Ponente
Descripción
The high prevalence of antibiotic resistance phenomenon in several environments is one of the greatest concerns threatening human health. Hence, it is vital to dispose of molecular tools that allow proper monitoring of the antibiotic resistant genes (ARGs) encoding resistances to these important therapeutic compounds. For accurate quantification of ARGs, there is a need for sensitive and robust qPCR assays support by a good design of primers and validated protocols. In this respect, eleven relevant ARGs were selected as target genes in this study, including aadA and aadB (conferring resistance to aminoglycosides); ampC, blaTEM, blaSHV and mecA (resistance to β-lactams); drfA1 (trimethoprim resistance); ermB (resistance to macrolides); fosA (resistance to fosfomycin); qnrS (resistance to quinolones) and tetA (resistance to tetracyclines). The in silico design of new primer sets was performed from the sequences targeting these ARGs deposited in the Kyoto Encyclopedia of Genes and Genome (KEGG) database. The adequate design and well performance of new molecular tools were validated in vivo in active sludge samples. The hallmarks of the optimized qPCR assays were high amplification efficiency (>90%), good linear standard curve (R2 > 0.980), high precision between experimental replicates, consistency across replicate experiments and a wide dynamic range. This work provided a well-proven methodology for quantification of ARGs from environmental samples and described relevant experimental conditions for utilization of qPCR, representing useful tools for monitoring dissemination of ARGs into the environment.
