First description of CTX-M-15-producing Klebsiella pneumoniae in Portugal

The first CTX-M-15 β-lactamase was found in several enterobacterial isolates from India in 1999 (4). Worldwide spread of CTX-M-15 β-lactamases is now well documented (1-5, 7, 8, 12). CTX-M β-lactamases confer high-level resistance to cefotaxime, ceftriaxone, and aztreonam and are well inhibited by clavulanate and tazobactam (10, 11). Klebsiella pneumoniae 193KFFUL was isolated from a blood culture in September 2003 at the Hospital de Santa Maria. The clinical isolate was resistant to cefotaxime and cefepime (MIC, >256 μg/ml) and ceftazidime and aztreonam (MIC, 96 μg/ml) and susceptible to imipenem (MIC, 0.125 μg/ml). Tazobactam and clavulanate restored the activities of piperacillin, cefotaxime, ceftazidime, and cefepime as determined by E-test strips (Table ​(Table1).1). Genomic DNA was prepared as described elsewhere (6), and PCR experiments were performed using specific primers in order to amplify bla genes coding for CTX-M β-lactamases. The set of primers CTX1 (5′-SCS ATG TGC AGY ACC AGT AA-3′) and CTX2 (5′-CCG CRA TAT GRT TGG TGG TG-3′), designed in accordance with consensus sequences from the blaCTX-M genes available at GenBank, produced an amplicon with 544 bp. In order to perform sequencing of the entire gene, PCR was performed with primers CTX-M-1F (5′-ATG GTT AAA AAA TCA CTG CGY C-3′) and CTX-M-1R (5′-TTA CAA ACC GTC GGT G-3′). The amplicon of 876 bp was cloned into the pCR2.1-TOPO vector, resulting in plasmid p193K1, and introduced into Escherichia coli TOP10 chemically competent cells. The sequenced gene shared 100% homology with blaCTX-M-15. E. coli 193K1 revealed MIC profiles similar to those of the parental strain, particularly for cefotaxime, whose MIC was >256 μg/ml. CTX-M-15 showed increased activity against ceftazidime because of a single nucleotide substitution (A-725→G) that has already been reported in CTX-M-16 (2, 4, 10). TABLE 1. MICs of β-lactam antibiotics alone or in association with β-lactam inhibitors for K. pneumoniae clinical isolate 193KFFUL and E. coli 193K1 harboring recombinant plasmid p193K1 To explore the surrounding regions of blaCTX-M-15, PCR was performed with internal primers CTX1 and CTX2 and primers hybridizing to the ends of the insertion sequences ISEcp1 and IS903 (2) and to the conserved regions of class 1 integrons, 5′-CS and 3′-CS (6). Positive PCR products were obtained with primers ISEcp1F and CTX2 (911 bp) and primers CTX1 and IS903R (1,430 bp). Nucleotide sequence analysis indicated that blaCTX-M-15 was flanked upstream by an ISEcp1-like element and downstream by an IS903-like element. Insertion sequences such as ISEcp1 or IS903 have already been described as flanking regions of blaCTX-M-14, blaCTX-M-17, and blaCTX-M-19 (2, 4, 9). A class 1 integron was identified containing an aadA1 and an aadA2 gene not associated with the blaCTX-M-15 gene. The −35 and −10 promoter sequences for blaCTX-M-15 expression are located at the end of an ISEcp1-like element upstream of its inverted repeat, which is 48 bp from the start codon ATG (data not shown), as already described for blaCTX-M-15 from India and Turkey and different from the blaCTX-M-15 gene described in Poland, in which the distance is 128 bp (4, 9). In addition, analysis of the downstream region of blaCTX-M-15 showed that 685 nucleotides had 97% similarity to IS903-C from blaCTX-M-17. This is the first report identifying an IS903-like element downstream of the blaCTX-M-15 gene in a K. pneumoniae isolate from a Portuguese hospital.