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

Abstract

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.

Ceftaroline Is Active against Heteroresistant Methicillin-Resistant Staphylococcus aureus Clinical Strains despite Associated Mutational Mechanisms and Intermediate Levels of Resistance

“…Second, in studies published by our group, we showed that ␤-lactam-mediated HeR/HoR selection required increases in both expression of mecA and mutagenesis in order to achieve the selection of the highly resistant HoR phenotype (13,14). In the current study, and based on the mechanism of action of CPT, we determined that mecA plays a pivotal role during the selection process.…”

“…Three independent cultures were sampled in triplicate to minimize error caused by inter-and intrasample variation during the HeR/HoR selection process. Inoculated flasks were incubated at 37°C with shaking at 145 rpm; aliquots of 100 l were taken at different time intervals, including 6, 27, and 33 h, as we previously described (13,14).…”

“…In previous studies, we have demonstrated that the highly resistant HoR-MRSA derivative strains are selected when HeR-MRSA undergoes exposure to subinhibitory concentrations of ␤-lactams (e.g., OXA, 0.5 g/ml) (13,14). Furthermore, we have shown that this process required both mecA expression and SOS-mediated mutagenesis (13).…”

“…CPT maintains activity against MRSA with reduced susceptibility to vancomycin (VAN), including heteroresistant vancomycin-intermediate S. aureus (VISA) and daptomycin (DAP) (11,12). In previous studies, we demonstrated that acquisition of the homoresistant (HoR) phenotype during exposure to subinhibitory concentrations of ␤-lactam antibiotics was associated with both increased expression of the mecA gene and activation of the lexA/recA-SOS response, a conserved regulatory network in bacteria that is induced in response to DNA damage, together leading to high levels of crossresistance to many ␤-lactam antibiotics (13,14). The present results demonstrate that HeR-MRSA, when exposed to subinhibitory concentrations of CPT, activates the SOS response and increases the mutation rate despite inhibition of PBP2a by CPT, a process that results in selection of CPT-selected HoR cells.…”

Antimicrobial Heteroresistance: an Emerging Field in Need of Clarity

“…Heteroresistance to homogeneous high-resistance selection (HeR-HoR selection) by oxacillin was associated with an increased mutation rate and expression of mecA and the SOS response lexA/recA gene regulators (129). Increased expression of the agr (accessory gene regulator) system during HeR-HoR selection was required to tightly modulate SOS-mediated mutation rates, which then led to full expression of homogeneous oxacillin resistance in very heterogeneous clinical MRSA strains (130). The PBP1 protein played a role in SOS-mediated RecA activation and HeR-HoR selection (131).…”