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Background <p>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 <i>Aeromonas hydrophila</i> KCTC 2358.</p> Methods and results <p>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.</p> Conclusions <p>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 <i>A. hydrophila</i> strains and aquaculture-derived isolates.</p> Graphical abstract <p></p>

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

  • 期刊论文

  • 发表日期：

    2026年7月18日

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  • Si-Heon Song,
  • Eon-Bee Lee

摘要

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.

Graphical abstract