A novel non-invasive biomarker based on oral

microbiome dysbiosis for detection of Community-

Acquired Pneumonia
Ni Sun 
Jinan University
Xuhan Zhang 
Jinan University
Yating Hou 
Jinan University
Ting Zhong 
(

tingzhong2020@163.com
)
Jinan University

Research Article

Keywords: Oral microbiome, Biomarker, Community-acquired pneumonia, 16S rRNA

Posted Date: November 9th, 2022

DOI: https://doi.org/10.21203/rs.3.rs-2217588/v1

License:


This work is licensed under a Creative Commons Attribution 4.0 International
License.
 
Read Full License

Page 1/17
Abstract
Background: Early diagnosis of pathogenic bacteria is crucial for the treatment of community-acquired
pneumonia (CAP), but conventional diagnostics are limited by sampling difficulties. Oral microbiota has
also been explored as a noninvasive biomarker of lung diseases, but it’s role in CAP has been neglected.
We aimed to investigate whether the oral bacteria can be novel non-invasive biomarkers for CAP.

Methods: Oral swab samples were collected from 29 patients with CAP and 26 healthy volunteers and
characterized based on clinical parameters and 16S rRNA profiling of oral bacteria. A predict functional
profiling was performed for the functional and metabolic changes in oral microbial communities.

Results: Oral microbial of patients with CAP had a lower diversity than healthy group. And the dominant
bacteria were Streptococcus, Prevotella and­Neisseria in CAP.  Higher abundance of­Prevotella
(particularly Prevotella_melaninogenica), ­Veillonella and ­Campylobacter, and lower abundance of
Neisseriaand ­Fusobacterium were detected in CAP group. Analysis of the functional potential of oral
microbiota demonstrated that the pathway involving infectious disease was overrepresented in the CAP
groups relative to that in the healthy controls.

Conclusions: Oral microbial dysbiosis was found in patients with CAP, supporting the use of this non-
invasive specimen for biomarkers of CAP.

Highlights
Oral microbial diversity was significantly lower in community-acquired pneumonia (CAP) patients
than healthy controls.
Genera Neisseria and Fusobacterium were decreased, while genera Prevotella, Veillonella and
Campylobacter were increased in CAP versus healthy controls.
Oral microbiota-based biomarkers can serve as a promising non-invasive tool for the detection of
CAP.

1. Introduction
Community-acquired pneumonia (CAP) is the leading cause of death among infectious diseases and the
third leading cause of death worldwide [1–3]. As we know, the most common pathogens of CAP are
Streptococcus pneumoniae, Haemophilus influenzae and Staphylococcus aureus, or viruses such as
influenza viruses [4]. Up to now, the cornerstone of treatment and management for patients with
pneumonia is empirical antibiotic therapy [5]. However, with the extensive use of antibiotics, microbiome
dysbiosis and antibiotic resistance has brought a huge clinical and economic burden to CAP [6].

Conventional diagnostics of pathogen and resistance determination still rely on culture-based methods.
These techniques have certain limitations, leaving correct initial antimicrobial therapy to chance [7, 8].
The main microbial assessment techniques are sputum culture, blood culture, invasive technique, and
Page 2/17
urine antigen detection [5]. However, these microbial assessment methods may be limited by sampling
difficulties, delayed results, and difficulties in interpretation [7, 9, 10]. There is a need for improved
microbiological diagnostic techniques for CAP to optimize future treatment choices.

Up to now, accumulating evidence shows that oral cavity is the site of the primary source community of
lung microbiota (acquired via micro aspiration and inhalation) [11, 12]. Kentaro et al. observed
associations between variations of oral microbiota in patients with CAP and aspiration risks [13]. Jankl et
al. identified the role of poor oral health and oral microbiome dysbiosis in the development of pneumonia
and exacerbation of pneumonia-related complications [9]. Recently, the oral microbiota has also been
explored as a biomarker of lung disease. The variation in oropharyngeal microbiome of COVID-19
patients may be used as a noninvasive biomarker for dysbiosis of the pulmonary microbiome or for
invasion of potential pathogens in the lungs [14].

The mouths of inpatients with CAP were more frequently colonized by respiratory pathogens [12]. Oral
microbial diversity may be used to develop microbiome-based biomarkers, which may have potential
implications for the prevention, early diagnosis, and treatment of diseases such as pneumonia [15]. But
oral factors have been relatively neglected in the etiology and diagnosis of CAP [12]. Little is known about
the characteristics of the oral microbiota in patients with CAP and whether it can be used as a marker for
early diagnosis.

Here, we used 16S rRNA gene sequencing to explore the typical profile oral bacteria in patients with CAP
and found the noninvasive biomarkers for CAP.

2. Methods
2.1 Study design and sample collection
Twenty-nine consecutive CAP patients in the First Affiliated Hospital, Jinan University were enrolled in this
study. CAP was defined according to the Infectious Diseases Society of America (IDSA) / American
Thoracic Society (ATS) guidelines for diagnosing CAP in adults [16]. This study was approved by the
Human and Animal Ethics Review Committee of Jinan University, China (JNUKY-2022-002). Written
informed consent was obtained from either the patients or their guardians. Finally, 55 eligible cases
including 29 patients with CAP and 26 matched healthy family members were included in this study
according to the recruitment process. Oral samples were collected from participants by rubbing the
insides of both cheeks, palate, tongue, and teeth with a swab as described previously [9, 17]. The swab
containing the specimen was placed in a sterile tube and then stored at -80°C until further analysis. The
blank control sample was obtained by opening the sterile tube and placing it in the sample collection
environment for 5 minutes. The following basic information was collected: age, sex, BMI, underlying
diseases, clinical manifestations, and oral health status.

Inclusion criteria of patients:

Page 3/17
1) Patients with clinical and imaging diagnosis of CAP.

2) Age 18–80, with independent reading and writing skills.

3) More than 20 natural teeth.

4) Sign informed consent and voluntarily join the experiment.

Exclusion criteria of patients:

1) Suffering from serious cardiovascular diseases, tumors and other diseases that cannot be controlled
by drugs, etc.

2) Patients with difficulty in completing questionnaire or oral examination.

2.2 DNA extraction for microbiome analysis

Genomic DNA from oral swab samples was extracted using CTAB [18]. DNA integrity and size were
verified by 1.0% agarose gel electrophoresis, and DNA concentrations were determined using the
spectrophotometer.

2.3 High throughput 16S ribosomal RNA gene sequencing

16S ribosomal RNA (rRNA) gene amplification was performed using the primers (515F:
GTGCCAGCMGCCGCGGTAA; 806R: GGACTACHVGGGTWTCTAAT)directionally targeting the V3 and V4
hypervariable regions of the 16S rRNA gene. To differentiate each sample and yield accurate
phylogenetic and taxonomic information, the gene products were attached with forward and reverse error-
correcting barcodes. The amplicons were quantified after purification. Then, the normalized equimolar
concentrations of each amplicon were pooled and sequenced on the NovaSeq6000 sequencing
instrument.

2.4 Sequencing data analysis

QIIME 2 software was used to conduct all diversity analyses of the sequenced microbial communities. Its
key plug-in is DATA 2 [19]. Different from the traditional OTU-based analysis methods, DADA2 method is
mainly used for noise reduction. It does not cluster with similarity but only with dereplication, which is
equivalent to clustering with 100% similarity, resulting in ASVs (Amplicon sequence variants).

The Shannon index was used to assess alpha diversity; whereas weighted and unweighted UniFrac
distance matrices were used to assess beta diversity, which was visualized through a principal coordinate
analysis (PCoA). Beta diversity dissimilarity between groups was tested via an analysis of similarity
(ANOSIM) with the R (v 3.4.1) vegan package. Bacterial taxa that differed significantly in abundance of
oral microbiome between the CAP group and control group (the core shared oral bacteria) were identified
via a linear discriminant analysis (LDA) effect size (LEfSe) approach [20]. Significant enrichment was
defined as an LDA > 3 and a p-value < 0.05.

Page 4/17
2.5 Predictive functional profiling
To study the functional and metabolic changes in oral microbial communities, all ASVs were aligned into
the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) built-in
reference database [21].

2.6 Statistical analysis

The SPSS software package (version 21) was used. Associations between the clinical characteristics
were performed by Pearson's Chi-square test or Fisher's exact test. P < 0.05 was considered significant.

3. Results
3.1 Participant information 

In total, 55 eligible cases including 26 healthy family numbers, 29 patients with CAP were included in this
study according to the recruitment process. The basic conditions of the two groups were in good
agreement. There were no significant differences in the basic information, oral hygiene habits and oral
health status between the two groups. Detailed clinical data for the studied individuals were shown in
Table 1.

Table 1

Clinical characteristics of the enrolled participants

Page 5/17
 Variables CAP group Control group P value

(n=29) (n=26)

age, years, mean ± SD 56.17±15.4 48.12±14.7 0.052

BMI, mean ± SD 21.84±4.2 21.11±4.2 0.525

Gender, male, n (%) 17（58.6） 9（34.6） 0.078

Environmental pollution, n (%) 9（31.0） 4（15.4） 0.173

Hypertension, n (%) 9（31.0） 6（23.1） 0.517

Smoke      

Current smokers, n (%) 5（17.2） 3（11.5） 0.155

Former smokers, n (%) 6（20.7） 1（3.8）

Never smokers, n (%) 18（62.1） 22（84.6）

Oral hygiene habits      

Teeth scaling within the past year, n (%) 3（10.3） 6（23.1） 0.219

Brush your teeth, n (%) 28（96.6） 26（100.0） 0.348

Use a toothpick, n (%) 22（75.9） 14（53.8） 0.093

Use dental floss, n (%) 7（24.1） 10（38.5） 0.263

Oral health status      

Bleeding gums, n (%) 12（41.4） 13（50.0) 0.530

Food impaction, n (%) 21（72.4） 22 (84.6) 0.277

Halitosis, n (%) 18（62.1） 14 (53.8) 0.546

Tooth sensitivity, n (%) 14（48.3） 11 (42.3) 0.664

Toothache, n (%) 12（41.4） 10（38.5） 0.829

Definition of abbreviations: SD standard deviation, CAP community-acquired pneumonia

3.2 Bacterial diversity of oral microbiota was significantly lower in patients with CAP 

The oral microbiota was assessed using 16S rRNA sequencing. A total of 3,762,330 high-quality 16S
rRNA gene sequences were identified, with a median read count of 68,141 (ranging from 60,123 to
76,354) per sample. After the taxonomic assignment, 4796 ASVs were obtained (Table S1). The species
accumulation curve of all samples basically approached the asymptote, supporting the adequacy of our
sampling efforts (Figure 1A). Likewise, pielou’s evenness was evaluated by rarefaction curve, exhibiting
similar patterns in all samples (Figure 1B), which reflected the evenness of the distribution of species in

Page 6/17
the microbial community. Alpha diversity indexes were calculated to assess the differences in bacterial
diversity between the two groups (Table S2). The results showed that oral microbial alpha diversity was
significantly lower in the CAP group than in the healthy controls (Figure 1C-D). Moreover, the Venn
diagram showed that 1832 of the total 4796 ASVs were shared between the two groups. Notably, 1080
ASVs were unique for the CAP group (Figure 1E). To display the microbiome space between samples,
beta diversity was calculated using the weighted UniFrac method and the principal coordinate analysis
(PCoA) was performed. The results showed a gradually separated distribution of the oral microbial
communities between these two groups (Figure 1F). 

3.3 Oral microbial composition is different between people with CAP and healthy controls

The average composition of bacterial communities at the phylum and genus levels was shown in Figures
2A&2B, respectively Proteobacteria, Firmicutes, Bacteroidota were the three dominant bacterial phyla in
the two groups (Table S3). At the phylum level, Proteobacteria was significantly lower in the CAP group
than in the control group (Figure 2A). At the genus level, the dominant bacteria in the two groups were ­-
Neisseria and Streptococcus. Compared with the control group, Neisseria was significantly decreased,
and­Prevotella was significantly increased (Figure 2B, Table S4). The top 5 bacterial genera contributing
to the difference between the two groups were Neisseria, Streptococcus, Haemophilus,­Prevotella,
Fusobacterium (Figure 2C-D, Table S5). At the genera level,­Prevotella, ­Veillonella, Campylobacter were
significantly enriched in the CAP group compared with the control group. And ­Neisseria, Fusobacterium
were significantly reduced in the CAP group than in the control group ( P＜0.05 )( Figure 2E, Table S6). At
the species level,­Prevotella_melaninogenica, Campylobacter_concisus were significantly enriched in the
CAP group compared with the control group.  And ­Fusobacterium_periodonticum,­Prevotella_nanceiensis,
Haemophilus_parainfluenzae were significantly lower in the CAP group than in the control group ( P＜0.05
)( (Figure 2F, Table S7).

3.4 Oral bacteria are associated with infectious disease in people with CAP 

To study the functional and metabolic changes in oral microbial communities, all ASVs were aligned into
the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) built-in
reference database [15, 20]. A principal component analysis was then performed using the total KEGG
pathways data generated from all samples (Figure 3A). PICRUSt analysis identified 11 KEGG pathways
with significant differential abundance between the CAP and control group (Figure 3B). As shown in
Figure 3B, the pathway involving infectious disease was overrepresented in the CAP groups compared
with the control group. Moreover, the pathways involving cell motility and energy metabolism were
overrepresented in the CAP groups compared with the control group (figure 3C). The association between
differential bacteria and metabolic pathways was investigated using correlation heatmaps. As the results
showed, genera ­Veillonella,­Prevotella and Campylobacter were positively correlated with infectious
disease, while genera Neisseria and Fusobacterium were negatively correlated with the infectious
diseases (Figure 3C-D).

Page 7/17
4. Discussion
Management of CAP focuses particularly on early administration of the appropriate antimicrobial agent
[22]. Current diagnostic methods may be limited by sampling difficulties, delayed results, and difficulties
in interpretation [5]. Our research used the oral flora as the diagnostic factor of CAP, which effectively
solves the problem of sampling difficulties. Our data discovered the significantly higher abundance of
Prevotella, Veillonella and Campylobacter, and lower abundance of Neisseria and Fusobacterium than
healthy group, which can be used as biomarkers in diagnosis of CAP.

The baselines of our study population were consistent. Specifically, the basic information and oral health
status were not statistically different. We recruited the accompanying family members of the patients as
a control group, and they could maintain good consistency in terms of living habits, eating habits,
environment, socioeconomic conditions, etc. This can greatly reduce the interference of confounding
factors on the oral flora and ensure the accuracy of the results.

The oral bacteria composition of patients with COPD also found the loss of microbiota diversity [23]. We
also found that CAP group had a lower diversity than healthy controls. Due to risk factors such as
increased inhalation and mechanical ventilation under abnormal conditions, oral microorganisms may
transfer to the respiratory tract and aggravate respiratory disease [24, 25].

Prevotella is consistently among the core bacterial communities of the respiratory tract [26, 27], which is
known to be associated with pneumonia [14, 28, 29]. Research found that the periodontopathic bacterial
genera Prevotella were increased in the BALF of patients with CAP [30]. We discovered an enrichment of
Prevotella, especially Prevotella_melaninogenica in oral cavity of patients with CAP. It suggests that the
mouth may be a reservoir of bacteria for the lungs. In a mouse lung co-infection model, the increased
abundance of Prevotella melaninogenica was found, which probably was a potentially beneficial role to
reduce infection [31]. The above suggests that there was consistency in the changes of oral and
pulmonary bacteria (Prevotella and Prevotella_melaninogenica) under lung inflammation.

We also observed that the abundance of Veillonella and Campylobacter were significantly higher in the
CAP group. Few studies have focused on their relationship with CAP. But in a lung cancer model, lower
airway dysbiosis with Veillonella led to decreased survival time and increased tumor burden [32].
Skallevold et al. found that Veillonella and Streptococcus in saliva could be valuable markers with
diagnostic and prognostic potentials in lung cancer [33]. On the other hand, in COPD, asthma, and cystic
fibrosis (CF) lung diseases, oral microbiota (e.g. Prevotella species, Veillonella species) were present in
the airways and associated with increased host inflammatory response [26–28]. Additionally, Veillonella
was identified as the most prominent biomarker for COVID-19 group sequencing analyses of oropharynx
swab specimens [29, 34]. Therefore, the potential of Veillonella and Campylobacter as a diagnostic
biomarker for lung disease is proposed.

In control group, Neisseria and Streptococcus were the core genus of oral flora. The abundance of
Neisseria and Fusobacterium was distinctly lower in CAP group than control group. Similarly, the levels of
Page 8/17
Neisseria in the pharynx were significantly lower in COPD patients compared to healthy people [35].
Lower lung function, COPD diagnosis, and greater symptoms were recognized an association positively
with Neisseria in lung microbiota [36]. LaMotte et al. reported that oral Neisseria spp. could be causative
bacteria in ventilator-associated pneumonia (VAP) patients [37]. Neisseria is possibly an infection-
promoting factor for lung disease. There are very few studies on the relationship between Neisseria and
CAP, which is in urgent need of illustration.

Fusobacterium, recognized as a periodontal pathogen, is considered as a biomarker of lung function

deterioration of COPD patients coinfected with Pseudomonas aeruginosa [38]. Similar to oral flora
changes in the present study, the Fusobacterium periodonticum resulted as the most significantly
reduced species in COVID-19 patients [39]. Consequently, Fusobacterium, especially Fusobacterium
periodonticum was a potential specific detection indicator.

The functional analysis showed that the genera Veillonella, Prevotella and Campylobacter were positively
correlated with infectious disease, while genera Neisseria and Fusobacterium were the negative
correlations. The result further illustrated that these microbiotas can potentially serve CAP as biomarkers.

Anyway, our results provide baseline associations between oral microbiota and CAP, which will be
important for further evaluation of treatment and prognosis.

5. Limitations
There are some limitations about this study. The sample size can be expanded, and we just conducted a
single-center study instead of a multi-center research. Further exploration about the verification of
biomarkers and potential mechanism involved in CAP are required in the future.

6. Conclusions
Oral microbial dysbiosis was found in patients with CAP. Dysbiosis of the oral microbiota (e.g. Prevotella
and Neisseria) and neglected oral bacterial genera (e.g. Veillonella, Campylobacter and Fusobacterium)
are important biomarkers for CAP.

Abbreviations
CAP   community-acquired pneumonia

ASVs   amplicon sequence variants

PCoA   principal coordinate analysis

PICRUSt   phylogenetic investigation of communities by reconstruction of unobserved states

BALF   bronchoalveolar lavage fluid

Page 9/17
VAP    ventilator-associated pneumonia

COPD   Chronic obstructive pulmonary disease

COVID-19   Coronavirus disease 2019

CF   cystic fibrosis 

Declarations
Ethical approval

This study was approved by the Institutional Review Board of IRB of Jinan University (JNUKY-2022-002).
The study was performed in accordance with the Helsinki Declaration and Rules of Good Clinical
Practice. All participants signed written informed consents after the study protocol was fully explained.

Consent for publication

All presentations of case reports have consent for publication.

Availability of data and materials

The data used to support the findings of this study are included within the article and supplementary file.

Declaration of competing interests

The authors declare that they have no known competing financial interests or personal relationships that
could have appeared to influence the work reported in this paper.

Funding

Supported by the Fundamental Research Funds for the Central Universities (11621038) and the Science
and Technology Program of Guangzhou (202201010401)

Authors’ contributions

Study concept and design: TZ, NS; Specimen provider: NS, XZ, YH; Acquisition of clinical data: NS, XZ,
YH; Data analysis and interpretation (including statistical analysis): NS, XZ; Drafting of the manuscript:
NS. All authors approved the current version of this manuscript.

Acknowledgments

We thank Dr Xingdong Cai (the First Affiliated Hospital, Jinan University) for help in samples collection
and thank Novogene Co. Ltd. for 16SrRNA sequencing. We also thank the generous volunteer subjects
who enrolled in the study.

Page 10/17
References
1. Ferreira-Coimbra J, Sarda C, Rello J. Burden of Community-Acquired Pneumonia and Unmet Clinical
Needs. Adv Ther. 2020;37(4):1302-18; doi: 10.1007/s12325-020-01248-7.
2. Aliberti S, Dela Cruz CS, Amati F, Sotgiu G, Restrepo MI. Community-acquired pneumonia. Lancet.
2021;398(10303):906-19; doi: 10.1016/s0140-6736(21)00630-9.
3. Rothberg MB. Community-Acquired Pneumonia. Ann Intern Med. 2022;175(4):Itc49-itc64; doi:
10.7326/aitc202204190.
4. Shoar S, Musher DM. Etiology of community-acquired pneumonia in adults: a systematic review.
Pneumonia (Nathan). 2020;12:11; doi: 10.1186/s41479-020-00074-3.
5. Prina E, Ranzani OT, Torres A. Community-acquired pneumonia. Lancet. 2015;386(9998):1097-108;
doi: 10.1016/s0140-6736(15)60733-4.
6. Peyrani P, Mandell L, Torres A, Tillotson GS. The burden of community-acquired bacterial pneumonia
in the era of antibiotic resistance. Expert Rev Respir Med. 2019;13(2):139-52; doi:
10.1080/17476348.2019.1562339.
7. Schulte B, Eickmeyer H, Heininger A, Juretzek S, Karrasch M, Denis O, et al. Detection of pneumonia
associated pathogens using a prototype multiplexed pneumonia test in hospitalized patients with
severe pneumonia. PLoS One. 2014;9(11):e110566; doi: 10.1371/journal.pone.0110566.
8. Metlay JP, Waterer GW, Long AC, Anzueto A, Brozek J, Crothers K, et al. Diagnosis and Treatment of
Adults with Community-acquired Pneumonia. An Official Clinical Practice Guideline of the American
Thoracic Society and Infectious Diseases Society of America. Am J Respir Crit Care Med.
2019;200(7):e45-e67; doi: 10.1164/rccm.201908-1581ST.
9. Pathak JL, Yan Y, Zhang Q, Wang L, Ge L. The role of oral microbiome in respiratory health and
diseases. Respir Med. 2021;185:106475; doi: 10.1016/j.rmed.2021.106475.
10. Sinclair A, Xie X, Teltscher M, Dendukuri N. Systematic review and meta-analysis of a urine-based
pneumococcal antigen test for diagnosis of community-acquired pneumonia caused by
Streptococcus pneumoniae. J Clin Microbiol. 2013;51(7):2303-10; doi: 10.1128/jcm.00137-13.
11. Bassis CM, Erb-Downward JR, Dickson RP, Freeman CM, Schmidt TM, Young VB, et al. Analysis of the
upper respiratory tract microbiotas as the source of the lung and gastric microbiotas in healthy
individuals. mBio. 2015;6(2):e00037; doi: 10.1128/mBio.00037-15.
12. Mammen MJ, Scannapieco FA, Sethi S. Oral-lung microbiome interactions in lung diseases.
Periodontol 2000. 2020;83(1):234-41; doi: 10.1111/prd.12301.
13. Akata K, Yatera K, Yamasaki K, Kawanami T, Naito K, Noguchi S, et al. The significance of oral
streptococci in patients with pneumonia with risk factors for aspiration: the bacterial floral analysis
of 16S ribosomal RNA gene using bronchoalveolar lavage fluid. BMC Pulm Med. 2016;16(1):79; doi:
10.1186/s12890-016-0235-z.
14. Ma S, Zhang F, Zhou F, Li H, Ge W, Gan R, et al. Metagenomic analysis reveals oropharyngeal
microbiota alterations in patients with COVID-19. Signal Transduct Target Ther. 2021;6(1):191; doi:
Page 11/17
 10.1038/s41392-021-00614-3.
15. Verma D, Garg PK, Dubey AK. Insights into the human oral microbiome. Arch Microbiol.
2018;200(4):525-40; doi: 10.1007/s00203-018-1505-3.
16. Mandell LA, Wunderink RG, Anzueto A, Bartlett JG, Campbell GD, Dean NC, et al. Infectious Diseases
Society of America/American Thoracic Society consensus guidelines on the management of
community-acquired pneumonia in adults. Clin Infect Dis. 2007;44 Suppl 2(Suppl 2):S27-72; doi:
10.1086/511159.
17. Yu G, Phillips S, Gail MH, Goedert JJ, Humphrys MS, Ravel J, et al. The effect of cigarette smoking on
the oral and nasal microbiota. Microbiome. 2017;5(1):3; doi: 10.1186/s40168-016-0226-6.
18. Kalendar R, Boronnikova S, Seppänen M. Isolation and Purification of DNA from Complicated
Biological Samples. Methods Mol Biol. 2021;2222:57-67; doi: 10.1007/978-1-0716-0997-2_3.
19. Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJ, Holmes SP. DADA2: High-resolution
sample inference from Illumina amplicon data. Nat Methods. 2016;13(7):581-3; doi:
10.1038/nmeth.3869.
20. Segata N, Izard J, Waldron L, Gevers D, Miropolsky L, Garrett WS, et al. Metagenomic biomarker
discovery and explanation. Genome Biol. 2011;12(6):R60; doi: 10.1186/gb-2011-12-6-r60.
21. Langille MG, Zaneveld J, Caporaso JG, McDonald D, Knights D, Reyes JA, et al. Predictive functional
profiling of microbial communities using 16S rRNA marker gene sequences. Nat Biotechnol.
2013;31(9):814-21; doi: 10.1038/nbt.2676.
22. Eshwara VK, Mukhopadhyay C, Rello J. Community-acquired bacterial pneumonia in adults: An
update. Indian J Med Res. 2020;151(4):287-302; doi: 10.4103/ijmr.IJMR_1678_19.
23. Melo-Dias S, Valente C, Andrade L, Marques A, Sousa A. Saliva as a non-invasive specimen for COPD
assessment. Respir Res. 2022;23(1):16; doi: 10.1186/s12931-022-01935-9.
24. Asakawa M, Takeshita T, Furuta M, Kageyama S, Takeuchi K, Hata J, et al. Tongue Microbiota and
Oral Health Status in Community-Dwelling Elderly Adults. mSphere. 2018;3(4); doi:
10.1128/mSphere.00332-18.
25. de Carvalho Baptista IM, Martinho FC, Nascimento GG, da Rocha Santos CE, Prado RFD, Valera MC.
Colonization of oropharynx and lower respiratory tract in critical patients: Risk of ventilator-
associated pneumonia. Arch Oral Biol. 2018;85:64-9; doi: 10.1016/j.archoralbio.2017.09.029.
26. Caverly LJ, Huang YJ, Sze MA. Past, Present, and Future Research on the Lung Microbiome in
Inflammatory Airway Disease. Chest. 2019;156(2):376-82; doi: 10.1016/j.chest.2019.05.011.
27. Goolam Mahomed T, Peters RPH, Allam M, Ismail A, Mtshali S, Goolam Mahomed A, et al. Lung
microbiome of stable and exacerbated COPD patients in Tshwane, South Africa. Sci Rep.
2021;11(1):19758; doi: 10.1038/s41598-021-99127-w.
28. Lin M, Li X, Wang J, Cheng C, Zhang T, Han X, et al. Saliva Microbiome Changes in Patients With
Periodontitis With and Without Chronic Obstructive Pulmonary Disease. Front Cell Infect Microbiol.
2020;10:124; doi: 10.3389/fcimb.2020.00124.

Page 12/17
29. Shi YL, He MZ, Han MZ, Gui HY, Wang P, Yu JL, et al. Characterization of Altered Oropharyngeal
Microbiota in Hospitalized Patients With Mild SARS-CoV-2 Infection. Front Cell Infect Microbiol.
2022;12:824578; doi: 10.3389/fcimb.2022.824578.
30. Imai K, Iinuma T, Sato S. Relationship between the oral cavity and respiratory diseases: Aspiration of
oral bacteria possibly contributes to the progression of lower airway inflammation. Jpn Dent Sci Rev.
2021;57:224-30; doi: 10.1016/j.jdsr.2021.10.003.
31. Horn KJ, Schopper MA, Drigot ZG, Clark SE. Airway Prevotella promote TLR2-dependent neutrophil
activation and rapid clearance of Streptococcus pneumoniae from the lung. Nat Commun.
2022;13(1):3321; doi: 10.1038/s41467-022-31074-0.
32. Tsay JJ, Wu BG, Sulaiman I, Gershner K, Schluger R, Li Y, et al. Lower Airway Dysbiosis Affects Lung
Cancer Progression. Cancer Discov. 2021;11(2):293-307; doi: 10.1158/2159-8290.Cd-20-0263.
33. Skallevold HE, Vallenari EM, Sapkota D. Salivary Biomarkers in Lung Cancer. Mediators Inflamm.
2021;2021:6019791; doi: 10.1155/2021/6019791.
34. Xiong D, Muema C, Zhang X, Pan X, Xiong J, Yang H, et al. Enriched Opportunistic Pathogens
Revealed by Metagenomic Sequencing Hint Potential Linkages between Pharyngeal Microbiota and
COVID-19. Virol Sin. 2021;36(5):924-33; doi: 10.1007/s12250-021-00391-x.
35. Guo MY, Chen HK, Ying HZ, Qiu FS, Wu JQ. The Role of Respiratory Flora in the Pathogenesis of
Chronic Respiratory Diseases. Biomed Res Int. 2021;2021:6431862; doi: 10.1155/2021/6431862.
36. Madapoosi SS, Cruickshank-Quinn C, Opron K, Erb-Downward JR, Begley LA, Li G, et al. Lung
Microbiota and Metabolites Collectively Associate with Clinical Outcomes in Milder Stage Chronic
Obstructive Pulmonary Disease. Am J Respir Crit Care Med. 2022;206(4):427-39; doi:
10.1164/rccm.202110-2241OC.
37. Lambotte O, Timsit JF, Garrouste-Orgeas M, Misset B, Benali A, Carlet J. The significance of distal
bronchial samples with commensals in ventilator-associated pneumonia: colonizer or pathogen?
Chest. 2002;122(4):1389-99; doi: 10.1378/chest.122.4.1389.
38. Li Q, Wang H, Tan L, Zhang S, Lin L, Tang X, et al. Oral Pathogen Fusobacterium nucleatum
Coaggregates With Pseudomonas aeruginosa to Modulate the Inflammatory Cytotoxicity of
Pulmonary Epithelial Cells. Front Cell Infect Microbiol. 2021;11:643913; doi:
10.3389/fcimb.2021.643913.
39. Nardelli C, Gentile I, Setaro M, Di Domenico C, Pinchera B, Buonomo AR, et al. Nasopharyngeal
Microbiome Signature in COVID-19 Positive Patients: Can We Definitively Get a Role to
Fusobacterium periodonticum? Front Cell Infect Microbiol. 2021;11:625581; doi:
10.3389/fcimb.2021.625581.

Figures

Page 13/17
Figure 1

Bacterial diversity of the oral microbiota. (A) Species accumulation curve between number of samples.
(B) The pielou’s evenness reflected the evenness of the distribution of species in the microbial
community. Oral microbial diversity was estimated by the Shannon index (C) and Simpson index (D). (E)
A Venn diagram displayed the overlaps between groups. (F) Beta diversity was calculated using weighted
UniFrac by PCoA.
Page 14/17
Figure 2

Oral microbiota composition. Average composition of bacterial community at the phylum (A) and family
(B) levels. Abundance of the different bacterial genera (C) and species (D) between the CAP group and
control group. The increased or decreased microbial community at the genus level (E) and species level
(F) in patients with CAP versus healthy controls.

Page 15/17
Figure 3

Oral microbial functional dysbiosis in patients with CAP and control group. Differential KEGG pathways
were analyzed using PICRUSt, and PCA (A) analysis was conducted for the two groups. PICRUSt analysis
identified 11 KEGG pathways (B) with significant differential abundance between the CAP and control
group. The heatmaps (C—D) show partial spearman correlation coefficients of significant bacterial
genera and differential KEGG pathways between CAP and control group.

Supplementary Files
This is a list of supplementary files associated with this preprint. Click to download.

Supplementarytables.docx
TableS1ASVfeaturetable.xlsx
TableS2alphaindex.xls
Page 16/17
TableS3asvtable.p10.group.relative.xls
TableS4asvtable.g30.group.relative.xls
TableS5A1B1.xls
TableS6A1vsB1.psig.xls
TableS7A1vsB1.psig.xls

Page 17/17