Efflux in Acinetobacter baumannii can be determined by measuring accumulation of H33342 (bis-benzamide)

Abstract

ObjectivesOverexpression of efflux pumps in Acinetobacter baumannii is a common mechanism of multidrug resistance in this nosocomial pathogen. Increased efflux pump expression is often assumed from MICs of antibiotics and dyes, without measurement of efflux levels. This study describes a safe, rapid and simple 96-well plate assay that measures the accumulation of a fluorescent dye, Hoechst (H) 33342.MethodsThe growth kinetics of three resistant and three susceptible Singaporean clinical isolates of A. baumannii in the presence of carbonyl cyanide-m-chlorophenylhydrazone (CCCP) and phenylalanine-arginine-β-naphthylamide (PAβN) were studied to determine non-inhibitory concentrations for use in the assay. Accumulation of H33342 was measured in these clinical isolates with and without efflux inhibitors. Accumulation was also measured in an adeB efflux pump deletion mutant and its parental strain to assess the ability of the assay to identify altered efflux in strains lacking efflux pumps. Results were compared with data from accumulation assays with ethidium bromide and norfloxacin.ResultsIncreased accumulation, indicative of reduced efflux, was observed in AB211ΔadeB compared with parental strain AB211. Clinical isolates demonstrated different levels of accumulation of H33342. The addition of both CCCP and PAβN significantly increased the accumulation of H33342. The pattern of norfloxacin accumulation broadly reflected H33342 accumulation. Ethidium bromide showed a different pattern of accumulation in clinical isolates.ConclusionsThe measurement of the intracellular accumulation of H33342 in real time allowed a comparison of efflux activity between strains of A. baumannii.

Assessment of Efflux Activity Using H33342 Accumulation in Tigecycline-ResistantAcinetobacter baumanniiClinical Isolates

“…The H33342 accumulation assay was also used to assess the roles of efflux pumps in antimicrobial resistance in A. baumannii clinical isolates [9].…”

“…The H33342 accumulation assay was carried out as described by Richmond et al [9], with the following modifications. For the rpoB gene, we used rpoB-for-1 (5′-ctcactatggtcgtgtttgtc-3′) and rpoB-rev-2 (5′-ccaagaaaccgaagtcattcg-3′).…”

“…It has been used for the assessment of cellular permeability and efflux activity in Salmonella enterica and Escherichia coli [6,7]. H33342 accumulation has also been used in A. baumannii to assess the contribution of efflux activity to antibiotic accumulation [8,9].…”

“…Addition of the efflux pump inhibitor, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), which dissipates the proton motive force required by several efflux pumps, causes a significant increase in H33342 accumulation, suggesting reduced efflux pump activity due to the inhibition of active efflux [9]. Although it was used effectively to assess efflux activity between several strains of bacteria including S. enterica, E. coli and A. baumannii [6,9], it is hard to find out studies using it as an assessing tool of efflux activity in a lot of clinical strains of A.…”

“…Although it was used effectively to assess efflux activity between several strains of bacteria including S. enterica, E. coli and A. baumannii [6,9], it is hard to find out studies using it as an assessing tool of efflux activity in a lot of clinical strains of A.…”