Ethidium Bromide MIC Screening for Enhanced Efflux Pump Gene Expression or Efflux Activity in Staphylococcus aureus

Abstract

Multidrug resistance efflux pumps contribute to antimicrobial and biocide resistance in Staphylococcus aureus. The detection of strains capable of efflux is time-consuming and labor-intensive using currently available techniques. A simple and inexpensive method to identify such strains is needed. Ethidium bromide is a substrate for all but one of the characterized S. aureus multidrug-resistant (MDR) efflux pumps (NorC), leading us to examine the utility of simple broth microtiter MIC determinations using this compound in identifying efflux-proficient strains. Quantitative reverse transcription-PCR identified the increased expression of one or more MDR efflux pump genes in 151/309 clinical strains (49%). Ethidium bromide MIC testing was insensitive (48%) but specific (92%) in identifying strains with gene overexpression, but it was highly sensitive (95%) and specific (99%) in identifying strains capable of ethidium efflux. The increased expression of norA with or without other genes was most commonly associated with efflux, and in the majority of cases that efflux was inhibited by reserpine. Ethidium bromide MIC testing is a simple and straightforward method to identify effluxing strains and can provide accurate predictions of efflux prevalence in large strain sets in a short period of time.

Molecular Characterization of Fluoroquinolone Resistance of Methicillin –Resistant Clinical Staphylococcus aureus Isolates from Rawalpindi, Pakistan

“…Additional mutations resulting in V615I, S437P, and D646Y substitutions were also observed. In addition to the mutations in gyrase and topoisomerase genes, the contribution of efflux pumps that confer resistance to multiple antimicrobial agents in S. aureus has also been reported [6,18,24,27]. We observed the presence of efflux pump activity in all S. aureus isolates by an increased EtBr uptake and a reduction of antibiotic MICs in the presence of efflux pump inhibitors, CCCP and RES, as reported earlier [8,24,30].…”

Chlorhexidine and Mupirocin Susceptibility of Methicillin-Resistant Staphylococcus aureus Isolates in the REDUCE-MRSA Trial

“…Our results are consistent with several other published surveys that infrequently identified phenotypic or genotypic evidence of reduced susceptibility to CHG in MRSA isolates from U.S. health care facilities (22,23,35). More recently, reports have emerged of (49).…”

“…Third, the intervention period was relatively short (18 months), and results may not be generalizable to longer periods of routine decolonization. Fourth, we looked for only two efflux pump genes; other efflux pumps that can use CHG as a substrate have been described in S. aureus (18,35).…”

“…A real-time PCR assay was developed and was used to identify qacA and qacB (see the Supplemental Data and Table S1 in the supplemental material) (34). Isolates were tested after overnight growth on tryptic soy agar with 25 g/ml ethidium bromide (SigmaAldrich), which was added to provide selective pressure for strains that harbored efflux pumps such as those encoded by qacA and qacB (35). S. aureus SK2355 (qacA), S. aureus SK2725 (qacB) (gifts from Arnold Bayer [36]), and S. aureus NRS123 (qac negative [37]) were included as control strains in every run.…”

The action mechanism of daptomycin

“…Propidium is very similar in structure to ethidium, which is widely used as a model substrate for bacterial efflux systems. 78 These efflux systems are driven by ion gradients or ATP, both of which vanish when daptomycin takes effect. 79 This suggests that penetration of the dye may measure the cell's residual metabolic capacity for powering efflux rather than membrane continuity.…”