Reduced susceptibility of Staphylococcus aureus to vancomycin and platelet microbicidal protein correlates with defective autolysis and loss of accessory gene regulator (agr) function

Abstract

Loss of agr function, vancomycin exposure, and abnormal autolysis have been linked with both development of the GISA phenotype and low-level resistance in vitro to thrombin-induced platelet microbicidal proteins (tPMPs). We examined the potential in vitro interrelationships among these parameters in well-characterized, isogenic laboratory-derived and clinical Staphylococcus aureus isolates. The laboratory-derived S. aureus strains included RN6607 (agrII-positive parent) and RN6607V (vancomycin-passaged variant; hetero-GISA), RN9120 (RN6607 agr::tetM; agr II knockout parent), RN9120V (vancomycin-passaged variant), and RN9120-GISA (vancomycin passaged, GISA). Two serial isolates from a vancomycin-treated patient with recalcitrant, methicillin-resistant S. aureus (MRSA) endocarditis were also studied: A5937 (agrII-positive initial isolate) and A5940 (agrII-defective/hetero-GISA isolate obtained after prolonged vancomycin administration). In vitro tPMP susceptibility phenotypes were assessed after exposure of strains to either 1 or 2 mug/ml. Triton X-100- and vancomycin-induced lysis profiles were determined spectrophotometrically. For agrII-intact strain RN6607, vancomycin exposure in vitro was associated with modest increases in vancomycin MICs and reduced killing by tPMP, but no change in lysis profiles. In contrast, vancomycin exposure of agrII-negative RN9120 yielded a hetero-GISA phenotype and was associated with defects in lysis and reduced in vitro killing by tPMP. In the clinical isolates, loss of agrII function during prolonged vancomycin therapy was accompanied by emergence of the hetero-GISA phenotype and reduced tPMP killing, with no significant change in lysis profiles. An association was identified between loss of agrII function and the emergence of hetero-GISA phenotype during either in vitro or in vivo vancomycin exposure. In vitro, these events were associated with defective lysis and reduced susceptibility to tPMP. The precise mechanism(s) underlying these findings is the subject of current investigations.

FIG. 1.

(A) Population analysis of MSSA laboratory strains. Strains: RN6607, agr group II wild-type parent; RN9120, agr group II knockout (agr::tetM derived from RN6607); RN6607V, RN6607 grown in vancomycin at 1 μg/ml; RN9120V, RN9120 grown in vancomycin at 1 μg/ml; RN9120-GISA, RN9120 serially passaged in increasing vancomycin concentrations of up to 10 μg/ml. (Reprinted from the Journal of Infectious Diseases [37] with permission of the publisher.) (B) Population analysis of serial clinical MRSA isolates from a patient with aortic valve endocarditis. Strains: A5937, initial bloodstream isolate; A5940, valve tissue isolate after 50 days of vancomycin therapy. In this and the subsequent figures, all data are presented as the means of at least two experimental runs. Standard error bars were not included to improve the clarity and presentation of these figures.

FIG. 2.

Lysis curves of laboratory strains in vancomycin at 50 μg/ml. The results give the OD of the bacterial suspension over time as a percentage of the OD at time zero.

FIG. 3.

Lysis curves of clinical MRSA strains A5937 (early infective endocarditis [IE]) and A5940 (late IE) in vancomycin at 50 μg/ml. The results denote the OD of the bacterial suspension over time as a percentage of the OD at time zero.

FIG. 4.

Triton lysis curves of study strains. (A) RN6607 and RN6607V; (B) RN9120, RN9120V, and RN9120-GISA; (C) A5937 and A5940.