Abstract

Shiga toxin-producing Escherichia coli (STEC) is a zoonotic pathogen of global relevance whose persistence in food processing environments is frequently mediated by biofilm formation. The acquisition of stx prophages and associated regulatory mutations can impose adaptive restrictions on this phenotype. This study analyzed 216 whole-genome sequences (WGS) to investigate the genomic determinants of biofilm formation in a diverse collection of E. coli isolates (STEC and non-STEC) isolated from cattle feedlots. Genomes were characterized for serogroup, MLST, stx subtyping, integrity of the mlrA and rpoS regulators, single nucleotide polymorphisms (SNPs) in the csg/bcs operons, antimicrobial resistance (AMR) genes, and plasmid replicons. The biofilm phenotype was quantitatively evaluated using a crystal violet assay at 15 °C for 96 h. Results demonstrated higher biofilm forming ability among non-STEC isolates compared to STEC strains, with strains simultaneously carrying both stx1 and stx2 exhibiting the lowest prevalence of biofilm formation (3.3%). Although the rpoS mutation did not show a significant overall association (p = 0.354) with biofilm formation, it was universally present across all O157:H7 isolates, and likely enhanced mlrA disruption toward csgD repression. In addition, stx-positive isolates accumulated significantly more SNPs in the curli (csg) and cellulose (bcs) structural genes. Exploratory gene-level association tests and multiple correspondence analysis further indicated that stx status was associated with broader differences in non-stx accessory gene composition. In addition, there was no statistical association between AMR classes and biofilm-forming capacity. Furthermore, STEC strains demonstrated a lower frequency of resistance to aminoglycosides, phenicols, and tetracyclines. Finally, the plasmid replicons IncX1 and IncFII(pSE11) were present and associated with biofilm-positive isolates. In conclusion, biofilm suppression in STEC suggests a multifactorial and evolutionary phage-induced regulatory trade-off, whereas biofilm persistence in non-STEC strains is independently driven by mobile genetic elements and potential accessory determinants.

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