Identification and Phenotypic Characterization of a β-Lactam-Dependent, Methicillin-Resistant
            <i>Staphylococcus aureus</i>
            Strain

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Methicillin-resistant Staphylococcus aureus (MRSA) strains are characterized by a heterogeneous expression of resistance. We have previously shown in clinical oxacillin-susceptible, mecA-positive MRSA strains that selection from a very heterogeneous (HeR) to highly homogeneous (HoR) resistant phenotype was mediated by acquisition of mutations through an oxacillin-induced SOS response. In the present study, we used a spotted DNA microarray to evaluate differential gene expression during HeR-HoR selection and found increased expression of the agr two-component regulatory system. We hypothesized that increased expression of agr represents a mechanistically relevant component of this process. We demonstrated that inactivation of agr during the HeR-HoR selection process results in a significant increase in mutation rate; these effects were reversed by complementing the agr mutant. Furthermore, we found that extemporal ectopic expression of agr and, more specifically, RNAII in agr-null mutant HeR cells suppressed mutation frequency and the capacity of these cells to undergo the HeR-HoR selection. These findings sustain the concept that increased expression of agr during HeR-HoR selection plays a critical role in regulating the ␤-lactam-induced increased mutation rate in very heterogeneous MRSA strains. Moreover, they indicate that a temporally controlled increase in agr expression is required to tightly modulate SOS-mediated mutation rates, which then allows for full expression of oxacillin homogeneous resistance in very heterogeneous clinical MRSA strains.

Section: Differential Gene Expression Analysis Between Sa13011-her Anmentioning

confidence: 61%

Section: Materials and Mediamentioning

confidence: 99%

Fate of Mutation Rate Depends on agr Locus Expression during Oxacillin-Mediated Heterogeneous-Homogeneous Selection in Methicillin-Resistant Staphylococcus aureus Clinical Strains

Plata

Rosato

2011

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“…SCCmec type IV lacks mecI, whereas SCCmec type II contains mecI and mecR1. However, most SCCmec type II-containing strains have defective mecA repression due to point mutations either in the mecA promoter or in the mecI open reading frame (ORF); the function of mecI and mecR1 is replaced by blaI and blaR1, which has less effective repression and more effective induction of mecA (13,16). It remains to be determined whether expression profiles of mecA, mecI and mecR1, or blaI VOL.…”

Section: Figmentioning

confidence: 99%

Daptomycin-Oxacillin Combinations in Treatment of Experimental Endocarditis Caused by Daptomycin-Nonsusceptible Strains of Methicillin-Resistant Staphylococcus aureus with Evolving Oxacillin Susceptibility (the “Seesaw Effect”)

Yang

Xiong

Boyle-Vavra

et al. 2010

117

109

In vivo development of daptomycin resistance (DAP r ) among Staphylococcus aureus strains, especially methicillin-resistant S. aureus (MRSA) strains, in conjunction with clinical treatment failures, has emerged as a major problem. This has raised the question of DAP-based combination regimens to enhance efficacy against such strains. We studied five recent DAP-susceptible (DAP s )/DAP r clinical MRSA strain pairs obtained from patients who failed DAP monotherapy regimens, as well as one DAP s /DAP r MRSA strain pair in which the resistant strain was generated by in vitro passage in DAP. Of note, we identified a DAP-oxacillin (OX) "seesaw" phenomenon in vitro in which development of DAP r was accompanied by a concomitant fall in OX resistance, as demonstrated by 3-to 4-fold decreases in the OX MIC, a susceptibility shift by population analyses, and enhanced early killing by OX in time-kill assays. In addition, the combination of DAP and OX exerted modest improvement in in vitro bactericidal effects. Using an experimental model of infective endocarditis and two DAP s /DAP r strain pairs, we demonstrated that (i) OX monotherapy was ineffective at clearing DAP r strains from any target tissue in this model (heart valve, kidneys, or spleen) and (ii) DAP-OX combination therapy was highly effective in DAP r strain clearances from these organs. The mechanism(s) of the seesaw effect remains to be defined but does not appear to involve excision of the staphylococcal cassette chromosome mec (SCCmec) that carries mecA.

“…RNA extractions for real-time reverse transcription-PCR (RT-PCR) and Northern blot analyses were performed as previously described (12,19,44). Total RNA was extracted using an RNeasy isolation kit (Qiagen); all RNA samples were analyzed by A 260 /A 280 spectrophotometry and gel electrophoresis to assess concentration and integrity and cleaned of potential DNA contamination by treating them with DNase as per manufacturer recommendations (Ambion, Inc., Austin, TX).…”

Section: Materials and Mediamentioning

confidence: 99%

“…Microarray transcriptional profiling was carried out as previously described (12,19) The comparisons were performed between DS and DR MRSA isogenic pairs grown in drug-free LB medium collected at exponential growth (log) phase (OD 600 , 0.6). The results are based on a series of statistical analyses (filtering) where ratios of Cy3 and Cy5 were converted to log 2 values and the cutoff was set at above 1 (present) or below Ϫ1 (absent) (8,12).…”

Section: Materials and Mediamentioning

confidence: 99%

VraSR Two-Component Regulatory System Contributes to mprF -Mediated Decreased Susceptibility to Daptomycin in In Vivo -Selected Clinical Strains of Methicillin-Resistant Staphylococcus aureus

Mehta

Cuirolo

Plata

et al. 2012

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112

118

Daptomycin (DAP) is a new class of cyclic lipopeptide antibiotic highly active against methicillin-resistant Staphylococcus aureus (MRSA) infections. Proposed mechanisms involve disruption of the functional integrity of the bacterial membrane in a Cadependent manner. In the present work, we investigated the molecular basis of DAP resistance in a group of isogenic MRSA clinical strains obtained from patients with S. aureus infections after treatment with DAP. Different point mutations were found in the mprF gene in DAP-resistant (DR) strains. Investigation of the mprF L826F mutation in DR strains was accomplished by inactivation and transcomplementation of either full-length wild-type or mutated mprF in DAP-susceptible (DS) strains, revealing that they were mechanistically linked to the DR phenotype. However, our data suggested that mprF was not the only factor determining the resistance to DAP. Differential gene expression analysis showed upregulation of the two-component regulatory system vraSR. Inactivation of vraSR resulted in increased DAP susceptibility, while complementation of vraSR mutant strains restored DAP resistance to levels comparable to those observed in the corresponding DR wild-type strain. Electron microscopy analysis showed a thicker cell wall in DR CB5012 than DS CB5011, an effect that was related to the impact of vraSR and mprF mutations in the cell wall. Moreover, overexpression of vraSR in DS strains resulted in both increased resistance to DAP and decreased resistance to oxacillin, similar to the phenotype observed in DR strains. These results support the suggestion that, in addition to mutations in mprF, vraSR contributes to DAP resistance in the present group of clinical strains. Staphylococcus aureus is the most common Gram-positive pathogen among skin and soft tissue infections (2). Methicillin resistance in S. aureus is mediated by the acquisition of a penicillin-binding protein (PBP), PBP 2a, which has decreased affinity for ␤-lactam antibiotics but can continue to cross-link the cell wall once the native PBPs (i.e., PBPs 1 to 4) have been inactivated (23). A distinctive feature for most methicillin-resistant S. aureus (MRSA) strains is the heterogeneous expression of resistance, characterized by a small proportion (Յ0.1%) of the population expressing a high level of homogeneous resistance while most of the other isolates in the population express resistance to 10 g/ml (12, 15, 43). Daptomycin (DAP) is a cyclic anionic lipopeptide antibiotic that is produced by Streptomyces roseosporus (3) and is approved for treatment of skin and skin structure infections as well as treatment of bacteremia and right-side endocarditis caused by MRSA (1). The mechanism of action involves disruption of cytoplasmic membrane function, resulting in depolarization and cell death due to disruption of critical metabolic functions, such as protein, DNA, and RNA synthesis (2).The incidence of DAP resistance in clinical isolates is very low, and resistant strains display small increases in MIC (2). The exact m...