Detection of clinically relevant antibiotic-resistance genes in municipal wastewater using real-time PCR (TaqMan)

Real-time PCR assays were developed for the quantifiable detection of the antibiotic-resistance genes vanA of enterococci, ampC of Enterobacteriaceae, and mecA of staphylococci in different municipal wastewater samples. Primer and probe designs for these resistance genes were constructed and optimised for application in standardised TaqMan PCR assays. Using reference strains, the linear measurement ranges of the assays were defined and covered concentration ranges of five to seven exponential values. Wastewater isolates of vancomycin-resistant enterococci (VRE) and beta-lactam-resistant Enterobacteriaceae were cultivated from municipal wastewaters in order to verify the specificity and sensitivity of the primer-probe systems. Additionally, clinical strains of staphylococci resistant to methicillin (MRSA) confirmed the applicability of the mecA-specific detection system. Total DNAs were extracted from five different wastewater treatment plants and used for direct TaqMan PCR detection of the resistance genes without prior cultivation. In municipal wastewater, the resistance gene vanA was detected in 2 1% of the samples, and ampC in 78%. The gene mecA was not found in municipal wastewater, but in two clinical wastewater samples. (C) 2003 Elsevier B.V. All rights reserved.