An optimal method for generating stable vancomycin heteroresistance and intermediate susceptibility in methicillin-resistant Staphylococcus aureus blood isolates under in-vitro vancomycin pressure

Abstract

Objectives: The relevance of vancomycin intermediately-susceptible (VISA) and hetero-resistant (hVISA) methicillin-resistant Staphylococcus aureus (MRSA) remains uncertain because of their low frequency. In vitro attempts to generate reduced susceptibility have been inconsistent. We describe a simple method for generating VISA/hVISA. Materials and methods: Twenty-four SCCmec type II and IV MRSA blood isolates plus USA100 and USA300 controls were cultured (107 CFU/ml) in BHI broth with 0, 2 and 3 mg/L vancomycin for 10 days followed by 10 days vancomycin-free-passage. We monitored vancomycin minimum inhibitory concentration (MIC; Etest) and population analysis profile-area under the curve (PAP-AUC) ratios of tested isolate/Mu3 during and after vancomycin pressure (VP). PAP-AUC ratios 1.3 were considered consistent with susceptible, hVISA and VISA, respectively. Results: VP at 2 mg/L (VP-2) increased MIC to 3 mg/L in 20 (83.3%) isolates and raised PAP-AUC ratio to hVISA (n=18; 75.0%) and VISA (six; 25.0%) ranges. VP-3 increased MIC to 3 and 4 mg/L in 19 (79.2%) and three (12.5%) isolates, respectively and raised PAP-AUC ratio to hVISA (n=3; 12.5%) and VISA (n=19; 79.2%) ranges. SCCmec IV isolates had lower pre-exposure PAP-AUC ratios (0.48&plusmn;0.14 vs. 0.69&plusmn;0.10; p<0.001) and lower MIC (1.6&plusmn;0.2 vs. 1.8&plusmn;0.2 mg/L; p=0.1) but MIC rise and hVISA/VISA emergence was comparable. MIC and PAP-AUC ratio rises were stable in 20/22 isolates during drug-free passages. Conclusions: VISA/hVISA readily emerges among MRSA SCCmec type II and IV isolates under VP. VP at the MIC level generates hVISA while pressure at slightly higher level results in the VISA phenotype.