Repeated sub-MIC exposure and biofilm derivation are associated with resistance development and reduced bactericidal susceptibility in Aeromonas hydrophila KCTC 2358.

BACKGROUND: Repeated exposure to sub-minimum inhibitory concentrations (sub-MICs) of antimicrobials can impose selective pressure on bacterial pathogens, potentially promoting adaptive responses beyond classical resistance. This study investigated whether sequential sub-MIC exposure, followed by biofilm derivation, is associated with resistance development and phenotypes consistent with reduced bactericidal susceptibility in Aeromonas hydrophila KCTC 2358.

METHODS AND RESULTS: A parental population (R0) was subjected to ten cycles of sub-MIC exposure to generate an adapted population (R10), followed by biofilm derivation. Antimicrobial susceptibility, growth kinetics, post-antibiotic effect (PAE), INT reduction activity, biofilm formation, and gene expression were evaluated under oxytetracycline, florfenicol, and enrofloxacin. Serial sub-MIC exposure resulted in stepwise increases in minimum inhibitory concentration and disproportionate increases in minimum bactericidal concentration, leading to elevated MBC/MIC ratios. Biofilm-derived populations exhibited further increases in bactericidal thresholds and sustained growth under high antimicrobial concentrations. Time-kill and PAE analyses revealed reduced growth suppression and accelerated regrowth, with consistently negative PAE observed in biofilm-derived R10 populations. Population-level INT reduction activity remained higher under antimicrobial stress. Notably, high oxytetracycline concentrations led to an increase in residual biofilm biomass, suggesting retention of biofilm-associated material rather than definitive evidence of tolerant subpopulations or a classical Eagle effect.

CONCLUSIONS: Collectively, these findings indicate that repeated sub-MIC exposure and biofilm-associated growth are associated with reduced bactericidal susceptibility and increased residual biofilm biomass. These findings provide a proof-of-concept laboratory observation that subinhibitory antimicrobial exposure may contribute to reduced susceptibility-associated phenotypes, highlighting the need for further validation using multiple A. hydrophila strains and aquaculture-derived isolates.