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 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.
β-Lactam | MIC (μg/ml)
|
|
---|---|---|
K. pneumoniae 193KFFUL | E. coli 193K1 | |
Amoxicillin | >256 | >256 |
Amoxicillin + CLa | 8 | 16 |
Piperacillin | >256 | >256 |
Piperacillin + TZb | 32 | 32 |
Ceftazidime | 96 | 24 |
Ceftazidime + CL | 0.5 | 1.0 |
Cefepime | >256 | 16 |
Cefepime + CL | 0.19 | 0.064 |
Cefotaxime | >256 | >256 |
Cefotaxime + CL | 0.125 | 0.094 |
CL, clavulanic acid.
TZ, tazobactam.
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.
REFERENCES
- 1.Baraniak, A., J. Fiett, W. Hryniewicz, P. Nordmann, and M. Gniadkowski. 2002. Ceftazidime-hydrolyzing CTX-M-15 extended-spectrum β-lactamase (ESBL) in Poland. J. Antimicrob. Chemother. 50:393-396. [DOI] [PubMed] [Google Scholar]
- 2.Eckert, C., V. Gautier, M. Saladin-Allard, N. Hidri, C. Verdet, Z. Ould-Hocine, G. Barnaud, F. Delisle, A. Rossier, T. Lambert, A. Philippon, and G. Arlet. 2004. Dissemination of CTX-M-type β-lactamases among clinical isolates of Enterobacteriaceae in Paris, France. Antimicrob. Agents Chemother. 48:1249-1255. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Edelstein, M., M. Pimkin, I. Palagin, I. Edelstein, and L. Stratchounski. 2003. Prevalence and molecular epidemiology of CTX-M extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae in Russian hospitals. Antimicrob. Agents Chemother. 47:3724-3732. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Karim, A., L. Poirel, S. Nagarajan, and P. Nordmann. 2001. Plasmid-mediated extended-spectrum β-lactamase (CTX-M-3 like) from India and gene association with insertion sequence ISEcp1. FEMS Microbiol. Lett. 201:237-241. [DOI] [PubMed] [Google Scholar]
- 5.Lartigue, M. F., L. Poirel, C. Heritier, V. Tolun, and P. Nordmann. 2003. First description of CTX-M-15-producing Klebsiella pneumoniae in Turkey. J. Antimicrob. Chemother. 52:315-316. [DOI] [PubMed] [Google Scholar]
- 6.Lévesque, C., L. Piché, C. Larose, and P. H. Roy. 1995. PCR mapping of integrons reveals several novel combinations of resistance genes. Antimicrob. Agents Chemother. 39:185-191. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Mulvey, M. R., E. Bryce, D. Boyd, M. Ofner-Agostini, S. Christianson, A. E. Simor, and S. Paton. 2004. Ambler class A extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella spp. in Canadian hospitals. Antimicrob. Agents Chemother. 48:1204-1214. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Mushtaq, S., N. Woodford, N. Potz, and D. M. Livermore. 2003. Detection of CTX-M-15 extended-spectrum β-lactamase in the United Kingdom. J. Antimicrob. Chemother. 52:528-529. [DOI] [PubMed] [Google Scholar]
- 9.Poirel, L., J.-W. Decousser, and P. Nordmann. 2003. Insertion sequence ISEcp1B is involved in expression and mobilization of a blaCTX-M β-lactamase gene. Antimicrob. Agents Chemother. 47:2938-2945. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Poirel, L., M. Gniadkowski, and P. Nordmann. 2002. Biochemical analysis of the ceftazidime-hydrolyzing extended-spectrum β-lactamase CTX-M-15 and of its structurally related β-lactamase CTX-M-3. J. Antimicrob. Chemother. 50:1031-1034. [DOI] [PubMed] [Google Scholar]
- 11.Tzouvelekis, L. S., E. Tzelepi, P. T. Tassios, and N. J. Legakis. 2000. CTX-M-type β-lactamases: an emerging group of extended-spectrum enzymes. Int. J. Antimicrob. Agents 14:137-142. [DOI] [PubMed] [Google Scholar]
- 12.Yu, W. L., K. C. Cheng, L. T. Wu, M. A. Pfaller, P. L. Winokur, and R. N. Jones. 2004. Emergence of two Klebsiella pneumoniae isolates harboring plasmid-mediated CTX-M-15 β-lactamase in Taiwan. Antimicrob. Agents Chemother. 48:362-363. [DOI] [PMC free article] [PubMed] [Google Scholar]