Metagenomic insights into fungal community composition of the nasopharyngeal region of COVID-19 associated mucormycosis patients from India.

Coronavirus disease 2019 (COVID-19) associated mucormycosis (CAM) was reported predominantly from India during the second wave of COVID-19  and has a high mortality rate. The present study aims to understand the fungal community composition of the nasopharyngeal region of CAM-infected individuals and compare it with severe COVID-19 patients and healthy controls. The fungal community composition was decoded by analyzing the sequence homology of the internal transcribed spacer-2-(ITS-2) region of metagenomic DNA extracted from the upper respiratory samples. The alpha-diversity indices were found to be significantly altered in CAM patients (p < 0.05). Interestingly, a higher abundance of Candida africana, Candida haemuloni, Starmerella floris, and Starmerella lactiscondensi was observed exclusively in CAM patients. The interindividual changes in mycobiome composition were well supported by beta-diversity analysis (p < 0.05). The current study provides insights into the dysbiosis of the nasal mycobiome during CAM infection. In conclusion, our study shows that severe COVID-19 and CAM are associated with alteration in mycobiome as compared to healthy controls. However, the sequential alteration in the fungal flora which ultimately leads to the development of CAM needs to be addressed by future studies.

Collinsella aerofaciens linked with increased ethanol production and liver inflammation contribute to the pathophysiology of NAFLD.

Non-alcoholic fatty liver disease (NAFLD) is an emerging global health problem and a potential risk factor for metabolic diseases. The bidirectional interactions between liver and gut made dysbiotic gut microbiome one of the key risk factors for NAFLD. In this study, we reported an increased abundance of Collinsella aerofaciens in the gut of obese and NASH patients living in India. We isolated C. aerofaciens from the fecal samples of biopsy-proven NASH patients and observed that their genome is enriched with carbohydrate metabolism, fatty acid biosynthesis, and pro-inflammatory functions and have the potency to increase ethanol level in blood. An animal study indicated that mice supplemented with C. aerofaciens had increased levels of circulatory ethanol, high levels of hepatic hydroxyproline, triglyceride, and inflammation in the liver. The present findings indicate that perturbation in the gut microbiome composition is a key risk factor for NAFLD.

Antimicrobial resistance heterogeneity among multidrug-resistant Gram-negative pathogens: Phenotypic, genotypic, and proteomic analysis.

Microbes evolve rapidly by modifying their genomes through mutations or through the horizontal acquisition of mobile genetic elements (MGEs) linked with fitness traits such as antimicrobial resistance (AMR), virulence, and metabolic functions. We conducted a multicentric study in India and collected different clinical samples for decoding the genome sequences of bacterial pathogens associated with sepsis, urinary tract infections, and respiratory infections to understand the functional potency associated with AMR and its dynamics. Genomic analysis identified several acquired AMR genes (ARGs) that have a pathogen-specific signature. We observed that blaCTX-M-15, blaCMY-42, blaNDM-5, and aadA(2) were prevalent in Escherichia coli, and blaTEM-1B, blaOXA-232, blaNDM-1, rmtB, and rmtC were dominant in Klebsiella pneumoniae. In contrast, Pseudomonas aeruginosa and Acinetobacter baumannii harbored blaVEB, blaVIM-2, aph(3'), strA/B, blaOXA-23, aph(3') variants, and amrA, respectively. Regardless of the type of ARG, the MGEs linked with ARGs were also pathogen-specific. The sequence type of these pathogens was identified as high-risk international clones, with only a few lineages being predominant and region-specific. Whole-cell proteome analysis of extensively drug-resistant K. pneumoniae, A. baumannii, E. coli, and P. aeruginosa strains revealed differential abundances of resistance-associated proteins in the presence and absence of different classes of antibiotics. The pathogen-specific resistance signatures and differential abundance of AMR-associated proteins identified in this study should add value to AMR diagnostics and the choice of appropriate drug combinations for successful antimicrobial therapy.

Association of gut microbial dysbiosis with disease severity, response to therapy and disease outcomes in Indian patients with COVID-19.

BACKGROUND: Severe coronavirus disease 2019 (COVID-19) is associated with systemic hyper-inflammation. An adaptive interaction between gut microbiota and host immune systems is important for intestinal homeostasis and systemic immune regulation. The association of gut microbial composition and functions with COVID-19 disease severity is sparse, especially in India. We analysed faecal microbial diversity and abundances in a cohort of Indian COVID-19 patients to identify key signatures in the gut microbial ecology in patients with severe COVID-19 disease as well as in response to different therapies. The composition of the gut microbiome was characterized using 16Sr RNA gene sequences of genomic DNA extracted from faecal samples of 52 COVID-19 patients. Metabolic pathways across the groups were predicted using PICRUSt2. All statistical analyses were done using Vegan in the R environment. Plasma cytokine abundance at recruitment was measured in a multiplex assay. RESULTS: The gut microbiome composition of mild and severe patients was found to be significantly different. Immunomodulatory commensals, viz. Lachnospiraceae family members and Bifidobacteria producing butyrate and short-chain fatty acids (SCFAs), were under represented in patients with severe COVID-19, with an increased abundance of opportunistic pathogens like Eggerthella. The higher abundance of Lachnoclostridium in severe disease was reduced in response to convalescent plasma therapy. Specific microbial genera showed distinctive trends in enriched metabolic pathways, strong correlations with blood plasma cytokine levels, and associative link to disease outcomes. CONCLUSION: Our study indicates that, along with SARS-CoV-2, a dysbiotic gut microbial community may also play an important role in COVID-19 severity through modulation of host immune responses.

Study on Effects of Probiotics on Gut Microbiome and Clinical Course in Patients with Critical Care Illnesses.

Ventilator-associated pneumonia (VAP) is a nosocomial infection contracted by ventilator patients in which bacteria colonize the upper digestive tract and contaminated secretions are released into the lower airway. This nosocomial infection increases the morbidity and mortality of the patients as well as the cost of treatment. Probiotic formulations have recently been proposed to prevent the colonization of these pathogenic bacteria. In this prospective observational study, we aimed to investigate the effects of probiotics on gut microbiota and their relation to clinical outcomes in mechanically ventilated patients. For this study, 35 patients were recruited (22 probiotic-treated and 13 without probiotic treatment) from a cohort of 169 patients. Patients in the probiotic group were given a dose of 6 capsules of a commercially available probiotic (VSL#3®:112.5 billion CFU/cap) in three divided doses for 10 days. Sampling was carried out after each dose to monitor the temporal change in the gut microbiota composition. To profile the microbiota, we used a 16S rRNA metagenomic approach, and differences among the groups were computed using multivariate statistical analyses. Differences in gut microbial diversity (Bray Curtis and Jaccard distance, p-value > 0.05) between the probiotic-treated group and the control group were not observed. Furthermore, treatment with probiotics resulted in the enrichment of Lactobacillus and Streptococcus in the gut microbiota of the probiotic-treated groups. Our results demonstrated that probiotics might lead to favorable alterations in gut microbiome characteristics. Future studies should focus on the appropriate dosages and frequency of probiotics, which can lead to improved clinical outcomes.

Region-specific genomic signatures of multidrug-resistant Helicobacter pylori isolated from East and South India.

Helicobacter pylori is a ubiquitous bacterium and contributes significantly to the burden of chronic gastritis, peptic ulcers, and gastric cancer across the world. Adaptive phenotypes and virulence factors in H. pylori are heterogeneous and dynamic. However, limited information is available about the molecular nature of antimicrobial resistance phenotypes and virulence factors of H. pylori strains circulating in India. In the present study, we analyzed the whole genome sequences of 143 H. pylori strains, of which 32 are isolated from two different regions (eastern and southern) of India. Genomic repertoires of individual strains show distinct region-specific signatures. We observed lower resistance phenotypes and genotypes in the East Indian (Kolkata) H. pylori isolates against amoxicillin and furazolidone antibiotics, whereas higher resistance phenotypes to metronidazole and clarithromycin. Also, at molecular level, a greater number of AMR genes were observed in the east Indian H. pylori isolates as compared to the southern Indian isolates. From our findings, we suggest that metronidazole and clarithromycin antibiotics should be used judicially in the eastern India. However, no horizontally acquired antimicrobial resistance gene was observed in the current H. pylori strains. The comparative genome analysis shows that the number of genes involved in virulence, disease and resistance of H. pylori isolated from two different regions of India is significantly different. Single-nucleotide polymorphisms (SNPs) based phylogenetic analysis distinguished H. pylori strains into different clades according to their geographical locations. Conditionally beneficial functions including antibiotic resistance phenotypes that are linked with faster evolution rates in the Indian isolates.

Antimicrobial resistance and virulence in Helicobacter pylori: Genomic insights.

Microbes evolve rapidly by modifying their genome through mutations or acquisition of genetic elements. Antimicrobial resistance in Helicobacter pylori is increasingly prevalent in India. However, limited information is available about the genome of resistant H. pylori isolated from India. Our pan- and core-genome based analyses of 54 Indian H. pylori strains revealed plasticity of its genome. H. pylori is highly heterogenous both in terms of the genomic content and DNA sequence homology of ARGs and virulence factors. We observed that the H. pylori strains are clustered according to their geographical locations. The presence of point mutations in the ARGs and absence of acquired genetic elements linked with ARGs suggest target modifications are the primary mechanism of its antibiotic resistance. The findings of the present study would help in better understanding the emergence of drug-resistant H. pylori and controlling gastric disorders by advancing clinical guidance on selected treatment regimens.

The Vaginal Microbial Signatures of Preterm Birth Delivery in Indian Women.

Background: The incidence of preterm birth (PTB) in India is around 13%. Specific bacterial communities or individual taxon living in the vaginal milieu of pregnant women is a potential risk factor for PTB and may play an important role in its pathophysiology. Besides, bacterial taxa associated with PTB vary across populations. Objective: Conduct a comparative analysis of vaginal microbiome composition and microbial genomic repertoires of women who enrolled in the Interdisciplinary Group for Advanced Research on Birth Outcomes - A DBT India Initiative (GARBH-Ini) pregnancy cohort to identify bacterial taxa associated with term birth (TB) and PTB in Indian women. Methods: Vaginal swabs were collected during all three trimesters from 38 pregnant Indian women who delivered spontaneous term (n=20) and preterm (n=18) neonates. Paired-end sequencing of V3-V4 region of 16S rRNA gene was performed using the metagenomic DNA isolated from vaginal swabs (n=115). Whole genome sequencing of bacterial species associated with birth outcomes was carried out by shotgun method. Lactobacillus species were grown anaerobically in the De Man, Rogosa and Sharpe (MRS) agar culture medium for isolation of genomic DNA and whole genome sequencing. Results: Vaginal microbiome of both term and preterm samples reveals similar alpha diversity indices. However, significantly higher abundance of Lactobacillus iners (p-value All_Trimesters<0.02), Megasphaera sp (p-value1st_Trimester <0.05), Gardnerella vaginalis (p-value2nd_Trimester= 0.01) and Sneathia sanguinegens (p-value2nd_Trimester <0.0001) were identified in preterm samples whereas higher abundance of L. gasseri (p-value3rd_Trimester =0.010) was observed in term samples by Wilcoxon rank-sum test. The relative abundance of L. iners, and Megasphaera sp. were found to be significantly different over time between term and preterm mothers. Analyses of the representative genomes of L. crispatus and L. gasseri indicate presence of secretory transcriptional regulator and several ribosomally synthesized antimicrobial peptides correlated with anti-inflammatory condition in the vagina. These findings indicate protective role of L. crispatus and L. gasseri in reducing the risk of PTB. Conclusion: Our findings indicate that the dominance of specific Lactobacillus species and few other facultative anaerobes are associated with birth outcomes.