Detection of erythromycin resistant methylase gene by the polymerase chain reaction

A polymerase chain reaction (PCR) protocol was developed that could specifically amplify a 520-bp region of the erythromycin resistant methylase (ermC) gene sequence. The identity of the PCR-amplified 520-bp DNA was confirmed by HinCII endonuclease restriction digestion, which produced the predicted 440-bp and 80-bp DNA fragments. A 20-mer (alpha-32P) oligonucleotide probe specifically hybridized with these amplified products confirming the specificity and reliability of this diagnostic assay. The assay could detect the ermC gene in bacterial suspensions containing as few as 10(3) cells ml-1. The assay was used to detect the presence of the ermC gene in several Gram-positive bacterial strains identified as Streptococcus sp., Staphylococcus sp., Micrococcus sp., Lactobacillus sp. and Enterococcus sp., isolated from water samples maintained in experimental animal cages and clinical sources. Only bacteria identified as Staphylococcus sp. were resistant to the antibiotic. Although 17 strains of Staphylococcus sp. isolated from clinical samples were resistant to erythromycin, only seven of these isolates tested positive for the presence of the ermC gene. Of these strains, five were identified as coagulase-positive S. aureus and the rest were identified as coagulase-negative S. epidermidis. The erythromycin resistance in all seven ermC positive isolates was constitutive. The entire diagnostic assay, including template preparation, amplification and electrophoresis can be completed within 6 h.