Gut microbiota Original research Gut microbiota composition reflects disease severity Gut: first published as 10.1136/gutjnl-2020-323020 on 11 January 2021. Downloaded from and dysfunctional immune responses in patients with COVID-19 Yun Kit Yeoh ,1,2 Tao Zuo ,2,3,4 Grace Chung- Yan Lui,3,5 Fen Zhang,2,3,4 Qin Liu,2,3,4 Amy YL Li,3 Arthur CK Chung,2,3,4 Chun Pan Cheung,2,3,4 Eugene YK Tso,6 Kitty SC Fung,7 Veronica Chan,6 Lowell Ling,8 Gavin Joynt,8 David Shu- Cheong Hui,3,5 Kai Ming Chow ,3 Susanna So Shan Ng,3,5 Timothy Chun- Man Li,3,5 Rita WY Ng,1 Terry CF Yip,3,4 Grace Lai- Hung Wong ,3,4 Francis KL Chan ,2,3,4 Chun Kwok Wong,9 Paul KS Chan,1,2,10 Siew C Ng 2,3,4 ► Additional material is ABSTRACT Significance of this study published online only. To view Objective Although COVID-19 is primarily a please visit the journal online respiratory illness, there is mounting evidence (http:// dx. doi. org/ 10. 1136/ What is already known on this subject? gutjnl- 2020- 323020). suggesting that the GI tract is involved in this disease. We investigated whether the gut microbiome is linked ► SARS- CoV-2 primarily infects the respiratory For numbered affiliations see tract, however, pathophysiology of COVID-19 end of article. to disease severity in patients with COVID-19, and whether perturbations in microbiome composition, if can be attributed to aberrant immune responses in clearing the virus. Correspondence to any, resolve with clearance of the SARS- CoV-2 virus. Professor Siew C Ng, Medicine Methods In this two- hospital cohort study, we ► Several lines of evidence such as replication of and Therapeutics, The Chinese obtained blood, stool and patient records from 100 SARS- CoV-2 in human enterocytes, detection University of Hong Kong, Hong patients with laboratory- confirmed SARS- CoV-2 of viruses in faecal samples and the altered Kong, Hong Kong; gut microbiota composition in patients with siewchienng@ cuhk. edu. hk infection. Serial stool samples were collected from 27 of the 100 patients up to 30 days after clearance COVID-19 suggest involvement of the GI tract. YKY, TZ and CKW contributed of SARS- CoV-2. Gut microbiome compositions were ► COVID-19 gut microbiota surveys are limited equally. characterised by shotgun sequencing total DNA and have not examined links between gut microbiome and disease pathophysiology. http://gut.bmj.com/ PKC and SCN are joint senior extracted from stools. Concentrations of inflammatory authors. cytokines and blood markers were measured from What are the new findings? plasma. ► Composition of the gut microbiota in Received 7 September 2020 Results Gut microbiome composition was patients with COVID-19 is concordant with Revised 27 October 2020 significantly altered in patients with COVID-19 Accepted 16 November 2020 disease severity and magnitude of plasma compared with non- COVID-19 individuals concentrations of several inflammatory irrespective of whether patients had received cytokines, chemokines and blood markers of on October 1, 2021 by guest. Protected copyright. medication (p<0.01). Several gut commensals tissue damage. with known immunomodulatory potential such as ► Patients with COVID-19 were depleted in Faecalibacterium prausnitzii, Eubacterium rectale and gut bacteria with known immunomodulatory bifidobacteria were underrepresented in patients and potential, such as Faecalibacterium prausnitzii, remained low in samples collected up to 30 days Eubacterium rectale and several bifidobacterial after disease resolution. Moreover, this perturbed species. composition exhibited stratification with disease ► The dysbiotic gut microbiota composition in severity concordant with elevated concentrations of patients with COVID-19 persists after clearance inflammatory cytokines and blood markers such as of the virus. C reactive protein, lactate dehydrogenase, aspartate aminotransferase and gamma- glutamyl transferase. © Author(s) (or their Conclusion Associations between gut microbiota employer(s)) 2021. Re- use composition, levels of cytokines and inflammatory INTRODUCTION permitted under CC BY- NC. No markers in patients with COVID-19 suggest that Infection by the SARS-CoV -2 induces an immune commercial re- use. See rights and permissions. Published the gut microbiome is involved in the magnitude response to eliminate the virus, but there is mounting by BMJ. of COVID-19 severity possibly via modulating host evidence suggesting that aberrant responses are immune responses. Furthermore, the gut microbiota responsible for severe outcomes and possibly To cite: Yeoh YK, Zuo T, other inflammatory conditions beyond COVID- Lui GC- Y, et al. Gut Epub dysbiosis after disease resolution could contribute ahead of print: [please to persistent symptoms, highlighting a need to 19. Patients with severe disease exhibit high blood include Day Month Year]. understand how gut microorganisms are involved in plasma levels of inflammatory cytokines and inflam- doi:10.1136/ inflammation and COVID-19. matory markers such as IL 6, 8 and 10 as well as C gutjnl-2020-323020 reactive protein (CRP) and lactate dehydrogenase Yeoh YK, et al. Gut 2021;0:1–9. doi:10.1136/gutjnl-2020-323020 1 Gut microbiota collected by hospital staff while discharged patients provided Significance of this study stools on day of follow- up or self- sampled at home. Stools were collected in collection tubes containing preservative media (cat. Gut: first published as 10.1136/gutjnl-2020-323020 on 11 January 2021. Downloaded from How might it impact on clinical practice in the foreseeable 63700, Norgen Biotek Corp, Ontario Canada) and stored at future? −80°C until processing. We have previously shown that gut ► These findings suggest that depletion of immunomodulatory microbiota composition generated from stools collected in this gut microorganisms contributes to severe COVID-19 disease. preservative medium was comparable with results obtained from ► The dysbiotic gut microbiota that persists after disease immediate freezing at −80°C.17 resolution could be a factor in developing persistent symptoms and/or multisystem inflammation syndromes that Stool DNA extraction and sequencing occur in some patients after clearing the virus. Detailed methods are described in Zuo et al.10 Briefly, DNA Bolstering of beneficial gut species depleted in COVID-19 ► was extracted from 0.1 g of homogenised faecal samples using could serve as a novel avenue to mitigate severe disease, the Maxwell RSC PureFood GMO and Authentication Kit underscoring importance of managing patients’ gut and a Maxwell RSC Instrument nucleic acid extraction plat- microbiota during and after COVID-19. form (Promega, Wisconsin, USA) according to manufacturer’s instructions. Sequencing libraries were prepared from extracted DNA using the Nextera DNA Flex Library Prep Kit (Illumina, (LDH) reflecting immune response and tissue damage from California, USA) and sequenced on an Illumina NovaSeq 6000 SARS- CoV-2 infection.1–3 Additionally, some patients develop System (2×150 bp) at the Centre for Gut Microbiota Research, autoinflammatory symptoms after recovery, most prominent Chinese University of Hong Kong. Raw sequence data generated being multisystem inflammatory syndrome and Kawasaki-like for this study are available in the Sequence Read Archive under disease in children.4–6 Several observations suggest a substantial BioProject accession PRJNA650244. involvement of the GI tract, such as the ability of SARS- CoV-2 to infect and replicate in human small intestine enterocytes,7 consistent detection of virus RNA in faecal samples8 9 and the Sequence data processing, inferring gut microbiota altered gut microbiota composition in SARS- CoV-2 infected composition and statistical analysis subjects.10 11 As the GI tract is the largest immunological organ in Raw sequence data were quality filtered using Trimmomatic V.39 the body and its resident microbiota are known to modulate host to remove adaptor and low- quality sequences. Following this, immune responses,12 we hypothesised that the gut microbiota is microbiota composition profiles were inferred from quality- 18 associated with host inflammatory immune responses in COVID- filtered forward reads using MetaPhlAn2 V.2.7.7 with the 19. Here, we characterised the gut microbiota and immune V.20 database. The site by species counts and relative abundance 19 response in 100 patients with COVID-19 during hospitalisation tables were input into R V.3.5.1 for statistical analysis. Principal and up to 30 days after recovery, showing that gut microbiota component analysis (PCA) ordinations were used to visualise the composition during hospitalisation is associated with disease clustering of samples based on their species level compositional severity and plasma concentrations of several cytokines and profiles. Associations between gut community composition and inflammatory markers. Moreover, gut microbiota composition patients’ parameters were assessed using permutational multi- http://gut.bmj.com/ in recovered patients remained significantly altered compared variate analysis of variance (PERMANOVA) and Procrustes with non- COVID-19 individuals, which could have important analyses. Associations of specific microbial species with patient implications in future health problems beyond COVID-19. parameters were identified using the linear discriminant analysis effect size (LEfSe) and the multivariate analysis by linear models MATERIALS AND METHODS (MaAsLin) statistical frameworks implemented in the Hutten- Subject recruitment and sample collection hower Lab Galaxy instance