Inability of a reserpine-based screen to identify strains overexpressing efflux pump genes in clinical isolates of Staphylococcus aureus

Frempong-Manso, Emmanuel; Raygada, Jose L.; DeMarco, Carmen E.; Seo, Susan M.; Kaatz, Glenn W.

doi:10.1016/j.ijantimicag.2008.10.016

Cited by 29 publications

(22 citation statements)

References 11 publications

(17 reference statements)

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“…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.…”

Section: Methodsmentioning

confidence: 99%

“…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.…”

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confidence: 99%

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

et al. 2013

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The expression of mepA, encoding the Staphylococcus aureus MepA multidrug efflux protein, is repressed by the MarR homologue MepR. MepR dimers bind differently to operators upstream of mepR and mepA, with affinity being greatest at the mepA operator. MepR substitution mutations may result in mepA overexpression, with A103V most common in clinical strains. Evaluation of the functional consequences of this and other MepR substitutions using a lacZ reporter gene assay revealed markedly reduced repressor activity in the presence of Q18P, F27L, G97E, and A103V substitutions. Reporter data were generally supported by susceptibility and efflux assays, and electrophoretic mobility shift assays (EMSAs) confirmed compromised affinities of MepR F27L and A103V for the mepR and mepA operators. One mutant protein contained two substitutions (T94P and T132M); T132M compensated for the functional defect incurred by T94P and also rescued that of A103V but not F27L, establishing it as a limited-range suppressor. The function of another derivative with 10 substitutions was minimally affected, and this may be an extreme example of suppression involving interactions among several residues. Structural correlations for the observed functional effects were ascertained by modeling mutations onto apo-MepR. It is likely that F27L and A103V affect the protein-DNA interaction by repositioning of DNA recognition helices. Negative functional consequences of MepR substitution mutations may result from interference with structural plasticity, alteration of helical arrangements, reduced protein-cognate DNA affinity, or possibly association of MepR protomers. Structural determinations will provide further insight into the consequences of these and other mutations that affect MepR function, especially the T132M suppressor.

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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

et al. 2013

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“…EPIs have contributed substantially to studies of the involvement of efflux pumps in the resistance of clinical isolates (50,125), by demonstrating their ability to reverse resistance. However, a recent study found that reserpine did not accurately predict the overexpression of efflux pumps in Staphylococcus (66).…”

Section: Negating Efflux Pump Activitymentioning

confidence: 96%

Adaptive and Mutational Resistance: Role of Porins and Efflux Pumps in Drug Resistance

2012

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SUMMARY The substantial use of antibiotics in the clinic, combined with a dearth of new antibiotic classes, has led to a gradual increase in the resistance of bacterial pathogens to these compounds. Among the various mechanisms by which bacteria endure the action of antibiotics, those affecting influx and efflux are of particular importance, as they limit the interaction of the drug with its intracellular targets and, consequently, its deleterious effects on the cell. This review evaluates the impact of porins and efflux pumps on two major types of resistance, namely, mutational and adaptive types of resistance, both of which are regarded as key phenomena in the global rise of antibiotic resistance among pathogenic microorganisms. In particular, we explain how adaptive and mutational events can dramatically influence the outcome of antibiotic therapy by altering the mechanisms of influx and efflux of antibiotics. The identification of porins and pumps as major resistance markers has opened new possibilities for the development of novel therapeutic strategies directed specifically against these mechanisms.

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“…Using quantitative reverse transcription-PCR (qRT-PCR), we found that the increased expression of one or more chromosomal MDR efflux pump genes was present in half of all S. aureus bloodstream isolates recovered in 2005 from Detroitarea patients (2,3). However, this approach requires specialized instrumentation and is expensive in terms of both supplies and hands-on time.…”

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confidence: 99%

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

Patel

Kosmidis

Seo

et al. 2010

Antimicrob Agents Chemother

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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.

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Inability of a reserpine-based screen to identify strains overexpressing efflux pump genes in clinical isolates of Staphylococcus aureus

Cited by 29 publications

References 11 publications

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

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

Adaptive and Mutational Resistance: Role of Porins and Efflux Pumps in Drug Resistance

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

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