Non-uniform susceptibility profiles observed in <i>Staphylococcus aureus</i> , <i>Pseudomonas aeruginosa</i> , and <i>Acinetobacter baumannii</i> toward both front-line and novel cationic biocides with diverse structural compositions

ABSTRACT Heteroresistance, a phenotype of bacterial subpopulations that display differential susceptibility to antimicrobial agents, is a pressing problem that results in antibiotic treatment failure and potentially in resistance development. However, this phenotype remains largely unexplored in the context of biocides. Here, using population analysis profiling (PAP) assays, we investigated susceptibility patterns in Staphylococcus aureus , Acinetobacter baumannii , and Pseudomonas aeruginosa toward four healthcare-associated commercial cationic biocides (benzalkonium chloride, didecyl dimethyl ammonium chloride, octenidine, and chlorhexidine) and four structurally diverse, non-commercial cationic compounds. These agents exhibited a broad range of antimicrobial activity, with minimal inhibitory concentrations (MICs) for commercial biocides ranging from 1 to 125 µM and for the novel compounds from 0.5 to 8 µM across the species tested. Despite this inhibitory activity, bacterial subpopulations capable of growth at concentrations ≥8-fold higher than the MIC of the dominant population were observed at frequencies of approximately 10⁻⁷ to 10⁻⁶, phenotypically analogous to heteroresistance. Loss of this heteroresistance-like phenotype in efflux-deficient mutants may suggest that efflux systems contribute to this phenotype. Notably, it was observed that a hospital-associated S. aureus strain exhibited higher frequencies of these less susceptible bacterial subpopulations compared to community-associated and laboratory strains. Overall, these findings demonstrate that non-uniform susceptibility to cationic biocides, similar to the heteroresistance phenotype observed toward antibiotics, can be detected under defined experimental conditions. Given that in-use biocide concentrations are typically substantially higher than the MICs reported here, the clinical and infection prevention implications of this phenotype remain uncertain and warrant further investigation. IMPORTANCE Heteroresistance is a phenotype of growing concern in healthcare settings, contributing to antimicrobial resistance and antibiotic treatment failure. Yet, the development of heteroresistance-like phenotypes toward commercial biocides, prevalent in healthcare settings, remains understudied. Herein, we show that heteroresistance-like phenotypes similar to those observed in response to antibiotic treatment are observed toward both commercial and non-commercial biocides, and these phenotypes warrant future investigation.