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3 agar plates containing ceftriaxone 2?��g/mL, and one isolated coliform colony per sample was collected. Identification was confirmed by the API20E identification system (bioM��rieux, Marcy l��?toile, France). ESBL confirmatory tests were performed according to CLSI [10]. Identification of CTX-M-type determinants and characterization of CTX-M groups was carried out by PCR [9,11], followed by complete sequencing of blaCTX-M genes. Primers designed in this study were used for amplification and sequencing of variants belonging to the CTX-M-9 group (5��-GATGTAACACGGATTGACC and 5��- GAACTTTTGCTGAGTTGAAGG) and CTX-M-8 group (5��-CACGGATTCAATTTTCAGGAG and 5��-GAGCGCTCCACATTTTTTAG), whereas other groups were sequenced as described previously [9]. Genotyping of CTX-M producers was performed by determination PD98059 ic50 of the main E.?coli phylogenetic groups (A, B1, B2, D) according to the Clermont method [9], random amplification of polymorphic Carnitine palmitoyltransferase II DNA (RAPD) with the 1290 and 1254 decamers [9], and multilocus sequence typing using protocols and conditions described on the E.?coli multilocus sequence typing website [1]. All CTX-M producers were also investigated for quinolone susceptibility by the disk diffusion method [10,12], and for the presence of plasmid-mediated quinolone resistance genes by PCR, as described previously (qnrA, qnrB, qnrS, aac(6��)-Ib-cr, qepA [13]; qnrC [14]; qnrD [15]). Data entry and analysis were performed with the Imatinib solubility dmso Epi Info software package version 2008 (Centers for Disease Control and Prevention, Atlanta, GA, USA). Statistical differences were determined by the chi-squared test. The 2011 survey confirmed the very high resistance rates to the old antibiotics (i.e. ampicillin, tetracycline, trimethoprim-sulphamethoxazole, chloramphenicol) recorded in the previous studies [5�C7], and showed an alarmingly relentless increase of resistance to quinolones (including fluoroquinolones) and expanded-spectrum cephalosporins (Table?1). In particular, E.?coli isolates with acquired resistance to nalidixic acid, ciprofloxacin and expanded-spectrum cephalosporins were found in 76%, 44% and 12.4% of enrolled children, respectively (p?