Multidrug efflux pump overexpression in Staphylococcus aureus after single and multiple in vitro exposures to biocides and dyes

Abstract

Biocides and dyes are commonly employed in hospital and laboratory settings. Many of these agents are substrates for multiple-drug resistance (MDR)-conferring efflux pumps of both Gram-positive and Gram-negative organisms. Several such pumps have been identified in Staphylococcus aureus, and mutants overexpressing the NorA and MepA MDR pumps following exposure to fluoroquinolones have been identified. The effect of exposure to low concentrations of biocides and dyes on the expression of specific pump genes has not been evaluated. Using quantitative reverse-transcription PCR we found that exposure of clinical isolates to low concentrations of a variety of biocides and dyes in a single step, or to gradually increasing concentrations over several days, resulted in the appearance of mutants overexpressing mepA, mdeA, norA and norC, with mepA overexpression predominating. Overexpression was frequently associated with promoter-region or regulatory protein mutations. Mutants having significant increases in MICs of common pump substrates but no changes in expression of studied pump genes were also observed; in these cases changes in expression of as-yet-unidentified MDR pump genes may have occurred. Strains of S. aureus that exist in relatively protected environments and are repeatedly exposed to sublethal concentrations of biocides can develop efflux-related resistance to those agents, and acquisition of such strains poses a threat to patients treated with antimicrobial agents that are also substrates for those pumps, such as ciprofloxacin and moxifloxacin.

Skin bacteria after chlorhexidine exposure—is there a difference in response to human β-Defensin-3?

“…Huet et al demonstrated that in S. aureus, sin-296 gle as well as multiple step-exposure to biocides, which were comparable to CH, resulted in 297 an increase in the MIC for CH and multiple other biocides mostly due to an upregulation of 298 the gene expression of different MDR efflux pumps like mepA and norA [32]. These results 299…”

Functional Consequences of Substitution Mutations in MepR, a Repressor of the Staphylococcus aureus mepA Multidrug Efflux Pump Gene

“…The same approach was used to quantitate mepR expression in strains found to overexpress mepA in conjunction with a MepR substitution mutation (Table 2). qRT-PCR data for the 2005 strain set and selected in vitro-generated mepA-overexpressing mutants have been reported previously (10)(11)(12). For in vitro-derived mutants, the same procedures were used with the exception that gene expression was normalized to that in the appropriate parent strain.…”

“…Clinical isolates of S. aureus (one per patient) were collected in 2005 and again in 2009 from the microbiology laboratories of the Detroit Medical Center and the John D. Dingell VA Medical Center in Detroit, MI (n ϭ 232 and 243, respectively). The 2005 strain set has been described previously, as have in vitro-derived mutants of selected strains from this set found to overexpress mepA (10,12). Another 446 unique clinical isolates were collected from centers in Boston, MA, Houston, TX, Omaha, NE, and Freiberg, Germany, in 2009 (14).…”

“…This procedure has been described previously and utilizes triplicate cultures of test organisms growing from exponential to stationary phase over a 10-h period. At specified time intervals (every 30 min from 1 to 5 h and then at 6, 8, and 10 h), duplicate aliquots are removed for a determination of ␤-galactosidase activity, which correlates with expression of the gene to which lacZ is transcriptionally fused (12). All strains except those possessing substitution mutations at position A103 were grown in the absence and presence of 0.05 g/ml of tetracycline to quantitate the effect of induction of plasmid-based mepR expression.…”

“…Cooperative binding of MepR dimers to each signature motif in the mepA operator may be the basis for both the greater affinity and easier substrate induction at this site, perhaps in a manner similar to the interaction of QacR with the qacA operator (9). Using quantitative reverse transcription-PCR (qRT-PCR) we have identified numerous clinical and laboratory strains of S. aureus that overexpress mepA (6,(10)(11)(12)(13)(14). Understanding the mechanism(s) of mepA overexpression may provide clues for the design of means to overcome its potential deleterious clinical consequences.…”