Accurate antibiotic accumulation in Enterobacteriaceae isolates expressing efflux pumps

Abstract

In Enterobacteriaceae, antibiotic susceptibility is frequently influenced by mechanisms such as membrane modifications, target site mutations, and enzymatic resistance barriers. Recently, there has been a notable rise in Klebsiella pneumoniae, Escherichia coli, and Enterobacter cloacae isolates exhibiting antibiotic resistance in hospital settings. Of particular concern, some resistant isolates employ membrane-associated resistance mechanisms that significantly lower intracellular antibiotic concentrations, reducing them below the threshold required for therapeutic efficacy. Advancements in methods for quantifying drug accumulation within bacterial cells have provided critical insights into these resistance mechanisms. A key step in these studies relies on cell lysis to release intracellular contents including antibacterial molecules for precise quantification. However, current lysis methods are often time-consuming, underscoring the need for a robust, efficient approach to accurately measure intracellular antibiotic concentrations in isolates exhibiting various levels of efflux pump activity. In this study, we developed a rapid and reliable lysis protocol that minimizes the risk of drug alteration while enabling precise and reproducible measurement of intracellular antibiotic concentrations allowing an evidence-based study of efflux in resistant clinical strains of Enterobacteriaceae. This approach holds significant promise for enhancing our understanding of membrane-associated resistance mechanisms and for informing the optimization of treatment strategies.