A cross-sectional study on the nasopharyngeal microbiota of individuals with SARS-CoV-2 infection across three COVID-19 waves in India.

Background: Multiple variants of the SARS-CoV-2 virus have plagued the world through successive waves of infection over the past three years. Independent research groups across geographies have shown that the microbiome composition in COVID-19 positive patients (CP) differs from that of COVID-19 negative individuals (CN). However, these observations were based on limited-sized sample-sets collected primarily from the early days of the pandemic. Here, we study the nasopharyngeal microbiota in COVID-19 patients, wherein the samples have been collected across the three COVID-19 waves witnessed in India, which were driven by different variants of concern. Methods: The nasopharyngeal swabs were collected from 589 subjects providing samples for diagnostics purposes at the Centre for Cellular and Molecular Biology (CSIR-CCMB), Hyderabad, India and subjected to 16s rRNA gene amplicon - based sequencing. Findings: We found variations in the microbiota of symptomatic vs. asymptomatic COVID-19 patients. CP showed a marked shift in the microbial diversity and composition compared to CN, in a wave-dependent manner. Rickettsiaceae was the only family that was noted to be consistently depleted in CP samples across the waves. The genera Staphylococcus, Anhydrobacter, Thermus, and Aerococcus were observed to be highly abundant in the symptomatic CP patients when compared to the asymptomatic group. In general, we observed a decrease in the burden of opportunistic pathogens in the host microbiota during the later waves of infection. Interpretation: To our knowledge, this is the first analytical cross-sectional study of this scale, which was designed to understand the relation between the evolving nature of the virus and the changes in the human nasopharyngeal microbiota. Although no clear signatures were observed, this study shall pave the way for a better understanding of the disease pathophysiology and help gather preliminary evidence on whether interventions to the host microbiota can help in better protection or faster recovery.

'GutFeel': an in silico method for predicting gut health status based on the metabolic functional capabilities of the resident microbiome.

Dysbiosis or imbalance in the gut microbiome has been correlated with the etiology of a number of diseases/disorders. Thus, gut microbial communities can potentially be utilized for assessing the health of the human gut. Although the taxonomic composition of the microbiomes is dependent on factors such as diet, lifestyle, and geography, these microbes perform a specific set of common functions in the gut. In this study, metabolic pathway-based markers (agnostic to above-mentioned factors) specific to commensals and those specific to pathogens are utilized as indicators of gut health. Furthermore, this gut health assessment requires only a small set of features rather than complete sequencing of metagenomes. The proposed scheme can also be used to design personalized biotherapeutics, depending on functional aspects observed in an individual.

Crosstalk Between Female Gonadal Hormones and Vaginal Microbiota Across Various Phases of Women's Gynecological Lifecycle.

Functional equilibrium between vaginal microbiota and the host is important for maintaining gynecological and reproductive health. Apart from host genetics, infections, changes in diet, life-style and hygiene status are known to affect this delicate state of equilibrium. More importantly, the gonadal hormones strongly influence the overall structure and function of vaginal microbiota. Several studies have attempted to understand (a) the composition of vaginal microbiota in specific stages of women's reproductive cycle as well as in menopause (b) their association with gonadal hormones, and their potential role in manifestation of specific health conditions (from the perspective of cause/consequence). However, a single study that places, in context, the structural variations of the vaginal microbiome across the entire life-span of women's reproductive cycle and during various stages of menopause is currently lacking. With the objective to obtain a holistic overview of the community dynamics of vaginal micro-environment 'across' various stages of women's reproductive and post-reproductive life-cycle, we have performed a meta-analysis of approximately 1,000 vaginal microbiome samples representing various stages of the reproductive cycle and menopausal states. Objectives of this analysis included (a) understanding temporal changes in vaginal community taxonomic structure and composition as women pass through various reproductive and menopausal stages (b) exploring correlations between the levels of female sex hormones with vaginal microbiome diversity (c) analyzing changes in the pattern of community diversity in cases of dysbiotic conditions such as bacterial vaginosis, and viewing the analyzed changes in the context of a healthy state. Results reveal interesting temporal trends with respect to vaginal microbial community diversity and its pattern of correlation with host physiology. Results indicate significant differences in alpha-diversity and overall vaginal microbial community members in various reproductive and post-reproductive phases. In addition to reinforcing the known influence/role of gonadal hormones in maintaining gynecological health, results indicate how hormonal level perturbations cause/contribute to imbalances in vaginal microbiota. The nature of resulting dysbiotic state and its influence on vaginal health is also analyzed and discussed. Results also suggest that elevated vaginal microbial diversity in pregnancy does not necessarily indicate a state of bacterial infection. The study puts forward a hormone-level driven microbiome diversity hypothesis for explaining temporal patterns in vaginal microbial diversity during various stages of women's reproductive cycle and at menopause.

Tryptophan Metabolism by Gut Microbiome and Gut-Brain-Axis: An in silico Analysis.

The link between gut microbiome and brain is being slowly acknowledged due to the speculated role of resident gut microbial community in altering the functions of gut-brain axis (GBA). Recently, a number of microbial metabolites (referred to as neuro-active metabolites) produced through tryptophan metabolism have been suggested to influence the GBA. In view of this, the current study focuses on microbial tryptophan metabolism pathways which produce neuro-active metabolites. An in silico analysis was performed on bacterial genomes as well as publicly available gut microbiome data. The results provide a comprehensive catalog of the analyzed pathways across bacteria. The analysis indicates an enrichment of tryptophan metabolism pathways in five gut-associated phyla, namely, Actinobacteria, Firmicutes, Bacteroidetes, Proteobacteria, and Fusobacteria. Further, five genera, namely, Clostridium, Burkholderia, Streptomyces, Pseudomonas, and Bacillus have been predicted to be enriched in terms of number of the analyzed tryptophan metabolism pathways, suggesting a higher potential of these bacterial groups to metabolize tryptophan in gut. Analysis of available microbiome data corresponding to gut samples from patients of neurological diseases and healthy individuals suggests probable association of different sets of tryptophan metabolizing bacterial pathways with the etiology of different diseases. The insights obtained from the present study are expected to provide directions toward designing of microbiome based diagnostic and therapeutic approaches for neurological diseases/disorders.

In Silico Analysis of Putrefaction Pathways in Bacteria and Its Implication in Colorectal Cancer.

Fermentation of undigested proteins in human gastrointestinal tract (gut) by the resident microbiota, a process called bacterial putrefaction, can sometimes disrupt the gut homeostasis. In this process, essential amino acids (e.g., histidine, tryptophan, etc.) that are required by the host may be utilized by the gut microbes. In addition, some of the products of putrefaction, like ammonia, putrescine, cresol, indole, phenol, etc., have been implicated in the disease pathogenesis of colorectal cancer (CRC). We have investigated bacterial putrefaction pathways that are known to be associated with such metabolites. Results of the comprehensive in silico analysis of the selected putrefaction pathways across bacterial genomes revealed presence of these pathways in limited bacterial groups. Majority of these bacteria are commonly found in human gut. These include Bacillus, Clostridium, Enterobacter, Escherichia, Fusobacterium, Salmonella, etc. Interestingly, while pathogens utilize almost all the analyzed pathways, commensals prefer putrescine and H2S production pathways for metabolizing the undigested proteins. Further, comparison of the putrefaction pathways in the gut microbiomes of healthy, carcinoma and adenoma datasets indicate higher abundances of putrefying bacteria in the carcinoma stage of CRC. The insights obtained from the present study indicate utilization of possible microbiome-based therapies to minimize the adverse effects of gut microbiome in enteric diseases.

Comparative In silico Analysis of Butyrate Production Pathways in Gut Commensals and Pathogens.

Biosynthesis of butyrate by commensal bacteria plays a crucial role in maintenance of human gut health while dysbiosis in gut microbiome has been linked to several enteric disorders. Contrastingly, butyrate shows cytotoxic effects in patients with oral diseases like periodontal infections and oral cancer. In addition to these host associations, few syntrophic bacteria couple butyrate degradation with sulfate reduction and methane production. Thus, it becomes imperative to understand the distribution of butyrate metabolism pathways and delineate differences in substrate utilization between pathogens and commensals. The bacteria utilize four pathways for butyrate production with different initial substrates (Pyruvate, 4-aminobutyrate, Glutarate and Lysine) which follow a polyphyletic distribution. A comprehensive mining of complete/draft bacterial genomes indicated conserved juxtaposed genomic arrangement in all these pathways. This gene context information was utilized for an accurate annotation of butyrate production pathways in bacterial genomes. Interestingly, our analysis showed that inspite of a beneficial impact of butyrate in gut, not only commensals, but a few gut pathogens also possess butyrogenic pathways. The results further illustrated that all the gut commensal bacteria (Faecalibacterium, Roseburia, Butyrivibrio, and commensal species of Clostridia etc) ferment pyruvate for butyrate production. On the contrary, the butyrogenic gut pathogen Fusobacterium utilizes different amino acid metabolism pathways like those for Glutamate (4-aminobutyrate and Glutarate) and Lysine for butyrogenesis which leads to a concomitant release of harmful by-products like ammonia in the process. The findings in this study indicate that commensals and pathogens in gut have divergently evolved to produce butyrate using distinct pathways. No such evolutionary selection was observed in oral pathogens (Porphyromonas and Filifactor) which showed presence of pyruvate as well as amino acid fermenting pathways which might be because the final product butyrate is itself known to be cytotoxic in oral diseases. This differential utilization of butyrogenic pathways in gut pathogens and commensals has an enormous ecological impact taking into consideration the immense influence of butyrate on different disorders in humans. The results of this study can potentially guide bioengineering experiments to design therapeutics/probiotics by manipulation of butyrate biosynthesis gene clusters in bacteria.