Multi-kingdom gut microbiota analyses define COVID-19 severity and post-acute COVID-19 syndrome.

Our knowledge of the role of the gut microbiome in acute coronavirus disease 2019 (COVID-19) and post-acute COVID-19 is rapidly increasing, whereas little is known regarding the contribution of multi-kingdom microbiota and host-microbial interactions to COVID-19 severity and consequences. Herein, we perform an integrated analysis using 296 fecal metagenomes, 79 fecal metabolomics, viral load in 1378 respiratory tract samples, and clinical features of 133 COVID-19 patients prospectively followed for up to 6 months. Metagenomic-based clustering identifies two robust ecological clusters (hereafter referred to as Clusters 1 and 2), of which Cluster 1 is significantly associated with severe COVID-19 and the development of post-acute COVID-19 syndrome. Significant differences between clusters could be explained by both multi-kingdom ecological drivers (bacteria, fungi, and viruses) and host factors with a good predictive value and an area under the curve (AUC) of 0.98. A model combining host and microbial factors could predict the duration of respiratory viral shedding with 82.1% accuracy (error ± 3 days). These results highlight the potential utility of host phenotype and multi-kingdom microbiota profiling as a prognostic tool for patients with COVID-19.

Faecal microbiome-based machine learning for multi-class disease diagnosis.

Systemic characterisation of the human faecal microbiome provides the opportunity to develop non-invasive approaches in the diagnosis of a major human disease. However, shared microbial signatures across different diseases make accurate diagnosis challenging in single-disease models. Herein, we present a machine-learning multi-class model using faecal metagenomic dataset of 2,320 individuals with nine well-characterised phenotypes, including colorectal cancer, colorectal adenomas, Crohn's disease, ulcerative colitis, irritable bowel syndrome, obesity, cardiovascular disease, post-acute COVID-19 syndrome and healthy individuals. Our processed data covers 325 microbial species derived from 14.3 terabytes of sequence. The trained model achieves an area under the receiver operating characteristic curve (AUROC) of 0.90 to 0.99 (Interquartile range, IQR, 0.91-0.94) in predicting different diseases in the independent test set, with a sensitivity of 0.81 to 0.95 (IQR, 0.87-0.93) at a specificity of 0.76 to 0.98 (IQR 0.83-0.95). Metagenomic analysis from public datasets of 1,597 samples across different populations observes comparable predictions with AUROC of 0.69 to 0.91 (IQR 0.79-0.87). Correlation of the top 50 microbial species with disease phenotypes identifies 363 significant associations (FDR < 0.05). This microbiome-based multi-disease model has potential clinical application in disease diagnostics and treatment response monitoring and warrants further exploration.

Prolonged Impairment of Short-Chain Fatty Acid and L-Isoleucine Biosynthesis in Gut Microbiome in Patients With COVID-19.

BACKGROUND AND AIMS: Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with altered gut microbiota composition. Phylogenetic groups of gut bacteria involved in the metabolism of short chain fatty acids (SCFAs) were depleted in SARS-CoV-2-infected patients. We aimed to characterize a functional profile of the gut microbiome in patients with COVID-19 before and after disease resolution. METHODS: We performed shotgun metagenomic sequencing on fecal samples from 66 antibiotics-naïve patients with COVID-19 and 70 non-COVID-19 controls. Serial fecal samples were collected (at up to 6 times points) during hospitalization and beyond 1 month after discharge. We assessed gut microbial pathways in association with disease severity and blood inflammatory markers. We also determined changes of microbial functions in fecal samples before and after disease resolution and validated these functions using targeted analysis of fecal metabolites. RESULTS: Compared with non-COVID-19 controls, patients with COVID-19 with severe/critical illness showed significant alterations in gut microbiome functionality (P < .001), characterized by impaired capacity of gut microbiome for SCFA and L-isoleucine biosynthesis and enhanced capacity for urea production. Impaired SCFA and L-isoleucine biosynthesis in gut microbiome persisted beyond 30 days after recovery in patients with COVID-19. Targeted analysis of fecal metabolites showed significantly lower fecal concentrations of SCFAs and L-isoleucine in patients with COVID-19 before and after disease resolution. Lack of SCFA and L-isoleucine biosynthesis significantly correlated with disease severity and increased plasma concentrations of CXCL-10, NT- proB-type natriuretic peptide, and C-reactive protein (all P < .05). CONCLUSIONS: Gut microbiome of patients with COVID-19 displayed impaired capacity for SCFA and L-isoleucine biosynthesis that persisted even after disease resolution. These 2 microbial functions correlated with host immune response underscoring the importance of gut microbial functions in SARS-CoV-2 infection pathogenesis and outcome.

Temporal landscape of human gut RNA and DNA virome in SARS-CoV-2 infection and severity.

BACKGROUND: Coronavirus disease 2019 (COVID-19) caused by the enveloped RNA virus SARS-CoV-2 primarily affects the respiratory and gastrointestinal tracts. SARS-CoV-2 was isolated from fecal samples, and active viral replication was reported in human intestinal cells. The human gut also harbors an enormous amount of resident viruses (collectively known as the virome) that play a role in regulating host immunity and disease pathophysiology. Understanding gut virome perturbation that underlies SARS-CoV-2 infection and severity is an unmet need. METHODS: We enrolled 98 COVID-19 patients with varying disease severity (3 asymptomatic, 53 mild, 34 moderate, 5 severe, 3 critical) and 78 non-COVID-19 controls matched for gender and co-morbidities. All subjects had fecal specimens sampled at inclusion. Blood specimens were collected for COVID-19 patients at admission to test for inflammatory markers and white cell counts. Among COVID-19 cases, 37 (38%) patients had serial fecal samples collected 2 to 3 times per week from time of hospitalization until after discharge. Using shotgun metagenomics sequencing, we sequenced and profiled the fecal RNA and DNA virome. We investigated alterations and longitudinal dynamics of the gut virome in association with disease severity and blood parameters. RESULTS: Patients with COVID-19 showed underrepresentation of Pepper mild mottle virus (RNA virus) and multiple bacteriophage lineages (DNA viruses) and enrichment of environment-derived eukaryotic DNA viruses in fecal samples, compared to non-COVID-19 subjects. Such gut virome alterations persisted up to 30 days after disease resolution. Fecal virome in SARS-CoV-2 infection harbored more stress-, inflammation-, and virulence-associated gene encoding capacities including those pertaining to bacteriophage integration, DNA repair, and metabolism and virulence associated with their bacterial host. Baseline fecal abundance of 10 virus species (1 RNA virus, pepper chlorotic spot virus, and 9 DNA virus species) inversely correlated with disease COVID-19 severity. These viruses inversely correlated with blood levels of pro-inflammatory proteins, white cells, and neutrophils. Among the 10 COVID-19 severity-associated DNA virus species, 4 showed inverse correlation with age; 5 showed persistent lower abundance both during disease course and after disease resolution relative to non-COVID-19 subjects. CONCLUSIONS: Both enteric RNA and DNA virome in COVID-19 patients were different from non-COVID-19 subjects, which persisted after disease resolution of COVID-19. Gut virome may calibrate host immunity and regulate severity to SARS-CoV-2 infection. Our observation that gut viruses inversely correlated with both severity of COVID-19 and host age may partly explain that older subjects are prone to severe and worse COVID-19 outcomes. Altogether, our data highlight the importance of human gut virome in severity and potentially therapeutics of COVID-19. Video Abstract.

Gut microbiota composition reflects disease severity and dysfunctional immune responses in patients with COVID-19.

OBJECTIVE: Although COVID-19 is primarily a respiratory illness, there is mounting evidence suggesting that the GI tract is involved in this disease. We investigated whether the gut microbiome is linked to disease severity in patients with COVID-19, and whether perturbations in microbiome composition, if any, resolve with clearance of the SARS-CoV-2 virus. METHODS: In this two-hospital cohort study, we obtained blood, stool and patient records from 100 patients with laboratory-confirmed SARS-CoV-2 infection. Serial stool samples were collected from 27 of the 100 patients up to 30 days after clearance of SARS-CoV-2. Gut microbiome compositions were characterised by shotgun sequencing total DNA extracted from stools. Concentrations of inflammatory cytokines and blood markers were measured from plasma. RESULTS: Gut microbiome composition was significantly altered in patients with COVID-19 compared with non-COVID-19 individuals irrespective of whether patients had received medication (p<0.01). Several gut commensals with known immunomodulatory potential such as Faecalibacterium prausnitzii, Eubacterium rectale and bifidobacteria were underrepresented in patients and remained low in samples collected up to 30 days after disease resolution. Moreover, this perturbed composition exhibited stratification with disease severity concordant with elevated concentrations of inflammatory cytokines and blood markers such as C reactive protein, lactate dehydrogenase, aspartate aminotransferase and gamma-glutamyl transferase. CONCLUSION: Associations between gut microbiota composition, levels of cytokines and inflammatory markers in patients with COVID-19 suggest that the gut microbiome is involved in the magnitude of COVID-19 severity possibly via modulating host immune responses. Furthermore, the gut microbiota dysbiosis after disease resolution could contribute to persistent symptoms, highlighting a need to understand how gut microorganisms are involved in inflammation and COVID-19.

Alterations in Gut Microbiota of Patients With COVID-19 During Time of Hospitalization.

BACKGROUND & AIMS: Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects gastrointestinal tissues, little is known about the roles of gut commensal microbes in susceptibility to and severity of infection. We investigated changes in fecal microbiomes of patients with SARS-CoV-2 infection during hospitalization and associations with severity and fecal shedding of virus. METHODS: We performed shotgun metagenomic sequencing analyses of fecal samples from 15 patients with Coronavirus Disease 2019 (COVID-19) in Hong Kong, from February 5 through March 17, 2020. Fecal samples were collected 2 or 3 times per week from time of hospitalization until discharge; disease was categorized as mild (no radiographic evidence of pneumonia), moderate (pneumonia was present), severe (respiratory rate ≥30/min, or oxygen saturation ≤93% when breathing ambient air), or critical (respiratory failure requiring mechanical ventilation, shock, or organ failure requiring intensive care). We compared microbiome data with those from 6 subjects with community-acquired pneumonia and 15 healthy individuals (controls). We assessed gut microbiome profiles in association with disease severity and changes in fecal shedding of SARS-CoV-2. RESULTS: Patients with COVID-19 had significant alterations in fecal microbiomes compared with controls, characterized by enrichment of opportunistic pathogens and depletion of beneficial commensals, at time of hospitalization and at all timepoints during hospitalization. Depleted symbionts and gut dysbiosis persisted even after clearance of SARS-CoV-2 (determined from throat swabs) and resolution of respiratory symptoms. The baseline abundance of Coprobacillus, Clostridium ramosum, and Clostridium hathewayi correlated with COVID-19 severity; there was an inverse correlation between abundance of Faecalibacterium prausnitzii (an anti-inflammatory bacterium) and disease severity. Over the course of hospitalization, Bacteroides dorei, Bacteroides thetaiotaomicron, Bacteroides massiliensis, and Bacteroides ovatus, which downregulate expression of angiotensin-converting enzyme 2 (ACE2) in murine gut, correlated inversely with SARS-CoV-2 load in fecal samples from patients. CONCLUSIONS: In a pilot study of 15 patients with COVID-19, we found persistent alterations in the fecal microbiome during the time of hospitalization, compared with controls. Fecal microbiota alterations were associated with fecal levels of SARS-CoV-2 and COVID-19 severity. Strategies to alter the intestinal microbiota might reduce disease severity.