The diversity and basic functional attributes of the gut microbiome of healthy Indians is not well understood. This study investigated the gut microbiome of three Indian communities: individuals residing in rural and urban (n = 49) sea level Ballabhgarh areas and in rural high altitude areas of Leh, Ladakh in North India (n = 35). Our study revealed that the gut microbiome of Indian communities is dominated by Firmicutes followed by Bacteroidetes, Actinobateria and Proteobacteria. Although, 54 core bacterial genera were detected across the three distinct communities, the gut bacterial composition displayed specific signatures and was observed to be influenced by the topographical location and dietary intake of the individuals. The gut microbiome of individuals living in Leh was observed to be significantly similar with a high representation of Bacteroidetes and low abundance of Proteobacteria. In contrast, the gut microbiome of individuals living in Ballabhgarh areas harbored higher number of Firmicutes and Proteobacteria and is enriched with microbial xenobiotic degradation pathways. The rural community residing in sea level Ballabhgarh areas has unique microbiome characterized not only by a higher diversity, but also a higher degree of interindividual homogeneity.

To explore the natural microbial community of any ecosystems by high-resolution molecular approaches including next generation sequencing, it is extremely important to develop a sensitive and reproducible DNA extraction method that facilitate isolation of microbial DNA of sufficient purity and quantity from culturable and uncultured microbial species living in that environment. Proper lysis of heterogeneous community microbial cells without damaging their genomes is a major challenge. In this study, we have developed an improved method for extraction of community DNA from different environmental and human origin samples. We introduced a combination of physical, chemical and mechanical lysis methods for proper lysis of microbial inhabitants. The community microbial DNA was precipitated by using salt and organic solvent. Both the quality and quantity of isolated DNA was compared with the existing methodologies and the supremacy of our method was confirmed. Maximum recovery of genomic DNA in the absence of substantial amount of impurities made the method convenient for nucleic acid extraction. The nucleic acids obtained using this method are suitable for different downstream applications. This improved method has been named as the THSTI method to depict the Institute where the method was developed.

SummaryRelA and SpoT of Gram-negative organisms critically regulate cellular levels of (p)ppGpp. Here, we have dissected the spoT gene function of the cholera pathogen Vibrio cholerae by extensive genetic analysis. Unlike Escherichia coli, V. cholerae DrelA DspoT cells accumulated (p)ppGpp upon fatty acid or glucose starvation. The result strongly suggests RelA-SpoT-independent (p)ppGpp synthesis in V. cholerae. By repeated subculturing of a V. cholerae DrelA DspoT mutant, a suppressor strain with (p)ppGpp 0 phenotype was isolated. Bioinformatics analysis of V. cholerae whole genome sequence allowed identification of a hypothetical gene (VC1224), which codes for a small protein (~29 kDa) with a (p)ppGpp synthetase domain and the gene is highly conserved in vibrios; hence it has been named relV. Using E. coli DrelA or DrelA DspoT mutant we showed that relV indeed codes for a novel (p)ppGpp synthetase. Further analysis indicated that relV gene of the suppressor strain carries a point mutation at nucleotide position 676 of its coding region (DrelA DspoT relV676), which seems to be responsible for the (p)ppGpp 0 phenotype. Analysis of a V. cholerae DrelA DspoT DrelV triple mutant confirmed that apart from canonical relA and spoT genes, relV is a novel gene in V. cholerae responsible for (p)ppGpp synthesis.

The Bay of Bengal is known as the epicenter for seeding several devastating cholera outbreaks across the globe. Vibrio cholerae, the etiological agent of cholera, has extraordinary competency to acquire exogenous DNA by horizontal gene transfer (HGT) and adapt them into its genome for structuring metabolic processes, developing drug resistance, and colonizing the human intestine. Antimicrobial resistance (AMR) in V. cholerae has become a global concern. However, little is known about the identity of the resistance traits, source of AMR genes, acquisition process, and stability of the genetic elements linked with resistance genes in V. cholerae. Here we present details of AMR profiles of 443 V. cholerae strains isolated from the stool samples of diarrheal patients from two regions of India. We sequenced the whole genome of multidrug-resistant (MDR) and extensively drug-resistant (XDR) V. cholerae to identify AMR genes and genomic elements that harbor the resistance traits. Our genomic findings were further confirmed by proteome analysis. We also engineered the genome of V. cholerae to monitor the importance of the autonomously replicating plasmid and core genome in the resistance profile. Our findings provided insights into the genomes of recent cholera isolates and identified several acquired traits including plasmids, transposons, integrative conjugative elements (ICEs), pathogenicity islands (PIs), prophages, and gene cassettes that confer fitness to the pathogen. The knowledge generated from this study would help in better understanding of V. cholerae evolution and management of cholera disease by providing clinical guidance on preferred treatment regimens. cholera | antimicrobial resistance | mobile genetic elements | genome | proteome

In bacteria, nutrient deprivation evokes the stringent response, which is mediated by the small intracellular signaling molecule ppGpp. In Gram negatives, the RelA enzyme synthesizes and SpoT hydrolyzes ppGpp, although the latter protein also has weak synthetase activity. DksA, a recently identified RNA polymerase binding transcription factor, acts as a coregulator along with ppGpp for controlling the stringent response. Recently, we have shown that three genes, relA, spoT, and relV, govern cellular levels of ppGpp during various starvation stresses in the Gram-negative cholera pathogen Vibrio cholerae. Here we report functional characterization of the dksA gene of V. cholerae (dksA Vc ), coding for the protein DksA Vc . Extensive genetic analyses of the ⌬dksA Vc mutants suggest that DksA Vc is an important component involved in the stringent response in V. cholerae. Further analysis of mutants revealed that DksA Vc positively regulates various virulence-related processes, namely, motility, expression of the major secretory protease, called hemagglutinin protease (HAP), and production of cholera toxin (CT), under in vitro conditions. We found that DksA Vc upregulates expression of the sigma factor FliA ( 28 ), a critical regulator of motility in V. cholerae. Altogether, it appears that apart from stringent-response regulation, DksA Vc also has important roles in fine regulation of virulence-related phenotypes of V. cholerae.

The gastric microbiome is suspected to have a role in the causation of diseases by Helicobacter pylori. Reports on their relative abundance vis-à-vis H. pylori are available from various ethnic and geographic groups, but little is known about their interaction patterns. Endoscopic mucosal biopsy samples from the gastric antrum and corpus of 39 patients with suspected H. pylori infection were collected and microbiomes were analyzed by 16S rDNA profiling. Four groups of samples were identified, which harbored Helicobacter as well as a diverse group of bacteria including Lactobacillus, Halomonas and Prevotella. There was a negative association between the microbiome diversity and Helicobacter abundance. Network analyses showed that Helicobacter had negative interactions with members of the gastric microbiome, while other microbes interacted positively with each other, showing a higher tendency towards intra-cluster co-occurrence/co-operation. Cross-geographic comparisons suggested the presence of region-specific microbial abundance profiles. We report the microbial diversity, abundance variation and interaction patterns of the gastric microbiota of Indian patients with H. pylori infection and present a comparison of the same with the gastric microbial ecology in samples from different geographic regions. Such microbial abundance profiles and microbial interactions can help in understanding the pathophysiology of gastric ailments and can thus help in development of new strategies to curb it.The acidic pH in the stomach lumen impedes bacterial growth 1 . However, it is now known that the human stomach is not sterile but is rather colonized by diverse microbiota 2 . High-throughput sequencing of gastric biopsy samples suggests that the human stomach may harbor 128 phyla, although mainly dominated by five phyla, namely, Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes and Fusobacteria 2-5 . Among these, several species of Helicobacter are known to be natural inhabitants of the human stomach. Helicobacter pylori (H. pylori), a Gram-negative bacterium and frequent isolate of stomach specimens, with reported links to the causation of chronic/atrophic gastritis, duodenal ulcers, gastric mucosa-associated lymphoid tissue (MALT) lymphoma and adenocarcinoma 2,6,7 , has coevolved with its human host.While H. pylori has been incriminated in disease, an aspect that has only been recently investigated is the role of the stomach microbiome in the causation of these diseases, especially adenocarcinoma. Studies focusing on the microbial composition of the stomach in healthy individuals 4,[8][9][10] have indicated the presence of genera like Streptococcus, Prevotella, Veillonella, Fusobacterium, Haemophilus and Clostridium. H. pylori is known to utilize specific molecular mechanisms to modulate host immune response and create a local micro-environment that aids its colonization 5,11 . The influence of Helicobacter abundance on the populations of other genera and vice versa, and any interactions between them in the causation ...

Antimicrobial resistance (AMR) among bacterial species that resides in complex ecosystems is a natural phenomenon. Indiscriminate use of antimicrobials in healthcare, livestock, and agriculture provides an evolutionary advantage to the resistant variants to dominate the ecosystem. Ascendency of resistant variants threatens the efficacy of most, if not all, of the antimicrobial drugs commonly used to prevent and/or cure microbial infections. Resistant phenotype is very common in enteric bacteria. The most common mechanisms of AMR are enzymatic modifications to the antimicrobials or their target molecules. In enteric bacteria, most of the resistance traits are acquired by horizontal gene transfer from closely or distantly related bacterial population. AMR traits are generally linked with mobile genetic elements (MGEs) and could rapidly disseminate to the bacterial species through horizontal gene transfer (HGT) from a pool of resistance genes. Although prevalence of AMR genes among pathogenic bacteria is widely studied in the interest of infectious disease management, the resistance profile and the genetic traits that encode resistance to the commensal microbiota residing in the gut of healthy humans are not well-studied. In the present study, we have characterized AMR phenotypes and genotypes of five dominant commensal enteric bacteria isolated from the gut of healthy Indians. Our study revealed that like pathogenic bacteria, enteric commensals are also multidrug-resistant. The genes encoding antibiotic resistance are physically linked with MGEs and could disseminate vertically to the progeny and laterally to the distantly related microbial species. Consequently, the AMR genes present in the chromosome of commensal gut bacteria could be a potential source of resistance functions for other enteric pathogens.

Foodborne illness caused by pathogenic Vibrios is generally associated with the consumption of raw or undercooked seafood. Fish and other seafood can be contaminated with Vibrio species, natural inhabitants of the marine, estuarine, and freshwater environment. Pathogenic Vibrios of major public health concerns are Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus. Common symptoms of foodborne Vibrio infection include watery diarrhea, stomach cramping, nausea, vomiting, fever, and chills. Administration of oral or intravenous rehydration salts solution is the mainstay for the management of cholera, and antibiotics are also used to shorten the duration of diarrhea and to limit further transmission of the disease. Currently, doxycycline, azithromycin, or ciprofloxacin are commonly used for V. cholerae, and doxycycline or quinolone are administered for V. parahaemolyticus, whereas doxycycline and a third-generation cephalosporin are recommended for V. vulnificus as initial treatment regimen. The emergence of antimicrobial resistance (AMR) in Vibrios is increasingly common across the globe and a decrease in the effectiveness of commonly available antibiotics poses a global threat to public health. Recent progress in comparative genomic studies suggests that the genomes of the drug-resistant Vibrios harbor mobile genetic elements like plasmids, integrating conjugative elements, superintegron, transposable elements, and insertion sequences, which are the major carriers of genetic determinants encoding antimicrobial resistance. These mobile genetic elements are highly dynamic and could potentially propagate to other bacteria through horizontal gene transfer (HGT). To combat the serious threat of rising AMR, it is crucial to develop strategies for robust surveillance, use of new/novel pharmaceuticals, and prevention of antibiotic misuse.