Comparative Genomics of DH5α-Inhibiting Escherichia coli Isolates from Feces of Healthy Individuals Reveals Common Co-Occurrence of Bacteriocin Genes with Virulence Factors and Antibiotic Resistance Genes

<b>Background/Objectives</b>: The presence of multi-drug-resistant (MDR) bacteria in healthy individuals poses a significant public health concern, as these strains may contribute to or even facilitate the dissemination of antibiotic resistance genes (ARGs) and virulence factors. In this study, we investigated the genomic features of antimicrobial-producing <i>Escherichia coli</i> strains from the gut microbiota of healthy individuals in Singapore. <b>Methods</b>: Using a large-scale screening approach, we analyzed 3107 <i>E. coli</i> isolates from 109 fecal samples for inhibitory activity against <i>E. coli</i> DH5α and performed whole-genome sequencing on 37 representative isolates. <b>Results</b>: Our findings reveal genetically diverse strains, with isolates belonging to five phylogroups (A, B1, B2, D, and F) and 23 unique sequence types (STs). Bacteriocin gene clusters were widespread (92% of isolates carried one or more bacteriocin gene clusters), with colicins and microcins dominating the profiles. Notably, we identified an <i>hcp-et3-4</i> gene cluster encoding an effector linked to a Type VI secretion system. Approximately 40% of the sequenced isolates were MDR, with resistance for up to eight antibiotic classes in one strain (strain D96). Plasmids were the primary vehicles for ARG dissemination, but chromosomal resistance determinants were also detected. Additionally, over 55% of isolates were classified as potential extraintestinal pathogenic <i>E. coli</i> (ExPEC), raising concerns about their potential pathogenicity outside the intestinal tract. <b>Conclusions</b>: Our study highlights the co-occurrence of bacteriocin genes, ARGs, and virulence genes in gut-residing <i>E. coli</i>, underscoring their potential role in shaping microbial dynamics and antibiotic resistance. While bacteriocin-producing strains show potential as probiotic alternatives, careful assessment of their safety and genetic stability is necessary for therapeutic applications.