Prevalence of noncanonical virulence factors in the adherence and invasion by enterotoxigenic Escherichia coli in the small and large intestines

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Introduction Enterotoxigenic Escherichia coli (ETEC) expresses a diverse set of noncanonical genes that increase its virulence and are involved in mucin degradation, epithelial invasion, and biofilm formation. Collectively, these factors enable effective intestinal colonization, leading to watery diarrhea. Aim This study investigated the prevalence of the noncanonical genes tia , tib A, and leo A and their effects on the ability of ETEC to adhere, invade, and form biofilms. Materials and methods A total of 244 ETEC strains were analyzed by PCR to confirm the presence of noncanonical virulence genes ( tia , tibA , and leoA ) and toxins, including heat-labile toxins (LTs) and/or heat-stable toxins (STs). The ability of the strains to adhere to and invade HT-29 colorectal adenocarcinoma cells (large intestine) and HuTu80 duodenal adenocarcinoma cells (small intestine) was evaluated, along with their ability to form biofilms. The susceptibility of these strains to 18 antibiotics was evaluated in accordance with the Clinical & Laboratory Standards Institute (CLSI) 2025 guidelines. Results Our data revealed that the tia , tibA and leoA genes were amplified in 11.34% (11/97), 15.46% (15/97), and 7.21% (7/97) of the ETEC strains, respectively. ETEC tia + strains showed adherence values ranging from 3.6 × 10 5 to 34.1 × 10 5 CFUs/mL in HT-29 cells and from 4.1 × 10 5 to 16.6 × 10 5 CFUs/mL in HuTu80 cells. ETEC tib A + strains exhibited adherence values ranging from 11.16 × 10 5 to 65.1 × 10 5 CFUs/mL in HT-29 cells and from 5.6 × 10 5 to 27.1 × 10 5 CFUs/mL in HuTu80 cells. Among the ETEC tia + strains, 54.54% (6/11) were invasive, with invasion frequencies ranging 0.17% and 1.01% in HT-29 cells and between 0.20% and 0.404% in HuTu80 cells. Between the strains expressing the tib A gene, 47.05% carried both LTs and STs. Among the ETEC tib A + strains, 20% were strongly biofilm-producing, 13.33% were moderately biofilm-producing, and 66.66% were weakly biofilm-producing. ETEC tia + /tib A + strains exhibited high rates of multidrug resistance (MDR). Conclusions Our data suggest that noncanonical ETEC strains are highly efficient because their adhesion genes ( tia , tib A, and leo A), toxins ( lt and st ), and adherence and biofilm-forming capabilities work together to cause infection, even in the absence of some classical virulence factors.

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