Simultaneous detection of erythromycin-resistant methylase genes ermA and ermC from Staphylococcus spp. by multiplex-PCR.
A comparative analysis of the two most dominant erythromycin-resistance determinant genes in Staphylococcus sppnamely, the ermA and ermC genes, was carried out. Sixty erythromycin-resistant strains of Staphylococcus spp. were tested, of which 24 were avian and 36 were clinical isolates. Our results indicated the prevalence of ermA over the ermC gene as opposed to the widely held opinion of the ermC gene being the most dominant resistance determinant gene. A multiplex-PCR assay was developed to detect the presence of ermA and ermC genes. Two pairs of primers, specific for the detection of ermA and ermC genes, were used in a multiplex-polymerase chain reaction (PCR) assay to yield amplified DNA products of 610 and 520 bp, respectively. Their digestion with restriction enzyme FokI that yielded a 477 bp and a 132 bp digestion product for ermA and a 333 bp and a 187 bp digestion product for ermC confirmed the authenticity of PCR products. The method could be used to amplify the ermA and ermC genes with as little as 5 pg of template DNA. The use of excess primers or the template DNA resulted in gene-specific amplification and no non-specific amplification was observed by changing the primer to primer or template to primer ratios. Furthermore, no amplification from erythromycin-sensitive S. aureus strain was observed. Using this assay, the poultry strains were found to contain either ermA alone (50%) or a combination of ermA (100%) and ermC (50%) both. The clinical strains contained either ermA (94.5%) or ermC (5.5%) but never both. The gene-specific internal probes were also used to verify the above findings and a high degree of correlation between the multiplex PCR and Southern hybridization data was observed.
Khan
S A
SA
Division of Microbiology, National Center for Toxicological Research, Food and Drug Administration, 3900 NCTR Road, Jefferson, AR 72079, USA.
Nawaz
M S
MS
Khan
A A
AA
Cerniglia
C E
CE
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D017931:DNA Primers; D004262:DNA Restriction Enzymes; D004352:Drug Resistance, Microbial / Q000235:genetics*; D004917:Erythromycin / Q000494:pharmacology*; D005798:Genes, Bacterial; D005819:Genetic Markers / Q000235:genetics; D008780:Methyltransferases / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D013210:Staphylococcus / Q000187:drug effects / Q000235:genetics*
D000900:Anti-Bacterial Agents; D017931:DNA Primers; D005819:Genetic Markers; D004917:Erythromycin; D008780:Methyltransferases; D004262:DNA Restriction Enzymes