Optimized In Vitro Antibiotic Susceptibility Testing Method for Small-Colony Variant Staphylococcus aureus

Abstract

bStaphylococcus aureus small-colony variants (SCVs) emerge frequently during chronic infections and are often associated with worse disease outcomes. There are no standardized methods for SCV antibiotic susceptibility testing (AST) due to poor growth and reversion to normal-colony (NC) phenotypes on standard media. We sought to identify reproducible methods for AST of S. aureus SCVs and to determine whether SCV susceptibilities can be predicted on the basis of treatment history, SCV biochemical type (auxotrophy), or the susceptibilities of isogenic NC coisolates. We tested the growth and stability of SCV isolates on 11 agar media, selecting for AST 2 media that yielded optimal SCV growth and the lowest rates of reversion to NC phenotypes. We then performed disk diffusion AST on 86 S. aureus SCVs and 28 isogenic NCs and Etest for a subset of 26 SCVs and 24 isogenic NCs. Growth and reversion were optimal on brain heart infusion agar and Mueller-Hinton agar supplemented with compounds for which most clinical SCVs are auxotrophic: hemin, menadione, and thymidine. SCVs were typically nonsusceptible to either trimethoprim-sulfamethoxazole or aminoglycosides, in accordance with the auxotrophy type. In contrast, SCVs were variably nonsusceptible to fluoroquinolones, macrolides, lincosamides, fusidic acid, and rifampin; mecA-positive SCVs were invariably resistant to cefoxitin. All isolates (both SCVs and NCs) were susceptible to quinupristin-dalfopristin, vancomycin, minocycline, linezolid, chloramphenicol, and tigecycline. Analysis of SCV auxotrophy type, isogenic NC antibiograms, and antibiotic treatment history had limited utility in predicting SCV susceptibilities. With clinical correlation, this AST method and these results may prove useful in directing treatment for SCV infections. Staphylococcus aureus causes diverse infections ranging in severity from benign to life-threatening. Persistent and relapsing S. aureus infections often occur despite prolonged antimicrobial therapy and have been linked to the emergence of small-colony variants (SCVs). S. aureus SCVs have been associated with a poor response to antibiotic treatment (1, 2) and are often recovered from many chronic infections, including endocarditis (3, 4), osteomyelitis (5), device infections (3), soft tissue infections (6), and airway infections (7,8). SCV airway infections are particularly prevalent among people with the genetic disease cystic fibrosis (CF), affecting between 8 and 33% of CF patients, often following prolonged antibiotic treatment (1, 7). In children with CF, SCV respiratory infection was found to be independently associated with worse lung function and faster lung function decline (9).SCVs are typically identified by distinctive phenotypic traits when grown on most agar-based media. SCVs produce colonies approximately 1/10 the size of normal-colony (NC) S. aureus isolates (Fig. 1), and they are characteristically nonhemolytic and nonpigmented and have diminished coagulase production (10). SCVs usually carry mutations in one of a few, ...

Multicenter Evaluation of a Modified Cefoxitin Disk Diffusion Method and PBP2a Testing To Predict mecA -Mediated Oxacillin Resistance in Atypical Staphylococcus aureus

“…However, we postulate that hemin levels may differ based on the age of the erythrocytes (and degree of lysing) in these media, accounting for the differences in performance. Similar manufacturer-to-manufacturer differences in the ability of BHI agar to support SCV growth have been observed (9). Furthermore, the medium developed by Precit and colleagues was designed to support the growth of SCV isolated from CF patients, since about one-third of CF isolates are thymidine dependent, and two-thirds are menadione and/or hemin dependent (3,14,15).…”

“…Even with these requirements, such a medium would be best suited for use by laboratories that serve large CF patient populations, orthopedic surgical centers, or large reference laboratories, since most clinical laboratories encounter atypical S. aureus isolates infrequently. Precit and colleagues developed an MHA supplemented with menadione, hemin, and thymidine for the testing of SCV (9), which yielded AST results similar to those observed for isogenic wild-type isolates (9). However, this medium requires further, multicenter evaluation, including evaluation of its performance when it is produced by more than one manufacturer.…”

“…While our goal was to evaluate media currently in use by clinical laboratories, thereby avoiding special orders and supplemental quality control testing by the laboratory, others have suggested that a novel medium be developed for AST of atypical S. aureus (9). In order for such a medium to achieve widespread utility for clinical laboratories, it should (i) support the growth of all (or nearly all) atypical S. aureus isolates, (ii) be commercially available, (iii) generate accurate results compared to a reference method (e.g., mecA PCR), and (iv) have good lot-to-lot and manufacturerto-manufacturer reproducibility.…”

“…Antimicrobial susceptibility testing (AST) of the atypical S. aureus variant is therefore necessary but poses a significant challenge to clinical laboratories, since many atypical S. aureus strains will not grow in commercial, automated AST systems. Similarly, many strains will not grow on Mueller-Hinton agar (MHA) or in cationadjusted Mueller-Hinton broth (CA-MHB), which are recommended by the Clinical and Laboratory Standards Institute (CLSI) for the testing of typical S. aureus isolates (9,10). Some clinical and research laboratories have evaluated AST performed on alternative test media that support the growth of SCV, based on supplementation for the typical auxotrophies observed (9,10).…”

“…Similarly, many strains will not grow on Mueller-Hinton agar (MHA) or in cationadjusted Mueller-Hinton broth (CA-MHB), which are recommended by the Clinical and Laboratory Standards Institute (CLSI) for the testing of typical S. aureus isolates (9,10). Some clinical and research laboratories have evaluated AST performed on alternative test media that support the growth of SCV, based on supplementation for the typical auxotrophies observed (9,10). However, determination of the accuracy of such testing is a challenge, because no gold-standard AST method exists for atypical S. aureus, and genotypic resistance mechanisms are not fully defined for all antimicrobials that might be considered for the treatment of these infections.…”