Derivatives of a vancomycin-resistant Staphylococcus aureus strain isolated at Hershey Medical Center

Abstract

Antimicrobial susceptibilities and genetic relatedness of the vancomycin-resistant Staphylococcus aureus strain (VRSA) isolated at Hershey, Pa. (VRSA Hershey), and its vancomycin-susceptible and high-level-resistant derivatives were studied and compared to 32 methicillin-resistant S. aureus strains (MRSA) isolated from patients and medical staff in contact with the VRSA patient. Derivatives of VRSA were obtained by subculturing six VRSA colonies from the original culture with or without vancomycin. Ten days of drug-free subculture caused the loss of vanA in two vancomycin-susceptible derivatives for which vancomycin MICs were 1 to 4 microg/ml. Multistep selection of three VRSA clones with vancomycin for 10 days increased vancomycin MICs from 32 to 1,024 to 2,048 microg/ml. MICs of teicoplanin, dalbavancin, and oritavancin were also increased from 4, 0.5, and 0.12 to 64, 1, and 32 microg/ml, respectively. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing analysis indicated that VRSA Hershey was the vanA-acquired variety of a common MRSA clone in our hospital with sequence type 5 (ST5). Three of five vancomycin-intermediate S. aureus strains tested from geographically different areas were also ST5, and the Michigan VRSA was ST371, a one-allele variant of ST5. Derivatives of VRSA Hershey had differences in PFGE profiles and the size of SmaI fragment that carries the vanA gene cluster, indicating instability of this cluster in VRSA Hershey. However induction with vancomycin increased glycopeptide MICs and stabilized the resistance.

FIG. 1.

PFGE patterns after digestion by SmaI of MRSA strains isolated from contacts. Seventeen of 20 strains were clonally related to the VRSA strain and had zero to three band differences. Strains studied for MLST (V3, VRS2.3; 12, S12; and 11, S11) are underlined. S. aureus NCTC 8325 was used as a molecular size marker (M).

FIG. 2.

Analysis of genomic DNA from VRSA strains isolated at Hershey and from Michigan by Southern blot hybridization. Panel A shows total DNA separated by PFGE after digestion by SmaI, and panel B shows hybridization of DNA transferred to a nylon sheet with biotin-labeled vanA probe. Some of the derivatives of VRSA Hershey show different PFGE profiles. Hybridization with vanA probe indicates that the size of the SmaI fragment, which carries vanA, may vary within the derivatives of the same strain. Strains VRS1 (lane MI), VRS2.1 (lane V1), VRS2.2 (lane V2), VRS2.3 (lane V3), VRS2.4 (lane V4), and VRS2.6 (lane V6) are derivatives of VRSA Hershey. Strains VRS2.11 (lane 11), VRS2.12 (lane 12), VRS2.21 (lane 21), VRS2.31 (lane 31), and VRS2.32 (lane 32) are vancomycin-induced derivatives of VRSA strains. Strains VRS2.S1 (lane S1) and VRS2.S2 (lane S2) are vancomycin-susceptible derivatives of VRSA Hershey.