Heterogeneous Macrolide Resistance in Methicillin-Resistant Staphylococcus aureus: Investigation of Resistance Mechanisms and Clonality

Staphylococcus aureus is one of the most important pathogens leading to hospital infections and has ability to gain multiple resistance to most of the antibiotics used as well as ability to spread clonally. In this study we aimed to investigate the mechanism of heterogenous macrolide resistance and clonality of 41 methicillin-resistant S.aureus (MRSA) strains isolated from clinical samples of inpatients between 2006 and 2008 in a tertiary care training hospital. Heterogenous macrolide resistance is defined as the isolates with colonies in the inhibiton zone of erythromycin, azithromycin, claritromycin discs, and named as hMLS(B) (heterogeneous macrolides, lincosamides, streptogramin-B) phenotype. A total of 63 macrolide-resistant MRSA strains isolated during the same period with non-hMLS(B) phenotype were included in the study as a control group. The susceptibilties of isolates to methicillin, erythromycin, azithromycin, claritromycin, clindamycin, quinupristin-dalfopristin, penicillin, vancomycin, teicoplanin, linezolide, gentamicin, amikacin, ciprofloxacin, trimethoprim-sulphamethoxazole, chloramphenicol, rifampin, tetracycline and telithromycin were determined by disc diffusion method according to the CLSI criteria. hMLS(B) isolates were also tested for erythromycin, clindamycin and quinupristin-dalfopristin susceptibility by Vitek-2 automated system (bioMerieux, France). Presence of erm(A), erm(B), erm(C), msr(A/B) resistance genes were investigated by PCR. The regulatory region of the erm(A) gene, which was found to be the only molecular mechanism of hMLS(B), was sequenced. Sequence analysis of regulatory regions showed deletion of "guanine" base at position 149 in 34 strains and two point mutations namely "C368G" and "T377C" in 16 strains. Pulsed-field gel electrophoresis (PFGE) analysis indicated presence of a common clone and its variants; however this clone was also common among control group strains. Interestingly all heterogeneous macrolide resistant isolates were reported as susceptible to erythromycin, clindamycin, quinupristin-dalfopristin by the automated system. As a result a clone specific to heterogenous macrolide resistant strains was not detected. Although all hMLS(B) strains had erm(A) gene, a specific and common genetic modification could not be found in regulatory region of the isolates. The most important finding of this study is the detection of inability of the automated system to properly reflect the hMLS(B) phenotype. In addition it was suggested that longer incubation (at least 24 hours) of the antibiotic susceptibility test plates could help identification of hMLS(B) phenotype in disc diffusion tests.