VanA-type vancomycin-resistant Staphylococcus aureus

Abstract

Since 2002, nine methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) strains that are also resistant to vancomycin (VRSA) have been reported in the United States, including seven clinical isolates from Michigan. The strains harbor a plasmid-borne Tn1546 element following conjugation from a glycopeptide-resistant Enterococcus strain. In the second step, Tn1546 transposed to a resident plasmid in five strains; the acquired plasmid behaved as a suicide gene delivery vector, and the incoming DNA had been rescued by illegitimate recombination. Surprisingly, combination of a glycopeptide with a beta-lactam has a strong synergistic effect against VRSA, both in vitro and in an animal model, despite resistance of the strains to both drug classes when administered separately. This results from the fact that the late peptidoglycan precursors ending in D-alanine-D-lactate (D-Ala-D-Lac) that are mainly synthesized in the presence of glycopeptide inducers are not substrates for PBP2', which is the only transpeptidase that remains active in the presence of oxacillin. One VRSA strain is partially dependent on vancomycin for growth due to a mutation in the host D-Ala:D-Ala ligase, thus having to rely on the inducible resistance pathway for cell wall synthesis. Competition growth experiments in the absence of inducer between the MRSA recipient and isogenic VRSA transconjugant revealed a disadvantage for the transconjugant, accounting, in part, for the low level of dissemination of the VRSA clinical isolates. The association of multiple molecular and environmental factors has been implicated in the regional emergence of VRSA in Michigan.

FIG. 1.

Comparison of Tn1546 elements. (A) Schematic representation of Tn1546. IR_L and IR_R, inverted left and right repeats, respectively. Open arrows represent coding sequences and indicate direction of transcription. Brown arrows, genes required for transposition; red arrows, regulatory genes; blue arrows, genes required for resistance; green arrow, accessory gene; pink arrow, gene of unknown function. (B) Organization of the Tn1546 element in strains VRSA-2 and VRSA-3; gray arrows, insertion sequences.

FIG. 2.

Schematic representation of Tn1546 transfer from Enterococcus spp. to S. aureus. Blue and red wavy lines represent chromosomal DNA of Enterococcus and S. aureus, respectively. Blue circle, enterococcal plasmid with a broad host range of transfer; red circle, resident staphylococcal plasmid. Acquisition of Tn1546 was obtained in one step by VRSA-3, -5, and -6 and in two steps by VRSA-1, -7, -8, -9, and -10. GRE, glycopeptide-resistant enterococci.

FIG. 3.

Oxacillin-glycopeptide synergism against HLR-VRSA as tested by (top) disk diffusion and (bottom) Etest. BHI, brain heart infusion agar. ERY, erythromycin (15 μg); OXA, oxacillin (5 μg); PEN, penicillin (6 μg); SPT, spectinomycin (100 μg); TEC, teicoplanin (30 μg); VAN, vancomycin (30 μg).

FIG. 4.

Schematic representation of peptidoglycan synthesis by a VRSA strain in the presence of vancomycin (top) or of vancomycin and methicillin (bottom). Top, in the presence of vancomycin, the resistance pathway is activated, leading to synthesis of peptidoglycan precursors ending in d-Ala-d-Lac and elimination of the d-Ala-d-Ala ending precursors. The depsipeptide late precursors are substrates for the transpeptidases (PBP) but are not a target for the glycopeptides. Bottom, in the presence of vancomycin and methicillin, the d-Ala-d-Lac terminating precursors, which are synthesized following induction by vancomycin, are not substrates of PBP2′, which is the only transpeptidase that remains active in the presence of methicillin. Thus, biosynthesis of the cell wall cannot proceed despite the fact that the strain is highly resistant to both drug classes when used separately.

FIG. 5.

Glycopeptide susceptibility of VRSA-7 as tested by disk diffusion. VAN, vancomycin (30 μg); TEC, teicoplanin (30 μg).