Dissemination in Portugal of CTX-M-15-, OXA-1-, and TEM-1-producing Enterobacteriaceae strains containing the aac(6')-Ib-cr gene, which encodes an aminoglycoside- and fluoroquinolone-modifying enzyme.

CTX-M-15, recently detected in Portugal, is an extended-spectrum -lactamase (ESBL) that has spread worldwide (3, 4, 9, 10, 15). The basis for the successful dissemination of blaCTX-M-15 is unknown, although the spread of epidemic Escherichia coli strains and/or conjugative plasmids harboring blaCTX-M-15 has been documented (3, 6). We report the dissemination of blaCTX-M-15 associated with strains and plasmids harboring blaOXA-1, blaTEM-1, and the recently described aac(6 )-Ib-cr gene, which encodes an aminoglycosideand fluoroquinolone-modifying enzyme (12) in different Portuguese regions. During a surveillance study of ESBL-producing isolates in two hospitals and one ambulatory laboratory in three different Portuguese cities (January 2003 to October 2004), we identified 17 E. coli and 2 Klebsiella pneumoniae isolates producing CTX-M-15 (Table 1). Susceptibility testing and characterization of -lactamases were performed by standard methods (2, 7). The presence of resistance genes such as aac(6 )-Ib-cr, qnrA, or qnrB or sequences linked to CTX-M-genetic environments such as ISEcp1, IS903, class 1 integrons, or CR1 (also named open reading frame 513 [ORF513]) was investigated by PCR and sequencing (5, 7, 8, 12, 13). PCR experiments based on published sequences (GenBank accession no. AY458016 and AY259086) were performed to search the vicinity of particular genes. Clonal relatedness was established by pulsedfield gel electrophoresis (PFGE) and identification of E. coli phylogenetic groups by a multiplex PCR assay (7). Conjugation experiments were carried out as described previously (7). Plasmid analysis included estimation of plasmid number and size and comparison of EcoRI-digested DNA patterns (1). All isolates were nonsusceptible to amoxicillin-clavulanic acid, cefotaxime, ceftazidime, aztreonam, cefepime, nalidixic acid, ciprofloxacin, and, with the exception of one K. pneumoniae isolate, kanamycin, gentamicin, and tobramycin. E. coli isolates were grouped into three PFGE types belonging to phylogenetic group B2. Isolates of PFGE type A were predominant, being recovered from different wards in hospitals in the north and center regions and from an outpatient living geographically apart from both institutions. The K. pneumoniae isolates showed identical PFGE patterns. Most strains simultaneously produced CTX-M-15, OXA-1, and TEM-1. The gene blaCTX-M-15 was located 49 nucleotides