Denaturing High-Performance Liquid Chromatography Detection of Ribosomal Mutations Conferring Macrolide Resistance in Gram-Positive Cocci

Abstract

ABSTRACT Mutations in genes coding for L4 ( rplD ) or L22 ( rplV ) ribosomal proteins or in 23S rRNA ( rrl gene) are reported as a cause of macrolide resistance in streptococci and staphylococci. This study was aimed at evaluating a denaturing high-performance liquid chromatography (DHPLC) technique as a rapid mutation screening method. Portions of these genes were amplified by PCR from total DNA of 48 strains of Streptococcus pneumoniae ( n = 22), Staphylococcus aureus ( n = 16), Streptococcus pyogenes ( n = 6), Streptococcus oralis ( n = 2), and group G streptococcus ( n = 2). Thirty-seven of these strains were resistant to macrolides and harbored one or several mutations in one or two of the target genes, and 11 were susceptible. PCR products were analyzed by DHPLC. All mutations were detected, except a point mutation in a pneumococcal rplD gene. The method detected one mutated rrl copy out of six in S. aureus . This automated method is promising for screening of mutations involved in macrolide resistance in gram-positive cocci.