Genomic profiling of ESBL/AmpC-producing <i>Escherichia coli</i> from backyard poultry: resistome, virulome, plasmidome, and CRISPR-Cas insights
Abstract
<h4>Introduction</h4>Antimicrobial resistance (AMR) is a major One Health concern driven by many factors including unregulated animal farming. This study aimed to perform a comprehensive phenotypic and genotypic characterization of <i>Escherichia coli</i> isolated from backyard poultry reared in households in rural setup in southern India.<h4>Methods</h4>A total of 48 cloacal samples were collected from poultry birds across 12 epidemiological units using a 30-cluster sampling strategy. <i>E. coli</i> isolation was performed using standard microbiological methods and confirmed by species-specific multiplex PCR. Of the 48 samples, 45 yielded confirmed <i>E. coli</i> isolates, among which 18 ESBL- and/or AmpC-positive isolates were selected for whole genome sequencing (WGS). Genomic analyses included detection of antimicrobial resistance genes, virulence-associated genes, mobile genetic elements, plasmid replicons, biofilm-associated genes, multilocus sequence typing, serotyping, CH typing, Clermont phylogrouping, CRISPR-Cas profiling, and SNP-based phylogenetic analysis.<h4>Results</h4>A total of 48 cloacal samples yielded 45 confirmed isolates of which 18 detected with ESBL genes including <i>bla</i> <sub>SHV</sub> (28.8%), <i>bla</i> <sub>TEM</sub> (26.6%), and <i>bla<sub>CTX-M</sub></i> (20%) along with plasmid-mediated <i>AmpC</i> genes (11.11%). Resistome profiling revealed diverse ARGs such as <i>bla</i> <sub><i>CTX-M-15</i>,</sub> <i>bla</i> <sub>DHA-1</sub>, <i>qnrS/B, tet(A), sul1/2/3, dfrA</i> var<i>iants,</i> and <i>mphA/B,</i> in addition to intrinsic efflux systems (<i>acr, emr, mdt, mar</i>). Virulome analysis showed conserved genes associated with adhesion <i>(fim, csg, ecp),</i> iron acquisition <i>(ent, fep, fes, ybt),</i> and stress response <i>(gad, hlyE, ompT, iss).</i> CRISPR-Cas analysis revealed a high prevalence of Type I-E arrays (83.3%) coexisting with multiple plasmids. MLST and CH typing revealed high genetic diversity across 12 sequence types, including ST10, ST48, ST3107, and ST226. SNP-based phylogeny placed isolates mainly within commensal phylogroups A and B1, with relatedness to global strains. All sequenced isolates were multidrug-resistant, with significant <i>β</i>-lactam resistance enrichment (Z = 3.46, <i>p</i> < 0.001). ARG distribution differed significantly among phylogroups (<i>p</i> = 0.036), and Simpson's diversity index (D = 0.94) indicated marked clonal heterogeneity. Strong plasmid-ARG associations (Cramér's V > 0.5) suggested plasmid-mediated resistome structuring.<h4>Discussion</h4>Backyard poultry-derived <i>E. coli</i> showed multidrug resistance, genetic diversity, and virulence-associated traits, highlighting backyard poultry as a potential reservoir for AMR dissemination at the human-animal-environment interface.