, Li Q, Liu C, Lin M, Li X, Xiao X, et al. Efficacy and
ing conditions like climate, soil quality, and other safety of probiotics in the treatment of Candida-
external factors. Another challenge is the safety associated stomatitis. Mycoses. 2014;57:141–6.
regarding allergic reaction and administration of Li S, Breaker RR. Fluoride enhances the activity of fungi-
cides that destabilize cell membranes. Bioorg Med
the essential oils (Palmeira-de-Oliveira et al. Chem Lett. 2012;22:3317–22.
2009). In two RCT with HIV-positive patients, Maubon D, Garnaud C, Calandra T, Sanglard D, Cornet
essential oils have been shown to reduce the can- M. Resistance of Candida spp. to antifungal drugs in
dida count and oral candidiasis (Lam et al. 2012). the ICU: where are we now? Intensive Care Med.
2014;40:1241–55.
Mendonca FH, Santos SS, Faria IS, Goncalvese Silva CR,
Jorge AO, Leao MV. Effects of probiotic bacteria on
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Index
A B
ACPAs. See Anti-citrullinated protein antibodies Bacillus bifidus communes, 125
(ACPAs) Bacteremia, 4, 23–24, 27, 28, 60, 133
Adjuvant arthritis, 48–49 Bacteria, 97–98
Advanced glycation end products (AGEs), 37–39 Bacteriophages, 98–99, 107
Aging Bifidobacterium, 109, 126, 143
cancer-related deaths, 60 Biopsy, 68, 69, 74
dental caries, 14–15 Bleeding on probing (BOP), 130
human oral microbiome, 97
Amphotericin B, 138–141
Angular cheilitis, 68, 81, 84, 138 C
Anti-citrullinated protein antibodies (ACPAs), 45–48 Cancer
Antifungal treatment acetaldehyde production, 60–61
dental caries, 83 dental caries, 14, 62–63
oral candidiasis epidemiology, 59–60
azoles, 138–139 infection-driven mechanisms, 60, 61
chlorhexidine, 143 periodontitis, 62–63
essential oils, 144 prevalence, 4
fluoride, 143 saliva, 63
host-sensitive parameters, 139–141 Cancer chemotherapy and oral microbiota
infection-sensitive parameters, 140–142 dental caries, 83–84
polyenes, 138 oral candidiasis, 71–72, 84–85
probiotics, 143–144 Candida albicans, 65–67, 69–72, 81, 82, 84, 86, 100,
prophylaxis, 140–142 142
specialist settings, 139 Candida infections, 60, 61, 66, 68, 70, 74
Archaea, 99–100, 106 Cardiovascular diseases, 19–20. See also Atherosclerosis
Asthma, 13, 87 Case–control studies
Atherogenesis, 21–24, 29–31 dental caries, 11–12, 14
Atherosclerosis periodontitis, 25, 29, 46, 47, 62
acute atherosclerotic events, 20–21 Cerebrovascular disease, 21
periodontitis Checkerboard DNA-DNA hybridization, 101
atherogenesis, 21–23 Chlorhexidine, 84, 143
bacteremia, 23–24 Chronic glomerulonephritis, 54
biologically plausible mechanisms, 31 Chronic kidney disease
common genetic risk factors, 29–30 glomerular filtration rate (GFR), 53
consequences, 21 oral infections, aspects of, 56
dyslipidemia, 29 periodontal disease, 54
overactive immunity, 24 prevalence, 53
pro-inflammatory state, 25–26 Chronic mucocutaneous candidiasis (CMC), 72
prothrombotic state, 26–29 Cleft lip palate, 14
Attention deficit hyperactivity disorder (ADHD), 14 Clotrimazole, 138
Autoimmune polyendocrinopathy-candidiasis- Cluster of differentiation 40 ligand (CD40L), 28
ectodermal dystrophy (APECED), 60 CMC. See Chronic mucocutaneous candidiasis (CMC)
Azoles, 74, 138–140, 142 Cognitive impairment and dental caries, 10, 14–15

DOI 10.1007/978-3-319-25091-5
146 Index
Commensal oral microorganism, 10, 65, 67, 97, 100, E

116, 131 Entamoeba gingivalis, 100
Community-wide transcriptome analysis, 105 Epstein-Barr virus, 99
Computer-based caries risk model, 11 Erythematous candidiasis, 67–68, 71, 81, 82
Concomitant human papillomavirus (HPV) Essential oils, 144
infection, 59, 62 European Federation of Periodontology/American
Congenital heart disease, 13–14 Academy of Periodontology (EFP/AAP), 19,
Coronary artery disease (CAD), 21, 25, 30 21, 31
C-reactive protein (CRP), 25, 39, 40 Exfoliative cytologic examination, 72
Culture sampling procedure, 72
Cytomegalovirus, 99
Cytosmears, 72 F
Fluconazole, 71, 138, 140, 142
Fluorescence in situ hybridization (FISH), 74, 107–109, 118
D Fluoride, 143
Decayed, missing, filled permanent teeth (DMFT), 14, 84 Fungi, 100, 137
Dental caries
aging and cognitive impairment, 14–15
asthma, 13 G
biofilm-mediated disease, 10 Gingivitis, 36, 62, 83, 128, 130
cancers, 14, 62–63 Glomerulonephritis, 54
cleft lip palate, 14
congenital heart disease, 13–14
diabetes, 13 H
general diseases Halitosis, 132
case–control studies, 11–12 Healthy oral cavity, 3
observational studies, 11–12 Heat shock proteins (HSP), 24
oral hygiene, 10 Helicobacter pylori, 59, 126
risk assessment, 10–11 High-density lipids (HDL), 29
incidence, 3 Highly active antiretroviral therapy (HAART), 4, 71
medically compromised patient management, 15 High sensitivity C-reactive protein (hsCRP), 25–26, 32,
neuropsychiatric disorders, 14 41
obesity and overweight, 12–13 High-throughput sequencing, 66, 102
oral hygiene, 10 Histatins, 66
oral microbiota, 108–109 HIV infection
biofilm architecture, 109–110 essential oils, 144
cancer chemotherapy, 83–84 oral candidiasis, 60, 70–71
radiotherapy, 85–86 HOMIM analysis. See Human Oral Microbe
salivary gland dysfunction, 87–88 Identification Microarray (HOMIM)
prevalency, 9–10 analysis
prevention and management, 15–16 HOMINGS, 102
risk assessment, 11 Human Microbiome Project (HMP), 107, 115, 116
salivary microbiota, 118–120 Human Oral Microbe Identification Microarray
SS, 80–81 (HOMIM) analysis, 102, 116, 118, 119
Denture stomatitis, 68, 70, 143 Human oral microbiome. See Oral microbiome/
Diabetes, 13 microbiota
Diabetes mellitus (DM) Human Oral Microbiome Database (HOMD), 98, 101,
forms, 35–36 102
oral candidiasis, 70 Hyperplastic candidiasis, 68–69, 74
periodontitis
biological similarities, 37
mechanisms of association, 37–40 I
population data, 36–37 Infection-driven carcinogenesis, 60, 61
pro-inflammatory cytokines, 37 Insidious dental infections, 4, 80
treatment outcome, 40–41 Itraconazole, 138, 140
Diabetic nephropathy, 54
DMFT. See Decayed, missing, filled permanent teeth
(DMFT) K
Dyslipidemia, 29, 31, 32 Ketoconazole, 138–139
Index 147
L molecular techniques, 74
Lactobacillus, 80, 81, 84, 85, 87, 89, 109, 118, 119, smears, 72
133, 143 medically compromised patient
Lipopolysaccharide (LPS), 23, 24, 29 adverse drug reaction, 69–70
Liver cirrhosis, 54 diabetes mellitus, 70
Liver diseases, 54–55 HIV Infection, 70–71
Low-density lipids (LDL), 29 solid organ transplants, 71–72
oral microbiota
cancer chemotherapy, 84–85
M radiotherapy, 86
Massive parallel RNA sequencing, 105 salivary gland dysfunction, 88
Matrix-assisted laser desorption ionization-time predisposing factors, 4, 69, 139,
of flight/mass spectrometry 140, 142
(MALDI-TOF/MS), 74 prevalence, 4
Mean platelet volume (MPV), 28 probiotics, 131–132
Median rhomboid glossitis, 67, 68 SS, 81–82
Medication-induced salivary gland dysfunction Oral microbiome/microbiota
(MISGD), 10 analytic techniques
Metatranscriptomics, 102, 105 community-wide transcriptome
Methicillin-resistant Staphylococcus aureus analysis, 105
(MRSA), 142 culture-independent methods, 101–102
Miconazole, 138, 140 high-throughput sequencing, 102
Model for End-Stage Liver Disease (MELD) HOMINGS, 102
score, 55 metatranscriptomics, 102, 105
Mucositis, 132 microscopy and culture
methods, 100–101
next-generation sequencing
N platforms, 103–104
Neuropsychiatric disorders, 14 oligotyping analysis, 102
Next-generation sequencing single-cell genome sequencing, 102
platforms, 103–104 WGS, 102
Normal blood clotting events, 26–27 archaea, 99–100
Nystatin, 138, 140, 143 bacteria, 97–98
bacteriophages/viruses, 98–99
biofilm architecture
O dental caries, 109–110
Obesity/overweight, 12–13 periodontal disease, 107–108
Oligotyping analysis, 102 cancer chemotherapy
Oral candidiasis, 4 dental caries, 83–84
antifungal treatment oral candidiasis, 84–85
azoles, 138–139 cancer epidemiology, 60
chlorhexidine, 143 carcinogenesis, 59–60
essential oils, 144 chairside diagnostics, 111
fluoride, 143 disruption factors, 3
host-sensitive parameters, 139–141 fungi, 100
infection-sensitive parameters, 140–142 vs. genomic composition, 97, 98
polyenes, 138 protozoa, 100
probiotics, 143–144 radiotherapy
prophylaxis, 140–142 dental caries, 85–86
specialist settings, 139 oral candidiasis, 86
clinicopathological aspects salivary gland dysfunction
histopathology, 68–69 dental caries, 87–88
immunological aspects, 69 oral candidiasis, 88
types and differential diagnostic considerations, periodontal disease, 88
67–68
diagnostic methods
advantages and disadvantages, 73 P
biopsy, 74 Peptidylarginine deiminases (PADs), 46
culture media sampling, 72 Periodontal diseases. See Periodontitis
148 Index
Periodontitis DM and, 37
atherosclerosis RA and, 47
atherogenesis, 21–23 Protozoa, 100
bacteremia, 23–24 P-selectin, 28
biologically plausible mechanisms, 31 Pseudomembranous candidiasis, 67, 71
common genetic risk factors, 29–30 Pyrosequencing, 102
consequences, 21
dyslipidemia, 29
overactive immunity, 24 R
pro-inflammatory state, 25–26 RA. See Rheumatoid arthritis (RA)
prothrombotic state, 26–29 Radiotherapy and oral microbiota
cancer, 62–63 dental caries, 10, 85–86
DM oral candidiasis, 86
biological similarities, 37 Recurrent oral candidiasis, 80, 81, 142
mechanisms of association, 37–40 Renal diseases, 54
population data, 36–37 Rheumatoid arthritis (RA)
pro-inflammatory cytokines, 37 ACPAs, 45–46
treatment outcome, 40–41 autoantibodies, 45
human clinical studies, 128–130 incidence, 45
incidence, 4 periodontitis
liver transplant recipients, 55 biological similarities, 47
oral microbiota, 106–107 mechanisms of association, 47–48
biofilm architecture, 107–108 population data, 46–47
community-wide transcriptome treatment outcome, 49
analysis, 105
probiotics, 128–131
RA S
biological similarities, 47 Salivary gland dysfunction and oral
mechanisms of association, 47–48 microbiota, 5, 15
population data, 46–47 biomarker, 120
treatment outcome, 49 carcinogenesis, 63
renal diseases, 54 causes, 80
salivary gland dysfunction and oral dental caries, 87–88, 118–120
microbiota, 88 oral candidiasis, 88
salivary microbiota, 117–118 periodontal disease, 88
Peripheral arterial disease (PAD), 21 periodontitis, 117–118
Polycystic kidney disease, 54 Secondary Sjögren’s syndrome (sSS), 80–83
Polyenes, 137, 138, 140, 142 Second-generation azoles, 139
Polymerase chain reaction (PCR) procedures, 74 Sialometry, 15
Porphyromonas gingivalis, 24, 28, 48–49, 55, 83, 88, Single-cell genome sequencing, 102
106, 107, 117, 118 Sjögren’s syndrome (SS)
Posaconazole, 139 dental caries, 80–81
Primary Sjögren’s syndrome (pSS), 80–83, 88 oral candidiasis, 81–82
Probiotics Smears, 72
adverse effects, 133 Sporadic oral candidiasis, 67, 72, 142
dental caries, 127–128 SS. See Sjögren’s syndrome (SS)
future studies, 133 Streptococcal glomerulonephritis, 54
gingivitis, 128 Streptococcus mutans, 54, 80, 81, 83–86, 100, 109,
halitosis, 132 118, 119
mechanisms of action, 126–127 Streptococcus viridans, 54, 55
microorganisms, 126 Supragingival plaque, 81, 82, 84, 88, 107, 118, 119
mucositis, 132 Systematic scaling and root planing
oral candidiasis, 131–132, 143–144 (SRP), 130–131
periodontitis, 128–131
safety, 132–133
Pro-inflammatory cytokines T
carcinogenesis, 60 Thrombopoietin, 27
oral candidiasis, 69 Tissue inhibitor of matrix metalloproteinases (TIMP-4),
periodontitis 48–49
Index 149
Toll-like receptors (TLRs), 28, 39 W

Torque Teno viruses (TTVs), 99 Whole-genome shotgun sequencing (WGS), 102
Transient ischemic accidents (TIA), 21 World Health Organization (WHO)
Trichomonas tenax, 100 cancer epidemiology, 59
Type 1 diabetes mellitus (T1DM), 13, 36–37 obesity and dental caries, 13
Type 2 diabetes mellitus (Type 2 DM), 36–37 oral cancer, 4
probiotic bacteria, 125
V
Very low-density lipoproteins ((v)LDL), 29 X
Viruses, 98–99 Xerogenic drugs, 10, 15, 69, 70, 83, 87–89
Voriconazole, 139 Xerostomia, 10, 16, 54, 80, 86, 87, 100, 126

Oral Infections

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Oral Infections

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What is the book about?

What is the book about?

What topics related to oral infections and general health are discussed?

What topics related to oral infections and general health are discussed?

Anne Marie Lynge Pedersen

ISBN 978-3-319-25089-2 ISBN 978-3-319-25091-5 (eBook)

Library of Congress Control Number: 2015957993

Springer Cham Heidelberg New York Dordrecht London

Printed on acid-free paper

Springer International Publishing AG Switzerland is part of Springer Science+Business Media

Part I Background Topics

Part II Oral Infections and General Health

2 Dental Caries and General Health in Children

Part III Future Diagnostic Methods and Techniques

10 The Oral Microbiome in Health and Disease . . . . . . . . . . . . . . . . 97

Part IV Future Perspectives in Management of Oral Infections

12 Use of Probiotics in Future Prevention and Treatment

Daniel Belstrøm, DDS, PhD Section 1, Periodontology and Oral

Claus H. Nielsen, MD, MSc, PhD Section 1, Periodontology

© Springer International Publishing Switzerland 2016 3

Dental caries is still the major cause of tooth loss,

© Springer International Publishing Switzerland 2016 9

Long-term survivors of malignant conditions are 2.10 Neuropsychiatric Disorders

gaps of knowledge concerning caries and gen-

Bruno G. Loos, Wijnand J. Teeuw,

3.1 Introduction has exponentially increased in this period.

© Springer International Publishing Switzerland 2016 19

Cardiovascular diseases include atherosclerosis, Traditionally, we differentiate between two

will increase the pro-inflammatory fibrous cap, weakening the strength

3.3 Bacteremia subgingival area (Loos 2005; Nesse et al. 2008).

et al. 2009; Wu et al. 2000). Studies on the effects

Chronic oral infection

Bacteria in atheromas Genetic background

4.1 Diabetes Mellitus and type 2 DM. While type 1 DM is due to an

© Springer International Publishing Switzerland 2016 35

4.2.2 Biological Similarities

RANKL/OPG axis AGE/RAGE axis Oxidative stress

Ecological Immune dysfunction, cellular stress &

© Springer International Publishing Switzerland 2016 45

experimental study in rats. These animals had Conclusion

6.1 Introduction that less than 25 % of the function remains.

© Springer International Publishing Switzerland 2016 53

Fig. 6.1 Dental infections 100

Tamaki N, Takaki A, Tomofuji T, Endo Y, Kasuyama K, Vesterinen M, Ruokonen H, Furuholm J, Honkanen E,

7.1 Introduction where, for example, local acetaldehyde production

© Springer International Publishing Switzerland 2016 59

IL: Interleukin Stimulation of B-cells

Fig. 7.1 Metabolic pathways in infection-driven carcinogenesis (Picture prepared by Bascones-Martinez)

Salivary acetaldehyde production in correlation with smoking and

8.1 Introduction studies due to geographical variations and varia-

© Springer International Publishing Switzerland 2016 65

8.2 Clinicopathological Aspects

In patients carrying Candida as part of the com-

Fig. 8.4 Chronic erythematous candidiasis presenting as

8.3 Oral Candidiasis

Oral candidiasis is the result of yeast overgrowth

8.3.1 Oral Candidiasis as Adverse

8.5.3 Biopsy a cheap and quick technique and is part of rou-

Ricker A, Vickerman M, Dongari-Bagtzoglou A. Thein ZM, Seneviratne CJ, Samaranayake YH,

Siri Beier Jensen and Anne Marie Lynge Pedersen

9.1 Introduction to formation of the dental pellicle, diluting food

© Springer International Publishing Switzerland 2016 79

9.3 Oral Microbiota in Patients