Abstract
Vancomycin is often the preferred treatment for invasive methicillin-resistant Staphylococcus aureus (MRSA) infection. With the increase in incidence of MRSA infections, the use of vancomycin has increased and, as feared, isolates of vancomycin-resistant Staphylococcus aureus (VRSA) have emerged. VRSA isolates have acquired the entercoccal vanA operon contained on transposon (Tn) 1546 residing on a conjugal plasmid. VraTSR is a vancomycin and β-lactam-inducible three-component regulatory system encoded on the S. aureus chromosome that modulates the cell-wall stress response to cell-wall acting antibiotics. Mutation in vraTSR has shown to increase susceptibility to β-lactams and vancomycin in clinical VISA strains and in recombinant strain COLVA-200 which expresses a plasmid borne vanA operon. To date, the role of VraTSR in vanA operon expression in VRSA has not been demonstrated. In this study, the vraTSR operon was deleted from the first clinical VRSA strain (VRS1) by transduction with phage harvested from a USA300 vraTSR operon deletion strain. The absence of the vraTSR operon and presence of the vanA operon were confirmed in the transductant (VRS1Δvra) by PCR. Broth MIC determinations, demonstrated that the vancomycin MIC of VRS1Δvra (64 µg/ml) decreased by 16-fold compared with VRS1 (1024 µg/ml). The effect of the vraTSR operon deletion on expression of the van gene cluster (vanA, vanX and vanR) was examined by quantitative RT-PCR using relative quantification. A 2–5-fold decreased expression of the vanA operon genes occured in strain VRS1Δvra at stationary growth phase compared with the parent strain, VRS1. Both vancomycin resistance and vancomycin-induced expression of vanA and vanR were restored by complementation with a plasmid harboring the vraTSR operon. These findings demonstrate that expression in S. aureus of the horizontally acquired enterococcal vanA gene cluster is enhanced by the staphylococcal three-component cell wall stress regulatory system VraTSR, that is present in all S. aureus strains.
Highlights
Invasive methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a major public health problem, implicated in 18,000 deaths annually with an estimated 94, 360 invasive MRSA infections in 2005 [1]
At a subinhibiitory vancomycin concentration (32 mg/ml), the duration of the lag phase increased by about 3.5 hrs in strain VRS1Dvra compared with VRS1; the growth rates of the two strains were similar in the presence of this amount of vancomycin
Complementation with the vraTSR operon decreased the duration of the lag phase of VRS1Dvra by 2 hrs when grown with 32 mg/ml of vancomycin, which is intermediate between the wildtype and mutant strains
Summary
Invasive methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a major public health problem, implicated in 18,000 deaths annually with an estimated 94, 360 invasive MRSA infections in 2005 [1]. Whereas vancomycin intermediate resistance involves chromosomal point mutations and a thicker cell wall [4,5,6], VRSA isolates to date have acquired the vanA operon contained on transposon (Tn)1546 residing on a conjugal plasmid [7,8]. The vanA locus typically confers high-level vancomycin resistance (MICs 512–1024 mg/ml) to enterococcal species [10] by encoding the genes necessary for producing an altered peptidoglycan precursor in which the final dipeptide, D-alanyl-D-alanine (D-Ala-D-Ala) is replaced by depsipeptide, D-alanyl-D-lactate (DAla-D-Lac). The vanA locus consists of seven genes, vanRSHAXYZ, whose expression is inducible by the glycopeptides, vancomycin and teicoplanin. Three of these genes (vanHAX) are necessary for production of the D-Ala-D-Lac containing peptidoglycan precur-
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