The prevalence of plasmid-mediated quinolone resistance genes [qnr, aac(69)-Ib-cr and qepA] was sought among Enterobacteriaceae strains obtained from the Children's Hospital of Tunis (Tunisia). Non-duplicate isolates (n5278) with resistance to extended-spectrum cephalosporins and collected in 2003, 2007, 2008 and 2009 were screened for qnr genes. Forty (14.4 %) isolates were qnr positive and were screened for the presence of the aac(69)-Ib-cr and qepA genes. qnrB was detected in 21 Klebsiella pneumoniae, 11 Escherichia coli and 6 Enterobacter cloacae isolates. Sequence analysis of the qnrB amplicons revealed variants including 24 qnrB1, 11 qnrB2 and 3 qnrB6. qnrS (qnrS1 allele) was detected only in K. pneumoniae isolates, either alone (two isolates) or with the qnrB gene (one isolate). The qnrA, qnrC and qnrD genes were not found in any of the 278 isolates. No qnr-positive isolates carried the qepA gene. Pyrosequencing results showed that aac(69)-Ib-cr, a variant of the aac(69)-Ib gene, was present in 31 qnr-positive isolates (21 K. pneumoniae isolates, seven Escherichia coli isolates and three Enterobacter cloacae isolates). aac(69)-Ib was also found either alone (two isolates) or in association with aac(69)-Ib-cr (one isolate). Of the 40 qnr-positive isolates, 92.5, 82.5, 57.5, 85 and 82.5 % were non-susceptible to nalidixic acid, ciprofloxacin, levofloxacin, ofloxacin and norfloxacin, respectively, and all were extended-spectrum b-lactamase producers. Random amplified polymorphic DNA-PCR typing of these isolates showed 16, 8 and 5 different genotypes in K. pneumoniae, Escherichia coli and Enterobacter cloacae isolates, respectively. Our study highlights the high prevalence of qnr in association with aac(69)-Ib-cr among Enterobacteriaceae isolates, even from children, who are patients not overtreated with quinolones.

Of forty-seven extended-spectrum cephalosporin-resistant Escherichia coli isolates, collected from children at the Children's Hospital in 2006 (Tunis, Tunisia), we analyzed 32 isolates that were genotypically different by enterobacterial repetitive intergenic consensus -polymerase chain reaction. For all isolates, the double-disk diffusion test revealed synergy between clavulanate and cefotaxime and/or ceftazidime, suggesting the production of extended-spectrum beta-lactamases. Polymerase chain reaction experiments, performed on plasmid DNA, and sequencing revealed the presence of bla(TEM-1B) (26 isolates, 81%), bla(TEM-34(IRT-6)) (3 isolates, 9%), bla(SHV-12) (2 isolates, 6%), and bla(CTX-M-15) (31 isolates, 97%). Further, the insertion sequence ISEcp1 was found upstream from the bla(CTX-M-15) gene in 11 isolates. The bla genes were found alone or in various combinations in a single isolate. bla(TEM-1B) and bla(CTX-M-15) genes were detected in 26 out of the 32 isolates. Three isolates harbored both bla(TEM-34(IRT-6)) and bla(CTX-M-15). bla(SHV-12) was identified either alone or with bla(CTX-M-15) in a single isolate. Our investigation showed the dominance of CTX-M-type extended-spectrum beta-lactamases, with CTX-M-15 particularly common, and to our best knowledge, this is the first report of the coexistence of CTX-M-15 and IRT-6 in E. coli isolates from children in Tunisia.

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