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. 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 β-lactams, aminoglycosides, quinolones and macrolides was found. Eschericia sp. was the major contributor of ARGs. 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.

中文翻译：

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对印度加尔各答腹泻粪便样本的肠道微生物组和耐药组进行宏基因组分析，揭示了核心和可变微生物群，包括微生物暗物质的特征。

肠道微生物组和耐药组的宏基因组分析有助于了解腹泻发病机制和抗菌素耐药性传播 (AMR) 的动态。对来自加尔各答的 20 份腹泻粪便样本进行宏基因组测序，以了解核心和可变的肠道微生物群。其中五个样品用于抗性组分析。该试点研究的目的是确定腹泻肠道中的微生物群特征和抗菌素耐药基因 (ARG) 的来源。使用 Illumina MiSeq 平台进行 16S rRNA 扩增子测序，并使用 MGnify 管道进行分析。基因组分类数据库（GTDB-Tk）用于细菌分类鉴定。通过培养法确定腹泻病因。厚壁菌门、拟杆菌门、变形菌门和放线菌门始终存在于 20 个样品中。厚壁菌门是 11 个样品中最丰富的门。18 个样品中拟杆菌门/厚壁菌门的比例小于 1。观察到584个属。所有 20 个样本中均存在其中 18 个。变形菌门是与霍乱弧菌感染相关的 6 个样本中的优势门。所有样本中操作分类单位（OTU）的保守表明核心微生物组的存在。发现无症状携带霍乱弧菌和幽门螺杆菌等病原体。出现了候选门或“微生物暗物质”的特征。发现共生菌和病原体的细菌家族的相对丰度存在显着相关性。在 Illumina MiSeq 系统上进行全基因组测序 (WGS)，并使用 metaSPAdes v3.9.1 组装原始读数，以研究 5 个样品的抗性组。ABRicate 用于分配 ARG 函数。鉴定出 491 个耐药性决定因素。在 80% 的样本中，四环素耐药性是最丰富的耐药性决定因素。发现了高丰度的针对 β-内酰胺类、氨基糖苷类、喹诺酮类和大环内酯类的 ARG。埃希氏菌属 是 ARG 的主要贡献者。这是第一个针对与不同腹泻病原体相关的肠道微生物组的比较研究。它提出了代表急性腹泻患者核心和可变微生物组的不同细菌分类群的第一个目录。该研究帮助确定了与腹泻相关的肠道微生物群特征的趋势，并揭示了哪些 ARG 大量存在以及导致 AMR 的宏基因组组装基因组 (MAG)。