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Metagenomic analysis of gut microbiome and resistome of diarrheal fecal samples from Kolkata, India, reveals the core and variable microbiota including signatures of microbial dark matter.

ABSTRACT: Background:Metagenomic analysis of the gut microbiome and resistome is instrumental for understanding the dynamics of diarrheal pathogenesis and antimicrobial resistance transmission (AMR). Metagenomic sequencing of 20 diarrheal fecal samples from Kolkata was conducted to understand the core and variable gut microbiota. Five of these samples were used for resistome analysis. The pilot study was conducted to determine a microbiota signature and the source of antimicrobial resistance genes (ARGs) in the diarrheal gut. Results:16S rRNA amplicon sequencing was performed using Illumina MiSeq platform and analysed using the MGnify pipeline. The Genome Taxonomy Database (GTDB-Tk) was used for bacterial taxonomic identification. Diarrheal etiology was determined by culture method. Phylum Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria were consistently present in 20 samples. Firmicutes was the most abundant phylum in 11 samples. The Bacteroidetes/Firmicutes ratio was less than 1 in 18 samples. 584 genera were observed. 18 of these were present in all the 20 samples. Proteobacteria was the dominant phylum in 6 samples associated with Vibrio cholerae infection. Conservation of operational taxonomic units (OTUs) among all the samples indicated the existence of a core microbiome. Asymptomatic carriage of pathogens like Vibrio cholerae and Helicobacter pylori was found. Signature of Candidate phyla or "microbial dark matter" occurred. Significant correlation of relative abundance of bacterial families of commensals and pathogens were found. Whole-genome sequencing (WGS) on Illumina MiSeq system and assembly of raw reads using metaSPAdes v3.9.1 was performed to study the resistome of 5 samples. ABRicate was used to assign ARG function. 491 resistance determinants were identified. In 80% of the samples tetracycline resistance was the most abundant resistance determinant. High abundance of ARGs against ?-lactams, aminoglycosides, quinolones and macrolides was found. Eschericia sp. was the major contributor of ARGs. Conclusions:This is the first comparative study of the gut microbiome associated with different diarrheal pathogens. It presents the first catalogue of different bacterial taxa representing the core and variable microbiome in acute diarrheal patients. The study helped to define a trend in the gut microbiota signature associated with diarrhea and revealed which ARGs are abundantly present and the metagenome-assembled genomes (MAGs) contributing to AMR.

SUBMITTER: De R

PROVIDER: S-EPMC7339541 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Metagenomic analysis of gut microbiome and resistome of diarrheal fecal samples from Kolkata, India, reveals the core and variable microbiota including signatures of microbial dark matter.

De Rituparna R Mukhopadhyay Asish Kumar AK Dutta Shanta S

Gut pathogens 20200707

<h4>Background</h4>Metagenomic analysis of the gut microbiome and resistome is instrumental for understanding the dynamics of diarrheal pathogenesis and antimicrobial resistance transmission (AMR). Metagenomic sequencing of 20 diarrheal fecal samples from Kolkata was conducted to understand the core and variable gut microbiota. Five of these samples were used for resistome analysis. The pilot study was conducted to determine a microbiota signature and the source of antimicrobial resistance genes ...[more]

PMID: 32655699

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Project description:Twenty-five diarrheal fecal samples from Kolkata were examined to determine the relative abundance of antimicrobial resistance genes (ARGs) against eight common classes of antibiotics with polymerase chain reaction (PCR) and Sanger sequencing. Relative abundance of an ARG was calculated as the percentage of fecal samples showing the presence of that particular ARG. The frequency of occurrence of resistance marker against each class of antibiotic was calculated as the percentage of fecal samples carrying at least one resistance marker for that particular class of antimicrobials. Antibiogram of Vibrio cholerae (V. cholerae) O1 strains isolated from four of these samples was obtained by disc diffusion method and was compared with the ARG profile of corresponding fecal samples from which the strains were isolated. A 464 bp amplicon of the V3-V4 region of bacterial 16S rDNA was obtained by PCR from 9 of these 25 samples using the primer pair S-D-Bact-0341-b-S-17 and S-D-Bact-0785-a-A-21 and sequenced to determine the major operational taxonomic unit (OTU). These 9 samples represented diarrhea due to diverse etiology and also unresolved etiology as determined by culture method. We conclude that the diarrheal intestinal microbiome has a common gene pool of ARGs against the major classes of antibiotics and may be serving as a reservoir of ARG dissemination. ARG profile of cholera stool showed that ARGs present in the gut of cholera patients may be transferred to the V. cholerae genome and pose a serious threat to the treatment of cholera by triggering resistance against potential drugs to which contemporary strains of V. cholerae were found to be sensitive in the present study. Fecal samples which were culture negative for diarrheal pathogens we tested also carried ARGs and OTU. Abundance of resistance markers against macrolides, tetracyclines, and aminoglycosides was the highest. Phylum Proteobacteria was the most abundant OTU suggesting proteobacterial blooms characteristic of disturbed gut microflora. Our study is the first comparative study of ARG profile of diarrheal samples with varying etiologic agent revealing the presence of ARGs against the most important classes of antibiotics in the gut of diarrheal patients by common, robust molecular methods, which are easily accessible by molecular epidemiological laboratories worldwide.

Project description:Phages, such as those infecting Bacteroides spp., have been proven to be reliable indicators of human fecal contamination in microbial source tracking (MST) studies, and the efficacy of these MST markers found to vary geographically. This study reports the application and evaluation of candidate MST methods (phages infecting previously isolated B. fragilis strain GB-124, newly isolated Bacteroides strains (K10, K29, and K33) and recently isolated Kluyvera intermedia strain ASH-08), along with non-source specific somatic coliphages (SOMCPH infecting strain WG-5) and indicator bacteria (Escherichia coli) for identifying fecal contamination pathways in Kolkata, India. Source specificity of the phage-based methods was first tested using 60 known non-human fecal samples from common animals, before being evaluated with 56 known human samples (municipal sewage) collected during both the rainy and dry season. SOMCPH were present in 40-90% of samples from different animal species and in 100% of sewage samples. Phages infecting Bacteroides strain GB-124 were not detected from the majority (95%) of animal samples (except in three porcine samples) and were present in 93 and 71% of the sewage samples in the rainy and dry season (Mean = 1.42 and 1.83 log10PFU/100mL, respectively), though at lower levels than SOMCPH (Mean = 3.27 and 3.02 log10PFU/100mL, respectively). Phages infecting strain ASH-08 were detected in 89 and 96% of the sewage samples in the rainy and dry season, respectively, but were also present in all animal samples tested (except goats). Strains K10, K29, and K30 were not found to be useful MST markers due to low levels of phages and/or co-presence in non-human sources. GB-124 and SOMCPH were subsequently deployed within two low-income neighborhoods to determine the levels and origin of fecal contamination in 110 environmental samples. E. coli, SOMCPH, and phages of GB-124 were detected in 68, 42, and 28% of the samples, respectively. Analyses of 166 wastewater samples from shared community toilets and 21 samples from sewage pumping stations from the same districts showed that SOMCPH were present in 100% and GB-124 phages in 31% of shared toilet samples (Median = 5.59 and <1 log10 PFU/100 mL, respectively), and both SOMCPH and GB-124 phages were detected in 95% of pumping station samples (Median = 5.82 and 4.04 log10 PFU/100 mL, respectively). Our findings suggest that GB-124 and SOMCPH have utility as low-cost fecal indicator tools which can facilitate environmental surveillance of enteric organisms, elucidate human and non-human fecal exposure pathways, and inform interventions to mitigate exposure to fecal contamination in the residential environment of Kolkata, India.

Project description:BackgroundAntibiotic-resistant pathogens pose high risks to human and animal health worldwide. In recent years, the role of gut microbiota as a reservoir of antibiotic resistance genes (ARGs) in humans and animals has been increasingly investigated. However, the structure and function of the gut bacterial community, as well as the ARGs they carry in migratory birds remain unknown.ResultsHere, we collected samples from migratory bird species and their associated environments and characterized their gut microbiomes and resistomes using shotgun metagenomic sequencing. We found that migratory birds vary greatly in gut bacterial composition but are similar in their microbiome metabolism and function. Birds from the same environment tend to harbor similar bacterial communities. In total, 1030 different ARGs (202 resistance types) conferring resistance to tetracycline, aminoglycoside, β-lactam, sulphonamide, chloramphenicol, macrolide-lincosamide-streptogramin (MLS), and quinolone are identified. Procrustes analysis indicated that microbial community structure is not correlated with the resistome in migratory birds. Moreover, metagenomic assembly-based host tracking revealed that most of the ARG-carrying contigs originate from Proteobacteria. Co-occurrence patterns revealed by network analysis showed that emrD, emrY, ANT(6)-Ia, and tetO, the hubs of ARG type network, are indicators of other co-occurring ARG types. Compared with the microbiomes and resistomes in the environment, migratory birds harbor a lower phylogenetic diversity but have more antibiotic resistance proteins. Interestingly, we found that the mcr-1 resistance gene is widespread among different birds, accounting for 50% of the total samples. Meanwhile, a large number of novel β-lactamase genes are also reconstructed from bird metagenomic assemblies based on fARGene software.ConclusionsOur study provides a comprehensive overview of the diversity and abundance of ARGs in migratory birds and highlights the possible role of migratory birds as ARG disseminators into the environment. Video abstract.

Dataset Information

Metagenomic analysis of gut microbiome and resistome of diarrheal fecal samples from Kolkata, India, reveals the core and variable microbiota including signatures of microbial dark matter.

Publications

Metagenomic analysis of gut microbiome and resistome of diarrheal fecal samples from Kolkata, India, reveals the core and variable microbiota including signatures of microbial dark matter.

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