Staphylococcus aureus VRSA-11B is a constitutive vancomycin-resistant mutant of vancomycin-dependent VRSA-11A

Abstract

Vancomycin-resistant Staphylococcus aureus VRSA-10 was isolated in 2009, whereas VRSA-11A and VRSA-11B were isolated from the same patient in 2010. Growth curves and determination of the nature of the peptidoglycan precursors and of the VanX d,d-dipeptidase activity in the absence and in the presence of vancomycin indicated that vancomycin resistance was inducible in VRSA-10, that VRSA-11A was partially dependent on glycopeptide for growth, and that VRSA-11B was constitutively resistant. Both VRSA-11A and -11B harbored an insertion sequence, ISEf1, at the same locus in the vanX-vanY intergenic region of Tn1546 and an S(183)A mutation in the chromosomal d-alanyl:d-alanine ligase (Ddl). This substitution has been shown to be responsible for a drastic diminution of the affinity of the enzyme for d-Ala at subsite 1 in Escherichia coli DdlB. VRSA-11B exhibited an additional mutation, P(216)T, in the transcriptional regulator VanR, most probably associated with constitutive expression of vancomycin resistance. It is thus likely that VRSA-11B is a constitutive derivative of VRSA-11A selected during prolonged vancomycin therapy. Synthesis of peptidoglycan precursors ending in d-Ala-d-lactate was responsible for oxacillin susceptibility of VRSA-11A and VRSA-11B despite the presence of a wild-type mecA gene in both strains.

Fig 1

Expression of PBP2′. Total proteins of methicillin-resistant COL and VRSA-10 and of methicillin-susceptible COLΔmecA, VRSA-11A, and VRSA-11B were isolated, subjected to SDS-PAGE, and probed with 14A9C9-C6 monoclonal antibody against PBP2′. Purified PBP2′ was used as a size marker.

Fig 2

Growth of S. aureus VRSA-10, VRSA-11A, and VRSA-11B in the absence (N.I., not induced) or the presence (Vm, 16 μg/ml) of vancomycin.

Fig 3

d,d-Dipeptidase (VanX) activity in cytoplasmic extracts from S. aureus VRSA-10, -11A, and -11B. Results are the means from three independent experiments. N.I., not induced; Vm, induced by growth in the presence of 16 μg/ml of vancomycin.