Differential antimicrobial responses in polymicrobial triple-species biofilms associated with persistent urinary tract infections.

Polymicrobial urinary tract infections (UTIs) pose significant treatment challenges due to interspecies interactions within biofilms. To address this, we developed a static, triple-species biofilm model comprising Escherichia coli, Enterococcus faecalis, and Candida albicans to simulate recurrent UTI communities. Biofilms were treated with ampicillin, ciprofloxacin, fluconazole, ethylenediaminetetraacetic acid (EDTA), and N-acetylcysteine (NAC), and evaluated for biomass (crystal violet assay) and viability (CFU/mL). Ampicillin and ciprofloxacin reduced bacterial viability by > 90%, but had limited impact on C. albicans, while EDTA disrupted biomass by ≈ 45% and variably reduced viability across species. Fluconazole elicited non-monotonic effects on biomass and viability, and NAC increased microbial viability despite structural disruption. These results reveal that biofilm disruption did not consistently correlate with reductions in microbial viability in this model, highlighting the importance of evaluating both biomass and viable cell counts when assessing antimicrobial effects on polymicrobial biofilms relevant to persistent UTIs.