Plasmid-mediated fluoroquinolone resistance in Enterobacteriaceae

Clinical isolates of fluoroquinolone-resistant Enterobacteriaceae are emerging worldwide. The traditional resistance mechanism is the accumulation of mutations in the chromosome coding for the target molecules of fluoroquinolone. All known plasmid-mediated quinolone resistance determinants - namely, Qnr determinants, aac(6’)-Ib-cr enzyme, qepA and oqxAB efflux pumps - can individually confer low-level resistance. In this condition, bacterial cells have increased mutation frequency, making it easier the selection of higher-level fluoroquinolone resistance. Our aims were to survey for plasmid-mediated quinolone resistance determinants a collection of Enterobacteriaceae clinical isolates originating from clinical microbiological laboratories of North-East Italy, and to characterize both the resistant strains and the plasmids harbouring quinolone resistance determinants and beta-lactamase genes. Altogether, 756 Enterobacteriaceae clinical isolates were collected: 497 Escherichia coli, 68 Klebsiella spp., 18 Citrobacter spp., 69 Proteus spp., 24 Morganella spp., 21 Providencia spp., 52 Enterobacter spp. and 7 Serratia marcescens. MIC values were determined by microdilution for ciprofloxacin and levofloxacin, showing values between 0.06 and 128 µg/ml. Screening by PCR for plasmid-mediated quinolone resistance determinants yielded 108 positives [38 qnr and 70 aac(6’)-Ib-cr] out of 104 isolates. Sequencing and analysis of PCR-positive products verified: 26 qnrS1, 2 qnrB2, 2 qnrB6, 2 qnrB8, 1 qnrB19 and 5 qnrD. Four Proteus mirabilis and one Morganella morganii isolates were qnrD positive. Plasmid extraction was performed, and Southern blot analysis verified the plasmid localization of the qnrD determinants. Inverse PCR-based sequencing of qnrD-harbouring plasmids, resulted in a 2687 bp and 2684 bp plasmid DNA for Proteus mirabilis and Morganella morganii, respectively. Both plasmid sequences are deposited at Genbank with accession numbers JN183060 and JN183061. Among the qnrS1 positive isolates, 10 E. coli, 15 Klebsiella spp. and one Proteus mirabilis were detected. Detection of beta-lactamase genes by PCR and sequencing yielded: 6 VIM-1, 7 SHV-12 and 3 LAP-2 positives. All four qnrS1 E. coli with the aac(6’)-Ib-cr variant were CTX-M-15, OXA-1 and TEM-1 positive. PCR-based replicon typing found incN type plasmid to be the most prevalent. Conjugation experiment found 3 qnrS1 and VIM-1 harbouring conjugable plasmids. We could also demonstrate in three Klebsiella pneumoniae ST147 strains that qnrS1 and blaLAP-2 are integrated in the same ISEcl2 genetic context. Southern blot verified that the qnrS1 gene is localized on incN or untypable plasmids. Altogether 70 aac(6’)-Ib-cr variant positive isolates were found: 60 E. coli, 8 Klebsiella spp., one P.mirabilis and one M.morganii. Replicon typing of plasmids found incFia and incF types to be common for E. coli and P. mirabilis while incColE type to be predominant in Klebsiella spp., whilst M.morganii was untypeable. A high correlation of the aac(6)-Ib-cr variant with OXA-1 and CTX-M-15 beta-lactamases was found in E. coli (42/60) and in Klebsiella spp. (8/8), whilst in P. mirabilis the aac(6)-Ib-cr variant was associated with TEM-52 and OXA-1. Our survey for plasmid-mediated quinolone resistance determinants found a 13.7% prevalence (104/756). MIC values for ciprofloxacin and levofloxacin showed uneven resistance levels among the qnr-positive isolates, with only 13.1% of strains (5/38) in the range of low-level resistance, while in case of the aac(6’)-Ib-cr variant all positive isolates were resistant to fluoroquinolones. Our survey found the first qnrD determinants in Europe, and we could also demonstrate the first LAP-2 beta-lactamase in Italy.