The mef(A)- and its subclass mef(E) systems had long been considered to constitute one of the primary macrolide-resistant mechanisms in Streptococcus pyogenes. However, we have previously demonstrated that the msr(D) gene located immediately downstream of the mef(A)/mef(E) genes plays a predominant role in these systems. In previous studies, furthermore, mef(A)-associated msr(D) of an S. pyogenes strain (10-85) exhibited a greater increase in clarithromycin minimum inhibitory concentration (MIC) than mef(E)-associated msr(D) of another strain (13-O-10). Both msr(D) genes encode 487 amino acid residues, 13 amino acid residues of which are different from each other. In this study, we performed mutational analysis of the msr(D) genes and showed that a single-nucleotide polymorphism to cause a substitution of Asp238 with Gly is mainly associated with the greater increase in clarithromycin MIC by the msr(D) than by the msr(D) allele. In addition, another substitution of Ser with Arg at codon 194 is partially associated with the greater increase by the msr(D) than by the msr(D) allele.
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