antibiotics

Article
Incidence and Clinical Characteristics of Anaerobic Bacteremia
at a University Hospital in Hungary: A 5-Year Retrospective
Observational Study
Krisztina Kovács 1 , Adrienn Nyul 1 , Zsolt Lutz 1 , Gyula Mestyán 1 , Márió Gajdács 2 , Edit Urbán 1, *
and Ágnes Sonnevend 1

1 Department of Medical Microbiology and Immunology, Clinical Centre, Medical School, University of Pécs,
Szigeti út 12, 7624 Pécs, Hungary
2 Department of Oral Biology and Experimental Dental Research, Faculty of Dentistry, University of Szeged,
6720 Szeged, Hungary
* Correspondence: urban.edit@pte.hu

Abstract: Strict anaerobes have been reported to account for 0.5–13% of episodes of bacteremia in
the adult population, with a growing awareness among clinicians regarding anaerobic bacteremia,
especially in patients with specific predisposing factors. The aim of our present study was to
assess the incidence and clinical characteristics of anaerobic bacteremia during a 5-year period
(2016–2020) at a tertiary care teaching hospital, and to compare our findings with other studies
in Hungary. Overall, n = 160 strict anaerobes were detected, out of which, 44.4% (n = 71; 0.1% of
positive blood cultures, 0.1/1000 hospitalizations, 3.3/100,000 patient days) were clinically significant,
while Cutibacterium spp. accounted for 55.6% (n = 89) of isolates. Among relevant pathogens, the
Bacteroides/Parabacteroides spp. group (32.4%; n = 23), Clostridium spp. (22.5%; n = 16) and Gram-
Citation: Kovács, K.; Nyul, A.; Lutz,
positive anaerobic cocci (15.5%; n = 11) were the most common. The mean age of patients was
Z.; Mestyán, G.; Gajdács, M.; Urbán,
E.; Sonnevend, Á. Incidence and
67.1 ± 14.1 years, with a male majority (59.2%; n = 42). A total of 38.0% of patients were affected by
Clinical Characteristics of Anaerobic a malignancy or immunosuppression, while an abscess was identified in 15.5% of cases. A total of
Bacteremia at a University Hospital 74.7% (n = 53) of patients received antibiotics prior to blood culture sampling; in instances where
in Hungary: A 5-Year Retrospective antimicrobials were reported, anaerobic coverage of the drugs was appropriate in 52.1% (n = 37) of
Observational Study. Antibiotics 2022, cases. The 30-day crude mortality rate was 39.4% (n = 28); age ≥ 75 years was a significant predictor
11, 1326. https://doi.org/10.3390/ of 30-day mortality (OR: 5.0; CI: 1.8–14.4; p = 0.003), while malignancy and immunosuppression, lack
antibiotics11101326 of anti-anaerobic coverage or female sex did not show a significant relationship with the mortality of
Academic Editor: Antonello these patients. Early recognition of the role played by anaerobes in sepsis and timely initiation of
Di Paolo adequate, effective antimicrobial treatment have proven efficient in reducing the mortality of patients
affected by anaerobic bacteremia.
Received: 17 August 2022
Accepted: 26 September 2022
Keywords: anaerobes; bacteremia; bloodstream infections; MALDI-TOF; blood cultures; clinical
Published: 28 September 2022
study; Bacteroides; Clostridium
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. 1. Introduction
Anaerobic bacteria make up the vast majority of the microbiome of human skin, mu-
cous membranes and gastrointestinal tract [1]. These microorganisms are characterized by
Copyright: © 2022 by the authors. a partial or complete intolerance to atmospheric oxygen, which may be explained by the
Licensee MDPI, Basel, Switzerland. lack of catalase, superoxide dismutase (SOD) and peroxidase enzymes, which are vital for
This article is an open access article the elimination of toxic reactive oxygen species [2,3]. With the exceptions of bite wound in-
distributed under the terms and fections and gas gangrene, most anaerobic infections are from endogenous origins (from the
conditions of the Creative Commons site of normal colonization), leading to local abscess formation or invasive infections, which
Attribution (CC BY) license (https:// may be severe or life-threatening infections [4,5]. In the age of evidence-based medicine
creativecommons.org/licenses/by/ (EBM) and antimicrobial stewardship, strict sample collection and transport and proper
4.0/).

Antibiotics 2022, 11, 1326. https://doi.org/10.3390/antibiotics11101326 https://www.mdpi.com/journal/antibiotics

Antibiotics 2022, 11, 1326 2 of 10

anaerobiosis during culture are needed for the identification and antimicrobial susceptibil-
ity testing of anaerobes, to guide therapeutic decisions [6]. The susceptibility of clinically
relevant anaerobes was thought to be predictable, allowing for safe empiric therapy with
the relevant anti-anaerobic drugs; resistance rates in most anaerobic bacteria showed a
considerable increase in the last 20–30 years [7]. Thus, rapid and accurate species-level iden-
tification of anaerobes aids clinicians to administer the best care for their patients, leading to
significantly reduced morbidity and mortality rates and hospital stays [8]. However, even
today, anaerobic bacteria are some of the most neglected, unrecognized pathogens, as their
cultivation requires extensive microbiological experience, and many facilities (especially in
developing countries) may not have the equipment (e.g., anaerobic chambers) that allow
for proper anaerobiosis [9]. As a result, failure to isolate or accurately identify these bacteria
may lead to inappropriate, unnecessary use of broad-spectrum antibiotics, subsequently
leading to a worsening antimicrobial resistance situation [10].
There is considerable variation in the reported prevalence of anaerobic bacteria in
bacteremia, which may be explained by different standard operating procedures in clinical
practice, the healthcare institution in question (e.g., primary vs. tertiary), patient population
and the capabilities of the diagnostic laboratory [11]. In addition, the need for performing
blood cultures for anaerobic bacteria is sometimes contested, especially by clinicians, due
to their opinion that the presence of anaerobes in blood may easily be suspected/predicted,
based on the patients’ characteristics, and if the source of infection is known to be an
anatomical area with a rich anaerobic flora [12]. Anaerobes have been reported to account
for 0.5–13% of episodes of bacteremia in the adult population (before the introduction
of various prophylactic measures, their prevalence was as high as 20%) [13]. It is crucial
to establish early and effective therapy for patients affected by anaerobic bacteremia, as
this condition has a considerable mortality rate (15–55%) [11,13]. There is, however, a
growing awareness of the role of anaerobes in bacteremia, especially in those patients
who have specific predisposing factors (e.g., advanced age, immunosuppression, cancer,
previous surgical intervention) [14]. There is a scarce amount of research comparing the
epidemiological features of anaerobic bacteremia in a multicenter fashion or within a
country, and many of these comparisons are limited by the use of different operating
standards and the laboratory methodologies used. The primary aim of our present study
was to assess the incidence and clinical characteristics of anaerobic bacteremia during a
5-year period in a tertiary care university teaching hospital in the Southern Transdanubia
region of Hungary. In addition, our secondary aim was to compare our findings to our
previous study in the Southern Great Plain region of Hungary [15]; the comparison of data
in these two studies is facilitated by a similar study setting and duration, similar patient
population and the same instrumentation used for microbiological identification.

2. Results and Discussion

During the 5-year study period, on average, 11,548.6 ± 253.6 blood culture bottles
were processed at the the Department of Medical Microbiology and Immunology per
year (n = 57,743 overall), out of which, 18.6% (n = 10,759) were culture positive (includ-
ing both contaminants and clinically relevant isolates). Overall, n = 160 strict anaerobes
were detected, out of which, Cutibacterium spp. accounted for 55.6% (n = 89) of isolates.
Based on our criteria, all of these isolates were considered as contaminants; therefore,
they were excluded from the analysis of clinical data. Clinically relevant anaerobes con-
stituted 0.1% (n = 71) of all processed blood cultures (0.7% of all positive blood cultures),
representing 1.2 isolates/1000 blood culture bottles, 0.1 isolates/1000 hospitalizations and
3.3 isolates/100,000 patient days, respectively. Details regarding the primary epidemio-
logical characteristics of anaerobic bacteremia in the present and comparator study are
presented in Table 1. The mean TAT (turnaround time) for the processing, identification
and reporting of anaerobic pathogens in the 5-year period was 7.8 ± 3.2 days.
Antibiotics 2022, 11, 1326 3 of 10

Table 1. Comparison of study settings and primary results of the present study with our previous
comparator study.

Present Study Gajdács et al. [15]

University of Pécs (PTE) Albert Szent-Györgyi Clinical
Study location Clinical Centre Center, University of Szeged
(Pécs, Hungary) (Szeged, Hungary)
Study period 2016–2020 2013–2017
Acute: 1705 Acute: 1465
Hospital bed count
Chronic: 20 Chronic: 355
Population served ~800,000 patients ~600,000 patients
Number of hospitalized patients/year
100,461 ± 9623 84,438 ± 1866
(Average ± SD)
Number of blood culture bottles processed during the
57,743 116,371
study period
Percentage of positive blood culture bottles for aerobic and
anaerobic bacteria overall 18.6 ± 2.1% 10.5 ± 0.3%
(including contaminants)
Percentage of positive blood culture bottles for clinically
0.1 ± 0.03% 0.2 ± 0.02%
relevant anaerobes
(0.3 ± 0.03%) (0.4 ± 0.1%)
(Percentage of positive blood culture bottles for all anaerobes)
Number of clinically relevant anaerobic isolates in bacteremia 71 176
(Number of all anaerobic isolates in bacteremia) (160) (423)
Number of clinically relevant anaerobic
0.1 0.4
isolates/1000 hospitalizations
(0.3) (1.0)
(Number of all anaerobic isolates/1000 hospitalizations)
Number of clinically relevant anaerobic
3.3 8.5
isolates/100,000 patient days
(7.4) (20.6)
(Number of all anaerobic isolates/100,000 patient days)
MALDI-TOF MS (Bruker Daltonics),
Methods used for microbial identification
extraction with formic acid before measurements
BD BactecTM BacT/Alert 3D
Blood culture detection system
(Becton Dickinson) (bioMérieux)

The demographic characteristics of patients affected by anaerobic bacteremia in the

present and comparator study are shown in Table 2; a male dominance (59.2%; n = 42) was
observed, while the mean age of patients was 67.1 ± 14.1 years. A total of 29.6% (n = 21) of
patients were affected by a solid tumor or a hematological malignancy (the most common
being colon adenocarcinoma, in n = 6 cases), in addition to 8.5% (n = 6) of patients who were
immunosuppressed due to other illnesses or organ transplantation. Sepsis, pneumonia,
cardiovascular illness or intervention, and pancreatitis were described in 11.3% (n = 8),
11.3% (n = 8), 11.3% (n = 8) and 4.2% (n = 3) of patients, respectively. An abscess was
identified in 15.5% of cases (n = 11, out of which, n = 10 were located in the abdomen or
the rectum), 5.6% (n = 4) had severe decubitus ulcers or gangrene, while in 4.2% (n = 3)
of patients, perforations in the large intestines were noted. Polymicrobial bloodstream
infections were observed in 12.7% (n = 9) of affected patients; Escherichia coli was the
co-pathogen in n = 3 cases, while Proteus mirabilis was the co-pathogen in n = 1 case.
A total of 74.7% (n = 53) of patients received antibiotics prior to blood culture sampling;
in instances where antimicrobials were reported, anaerobic coverage of the drugs was
appropriate in 52.1% (n = 37) of cases (i.e., the patient received antibiotics that are effective
against anaerobes). The 30-day crude mortality rate was 39.4% (n = 28) among patients
with clinically significant anaerobic bacteremia. Age ≥ 75 years was a significant predictor
of 30-day mortality (OR: 5.0; CI: 1.8–14.4; p = 0.003), while malignancy (OR: 0.5; CI: 0.2–1.5;
Antibiotics 2022, 11, 1326 4 of 10

p = 0.229), malignancy and immunosuppression (OR: 0.9; CI: 0.3–2.3; p = 0.746), lack of
anti-anaerobic coverage (OR: 0.5; CI: 0.2–1.2; p = 0.200) and female sex (OR: 1.9; CI: 0.7–4.9;
p = 0.208) did not show a significant relationship with the mortality of patients.

Table 2. Demographic characteristics of patients affected by anaerobic bacteremia in the present

study and in our previous comparator study.

Study Year Present Study Gajdács et al. [15]

Number of affected patients 71 187
Male-to-female ratio 1.45 0.60
Mean age [year ± SD] 67.1 ± 14.1 71.9 ± 16.7
Age range [years] 25–97 18–102

The detailed species distribution of anaerobic isolates in clinically relevant anaerobic

bacteremia is presented in Table 3, while a comparative representation of data with the
reference study and other literature findings is shown in Table 4. Overall, the majority of
anaerobic pathogens were from the Bacteroides/Parabacteroides spp. group (32.4%; n = 23)
and Clostridium spp. (22.5%; n = 16); the distribution among Gram-positive and Gram-
negative anaerobes was almost equal (36 vs. 35).
Anaerobic bacteria are important constituents of the normal human microbiota, in
addition to having the potential of becoming relevant pathogens when displaced into
normally sterile anatomical sites in the body [16]. These bacteria may be important etio-
logical agents in bloodstream infections and/or other severe invasive infections, although
their reported prevalence is relatively low (which may also be explained by a bias of
under-reporting) [17]. In the present study, n = 71 cases of clinically relevant anaerobic
bacteremia were identified, over a period of 5 years (2016–2020); this corresponded to
0.7% of all positive blood cultures or 1.2 isolates/1000 blood culture bottles, which is at the
lower end of the range currently reported in the literature (0.5–13%) [11,13]. Similarly to
other literature sources, Cutibacterium spp. accounted for the majority (>50%) of anaerobes
in the study period, representing over half of the isolates both here and in our previous
study [15]. In contrast to the comparator study from Szeged—which reported a 2.5-times
higher prevalence of anaerobic bacteremia between 2013 and 2017, and a pronounced fe-
male dominance in affected patients—herein, male patients were more commonly affected.
Our study population showed the characteristics of patients at risk for anaerobic blood-
stream infections [18]; most of them were elderly, and over 38% of patients were affected by
malignancy and/or immunosuppression (vs. 8% [15]). In line with our previous study (and
in line with the summary of other literature reports), Bacteroides/Parabacteroides species
(among Gram-negatives) and Clostridium spp. (among Gram-positives) were the most com-
mon clinically relevant pathogens detected (corresponding to 60–80% of the isolates, see
Table 4), while a new species (A. schaali), not previously reported in the region, has also been
described. GPAC, and Gram-positive and Gram-negative rods other than clostridia and Bac-
teroides/Parabacteroides spp., represented the minority of anaerobic isolates, which, barring a
report from Sweden [19], is in line with the literature. Similarly to previous findings from
the region, Gram-negative anaerobic cocci were uncommonly isolated, with a 2–3% share in
anaerobic bloodstream infections [20]. Although in the study from Szeged, Gram-positive
anaerobic bacteria dominated during the 5-year period (61.1% vs. 49.3% in the present
study), we were faced with the dominance of Gram-negative anaerobes (50.7% vs. 38.9% in
Szeged). One of the main differences between the two clinical centers was the isolation rate
of Fusobacterium spp.; in the present study, their rate was 9.9% (n = 7 patients), while this
was only 1.2% (n = 2 patients) in the previous study [20]. Out of these patients, four had a
malignant underlying disease (lymphoma, colon carcinoma, supraglottic squamous cell
carcinoma and carcinoma of the appendix), with three out of seven patients recovering.
Antibiotics 2022, 11, 1326 5 of 10

Table 3. Detailed species distribution of clinically relevant anaerobic isolates from anaerobic bac-
teremia in our study (2016–2020).

Study Year 2016 2017 2018 2019 2020 Overall

Number of Affected Patients 15 10 19 12 15 71 (100%)
Gram-positive, spore-forming anaerobic rods 3 3 6 0 4 16 (22.5%)
Clostridium sp. (genus level) 1 1
C. baratii 1 1
C. hathewayi (Hungatella hathewayi) 1 1
C. paraputrificum 1 1
C. perfringens 1 4 1 6
C. ramosum 1 1 1 3
Paeniclostridium sordellii 2 2
C. septicum 1 1
Gram-positive, non-spore-forming anaerobic rods 0 1 1 5 1 8 (11.3%)
Actinotignum schaali 2 2
Actinomyces naeslundii 1 1
A. neuii 1 1
A. odontolyticus (Schaalia odontolytica) 2 1 3
A. turicensis (Schaalia turicensis) 1 1
Eggerthella lenta 2 2
Lactobacillus fermentum 1 1
Weissella viridescens 1 1
Gram-positive anaerobic cocci (GPAC) 5 1 3 0 2 11 (15.5%)
Anaerococcus octavius 1 1
A. tetradius 1 1
Parvimonas micra 1 2 3
Peptinophilus harei 1 1
Peptococcus niger 2 2
Peptostreptococcus sp. (genus level) 3 3
Gram-negative anaerobic rods 7 5 8 6 8 34 (47.9%)
Bacteroides sp. (genus level) 5 1 6
B. caccae 1 1
B. fragilis 1 3 2 2 4 12
B. faecis 1 1
B. thetaiotaomicron 1 1 2
Fusobacterium sp. (genus level) 1 1
F. nucleatum 2 3 5
F. periodonticum 1 1
Parabacteroides distasonis 1 1
Prevotella sp. (genus level) 1 1
P. bivia 1 1
P. intermedia 1 1
P. melaninogenica 1 1
Gram-negative anaerobic cocci 0 0 1 1 0 2 (2.8%)
Veillonella parvula 1 1 2

Many studies have called into question the relevance of blood cultures for anaerobic
bacteria; on one hand, several papers reported a considerable and sustained decrease in
the prevalence of anaerobic bacteremia [21,22]. In addition, clinical studies have shown
that patients’ characteristics (e.g., the presence of neutropenia) were indicative of anaerobic
bacteremia, and clinical cure was achieved without the need of blood cultures, as the
source of infection (most commonly from the gastrointestinal region, the urogenital region
or the oropharynx) was obvious [23,24]. For example, De Keukeleire et al. followed the
epidemiology of anaerobic bacteremia for a 10-year period [25] and found a decreasing
trend from 2004 to 2008 and 2009–2013, with 17.3/100,000 patient days to 13.7/100,000 pa-
Antibiotics 2022, 11, 1326 6 of 10

tient days, and 1.3/1000 patients to 0.9/1000 patients, respectively. Similarly, Morris et al.
showed that in their study, the source of bacteremia was evident in >80% of cases [26].
Anaerobic bacteremia is almost invariably secondary to a focal primary infection; therefore,
the anaerobic strains recovered often depend on the portal of entry and the underlying
disease of the patients [27]. Nevertheless, both of the abovementioned points were con-
tested by other reports, highlighting that: (i) with the increasing prevalence of patients with
cancer/immunosuppression, and with the increasing use of invasive medical technologies,
a parallel increase in anaerobic bacteremia was observed [28], (ii) misinterpretation of
the infection source or clinical findings may affect outcomes [29], (iii) novel, previously
unreported pathogens are increasingly being described as clinically significant [30], and
(iv) without isolation and susceptibility testing, therapy may fail if an antibiotic-resistant
isolate is the cause of the infection [31].

Table 4. Percentage distribution of anaerobes in the two respective studies and in comparison with
the literature findings.

Percentage of Anaerobes According

Anaerobic Isolates Present Study Gajdács et al. [15]
to Literature Data [11,13]
30–80%
Cutibacterium spp. * 55.6% (n = 89) 54.0% (n = 247)
(of anaerobes isolated)
All other isolates excluding Cutibacterium spp.:
Gram-negative anaerobes 50.7% (n = 36) 38.9% (n = 69)
Bacteroides/Parabacteroides spp. 26–75% 32.4% (n = 23) 34.2% (n = 54)
Fusobacterium spp. 4–15% 9.9% (n = 7) 1.2% (n = 2)
Prevotella and Porphyromonas spp. 0.5–10% 5.6% (n = 4) 1.2% (n = 2)
Veillonella spp. 0.5–2% 2.8% (n = 2) 2.3% (n = 4)
Gram-positive anaerobes 49.3% (n = 35) 61.1% (n = 107)
Clostridium spp. 8–46% 22.5% (n = 16) 33.3% (n = 59)
Gram-positive anaerobic cocci (GPAC) 8–20% 15.5% (n = 11) 12.0% (n = 21)
Gram-positive non-spore-forming rods
0.5–14% 11.3% (n = 8) 15.8% (n = 27)
(excluding: Cutibacterium spp.)
* Usually not considered clinically relevant (contaminants).

The 30-day crude mortality rate for our patient sample was 39.44%; advanced age was a
risk factor for mortality, while no significant associations were shown with the other studied
correlates. These findings were similar to the study of Kim et al., where cardiovascular
disease was a main risk factor for mortality [32], and Blairon et al., where overall mortality
was 13%, and the fatal outcome was mainly influenced by severe underlying diseases,
but not by antimicrobial coverage or the causative pathogen [33]. On the other hand,
the study of Salonen et al. highlighted the importance of appropriate anti-anaerobic
coverage in anaerobic bacteremia; in their study, only 50% of patients received correct
antibiotics initially, while the mortality rates among patients with appropriate therapy
(or therapy that had been changed after culture results) vs. the ones without coverage
for anaerobes were 18%/17% vs. 55%, respectively [29]. In the study of Ramos et al.,
mortality attributable to anaerobic bacteremia was 32.0%, where septic shock, kidney
failure, failure to perform drainage and inappropriate antimicrobial therapy were the
principal risk factors for mortality [34]. Finally, in a recent publication by Cobo et al.,
Bacteroides (43.9%), Clostridium (24.1%) species and GPAC (15.6%) were among the most
common pathogens in anaerobic bacteremia, with an attributable mortality rate of 20%.
In addition, their study highlighted the considerable increase in nonsusceptibility rates
of anaerobes against commonly used “anti-anaerobic” antimicrobials [35]. Some key
limitations need to be addressed: (i) the retrospective, single-center study design, leading
to a relatively low number of clinically relevant infections to be analyzed; (ii) selection bias,
as the study population originated from a tertiary care center, corresponding to patients
with more severe conditions or underlying illnesses; (iii) the lack of phenotypic antibiotic
Antibiotics 2022, 11, 1326 7 of 10

susceptibility data or genetic characterization for the respective isolates; and (iv) the lack of
the patients’ laboratory results (e.g., white blood cell count, coagulation parameters, liver
enzymes, inflammatory markers or other biomarkers) included in the analysis.

3. Conclusions
Despite their relatively low prevalence, anaerobic bacteria may be important causative
agents of severe infections in patients with characteristic underlying conditions, which are
often associated with a high mortality rate. Based on these known facts, it would be ex-
tremely important to review, analyze and compare the trends in the occurrence of anaerobic
bacteremia locally. Overall, early recognition of the role played by anaerobes in sepsis and
timely initiation of adequate, effective antimicrobial treatment, recognition of the source of
infection and proper control have proven efficient in reducing the mortality of patients af-
fected by anaerobic bacteremia. The increasing awareness of clinicians regarding anaerobes
may be partly due to the emergence of effective microbial identification methods—such as
matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF
MS)—which have allowed the identification of these pathogens in a clinically relevant time
frame. MALDI-TOF MS should replace all other identification techniques for the routine
identification of anaerobes in clinical microbiology laboratories. Developments such as
MALDI-TOF MS—in addition to the introduction of 16S rRNA sequencing platforms, as
a relevant identification method in routine laboratories—will ensure timely and accurate
diagnostics for patients affected by anaerobes, and may also allow for the rapid detection of
resistance mechanisms. The cooperation of microbiology laboratories with the clinical sus-
picion of the treating physicians will safeguard the appropriate selection of antimicrobials
for these infections.

4. Materials and Methods

4.1. Study Design, Collection of Data
The present retrospective observational study was carried out on the basis of mi-
crobiological and clinical patient data, collected corresponding to the 5-year time period
between 1 January 2016 and 31 December 2020. The clinical records of all patients with
positive BCs in the study period were reviewed retrospectively. During the study period,
the Department of Medical Microbiology and Immunology provided routine diagnostic
microbiological services at the University of Pécs (PTE) Clinical Centre (CC). The CC is a
1725-bed tertiary care university teaching hospital in the Southern Transdanubia region
of Hungary, serving as a primary facility to more than 800,000 inhabitants (~17% of the
population is aged 65 years or older), according to the National Health Insurance Fund
data for 2020 [36,37]. The CC maintains four adult intensive care units (ICUs; cardiology,
hematology, trauma and surgical) and two pediatric ICUs (pediatric and neonatal), with
different profiles.
Microbiological culture results for anaerobic bacteremia for adult patients (≥18 years)
were collected via an electronic search of the laboratory information system (LIS) of the
Department of Medical Microbiology and Immunology, related to the 5-year study period.
Data were collected for samples originating from inpatient departments and the emergency
department, while outpatient clinics were excluded. According to the case definition used
in the literature, isolates were considered separate if they occurred more than 14 days apart,
otherwise they were excluded [19]. Data were collected regarding the age, sex, anamnes-
tic data (e.g., underlying conditions, presence of known immunosuppression or cancer,
presence of a known abscess or focal infection), previous antimicrobial therapy and the
30-day crude mortality rate of the affected patients; data on turnaround times (TAT) were
also noted in each case. Although Cutibacterium spp. isolates are not usually considered as
causative agents of bacteremia, their relevance was ascertained based on clinical patient
data, according to the criteria described previously [15]. If Cutibacterium spp. was not
considered clinically relevant, clinical data corresponding to those cases were excluded.
Antibiotics 2022, 11, 1326 8 of 10

4.2. Sample Processing, Microbiological Identification

Blood culture sampling was performed at the request of the attending physicians, who
were responsible for the principal decisions concerning the diagnosis and treatment of
patients. Blood culture processing was carried out according to national and international
recommendations [38,39]. Clinicians routinely used parallel aerobic and anaerobic blood
culture bottles in pairs, with most blood cultures ordered as two sets, with one aerobic and
one anaerobic bottle per set. Blood cultures were analyzed in the laboratory by the BD
BactecTM automated system (Becton Dickinson, Franklin Lakes, NJ, USA), following inocu-
lation of 5–10 mL of blood into aerobic and anaerobic bottles, respectively. Incubation of
the blood culture bottles was performed for 7 days (21 days, if endocarditis was suspected)
with constant shaking, according to the manufacturer’s instructions.
Samples from positive bottles were plated onto Schaedler agar (bioMérieux, Marcy
l’Etoile, France) containing 5% v/v horse blood, haemin and Vitamin K1 to isolate anaer-
obic bacteria; plates were incubated in an anaerobic environment in an atmosphere of
90% N2 , 5% H2 and 5% CO2 , with the aid on an anaerobic chamber (Concept 400 anaer-
obic incubator, Biotrace International Plc., UK). The incubation time generally lasted for
2–5 days at 37 ◦ C [40]. Identification of the isolates was carried out with matrix-assisted
laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), using a
microFlex LT Biotyper (Bruker Daltonics, Bremen, Germany), the MALDI Biotyper RTC
3.1 software (Bruker Daltonics, Germany) and the MALDI Biotyper Library 3.1 for spectra
analysis. To facilitate successful identification, a formic acid extraction step was included
before the measurements. The sample preparation methodology and the technical details
of MS measurements were described elsewhere [41]. Genus-level identification was con-
sidered reliable for log(score) ≥ 1.7, while the value for species-level identification was
log(score) ≥ 2.0.

4.3. Statistical and Comparative Analysis

Descriptive statistical analysis (including means with ranges and percentages to
characterize the data) was performed using Microsoft Excel 2013 (Microsoft Corp. Red-
mond, WA, USA). The relationships between the 30-day crude mortality rate and other
relevant study correlates were expressed as odds ratios (OR ± 95% confidence interval
[CI]), which were calculated by SPSS software version 22 (IBM Corp., Endicott, NY, USA).
p values < 0.05 were considered statistically significant.
For the sake of comparison, and to present the results in a national context, the
findings of this study were directly compared to a similar retrospective epidemiological
study regarding anaerobic bacteremia, performed by the authors in the Southern Great
Plain of Hungary (at the Albert Szent-Györgyi Clinical Center, University of Szeged) [15].
The bases of the comparison are: the same study design and duration (2013–2017), similar
demographic characteristics of the population, similar tertiary care university hospital
setting, the same guidelines used for sampling and the same instrumentation used for
microbiological identification.

4.4. Ethical Considerations

The study was conducted in accordance with the Declaration of Helsinki, and national
and institutional ethical standards. Ethical approval for the study protocol was obtained
from the Human Institutional and Regional Committee for Research Ethics, University of
Pécs (registration number: KK-164-2/2021).

Author Contributions: E.U. and M.G. conceived and designed the study. K.K., A.N., Z.L., G.M. and
Á.S. were the senior microbiologists and performed the identification of the bacterial isolates during
the study period. K.K. and Á.S. performed microbiological and clinical data collection. M.G. and E.U.
performed the primary analysis, wrote the initial draft, and revised the full paper. K.K., A.N., Z.L.,
G. M. and Á.S. wrote and revised the full paper. All authors have read and agreed to the published
version of the manuscript.
Antibiotics 2022, 11, 1326 9 of 10

Funding: M.G. was supported by the János Bolyai Research Scholarship (BO/00144/20/5) of the
Hungarian Academy of Sciences. The research was supported by the ÚNKP-22-5-SZTE-107 New
National Excellence Program of the Ministry for Innovation and Technology from the source of the
National Research, Development and Innovation Fund. M.G. would also like to acknowledge the
support of ESCMID’s “30 under 30” Award. The APC was kindly funded by the Antibiotics Editorial
Office (MDPI).
Institutional Review Board Statement: The study was conducted in accordance with the Declaration
of Helsinki, and national and institutional ethical standards. Ethical approval for the study protocol
was obtained from the Human Institutional and Regional Committee for Research Ethics, University
of Pécs (registration number: KK-164-2/2021).
Informed Consent Statement: Informed consent was not obtained as anonymity was maintained
during data collection and due to the retrospective nature of the study.
Data Availability Statement: All data generated during the study are presented in this paper.
Acknowledgments: The authors would like to acknowledge the staff of the Department of Medical
Microbiology and Immunology, University of Pécs Medical School.
Conflicts of Interest: The authors declare no conflict of interest, monetary or otherwise. The authors
alone are responsible for the content and writing of this article.

References
1. Finegold, S.M. Overview of clinically important anaerobes. Clin. Infect. Dis. 1995, 20, S205–S207. [CrossRef] [PubMed]
2. Finegold, S.M. Anaerobic Infections: General Concepts. In Principles and Practice of Infectious Diseases; Mandell, G.L., Bennett, J.E.,
Dolin, R., Eds.; Churchill Livingstone: London, UK, 2000; Volume 2.
3. La Scola, B.; Fournier, P.E.; Raoult, D. Burden of emerging anaerobes in the MALDI-TOF and 16S rRNA gene sequencing era.
Anaerobe 2011, 17, 106–112. [CrossRef] [PubMed]
4. Hecht, D.W. Anaerobes: Antibiotic resistance, clinical significance and the role of susceptibility testing. Anaerobe 2006, 12, 115–121.
[CrossRef] [PubMed]
5. Cobo, F.; Guillot, V.; Navarro-Marí, J.M. Breast Abscesses Caused by Anaerobic Microorganisms: Clinical and Microbiological
Characteristics. Antibiotics 2020, 9, e341. [CrossRef] [PubMed]
6. Woerther, P.L.; d’Humiéres, C.; Lescure, X.; Dubreuil, L.; Rodriguez, C.; Barbier, F.; Fihmann, V.; Ruppé, E. Is the term “anti-
anaerobic” still relevant? Int. J. Infect. Dis. 2021, 102, 178–180. [CrossRef] [PubMed]
7. Nagy, E.; Urban, E.; Nord, C.E.; ESCMID Study Group on Antimicrobial Resistance in Anaerobic Bacteria. Antimicrobial
susceptibility of Bacteroides fragilis group isolates in Europe: 20 years of experience. Clin. Microbiol. Infect. 2011, 17, 371–379.
[CrossRef]
8. Brook, I. Antimicrobial treatment of anaerobic infections. Expert Opin. Pharmacother. 2011, 12, 1691–1707. [CrossRef]
9. Nagy, E.; Boyanova, L.; Justesen, U.S. How to isolate, identify and determine antimicrobial susceptibility of anaerobic bacteria in
routine laboratories? Clin. Microbiol. Infect. 2018, 24, 1139–1148. [CrossRef]
10. Shafiq, N.; Kumar, M.P.; Gautam, V.; Negi, H.; Roat, R.; Malhotra, S.; Ray, P.; Agarwal, R.; Bhalla, A.; Sharma, N.; et al. Antibiotic
stewardship in a tertiary care hospital of a developing country: Establishment of a system and its application in a unit—GASP
Initiative. Infection 2016, 44, 651–659. [CrossRef]
11. Gajdács, M.; Urbán, E. Relevance of anaerobic bacteremia in adult patients: A never-ending story? Eur. J. Microbiol. Immunol.
2020, 10, 64–75. [CrossRef]
12. Grohs, P.; Mainardi, J.L.; Podglajen, I.; Hanras, X.; Eckert, C.; Buu-Hoi, A.; Varon, E.; Gutmann, L. Relevance of Routine Use of the
Anaerobic Blood Culture Bottle. J. Clin. Microbiol. 2007, 45, 2711–2715. [CrossRef] [PubMed]
13. Brook, I. The role of anaerobic bacteria in bacteremia. Anaerobe 2010, 16, 183–189. [CrossRef] [PubMed]
14. Vena, A.; Munoz, P.; Alcala, L.; Fernandez-Cruz, A.; Sanchez, C.; Valerio, M.; Bouza, E. Are incidence and epidemiology of
anaerobic bacteremia really changing? Eur. J. Clin. Microbiol. Infect. Dis. 2015, 34, 1621–1629. [CrossRef] [PubMed]
15. Gajdács, M.; Ábrók, M.; Lázár, A.; Terhes, G.; Urbán, E. Anaerobic blood culture positivity at a University Hospital in Hungary: A
5-year comparative retrospective study. Anaerobe 2020, 63, e102200. [CrossRef]
16. Shenoy, P.A.; Vishwanath, S.; Gawda, A.; Shetty, S.; Anegundi, R.; Varma, M.; Mukhopadhyay, C.; Chawla, K.J. Anaerobic bacteria
in clinical specimens–frequent, but a neglected lot: A five year experience at a tertiary care hospital. Clin. Diagn. Res. 2017, 11,
DC44–DC48.
17. Garg, R.; Kaistha, N.; Gupta, V.; Chander, J. Isolation, identification and antimicrobial susceptibility of anaerobic bacteria: A study
reemphasizing its role. J. Clin. Diag. Res. 2014, 8, DL01–DL02. [CrossRef]
18. Wilson, J.R.; Limaye, A.P. Risk Factors For Mortality In Patients With Anaerobic Bacteremia. Eur. J. Clin. Microbiol. Infect. Dis.
2004, 23, 310–316.
Antibiotics 2022, 11, 1326 10 of 10

19. Badri, M.; Nilson, B.; Ragnarsson, S.; Senneby, E.; Rasmussen, M. Clinical and microbiological features of bacteraemia with
Gram-positive anaerobic cocci: A population-based retrospective study. Clin. Microbiol. Infect. 2019, 25, 760.e1–760.e6. [CrossRef]
20. Gajdács, M.; Urbán, E. Epidemiology and species distribution of anaerobic Gram-negative cocci: A 10-year retrospective survey
(2008-2017). Acta Pharm. Hung. 2019, 89, 84–87. [CrossRef]
21. Goldstein, E.J. Anaerobic bacteremia. Clin. Infect. Dis. 1996, 23, S97–S101. [CrossRef]
22. Gransden, W.R.; Eykyn, S.J.; Phillips, I. Anaerobic bacteremia: Declining rate over a 15-year period. Rev. Infect. Dis. 1991, 13,
1255–1256. [CrossRef] [PubMed]
23. Bartlett, J.G.; Dick, J. The controversy regarding routine anaerobic blood cultures. Am. J. Med. 2000, 108, 505–506. [CrossRef]
24. Fenner, F.; Widmer, A.D.; Straub, C.; Frei, R. Is the incidence of anaerobic bacteremia decreasing? Analysis of 114,000 blood
cultures over a ten-year period. J. Clin. Microbiol. 2008, 46, 2432–2434. [CrossRef] [PubMed]
25. De Keukeleire, S.; Wybo, I.; Naessens, A.; Echahidi, F.; Van der Beken, M.; Vandoorslaer, K. Anaerobic bacteremia: A 10-year
retrospective epidemiological survey. Anaerobe 2016, 39, 54–59. [CrossRef]
26. Morris, A.J.; Wilson, M.L.; Mirrett, S.; Reller, B.L. Rationale for selective use of anaerobic blood cultures. J. Clin. Microbiol. 1991,
31, 2110–2113. [CrossRef]
27. Strohaker, J.; Bareiss, S.; Nadalin, S.; Königsrainer, A.; Ladurner, R.; Meier, A. The Prevalence and Clinical Significance of
Anaerobic Bacteria in Major Liver Resection. Antibiotics 2021, 10, e139. [CrossRef]
28. Giorgio, A.; Merola, M.G.; Montesarchio, L.; Merola, F.; Gatti, P.; Coppola, C.; Calisti, G. Percutaneous radiofrequency ablation of
hepatocellular carcinoma in cirrhosis: Analysis of complications in a single centre over 20 years. Br. J. Radiol. 2017, 90, e20160804.
[CrossRef]
29. Salonen, J.H.; Eerola, E.; Meurman, O. Clinical significance and outcome of anaerobic bacteremia. Clin. Infect. Dis. 1998, 26,
1413–1417. [CrossRef]
30. Boiten, K.E.; Jean-Pierre, H.; Veloo, A.C.M. Assessing the clinical relevance of Fenollaria massiliensis in human infections, using
MALDI-TOF MS. Anaerobe 2018, 54, 240–245. [CrossRef]
31. Boyanova, L.; Kolarov, R.; Mitov, I. Recent evolution of antibiotic resistance in the anaerobes as compared to previous decades.
Anaerobe 2015, 31, 4–10. [CrossRef]
32. Kim, J.; Lee, Y.; Park, Y.; Kim, M.; Choi, J.Y.; Yong, D. Anaerobic bacteremia: Impact of inappropriate therapy on mortality. Infect.
Chemother. 2016, 48, 91–98. [CrossRef] [PubMed]
33. Blairon, L.; De Gheldre, Y.; Delaere, B.; Sonet, A.; Bosly, A.; Glupczynski, Y. A 62-month retrospective epidemiological survey of
anaerobic bacteremia in a university hospital. Clin. Microbiol. Infect. 2006, 12, 527–532. [CrossRef] [PubMed]
34. Ramos, J.M.; García-Corbeira, P.; Fernández-Roblas, R.; Soriano, F. Bacteremia caused by anaerobes: Analysis of 131 episodes.
Enferm. Infec. Microbiol. Clin. 1994, 12, 9–16.
35. Cobo, F.; Boorego, J.; Gómez, E.; Casanovas, I.; Calatrava, E.; Foronda, C.; Navarro-Marí, J.M. Clinical Findings and Antimicrobial
Susceptibility of Anaerobic Bacteria Isolated in Bloodstream Infections. Antibiotics 2020, 9, e345. [CrossRef] [PubMed]
36. Hospital Bed Count and Patient Turnover Report. National Health Insurance Fund of Hungary. Available online:
http://www.neak.gov.hu/felso_menu/szakmai_oldalak/publikus_forgalmi_adatok/gyogyito_megelozo_forgalmi_adat/
fekvobeteg_szakellatas/korhazi_agyszam.html (accessed on 16 July 2022).
37. Hungarian Central Statistical Office. Regional Statistical Yearbook of Hungary; Demográfiai Évkönyv; Központi Statisztikai Hivatal:
Budapest, Hungary, 2020. (In Hungarian)
38. Hungarian Professional Committee of Medical Microbiology: Guidelines for the Microbiological Diagnosis of Bloodstream Infec-
tions. [Orvosi Mikrobiológiai Szakmai Kollégium. Módszertani Ajánlás: A Véráram Infekciók Mikrobiológiai Diagnosztikájára].
Available online: http://infektologia.hu/upload/infektologia/document/hemokultura_modszertani_ajanlas_2018_december.
pdf?web_id= (accessed on 16 July 2022). (In Hungarian)
39. Opota, O.; Croxatto, A.; Prod’hom, G.; Greub, G. Blood culture-based diagnosis of bacteraemia: State of the art. Clin. Microbiol.
Infect. 2015, 21, 313–322. [CrossRef] [PubMed]
40. Jousimies-Somer, H.; Summanen, P.; Citron, D.M.; Baron, E.J.; Wexler, H.M.; Finegold, S.M. (Eds.) Wadsworth-KTL Anaerobic
Bacteriology Manual, 6th ed.; Star Publishing Company: Belmont, CA, USA, 2003.
41. Urbán, E.; Gajdács, M.; Torkos, A. The incidence of anaerobic bacteria in adult patients with chronic sinusitis: A prospective,
single-centre microbiological study. Eur. J. Microbiol. Immunol. 2020, 10, 107–114. [CrossRef]