Characterization of ERM (B), MEF and MEL genes in macrolide-resistant Streptococcus Pneumoniae isolated from patients at King Chulalongkorn Memorial Hospital

Abstract

Macrolide resistance in Streptococcus pneumoniae is an increasingly recognized problem in many parts of the world including Thailand. Two main mechanisms of macrolide resistance in S. pneumoniae were due to alteration of drug target by methylation of 23S rRNA encoded by erm (B) and macrolide efflux encoded by mef gene. The purpose of this study was to investigate the prevalence of macrolide resistance and characterize the resistance gene including erm (B), mef and mel genes in S. pneumoniae. A total of 385 S. pneumoniae isolates were collected from patients at King Chulalongkorn Memorial Hospital between 2003-2007. Erythromycin, clarithromycin and clindamycin susceptibility were determined by agar dilution method. Prevalence of macrolide resistance was 54.02% for erythromycin and 53.76% for clarithromycin. Prevalence of clindamycin resistance was 25.20%. Macrolide resistance phenotypes were identified by double disc diffusion. Among the 208 erythromycin-resistant isolates, 96(46.15%) were resistant to both erythromycin and clindamycin and showed cMLS[subscript B] phenotype, whereas 112(53.85%) were resistant to erythromycin but susceptible to clindamycin and exhibited the M phenotype. The iMLS[subscript B] phenotype were not detected. Detection of macrolide resistance genes by multiplex PCR revealed that the erm (B) gene was found in 95 isolates (45.67%) and the mef gene was identified in 112 isolates (53.85%). One isolate (0.48%) carried both mef and erm (B) genes. Detection of mef (A/E) type genes was investigated by PCR-RFLP. The mef (E) gene was detected in all M phenotype isolates. The erythromycin MIC of 112 M-phenotype S. pneumoniae were decreased 6-9 fold in the presence of CCCP, an efflux pump inhibitor, confirming the presence of an efflux mechanism in these isolates. DNA sequence analysis of mef (E) and mel genes in 10 M-phenotype S. pneumoniae (MIC range 1-16 ?g/ml) revealed a 1,218-bp ORF of entire mef (E) gene, encoding 405 amino acids and 1,464-bp ORF of entire mel gene, encoding 487 amino acids. All 10 sequences of entire mef and mel genes were identical to each other at the DNA and amino acid levels and also identical with the mef (E) published sequences in GenBank. Analysis of a 630 bp upstream region of mef (E) gene showed 23 nucleotide changes ; T-31C, T-54G, T deletion at position -63, A-78T, T-81G, A-82G, T-345A and 16 bp deletion at position -155 upstream of mef (E) gene in 10 M-phenotype S. pneumoniae. All ten isolates had 22 nucleotide changes. Four isolates with the MIC range of 2-16 μg/ml carried an additional mutation at T-345A whereas the other six isolates with the MIC range of 1-4 μg/ml had no substitution at this position. The results demonstrated that mutation at T-345A may be associated with increased erythromycin MIC in M-phenotype S. pneumoniae.