RAPID MULTIPLEX PCR ASSAY FOR SIMULTANEOUS DETECTION OF MAJOR ANTIBIOTIC RESISTANCE DETERMINANTS IN CLINICAL ISOLATES OF BACTEROIDES FRAGILIS

This study developed a rapid and cost-effective multiplex polymerase chain reaction assay (M-PCR) for simultaneous detection of major clinically relevant antibiotic resistance genes in Bacteroides fragilis. The M-PCR optimized amplification conditions with primers designed for detection of five resistance genes: carbapenems (cfiA) and cephalosporins (cepA), clindamycin (ermF), metronidazole (nimA-F) and tetracycline (tetQ), plus a set of primers for the B. fragilis 16S rRNA gene (positive control). An initial single PCR was performed for each gene, followed by a gradient PCR to determine the optimal PCR conditions for each primer set. This was followed by several test M-PCRs using the Qiagen multiplex PCR kit (Valencia, CA) with 60 ng of a template DNA mastermix containing all genes of interest and 0.2 mu M of each primer set. Multiplex PCR products were detectable at annealing temperatures ranging from 53-57C for 30-35 cycles. The final optimized M-PCR was then used to evaluate 21 different B. fragilis isolates from various patients. Eleven different M-PCR genotypes were obtained, which correlated with phenotypic antibiograms and nitrocefin beta-lactamase production assays. This M-PCR provides an accurate, rapid, sensitive and cost-effective tool for detecting clinically relevant antibiotic resistance genes in B. fragilis, and owing to its high sensitivity, it could also be used to investigate the prevalence of such genetic determinants in mixed bacterial specimens.