Metagenomics reveals impact of geography and acute diarrheal disease on the Central Indian human gut microbiome

© 2020, © 2020 The Author(s). Published with license by Taylor & Francis Group, LLC. Background: The Central Indian gut microbiome remains grossly understudied. Herein, we sought to investigate the burden of antimicrobial resistance and diarrheal diseases, particularly Clostridioides difficile, in rural-agricultural and urban populations in Central India, where there is widespread unregulated antibiotic use. We utilized shotgun metagenomics to comprehensively characterize the bacterial and viral fractions of the gut microbiome and their encoded functions in 105 participants. Results: We observed distinct rural-urban differences in bacterial and viral populations, with geography exhibiting a greater influence than diarrheal status. Clostridioides difficile disease was more commonly observed in urban subjects, and their microbiomes were enriched in metabolic pathways relating to the metabolism of industrial compounds and genes encoding resistance to 3rd generation cephalosporins and carbapenems. By linking phages present in the microbiome to their bacterial hosts through CRISPR spacers, phage variation could be directly related to shifts in bacterial populations, with the auxiliary metabolic potential of rural-associated phages enriched for carbon and amino acid energy metabolism. Conclusions: We report distinct differences in antimicrobial resistance gene profiles, enrichment of metabolic pathways and phage composition between rural and urban populations, as well as a higher burden of Clostridioides difficile disease in the urban population. Our results reveal that geography is the key driver of variation in urban and rural Indian microbiomes, with acute diarrheal disease, including C. difficile disease exerting a lesser impact. Future studies will be required to understand the potential role of dietary, cultural, and genetic factors in contributing to microbiome differences between rural and urban populations

Metagenomics Reveals Impact of Geography and Acute Diarrhoeal Disease on the Central Indian Human Gut Microbiome

Background: The Central Indian gut microbiome remains grossly understudied. Herein, we sought to investigate the burden of antimicrobial resistance and diarrhoeal diseases, particularly Clostridioides difficile, in rural-agricultural and urban populations in Central India, where there is widespread unregulated antibiotic use. We utilised shotgun metagenomics to comprehensively characterise the bacterial and viral fractions of the gut microbiome and their encoded functions in 105 participants. Results: We observed distinct rural-urban differences in bacterial and viral populations, with geography exhibiting a greater influence than diarrhoeal status. Clostridioides difficile disease was more commonly observed in urban subjects, and their microbiomes were enriched in metabolic pathways relating to the metabolism of industrial compounds and genes encoding resistance to 3rd generation cephalosporins and carbapenems. By linking phages present in the microbiome to their bacterial hosts through CRISPR spacers, phage variation could be directly related to shifts in bacterial populations, with the auxiliary metabolic potential of rural-associated phages enriched for carbon and amino acid energy metabolism.Conclusions: We report distinct differences in antimicrobial resistance gene profiles, enrichment of metabolic pathways and phage composition between rural and urban populations, as well as a higher burden of Clostridioides difficile disease in the urban population. Our results reveal that geography is the key driver of variation in urban and rural Indian microbiomes, with acute diarrhoeal disease, including C. difficile disease exerting a lesser impact. Future studies will be required to understand the potential role of dietary, cultural and genetic factors in contributing to microbiome differences between rural and urban populations

RNA-Seq of untreated wastewater to assess COVID-19 and emerging and endemic viruses for public health surveillance

BackgroundThe COVID-19 pandemic showcased the power of genomic sequencing to tackle the emergence and spread of infectious diseases. However, metagenomic sequencing of total microbial RNAs in wastewater has the potential to assess multiple infectious diseases simultaneously and has yet to be explored.MethodsA retrospective RNA-Seq epidemiological survey of 140 untreated composite wastewater samples was performed across urban (n = 112) and rural (n = 28) areas of Nagpur, Central India. Composite wastewater samples were prepared by pooling 422 individual grab samples collected prospectively from sewer lines of urban municipality zones and open drains of rural areas from 3rd February to 3rd April 2021, during the second COVID-19 wave in India. Samples were pre-processed and total RNA was extracted prior to genomic sequencing.FindingsThis is the first study that has utilised culture and/or probe-independent unbiased RNA-Seq to examine Indian wastewater samples. Our findings reveal the detection of zoonotic viruses including chikungunya, Jingmen tick and rabies viruses, which have not previously been reported in wastewater. SARS-CoV-2 was detectable in 83 locations (59%), with stark abundance variations observed between sampling sites. Hepatitis C virus was the most frequently detected infectious virus, identified in 113 locations and co-occurring 77 times with SARS-CoV-2; and both were more abundantly detected in rural areas than urban zones. Concurrent identification of segmented virus genomic fragments of influenza A virus, norovirus, and rotavirus was observed. Geographical differences were also observed for astrovirus, saffold virus, husavirus, and aichi virus that were more prevalent in urban samples, while the zoonotic viruses chikungunya and rabies, were more abundant in rural environments.InterpretationRNA-Seq can effectively detect multiple infectious diseases simultaneously, facilitating geographical and epidemiological surveys of endemic viruses that could help direct healthcare interventions against emergent and pre-existent infectious diseases as well as cost-effectively and qualitatively characterising the health status of the population over time

Clostridioides difficile epidemiology in India

Clostridioides difficile infection (CDI) continues to affect hospitalized patients and community populations worldwide. In contrast to the substantial resources invested in the diagnosis and prevention of CDI in high-income countries, this anaerobic toxigenic bacterium has been largely overlooked in low-and-middle-income countries (LMICs) such as India, where there remains a paucity of epidemiologic data evaluating the burden of CDI. Extensive multi-institutional studies describing C. difficile epidemiology in India have not yet been performed. Given recent economic growth in many Asian countries, with aging populations, increased access to healthcare and widespread inappropriate use of antimicrobials, C. difficile is likely to be highly prevalent and causing significant disease burden. Greater efforts are required to enhance awareness of this neglected pathogen, through educating healthcare practitioners to test for CDI. There is also an urgent need to strengthen laboratory capacity, and ideally establish a national reference laboratory, to help facilitate a greater understanding of the molecular epidemiology of CDI in India and other LMICs.This mini-review aims to summarize the existing research evaluating the burden of CDI in humans and the environment in India

Modelling of cerebral tuberculosis in BALB/c mice using clinical strain from patients with CNS tuberculosis infection

Background & objectives: Central nervous system (CNS) infection caused by Mycobacterium tuberculosis (MTB) is the most severe form of extrapulmonary tuberculosis (EPTB) due to a high level of mortality and morbidity. Limited studies are available on CNS-TB animal model development. The present study describes the development of a murine model of CNS-TB using a clinical strain (C3) isolated from the cerebrospinal fluid (CSF) of CNS-TB patients. Methods: Groups of mice were infected by the intravenous route with MTB C3 strain isolated from the CSF of CNS-TB patients. Brain and lung tissue were evaluated for bacterial burden, histopathology and surrogate markers of TB infection at 30 and 50 days post-infection. Results: Mice infected intravenously with MTB C3 strains showed progressive development of CNS disease with high bacillary burden in lungs at the initial stage (30 days), which eventually disseminated to the brain at a later stage (50 days). Similarly, high mortality (60%) was associated in mice infected with C3 strain compared to control. Interpretation & conclusions: The study showed development of a novel murine model of CNS-TB using the C3 strain of MTB that replicated events of extrapulmonary dissemination. The developed model would be helpful in understanding the pathogenesis of CNS-TB infection for the development of improved therapeutic interventions in future

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Not AvailableWe present here the draft genome sequence of Listeria monocytogenes CIIMS-NV-3, a serovar 4b strain isolated from the vaginal swab of a female patient from central India. The availability of this genome may provide useful information on virulence characteristics for comparative genomic analysis.Not Availabl

ESX-5-targeted export of ESAT-6 in BCG combines enhanced immunogenicity & efficacy against murine tuberculosis with low virulence and reduced persistence

Tuberculosis (TB) is the leading infectious cause of death globally. The only licensed TB vaccine, Bacille Calmette–Guérin (BCG), has low efficacy against TB in adults and is not recommended in people with impaired immunity. The incorporation of the Mycobacterium tuberculosis (Mtb) secretion system ESX-1 into BCG improves immunogenicity and protection against TB in animal models, which is associated with the secretion of the ESX-1-dependent protein ESAT-6. However, the resulting strain, BCG::ESX1Mtb, has been deemed unsafe as a human vaccine, due to prolonged persistence and increased virulence in immunocompromised mice. In this study, we describe a new recombinant BCG strain that uncouples the beneficial aspects of ESAT-6 secretion from the detrimental ESX-1effects on virulence and persistence. The strain was constructed by fusing the ESAT-6-encoding gene esxA to the general secretion signal for the mycobacterial type VII secretion pathway protein PE25. This new strain, BCG::ESAT6-PE25SS, secretes full-length ESAT-6 via the ESX-5 secretion system, which in contrast to ESX-1 is also present in BCG. In vivo testing revealed that ESX-5-targeted ESAT-6 export, induces cytosolic contact, generates ESAT-6-specific T cells and enhances the protective efficacy against TB disease, but is associated with low virulence and reduced persistence in immunocompetent and immunocompromised mice. Additionally, compared to BCG::ESX1Mtb and parental BCG, mucosal administration of BCG::ESAT6-PE25SS is associated with more rapid clearance from the lung. These results warrant further studies to evaluate BCG::ESAT6-PE25SS as a potential live attenuated vaccine candidate for TB