Types I, II, V, and VI secretion system genes in clinical uropathogenic Escherichia coli associate with antibiotic susceptibility, biofilm formation, and persister cells

Abstract

ABSTRACT Uropathogenic Escherichia coli (UPEC) often causes recurrent urinary tract infections, where biofilms and persister cells can promote recalcitrance. This study evaluated whether secretion-system marker genes associate with antimicrobial susceptibility, biofilm formation, and biofilm-associated persistence in clinical urinary isolates. A total of 71 isolates were screened by PCR for fimH and markers of secretion systems: type I (hlyA), type II (gspD), type V (ag43), and type VI (hcp). Minimum inhibitory concentrations (MICs) were measured by Etest, biofilm biomass by crystal violet assay, and persister survival in strong biofilm producers after antibiotic challenge. fimH was detected in 91.5% isolates; gspD and ag43 were common (71.8% and 80.3%), while hlyA and hcp occurred in 32.4% and 46.5%. Marker co-occurrence was frequent, with 19.7% carrying all four secretion markers, and 8.5% with no secretion markers detected. A total of 81.7% isolates formed biofilm, including 18.3% strong producers. Individual marker prevalence did not differ across biofilm categories. Carriage of hlyA and hcp was associated with lower MIC distributions for selected antibiotics (including fosfomycin, ciprofloxacin, and ampicillin-sulbactam), whereas gspD and ag43 showed no consistent MIC associations. The biofilm category was linked to non-susceptibility to ampicillin-sulbactam, but not the other agents tested. Strong biofilm producers showed biphasic killing consistent with persister formation, and hcp carriage tended to align with higher ciprofloxacin survival. These findings map secretion-system markers in clinical UPEC and suggest that a type VI secretion system may track with biofilm-associated persistence. IMPORTANCE Recurrent urinary tract infections (rUTI) often relapse after antibiotic treatment because bacteria can generate persister cells—temporary survivors that tolerate antibiotics without being genetically resistant. Clinicians, therefore, lack markers that flag isolates likely to persist. In 71 clinical urinary Escherichia coli isolates, this study surveyed genes encoding several secretion systems, molecular machines that help bacteria interact with each other and with host tissues. These markers were widespread and frequently co-occurred, but they did not track with stronger biofilm formation. However, a type VI secretion system marker (hcp) was associated with higher survival during ciprofloxacin exposure in strong-biofilm isolates, linking virulence-associated machinery with biofilm-associated persistence. Genetic signatures that reflect persistence, rather than routine resistance, could help prioritize follow-up testing and support development of strategies that target hard-to-eradicate rUTI infections. Recurrent urinary tract infections (rUTI) often relapse after antibiotic treatment because bacteria can generate persister cells—temporary survivors that tolerate antibiotics without being genetically resistant. Clinicians, therefore, lack markers that flag isolates likely to persist. In 71 clinical urinary Escherichia coli isolates, this study surveyed genes encoding several secretion systems, molecular machines that help bacteria interact with each other and with host tissues. These markers were widespread and frequently co-occurred, but they did not track with stronger biofilm formation. However, a type VI secretion system marker (hcp) was associated with higher survival during ciprofloxacin exposure in strong-biofilm isolates, linking virulence-associated machinery with biofilm-associated persistence. Genetic signatures that reflect persistence, rather than routine resistance, could help prioritize follow-up testing and support development of strategies that target hard-to-eradicate rUTI infections.