Role of stop codons in development and loss of vancomycin non-susceptibility in methicillin-resistant Staphylococcus aureus.

OBJECTIVES: Problems of vancomycin non-susceptible Staphylococcus aureus (VISA) and subsequent treatment failure are increasing. This study aimed to observe development and loss of vancomycin non-susceptibility, determine exposure time needed for resistance development, and follow mutations in the VraSR and GraSR two-component systems during these processes. METHODS: Sequences of vraS, graR and rpoB, proposed as critical sites of mutation associated with non-susceptibility development, were compared in susceptible clinical methicillin-resistant S. aureus isolates both initially and following vancomycin induction and its withdrawal, to identify mutations. Mutations were correlated with exposure time, increase in vancomycin MIC and phenotypic changes. RESULTS: Both time required for heterogeneous VISA and VISA development, and maximum MIC attained (6-20 mg/L) varied between strains. Sequence analysis revealed the presence of stop codons in an initial strain with delayed non-susceptibility development. Other changes in vraS and graR occurred during VISA development in all isolates. After removal of vancomycin pressure, most strains reverted to susceptibility accompanied by emergence of stop codons in both vraS and graR. One strain not displaying stop codons remained resistant in the absence of vancomycin pressure. A substitution in GraR (D148Q) appeared to be associated with an elevated MIC (20 mg/L). No rpoB mutations were observed throughout VISA development. CONCLUSIONS: Vancomycin non-susceptibility developed in all strains tested. Mutations in vraS and graR appeared to be essential for VISA development, with stop codons playing an important role in delaying non-susceptibility development and reversion. Absence of mutations in rpoB suggests that these are not essential for vancomycin resistance. Further work is required to confirm consistent changes involved in non-susceptibility development.

Importance of early detection of vancomycin-resistant subpopulations in apparently susceptible meticillin-resistant Staphylococcus aureus clinical isolates.

This study aimed to investigate the presence of vancomycin-non-susceptible subpopulations in apparently susceptible meticillin-resistant Staphylococcus aureus (MRSA) and the ability of these isolates to develop into homogeneously resistant strains. Vancomycin MICs of 200 clinical MRSA isolates were determined using agar dilution (AD) and spiral gradient endpoint (SGE) technique. Isolates with an MIC≤2mg/L but displaying subpopulations with an MIC>2mg/L by SGE were re-tested by Etest and PAP-AUC and were incubated with 2mg/L vancomycin for 2 weeks. MIC testing was repeated weekly by AD, Etest and SGE to observe progression to non-susceptibility. A total of 17.5% and 16.0% of isolates were non-susceptible to vancomycin (MIC>2mg/L) by SGE and AD, respectively. Eight isolates (4%) displayed a resistant subpopulation; five met the definition of hVISA by PAP-AUC. The initial Etest MIC for these isolates was 2mg/L, but resistant subpopulations were observed in only three isolates on prolonged incubation. MICs of all eight isolates increased rapidly in the presence of vancomycin, reaching ≥3.0mg/L by Day 7 and ≥4mg/L after 14 days by all three methods. The prevalence of vancomycin-non-susceptible MRSA was high, and non-susceptibility developed rapidly in seemingly susceptible isolates with covert subpopulations. These were effectively detected by SGE. With increasing reports of vancomycin clinical failure, early detection of potentially non-susceptible isolates before or early in vancomycin therapy is essential to avoid further resistance development and poor clinical outcomes. SGE offers a novel and cost-effective technique for detection of potentially non-susceptible strains.

Tracking changes in the vraSR and graSR two component regulatory systems during the development and loss of vancomycin non-susceptibility in a clinical isolate.

We investigated changes in regulatory genes, vraS and graR, during development of vancomycin non-susceptibility in a patient with methicillin-resistant Staphylococcus aureus who failed therapy and following in-vitro vancomycin exposure and a subsequent drug-free growth period. Minimum Inhibitory Concentration (MICs) were determined and genes sequenced at each stage. After 30 days of vancomycin exposure, the strain attained maximum MIC (20 mg/L) and was resistant to all antibiotics. Reversion to vancomycin susceptibility occurred 21 days after removal. We observed mutations in vraS and graR during non-susceptibly development and novel stop codons in the reverted strain. Mutations in graR appear important for development of intermediate susceptibility to vancomycin. The results suggest that monitoring of vancomycin therapy could allow earlier change to appropriate agents.