Short report
Nasopharyngeal Microbiota Profiling of SARS-CoV-2 Infected Patients
https://doi.org/10.21203/rs.3.rs-27326/v1
This work is licensed under a CC BY 4.0 License
published 25 Jul, 2020
Read the published version in Biological Procedures Online →
You are reading this older preprint version
We analyzed the bacterial communities of the nasopharynx in 40 SARS-CoV-2 infected and uninfected patients. All patients had a mild COVID-19 disease. We did not find statistically significant differences in either bacterial richness and diversity or composition. These findings suggest a nasopharyngeal microbiota at least early resilient to SARS-CoV-2 infection.
Nasopharynx
Bacterial community
SARS-CoV-2 infection
16S rRNA sequencing
The human upper respiratory tract is the major portal of entry for aerosol-transmitted microorganisms, including the 2019 emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [1]. Consistent with an ongoing rise in the number of coronavirus disease 2019 (COVID-19) cases worldwide, the updated World Health Organization estimates on 29 April 2020 reported 3,023,788 confirmed cases, including 208,112 deaths, over time (https://covid19.who.int/). Because of apparently absent cross-protective immunity from related viral infections, SARS-CoV-2 transmissibility is high, hence facilitating widespread person-to-person transmission [2]. Both asymptomatic and symptomatic patients may transmit the virus [3], consistent with similar viral loads detected in the nasal (sampled from the nasopharynx) and throat swabs from both patients [4].
As with influenza virus, respiratory samples provide the greatest yield of SARS-CoV-2 nucleic acid shedding [4, 5]. In one study analyzing nine patients with mild courses of COVID-19, swab samples taken during the first week of symptoms displayed high loads and successful isolation of the virus and, importantly, the presence of viral replicative RNA intermediates confirmed the active replication of SARS-CoV-2 in the throat [5]. The virus uses the host receptor angiotensin-converting enzyme 2 (ACE2), which is predominantly expressed in the lung, to interact with its spike protein [6]. This interaction would be responsible of the extension of tropism to the throat, that is anyhow a tissue with low expression of ACE2 [5], following a fusion activity gain of SARS-CoV-2 spike protein at the S1-S2 junction that is not present in SARS-CoV [7].
Unlike influenza virus [8, 9], no studies have explored the relationship between SARS-CoV-2 infection and the bacterial community (also referred to as microbiota) within the nasopharynx. Therefore, we investigated whether the presence of the virus in the nasopharynx might reflect alterations of the resident microbiota by comparing the bacterial communities of SARS-CoV-2 infected and uninfected patients.
We collected nasopharynx samples from patients who were (n = 18) or were not (n = 22) diagnosed with COVID-19 based on the SARS-CoV-2 RNA detection using a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay [10].
Patients with samples collected based on the clinical COVID-19 suspicion (i.e., who presented symptoms of acute respiratory infection in the emergency department of the Fondazione Policlinico Universitario A. Gemelli IRCCS Hospital of Rome, Rome, Italy) [11] from March 6 through March 9, 2020, were included. Nasopharyngeal swabs were tested for the RT-PCR based COVID-19 diagnosis confirmation [10] using the Korean Ministry of Food and Drug Safety approved Allplex 2019-nCoV assay (Arrow Diagnostics S.r.l., Genova, Italy). The same samples were used to profile the nasopharyngeal microbiota in all 40 patients concomitantly.
Genomic DNA was extracted using the QIAGEN EZ1 Advanced XL system and the V5–V6 hypervariable region of the bacterial 16S ribosomal RNA (rRNA) gene was sequenced using an Illumina MiSeq V2 chemistry (2 × 250 bp) as described [8]. Sequencing data were processed for alignment and quality filtering in QIIME2 v2019.1, and representative amplicon sequence variants were obtained by DADA2 algorithm (https://doi.org/10.1038/s41587-019-0209-9). Samples were rarefied to the minimum number of sequence reads obtained totally (range, 107933–165427) to perform subsequent analyses. Taxonomic annotation was performed using both VSearch and SILVA database v132. Statistical tests, including Wilcoxon signed rank test and permutational analysis of variance (PERMANOVA) were performed in R.
No patients developed pneumonia as documented in the absence of significant abnormalities (i.e., multifocal ground-glass opacities) on chest computed tomography images.
The microbiota of the nasopharynx was not different in patients positive for SARS-CoV-2 RNA compared to the microbiota of patients negative for SARS-CoV-2 RNA (Fig. 1). No significant differences between the patient groups in either bacterial richness and diversity (observed species, Shannon index, and inverse Simpson index were assessed; P > 0.05 by Wilcoxon signed rank test) or composition (no samples clustering within each group, as visualized by multidimensional scaling; P > 0.05 by PERMANOVA) was noticed.
We analyzed the relative abundance of bacterial taxa most represented in both patient groups and did not observe significant differences between the groups. Most sequences in all samples (98% in both SARS-CoV-2 infected and uninfected patients) belonged to five phyla, namely Firmicutes (42% and 51%, respectively), Bacteroidetes (25% and 20%, respectively), Proteobacteria (18% and 16%, respectively), Actinobacteria (8% and 6%, respectively), and Fusobacteria (5% and 5%, respectively). These findings were consistent with the high similarity shared by the bacterial communities in both SARS-CoV-2 infected and uninfected patients.
In this study, SARS-CoV-2 infection was not associated with a different profile of the nasopharynx bacterial community analyzed at COVID-19 diagnosis, which is when the patients had a mild disease. This suggests that no microbiota compositional alterations occurred in response to SARS-CoV-2 or that SARS-CoV-2 was unable to induce these alterations in our patients. Both suggestions lend support to a nasopharyngeal microbiota at least early resilient to SARS-CoV-2 infection.
As nasopharyngeal swabs can be a reasonable proxy for lung samples [12], resilience of the nasopharynx bacterial community would also imply robustness of lung bacterial community in restricting SARS-CoV-2 growth or attachment. This would mirror a lack of effect on the oropharyngeal microbiota in volunteers challenged with H3N2 influenza virus, many of which developed very mild disease [9]. Thus, no bacterial taxa were identified for which relative abundance was significantly higher in SARS-CoV-2 infected patients. Conversely, taxa such as Dolosigranulum and Staphylococcus (also detected in our study) were found as enriched in the nasopharynx microbiota of H3N2 influenza virus infected patients compared to healthy control individuals [8].
The limitations of this study are that SARS-CoV-2 infections were defined by RT-PCR positivity, with no culture results, likely leading to include infections with low infectiousness and the number of analyzed samples was small. Further investigation of patients with detailed viral shedding data and sequential respiratory samples is warranted.
COVID-19: Coronavirus Disease 2019; PERMANOVA: Permutational Analysis Of Variance; rRNA: ribosomal RNA; RT-PCR: Reverse Transcriptase-Polymerase Chain Reaction; SARS-CoV-2: Severe Acute Respiratory Syndrome Coronavirus 2
Ethics approval and consent to participate: This study received appropriate ethical review committee approval, with a waiver of informed consent.
Consent for publication: Not applicable.
Availability of data and materials: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Competing interests: The authors declare that they have no competing interests.
Funding: The Reale Group and the Fondazione Valentino Garavani & Giancarlo Giammetti, to support the COVID-19 Research in our Institution, financed this work. The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.
Authors' contributions: FDM and MS had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. FDM, BP, and MS conceived and designed the study. FDM, BP, FRP, and MS acquired, analyzed, and interpreted the data. FDM and FRP performed statistical analyses. AG and MS supervised the work. BP was the major contributor in writing the manuscript. PC, AG, and MS critically revised the manuscript for important intellectual concept. All authors read and approved the final manuscript.
Acknowledgements: We thank Franziska Lohmeyer, PhD (Scientific Direction of the Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy) for English revision of the manuscript, and clinical staff members in the Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy for collection of samples. None of these individuals received compensation for their contributions.
published 25 Jul, 2020
Read the published version in Biological Procedures Online →
Review #3 received at journal
11 Jun, 2020
Editorial decision: Major revision
11 Jun, 2020
Reviewer #3 agreed at journal
28 May, 2020
Review #2 received at journal
16 May, 2020
Review #1 received at journal
16 May, 2020
Reviewer #2 agreed at journal
09 May, 2020
Reviewers invited by journal
07 May, 2020
Reviewer #1 agreed at journal
07 May, 2020
First submitted to journal
06 May, 2020
Editor assigned by journal
06 May, 2020
Submission checks completed at journal
05 May, 2020
Editor invited by journal
05 May, 2020
You are reading this older preprint version