A rapid two-step algorithm detects and identifies macrolide and ß-lactam antibiotics resistance genes

Abstract

Background: Antibiotic resistance has rapidly grown into a worldwide problem that threatens to compromise the effective treatment of a plethora of diseases. A two-step test algorithm was developed for detection and identification of macrolide and ß-lactamantibiotics resistance genes.Methods & Materials: Bacterial isolates were first tested by a multiplex real-time PCR (mRT-PCR) covering eight genes in relation to macrolide (msrA, ermA, ermB, and ermC) and ß-lactam (blaTEM, blaSHV, blaCTX-M-1, blaCTX-M-9) resistances and the presence of one or multiple resistance genes were determinedbymelting temperature (Tm) profile analyses. Amplification products with indistinguishable Tm profiles were tested subsequently by a liquid beads microarray assay (LBMA) assay with the eight resistance gene-specific probes. The clinical validity was evaluated on 340 clinical isolates with phenotypic antimicrobial susceptibility results used as the standard comparison methods.Results: It has been shown that 75% of isolates can be identify by mRT-PCR and melting temperature analyses only, while the rest 25% need both two approaches. An overall agreement of 96.6% (kappa = 0.93, 95% CI = 0.89-0.96)was observed between the mRT-PCR-LBMA and phenotypic methods. The sensitivities and specificities were 94.5% and 97.5% for macrolide and 92.6% and 98.4% for ß-lactam resistance determination, respectively. Conclusion:The two-step mRT-PCR-LBMA assay provided a cost-effective toolfor rapid determination of macrolide and ßlactam antibiotic resistances.