Цель. Выявить особенности таксономического состава микробиоты ротоглотки пациентов с COVID-19 с различной степенью тяжести заболевания. Материалы и методы. Исследуемая группа пациентов включала в себя 156 пациентов, госпитализированных с подтвержденным диагнозом COVID-19 в клинический медицинский центр ФГБОУ ВО «МГМСУ им. А.И. Евдокимова» в период с апреля по июнь 2021 г., из них 77 пациентов – с легкой формой пневмонии по данным компьютерной томографии (КТ) – КТ-1 и 79 пациентов с умеренной и среднетяжелой формой пневмонии (КТ-2 и КТ-3). Отбор мазков из ротоглотки осуществляли при поступлении пациента в стационар. Из образцов выделяли тотальную ДНК, затем производили амплификацию V3–V4 регионов гена 16s рРНК с последующим секвенированием на приборе Illumina HiSeq 2500. Для получения вариантов ампликонных последовательностей (ASV) использовался алгоритм DADA2. Результаты. При сравнении микробного состава ротоглотки пациентов с различной формой пневмонии обнаружены ASV, ассоциированные с развитием как легкой, так и тяжелой форм пневмонии вне лечения в стационаре. Исходя из полученных результатов можно заключить, что ASV, ассоциированные с меньшей степенью поражения легких, в основном относятся к классу грамотрицательных фирмикут (Negativicutes), к различным классам протеобактерий, а также к порядку Fusobacteria. В свою очередь ASV, ассоциированные с большей степенью поражения легких, относятся преимущественно к грамположительным фирмикутам классов Bacilli и Clostridia. При нахождении в стационаре пациенты с тяжелой формой пневмонии достоверно чаще демонстрировали отрицательную динамику на фоне проводимых терапевтических мероприятий. Заключение. Различия в таксономическом составе микробиоты ротоглотки, наблюдающиеся у пациентов с различной формой пневмонии, развившейся вне стационарного лечения на фоне COVID-19, могут быть связаны с предположительной барьерной функцией микробиоты ротоглотки, которая позволяет снизить риск нарастания титра вируса.
Aim. To identify features of the taxonomic composition of the oropharyngeal microbiota of COVID-19 patients with different disease severity. Materials and methods. The study group included 156 patients hospitalized with confirmed diagnosis of COVID-19 in the clinical medical center of Yevdokimov Moscow State University of Medicine and Dentistry between April and June 2021. There were 77 patients with mild pneumonia according to CT (CT1) and 79 patients with moderate to severe pneumonia (CT2 and CT3). Oropharyngeal swabs were taken when the patient was admitted to the hospital. Total DNA was isolated from the samples, then V3–V4 regions of the 16s rRNA gene were amplified, followed by sequencing using Illumina HiSeq 2500 platform. DADA2 algorithm was used to obtain amplicon sequence variants (ASV). Results. When comparing the microbial composition of the oropharynx of the patients with different forms of pneumonia, we have identified ASVs associated with the development of both mild and severe pneumonia outside hospital treatment. Based on the results obtained, ASVs associated with a lower degree of lung damage belong predominantly to the class of Gram-negative Firmicutes (Negativicutes), to various classes of Proteobacteria, as well as to the order Fusobacteria. In turn, ASVs associated with a greater degree of lung damage belong predominantly to Gram-positive classes of Firmicutes – Bacilli and Clostridia. While being hospitalized, patients with severe pneumonia demonstrated negative disease dynamics during treatment significantly more often. Conclusion. We have observed differences in the taxonomic composition of the oropharyngeal microbiota in patients with different forms of pneumonia developed outside hospital treatment against COVID-19. Such differences might be due to the presumed barrier function of the oropharyngeal microbiota, which reduces the risk of virus titer increase.
1. Caselli E, Fabbri C, D'Accolti M, et al. Defining the oral microbiome by whole-genome sequencing and resistome analysis: the complexity of the healthy picture. BMC Microbiol. 2020;20(1):120. DOI:10.1186/s12866-020-01801-y
2. Pace CC, McCullough GH. The association between oral microorgansims and aspiration pneumonia in the institutionalized elderly: review and recommendations. Dysphagia. 2010;25(4):307-22. DOI:10.1007/s00455-010-9298-9
3. 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
4. Callahan BJ, McMurdie PJ, Rosen MJ, et al. DADA2: High-resolution sample inference from Illumina amplicon data. Nat Methods. 2016;13(7):581-3. DOI:10.1038/nmeth.3869
5. Davis NM, Proctor DM, Holmes SP, et al. Simple statistical identification and removal of contaminant sequences in marker-gene and metagenomics data. Microbiome. 2018;6(1):226. DOI:10.1186/s40168-018-0605-2
6. McMurdie PJ, Holmes S. Phyloseq: a bioconductor package for handling and analysis of high-throughput phylogenetic sequence data. Pac Symp Biocomput. 2012:235-46.
7. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15(12):550. DOI:10.1186/s13059-014-0550-8
8. Castro-Nallar E, Bendall ML, Pérez-Losada M, et al. Composition, taxonomy and functional diversity of the oropharynx microbiome in individuals with schizophrenia and controls. PeerJ. 2015;3:e1140. DOI:10.7717/peerj.1140
9. Li Q, Pu Y, Lu H, et al. Porphyromonas, Treponema, and Mogibacterium promote IL8/IFNγ/TNFα-based pro-inflammation in patients with medication-related osteonecrosis of the jaw. J Oral Microbiol. 2021;13(1). DOI:10.1080/20002297.2020.1851112
10. Nguyen L, McCord KA, Bui DT, et al. Sialic acid-containing glycolipids mediate binding and viral entry of SARS-CoV-2. Nat Chem Biol. 2022;18(1):81-90.
DOI:10.1038/s41589-021-00924-1
11. Bouchet V, Hood DW, Li J, et al. Host-derived sialic acid is incorporated into Haemophilus influenzae lipopolysaccharide and is a major virulence factor in experimental otitis media. Proc Natl Acad Sci USA. 2003;100(15):8898-903. DOI:10.1073/pnas.1432026100
12. Honarmand Ebrahimi K. SARS-CoV-2 spike glycoprotein-binding proteins expressed by upper respiratory tract bacteria may prevent severe viral infection. FEBS Lett. 2020;594(11):1651-60. DOI:10.1002/1873-3468.13845
13. Nardelli C, Gentile I, Setaro M, 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
14. Liu J, Liu S, Zhang Z, et al. Association between the nasopharyngeal microbiome and metabolome in patients with COVID-19. Synth Syst Biotechnol. 2021;6(3):135-43. DOI:10.1016/j.synbio.2021.06.002
15. Zhang L, Fan Y, Su H, et al. Chlorogenic acid methyl ester exerts strong anti-inflammatory effects via inhibiting the COX-2/NLRP3/NF-κB pathway. Food Funct. 2018;9(12):6155-64. DOI:10.1039/c8fo01281d
16. Brinig MM, Lepp PW, Ouverney CC, et al. Prevalence of bacteria of division TM7 in human subgingival plaque and their association with disease. Appl Environ Microbiol. 2003;69(3):1687-94. DOI:10.1128/AEM.69.3.1687-1694.2003
17. Chipashvili O, Utter DR, Bedree JK, et al. Episymbiotic Saccharibacteria suppresses gingival inflammation and bone loss in mice through host bacterial modulation. Cell Host Microbe. 2021;29(11):1649-62.e7. DOI:10.1016/j.chom.2021.09.009
18. He X, McLean JS, Edlund A, et al. Cultivation of a human-associated TM7 phylotype reveals a reduced genome and epibiotic parasitic lifestyle. Proc Natl Acad Sci USA. 2015;112(1):244-9. DOI:10.1073/pnas.1419038112
19. Bor B, Bedree JK, Shi W, et al. Saccharibacteria (TM7) in the Human Oral Microbiome. J Dent Res. 2019;98(5):500-9. DOI:10.1177/0022034519831671
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1. Caselli E, Fabbri C, D'Accolti M, et al. Defining the oral microbiome by whole-genome sequencing and resistome analysis: the complexity of the healthy picture. BMC Microbiol. 2020;20(1):120. DOI:10.1186/s12866-020-01801-y
2. Pace CC, McCullough GH. The association between oral microorgansims and aspiration pneumonia in the institutionalized elderly: review and recommendations. Dysphagia. 2010;25(4):307-22. DOI:10.1007/s00455-010-9298-9
3. 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
4. Callahan BJ, McMurdie PJ, Rosen MJ, et al. DADA2: High-resolution sample inference from Illumina amplicon data. Nat Methods. 2016;13(7):581-3. DOI:10.1038/nmeth.3869
5. Davis NM, Proctor DM, Holmes SP, et al. Simple statistical identification and removal of contaminant sequences in marker-gene and metagenomics data. Microbiome. 2018;6(1):226. DOI:10.1186/s40168-018-0605-2
6. McMurdie PJ, Holmes S. Phyloseq: a bioconductor package for handling and analysis of high-throughput phylogenetic sequence data. Pac Symp Biocomput. 2012:235-46.
7. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15(12):550. DOI:10.1186/s13059-014-0550-8
8. Castro-Nallar E, Bendall ML, Pérez-Losada M, et al. Composition, taxonomy and functional diversity of the oropharynx microbiome in individuals with schizophrenia and controls. PeerJ. 2015;3:e1140. DOI:10.7717/peerj.1140
9. Li Q, Pu Y, Lu H, et al. Porphyromonas, Treponema, and Mogibacterium promote IL8/IFNγ/TNFα-based pro-inflammation in patients with medication-related osteonecrosis of the jaw. J Oral Microbiol. 2021;13(1). DOI:10.1080/20002297.2020.1851112
10. Nguyen L, McCord KA, Bui DT, et al. Sialic acid-containing glycolipids mediate binding and viral entry of SARS-CoV-2. Nat Chem Biol. 2022;18(1):81-90.
DOI:10.1038/s41589-021-00924-1
11. Bouchet V, Hood DW, Li J, et al. Host-derived sialic acid is incorporated into Haemophilus influenzae lipopolysaccharide and is a major virulence factor in experimental otitis media. Proc Natl Acad Sci USA. 2003;100(15):8898-903. DOI:10.1073/pnas.1432026100
12. Honarmand Ebrahimi K. SARS-CoV-2 spike glycoprotein-binding proteins expressed by upper respiratory tract bacteria may prevent severe viral infection. FEBS Lett. 2020;594(11):1651-60. DOI:10.1002/1873-3468.13845
13. Nardelli C, Gentile I, Setaro M, 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
14. Liu J, Liu S, Zhang Z, et al. Association between the nasopharyngeal microbiome and metabolome in patients with COVID-19. Synth Syst Biotechnol. 2021;6(3):135-43. DOI:10.1016/j.synbio.2021.06.002
15. Zhang L, Fan Y, Su H, et al. Chlorogenic acid methyl ester exerts strong anti-inflammatory effects via inhibiting the COX-2/NLRP3/NF-κB pathway. Food Funct. 2018;9(12):6155-64. DOI:10.1039/c8fo01281d
16. Brinig MM, Lepp PW, Ouverney CC, et al. Prevalence of bacteria of division TM7 in human subgingival plaque and their association with disease. Appl Environ Microbiol. 2003;69(3):1687-94. DOI:10.1128/AEM.69.3.1687-1694.2003
17. Chipashvili O, Utter DR, Bedree JK, et al. Episymbiotic Saccharibacteria suppresses gingival inflammation and bone loss in mice through host bacterial modulation. Cell Host Microbe. 2021;29(11):1649-62.e7. DOI:10.1016/j.chom.2021.09.009
18. He X, McLean JS, Edlund A, et al. Cultivation of a human-associated TM7 phylotype reveals a reduced genome and epibiotic parasitic lifestyle. Proc Natl Acad Sci USA. 2015;112(1):244-9. DOI:10.1073/pnas.1419038112
19. Bor B, Bedree JK, Shi W, et al. Saccharibacteria (TM7) in the Human Oral Microbiome. J Dent Res. 2019;98(5):500-9. DOI:10.1177/0022034519831671
1 ФБУН «Научно-исследовательский институт системной биологии и медицины» Роспотребнадзора, Москва, Россия;
2 ФГБОУ ВО «Московский государственный медико-стоматологический университет им. А.И. Евдокимова» Минздрава России, Москва, Россия;
3 ФГБУ «Федеральный научно-клинический центр физико-химической медицины» ФМБА России, Москва, Россия
*olgagaleeva546@gmail.com
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Elizaveta V. Starikova1, Julia S. Galeeva*1, Dmitry N. Andreev2, Philipp S. Sokolov2, Dmitry E. Fedorov1, Aleksander I. Manolov1, Alexander V. Pavlenko1, Ksenia M. Klimina3, Vladimir A. Veselovsky3, Andrew V. Zaborovsky2, Vladimir V. Evdokimov2, Nikolai G. Andreev2, Mikhail K. Devkota2, Aleksei K. Fomenko2, Vadim A. Khar'kovskii2, Pavel O. Asadulin2, Sergey A. Kucher2, Aleksandra S. Cheremushkina2, Oleg O. Yanushevich2, Igor V. Maev2, Natella I. Krikheli2, Oleg V. Levchenko2, Elena N. Ilina1, Vadim M. Govorun1
1 Research Institute of Systemic Biology and Medicine, Moscow, Russia;
2 Yevdokimov Moscow State University of Medicine and Dentistry, Moscow, Russia;
3 Federal Scientific and Clinical Center for Physical and Chemical Medicine, Moscow, Russia
*olgagaleeva546@gmail.com