Resistance elicited by sub-lethal concentrations of ampicillin is partially mediated by quorum sensing in Pseudomonas aeruginosa

Abstract

The rapid increase of antibiotic resistance is a serious challenge around the world. Antibiotics are present in various environments at sub-lethal concentrations, but how resistance emerges under sub-lethal conditions is not fully clear. In this study, we evolved Pseudomonas aeruginosa PAO1 under sub-lethal conditions, in the presence of either 15-30 μg/mL or 150-300 μg/mL of ampicillin. We found a ~ 5-6 fold increase in the minimum inhibitory concentration (MIC) among evolved isolates exposed to 15-30 μg/mL of ampicillin, and more than a 19-fold of increase in 150-300 μg/mL of ampicillin exposure. DNA sequencing revealed that mpl and ampD were frequently mutated in these resistant strains. We performed a transcriptome analysis of deletion mutations of mpl or ampD, compared to PAO1. Both showed a two-fold increase in expression of quorum sensing (QS) genes including lasR and rhlI/R; the heightened expression was positively correlated with the expression of the ampicillin resistance gene ampC. We queried if quorum sensing contributes to the increase in the ampicillin MIC. After adding the quorum quencher acylase I, the growth yield both decreased by roughly 50% for Δmpl in 2000 μg/mL of ampicillin and ΔampD in 4000 μg/mL of ampicillin. Addition of the QS signals into synthase mutants restored the higher MIC, but only for the rhlI/R circuit. This study highlights the involvement of QS in antibiotic resistance evolution, and shows the multifactorial contributors to the observed phenotypes.

Keywords: Ampicillin; Pseudomonas aeruginosa; Quorum sensing; Resistance evolution; Sub-lethal exposure.