84 An optimized in vitro antibiotic susceptibility testing method for Staphylococcus aureus small colony variants

Abstract

Objectives S. aureus small colony variants (SCVs) frequently infect people with CF and are associated with worse lung disease. Because SCVs grow poorly and frequently revert to wild-type (WT) growth on standard laboratory media, there are currently no standardized methods for antibiotic susceptibility testing (AST) of SCVs to guide treatment. In this study, we sought to identify conditions that would support in vitro growth of SCVs, allowing for reproducible AST. Methods We tested growth and stability of SCVs on 11 agar media, selecting two for AST that yielded best SCV growth and lowest reversion rates. We performed AST by disk diffusion on 112 S. aureus CF respiratory isolates from 24 patients, comprising 85 SCVs and 27 isogenic WT isolates. Etest AST for a subset of isolates, 26 SCVs and 24 WT pairs, was also completed. Results Growth and reversion were optimal on brain-heart infusion agar (BHI) and cation-adjusted Mueller-Hinton agar supplemented with the 3 compounds for which most CF SCVs are auxotrophic: hemin, menadione, and thymidine (sMHA). AST yielded similar results for the majority of SCVs and isogenic WT pairs on both media. 81 SCVs and 19 WTs were not susceptible to at least 1 of the following: b-lactams, fluoroquinolones, macrolides, lincosamides, or rifampicin. All isolates were susceptible to quinupristin/dalfopristin, minocycline, and linezolid. Etest MICs were comparable to disk diffusion results. AST results also predicted MRSA status, which was confirmed by mecA PCR. Conclusion We have developed an optimized AST method for SCVs. With clinical correlation, AST profiles defined in this study may be useful in directing treatment for SCV infections.