Heterologous expression of the enterococcal vanA operon in methicillin-resistant Staphylococcus aureus

Abstract

Two methicillin- and vancomycin-resistant Staphylococcus aureus strains, MI-VRSA and PA-VRSA, and Enterococcus faecalis DMC83006B, considered to be the potential donor of glycopeptide resistance to MI-VRSA, were studied. MI-VRSA is highly resistant to both glycopeptides, whereas PA-VRSA displays low-level resistance to vancomycin and reduced susceptibility to teicoplanin. We have analyzed the expression of the vanA operon in the three clinical isolates. Determination of the relative amounts of late peptidoglycan precursors and quantification of the d,d-peptidase activities, in the absence or after induction by glycopeptides, revealed that the resistance genes were expressed at similarly high levels in the three strains. Glycopeptide resistance stability in the three strains was studied by replica plating. Resistance was lost at high frequency, ca. 50%, after overnight growth of PA-VRSA in the absence of antibiotics, whereas it was fully stable in MI-VRSA and E. faecalis DMC83006B. Induction of resistance by vancomycin was significantly delayed in PA-VRSA relative to MI-VRSA. Low-level glycopeptide resistance of S. aureus PA-VRSA is thus likely due to instability of the genetic element, plasmid or transposon, carrying the vanA operon associated with a longer lag phase before growth resumes after induction by vancomycin.

FIG. 1.

Schematic representation of Tn1546 insertion site. The 5-bp direct repeats of target DNA are indicated in boldface characters and are boxed. The left and right IR_L and IR_R imperfect terminal 38-bp repeats of Tn1546 are indicated by double arrows. Tn1546 is indicated by a thin line.

FIG. 2.

Cytoplasmic peptidoglycan precursors in E. faecalis DMC83006B and S. aureus MI-VRSA and PA-VRSA. Peptidoglycan synthesis was inhibited by addition of ramoplanin to the cultures for 15 min. NI, not induced; I, induced by growth in the presence of 4 μg of vancomycin/ml.

FIG. 3.

D,D-peptidase activities in extracts from E. faecalis DMC83006B and S. aureus MI-VRSA and PA-VRSA. D,D-dipeptidase (VanX) (top) and D,D-carboxypeptidase (VanY) (bottom) activities were measured in the cytoplasmic extracts and in the membrane fractions, respectively. The results are the means from two independent experiments. NI, not induced; I, induced by growth in the presence of 4 μg of vancomycin (Vm) or teicoplanin (Te)/ml.

FIG. 4.

Effect of overnight growth in the presence of vancomycin (6 μg/ml) on growth of S. aureus MI-VRSA (A) and PA-VRSA (B) in the absence or presence (6 μg/ml) of vancomycin. Vancomycin (Vm) concentration in the overnight culture/in the culture medium. O.D., optical density.

FIG. 5.

Vancomycin MIC determination by E-test of the PA-VRSA isolate after 24 h of incubation in the absence (A) or in the presence (6 μg/ml) (B) of vancomycin.