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, 60 (4), 2311-7

Role of the Stringent Stress Response in the Antibiotic Resistance Phenotype of Methicillin-Resistant Staphylococcus Aureus

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Role of the Stringent Stress Response in the Antibiotic Resistance Phenotype of Methicillin-Resistant Staphylococcus Aureus

Sandra Aedo et al. Antimicrob Agents Chemother.

Abstract

Resistance to beta-lactam antibiotics in methicillin-resistantStaphylococcus aureus(MRSA) requires the presence of an acquired genetic determinant,mecAormecC, which encode penicillin-binding protein PBP2A or PBP2A', respectively. Although all MRSA strains share a mechanism of resistance, the phenotypic expression of beta-lactam resistance shows considerable strain-to-strain variation. The stringent stress response, a stress response that results from nutrient limitation, was shown to play a key role in determining the resistance level of an MRSA strain. In the present study, we validated the impact of the stringent stress response on transcription and translation ofmecAin the MRSA clinical isolate strain N315, which also carries known regulatory genes (mecI/mecR1/mecR2andblaI/blaR1) formecAtranscription. We showed that the impact of the stringent stress response on the resistance level may be restricted to beta-lactam resistance based on a "foreign" determinant such asmecA, as opposed to resistance based on mutations in the nativeS. aureusdeterminantpbpB(encoding PBP2). Our observations demonstrate that high-level resistance mediated by the stringent stress response follows the current model of beta-lactam resistance in which the native PBP2 protein is also essential for expression of the resistance phenotype. We also show that theStaphylococcus sciuri pbpDgene (also calledmecAI), the putative evolutionary precursor ofmecA, confers oxacillin resistance in anS. aureusstrain, generating a heterogeneous phenotype that can be converted to high and homogenous resistance by induction of the stringent stress response in the bacteria.

Figures

FIG 1
FIG 1
Impact of the stringent response on expression of PBP2A in S. aureus N315. Membrane proteins were isolated from MRSA strain N315 grown in the absence or presence of a sub-MIC of oxacillin (0.1 μg/ml) and/or mupirocin (0.03 μg/ml) and analyzed by SDS-PAGE (top panel). Relative amounts of PBP2A in the membrane preparations were determined by Western blotting with a monoclonal antibody against PBP2A (bottom panel).
FIG 2
FIG 2
Impact of the stringent stress response on beta-lactam resistance mediated by a native and a foreign determinant of resistance. A population analysis profile of MSSA strain 27s (circles), ceftizoxime-resistant mutant ZOX3 (squares), and strain ZOX3 carrying plasmid-borne mecA (triangles) is shown. Overnight cultures were plated on TSA plates containing various concentrations of ceftizoxime in the absence (solid symbols) or presence (empty symbols) of a constant sub-MIC (0.03 μg/ml) of mupirocin. Colonies were counted after 72 h of incubation at 37°C or 30°C as indicated in Materials and Methods.
FIG 3
FIG 3
High-level resistance mediated by the stringent stress response requires the expression of the native PBP2. (A) MICs of oxacillin were determined at increasing IPTG concentrations in the absence or presence of a constant sub-MIC (0.03 μg/ml) of mupirocin in strain 27sspac::pbpB(pmecA). (B). Membrane proteins were isolated from 27sspac::pbpB(pmecA) grown in the absence or presence of the optimal IPTG concentration (500 μM) and treated with mupirocin or left untreated. The relative amounts of PBP2A in the membrane fractions by Western blotting using a monoclonal antibody against PBP2A were determined.
FIG 4
FIG 4
Transglycosylase activity of PBP2 remains essential for high-level resistance under the stringent stress response. (A) Etests for oxacillin were done in the absence or presence of a sub-MIC (0.03 μg/ml) of mupirocin in the agar medium. Strain COL and its derivative COLTG42 lacking transglycosylase activity of PBP2 were tested. (B) Membrane proteins were isolated from COL and COLTG42 grown in the absence or presence of a sub-MIC (0.03 μg/ml) of mupirocin in the culture. The relative amounts of PBP2A in the membrane fractions by Western blotting using a monoclonal antibody against PBP2A were determined.
FIG 5
FIG 5
Impact of the stringent stress response on beta-lactam resistance mediated by the putative mecA precursor S. sciuri pbpD. A population analysis profile of MSSA strain 476 (squares), strain 476 carrying plasmid-borne S. sciuri pbpD (triangles), and strain 476 carrying plasmid-borne S. sciuri pbpD (circles) plated on TSA plates containing a constant sub-MIC (0.03 μg/ml) of mupirocin is shown. Overnight cultures were plated on TSA plates containing various concentrations of oxacillin. Colonies were counted after 72 h of incubation at 30°C.

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