CTX-M-15, OXA-30 and TEM-1-producing Escherichia coli in two Portuguese regions

Sir, Currently, CTX-M-type enzymes are the most common group of extended-spectrum β-lactamases (ESBLs) not belonging to the TEM and SHV families.1 Here, we report the phenotypic and molecular characterization of two Portuguese strains of Escherichia coli harbouring CTX-M-15. Both strains were isolated in 2004. One strain (INSRA5753), isolated at Hospital Garcia de Orta in Lisbon, was from the urine of a 78-year-old woman who was living in a residential care home, using a catheter. The other strain (INSRA5754) was isolated from the blood of a 90-year-old woman 1 day after hospitalization in Vila Real. These two hospitals are about 400 km apart. The strains were detected as ESBL-producers in the hospitals, using the VITEK 1 system and ATB G-5, respectively. In the Antibiotic Resistance Unit, at the National Institute of Health in Lisbon, β-lactamase production was confirmed using the Etest ESBL screen method. Using PCR, fragments indicating the presence of the blaTEM gene, the blaOXA gene, the blaCTX gene and the ubiquitous ampC gene were amplified from both strains.2–4 The amplification products from blaTEM, blaOXA and blaCTX genes from both strains were then purified and sequenced. For sequencing of the blaCTX-M-15 gene, we used primers specific for the consensus of CTX-M group I: CTX-M-15F, 5′-AGAATAAGGAATCCCATGGTT-3′ and CTX-M-15R, 5′-ACCGTCGGTGACGATTTTAG-3′. Sequencing confirmed three resistance genes in both strains, namely blaTEM-1B, blaOXA-30 and blaCTX-M-15. Sequence analysis using CTX-M-15 primers and ISEcp1, IS26 or IS903r primers5 indicated the presence of an ISEcp1-like element in both strains although neither had IS903, thus differing from the mobile elements of the blaCTX-M-15 gene detected previously in Klebsiella pneumoniae.6 The IS26 element was also not present in our strains. These findings suggest that CTX-M-15 enzymes in these two species in Portugal might have emerged in multiple places by plasmid acquisition of blaCTX-M-15 genes, with different elements implicated in the dissemination of these β-lactamase genes.6 This was corroborated by the PFGE analysis, which showed different clonal origins for each isolate (data not shown). The transconjugant C600-URA5753 was a CTX-M-15-positive-transconjugant from E. coli INSRA5753 derived using C600 as the recipient. The transconjugant C600-URA5754 co-expressed CTX-M-15, OXA-30 and TEM-1 enzymes; indeed, none of these enzymes was transferable alone from E. coli INSRA5754 strain to E. coli C600 (Table 1), suggesting that the blaCTX-M-15 gene was carried in only one transferable plasmid with blaTEM-1B and blaOXA-30. MICs of various β-lactams, alone or in combination with β-lactamase inhibitors (Table 1), were determined using an agar dilution method,2 and MICs of other antimicrobial agents were determined using a broth microdilution method. The clinical isolates and the transconjugants showed a higher level of resistance to cefotaxime, ceftriaxone and aztreonam than to ceftazidime, which is characteristic of CTX-M producers. Isoelectric focusing revealed that both the clinical strain and transconjugant C600-URA5754 produced β-lactamases with pIs of 5.4 (TEM-1), 7.5 (OXA-30) and 8.9 (CTX-M-15). Transconjugant C600-URA5753 produced a β-lactamase with a pI of 8.9. MICs of β-lactam antibiotics for E. coli strains isolated in two hospitals, transconjugants and recipientsa E. coli C600-URA5753 (harbouring the CTX-M-15 enzyme) and E. coli C600-URA5754 (harbouring TEM-1B, OXA-30 and CTX-M-15 enzymes) were transconjugants of E. coli INSRA5753 (harbouring TEM-1B, OXA-30 and CTX-M-15 enzymes) and INSRA5754 (harbouring TEM-1B, OXA-30 and CTX-M-15 enzymes), respectively; E. coli C600 was the recipient; E. coli K12 J53 R111 (harbouring TEM-1 enzyme) was used as the control. CLA, clavulanic acid at a fixed concentration of 2 mg/L. TAZ, tazobactam at a fixed concentration of 4 mg/L. MICs of β-lactam antibiotics for E. coli strains isolated in two hospitals, transconjugants and recipientsa E. coli C600-URA5753 (harbouring the CTX-M-15 enzyme) and E. coli C600-URA5754 (harbouring TEM-1B, OXA-30 and CTX-M-15 enzymes) were transconjugants of E. coli INSRA5753 (harbouring TEM-1B, OXA-30 and CTX-M-15 enzymes) and INSRA5754 (harbouring TEM-1B, OXA-30 and CTX-M-15 enzymes), respectively; E. coli C600 was the recipient; E. coli K12 J53 R111 (harbouring TEM-1 enzyme) was used as the control. CLA, clavulanic acid at a fixed concentration of 2 mg/L. TAZ, tazobactam at a fixed concentration of 4 mg/L. To our knowledge the combination of CTX-M-15, TEM-1 and OXA-30 β-lactamases, here reported, was described previously only in strains from Senegal.7 However, the combination of CTX-M-15 and OXA-1 (similar to OXA-30) has been described in the UK, Canada and India.1,8 The spread of resistance to extended-spectrum cephalosporins, their extensive use, and the coexistence of strains co-expressing the same TEM, OXA and CTX-M enzymes in distant regions of Portugal may impede the use of β-lactams in other regions of the country. These cases involving hospital and community environments are consistent with dissemination either from or to the community, which is of high concern. None to declare. Nuno Mendonça was supported by the Portuguese ‘NIH Scientific Research Ricardo-Jorge’ grant. This work was supported financially by the POCTI/2001/ESP/43037 (38% FEDER) grant from Fundação para a Ciência e a Tecnologia, Portugal. 1Antibiotic Resistance Unit, Centre of Bacteriology, National Institute of Health Dr Ricardo Jorge, Avenida Padre Cruz, 1649-016 Lisboa, Portugal; 2Laboratory of Microbiology, Centro Hospitalar Vila Real-Peso da Régua S.A., Portugal; 3Laboratory of Microbiology, Hospital Garcia de Orta S.A., Portugal