Prevalence of aac(6')-Ib-cr plasmid-mediated and chromosome-encoded fluoroquinolone resistance in Enterobacteriaceae in Italy

The spread of aac(6')-Ib-cr plasmid-mediated quinolone resistance determinants was evaluated in 197 enterobacterial isolates recovered in an Italian teaching hospital. The aac(6')-Ib-cr gene was found exclusively in Escherichia coli strains. The gene was located on a plasmid which presented additional ESBL genes. Most of the clinical strains were clonally related and displayed three point mutations at the topoisomerase level which conferred high resistance to fluoroquinolones.

The aminoglycoside acetyltransferase Aac(6')-Ib-cr variant, an enzyme usually encoded by a plasmid-borne gene, extends its drug targets to include fluoroquinolones (FQs) in addition to aminoglycosides. It is characterized by amino acid changes at codon 102 (Trp Arg) and codon 179 (Asp Tyr). The Aac(6')-Ib-cr protein is able to specifically acetylate hydrophilic FQs presenting a free piperazinyl amine (i.e. ciprofloxacin and norfloxacin) [1].
The aac(6')-Ib-cr gene has spread rapidly among Enterobacteriaceae, and although only conferring a low-level resistance, it may create an environment facilitating the selection of more highly resistant determinants, especially those harbouring topoisomerase mutations. This fact is particularly worrisome at the nosocomial level, where aac(6')-Ib-cr containing strains should be promptly detected and treated with non-hydrophilic FQs, such as levofloxacin or ofloxacin, or other classes of antibiotics to prevent high-level resistance onset and spread.
In this work we determined the prevalence of the aac (6')-Ib-cr gene variant among clinical isolates of Enterobacteriacea collected at the teaching Hospital of Padua, Italy Table 1.
All aac(6')-Ib were tested for other plasmid-mediated quinolone-resistence genes, i.e. qnr and qepA, by PCR amplification and sequencing using published procedures [3]. None of these was found in the aac(6')-Ib-cr-positive samples, while 5 out of 9 aac(6')-Ib-positive strains presented the qnrB19 gene, indicating that just one of the  reported plasmid-encoded mechanisms of FQ resistance was acquired/maintained in the clinical isolates. The presence of mechanisms of chromosomial resistance to FQ was assessed on aac(6')-Ib-cr-positive strains. The genotypic analysis, performed with universal primers [4], revealed that all samples, but #44, coded for two mutations in GyrA (S83L, D87N) and one mutation in ParC (E84V). Phenotypic analysis of resistance to nalidixic acid and FQs confirmed the above results: 15 samples were resistant to all tested drugs, while one (#44) was fully susceptible.
Plasmid DNA was extracted from aac(6')-Ib-cr-positive strains and run on agarose gel, to confirm the presence of the aac(6')-Ib-cr gene on a mobile element: all samples presented two main electrophoretic band corresponding to > 100 Kbp and 25 Kbp ( Figure 1A). In each case the aac(6')-Ib-cr gene was located in the > 100 Kbp band, as demonstrated by PCR gene amplification, using DNA extracted from each band as template. Plasmid localization of the aac(6')-Ib-cr gene was further confirmed by successful transformation into E. coli Top10 strain of all 16 sample-plasmid DNA, extracted according the Kieser protocol [10]. Transferability of the resistance gene of three clinical isolates was tested by conjugation in a kanamycin/sodium azide resistant E. coli J53 strain [3]. All three samples were successfully conjugated. The presence of the aac(6')-Ib-cr was confirmed by PCR in both transformants and transconjugants. In addition, the ESBL genes previously detected in the clinical isolate were always found on the same plasmid as the aac(6')-Ib-cr gene: just in one case (sample #164) TEM-1 was not co-transformed along with aac(6')-Ib-cr and CTX-M-1.  MIC analysis of FQs in transformants and tranconjugants compared to the wild type isolates showed a drastic decrease in the MIC values of all tested antibiotics. However, MIC of ciprofloxacin increased of 2-4 times in transformants and transconjugats, compared to the wild type recipient strains, E. coli Top10 and J53. These results are in line with the notion that the aac(6')-Ib-cr alone does not confer high level resistance to the drugs, but it stimulates chromosomal mutations on the FQ targets, i.e. gyrase and topoisomerase IV, which in turn dramatically increase resistance to these drugs.
We have shown for the first time the presence of the aac(6')-Ib-cr gene limited to E. coli species in North-East Italy. Like other plasmid-mediated resistance genes, i.e. qnr, the aac(6')-Ib-cr gene does not significantly increment MIC values, but it seriously increases the mutant prevention concentration (MPC) with final production of remarkably resistant strains upon treatment with standard FQ dosage [1]. Indeed, we found that all but one clinical isolate presented three mutations at the topoisomerase level (2 in GyrA and 1 in ParC) with consequent generation of very resistant strains (MIC of ciprofloxacin ≥ 32). These mainly derived by clonal expansion, as demonstrated by ERIC subgrouping. Interestingly, sample #44 did not show any mutation in the topoisomerase genes, and so retained full susceptibility to FQs. However, #44 resulted clonally related to sample #39 which instead was fully resistant, indicating that transition from susceptibily to resistance probably occurred in a very limited time interval.
Finally, aac(6')-Ib-cr-positive strains, which were strongly associated with ESBL, were collected mainly by inpatients and the proven plasmid localization and conjugation underline a very efficient mechanism of 1000 bp 500 bp 25 Kb   6  13  23  37  39  40  44  51  M  52  53  164  175  178  182  184  horizontal transferability of these multiresistant strains. Therefore, the presence of aac(6')-Ib-cr-positive strains must be promptly detected and referred to clinicians in order to avoid use of FQs which would augment drug resistance and impair therapy.