Genomic features of three major diarrhoeagenic Escherichia coli pathotypes in India

Abstract

Background. Diarrhoea remains a major threat to children in developing nations, with diarrhoeagenic Escherichia coli (DEC) being the primary causative agent. Characterizing prevalent DEC strains is crucial, yet comprehensive genomic analyses of major DEC strains, including enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC) and enterotoxigenic E. coli (ETEC), are lacking in India.Methods. We sequenced 24 EAEC and 23 EPEC strains from Indian patients with diarrhoea and conducted an extensive database search for DEC human isolates from India. Detailed phylogenetic analyses, virulence gene subtyping and examinations of accessory virulence and antimicrobial resistance (AMR) genes were performed.Results. The analysed DEC strains included 32 EAEC, 25 EPEC, 32 ETEC and 1 each of the EPEC/ETEC-hybrid and ETEC/EAEC-hybrid pathotypes. These strains were predominantly classified into phylogroups A (35.2%) and B1 (41.8%) and dispersed within these phylogroups without pathotype-specific clustering. One ETEC strain was classified into cryptic clade 1. Subtypes of hallmark virulence genes varied substantially amongst strains in each pathotype, and 31 accessory virulence genes were detected either specifically within certain pathotypes or across multiple pathotypes at varying frequencies, indicating diversification of the virulence gene repertoire within each pathotype. Acquired AMR genes were found in 73.6% of the strains, with frequent identification of AMR genes for aminoglycosides (40.0%), β-lactams (64.8%), sulphonamides (49.5%) and trimethoprim (42.9%). Known quinolone-resistant mutations were found in 74.7% of the strains, whereas AMR genes for macrolide (30.8%), phenicol (11.0%) and tetracycline (27.4%) were less frequent.Conclusions. The diverse virulence potential and trends in AMR gene prevalence amongst major DEC strains in India are highlighted in this study. Continuous monitoring of DEC strain characteristics is essential for the effective control and treatment of DEC infections in India.

Keywords: India; antimicrobial resistance; diarrhoeagenic Escherichia coli; genome; virulence gene.

Fig. 1.. Phylogenomic relationships of Indian DEC strains and the distribution of virulence genes, AMR genes and plasmid replicons in these strains. The ML tree of 91 isolates from ETEC, EPEC and EAEC was constructed based on 212,286 SNP sites located on 2,917 core genes. Information about the phylogroup, ST and serotype and the distribution of virulence genes, acquired AMR genes, plasmid replicons and QRDR mutations for each strain is also provided.

Fig. 2.. Distribution of accessory virulence genes in the DEC strains. The distribution of 31 accessory virulence genes across each DEC pathotype and non-DEC isolate is illustrated. Gene presence or absence was determined through blastn homology analysis, employing thresholds of 50% sequence identity and 50% coverage.

Fig. 3.. Distribution of non-LEE effectors in the EPEC strains. The prevalence of non-LEE-encoded effectors in the EPEC strains is depicted. Gene presence or absence was determined through blastn homology analysis, employing thresholds of 50% sequence identity and 50% coverage.

Fig. 4.. Prevalence of strains carrying AMR genes amongst the EAEC, EPEC and ETEC strains. Bar graphs indicate the percentage of strains carrying different numbers of AMR genes in each pathotype of strains. The acquired AMR genes were identified using ABRicate with the default settings and the ARG-ANNOT database.

Fig. 5.. Distribution of acquired AMR genes and mutations in the QRDRs of DEC strains. Bar graphs show the prevalence of each resistance gene in the DEC and non-DEC isolates. The acquired AMR genes were identified using ABRicate with the default settings and the ARG-ANNOT database. Mutations within the QRDRs of gyrA, gyrB, parC and parE were analysed using the NCBI Antimicrobial Resistance Gene Finder with default settings. Only mutations with experimentally confirmed quinolone resistance are shown.

Fig. 6.. Co-occurrence network analysis of 35 AMR genes and 26 plasmid replicons identified in 91 DEC isolates. The network diagram was edge-weighted according to the frequency of gene co-occurrences, with edge widths representing co-occurrence strength and node sizes reflecting the detection frequency of each gene. The four communities identified are indicated by differently coloured edges.