Emerging resistance mechanisms in ESBL-producing strains

Objective: ESBL producing strains are increasingly reported to present additional resistance mechanisms. These multidrug resistant strains should be detected early to rationalize drug treatment and avoid increased selection of resistance. The aim of this study was to detect the presence of AmpC, carbapenemases and plasmid-mediated quinolone resistance (PMQR) (qnr, aac(6)-Ib-cr and qepA) mechanisms in ESBLproducing strains by genotypic assays and compare their efficiency versus phenotypic methods. Methods: ESBL- and AmpC-producing strains were identified by the double-disk test and double disk synergy test, respectively. Carbapenemases were phenotypically detected by the Hodge test. MIC of fluoroquinolones was detected by Etest. AmpC, carbapenemase, qnr, aac(6)-Ib-cr and qepA genes were identified by multiplex PCR and sequencing. Topoisomerase II mutations were detected by sequencing of the quinolone-resistant determining region. Results: In 2009, 200 ESBL-producing Enterobacteriaceae isolates were collected at the Microbiology Unit of the Padua Hospital. ESBL belonging to different classes (TEM, SHV, CTX-M and OXA) were characterized by genotypic analysis. Qnr and aac(6)-Ib-cr genes were found in 26% and 8% isolates, respectively. Qnr was mostly present in Klebsiella pneumoniae, while aac(6)-Ib-cr was found exclusively in Escherichia coli. QepA was not found. Both genes were localized on plasmids and could be both transformed and trans-conjugated in acceptor strains. MIC of fluoroquinolones on these acceptor strains indicated a 20–100 increased resistance due to the plasmid-mediated mechanism. However, high-level resistance to fluoroquinolone in the wild-type strains was due to the additional presence of topoisomerase mutations in strains presenting both ESBL and PMQR. AmpC were detected on 5.5% isolates of Enterobacter spp. and Proteus mirabilis. Carbapenemases were found in 3% isolates of E. aerogenes, E. coli and K. pneumoniae. Carbapenemases were subsequently genotypically characterized as IMP, VIM, OXA, KPC CMY or SME types. Conclusions: Emerging resistance mechanisms were found in ESBLproducing strains, with PMQR being the most frequent. While genotypic assays implement phenotypic testing of AmpC and carbapenemases, they are the only methods available up to date for detection of PMQR. Hence, both phenotypic and genotypic methods should be employed to rationally direct the pharmacological treatment.