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Antimicrobial Agents and Chemotherapy, January 2005, p. 477-478, Vol. 49, No. 1
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.1.477-478.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

LETTER TO THE EDITOR

First Description of CTX-M-15-Producing Klebsiella pneumoniae in Portugal


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LETTER
 
The first CTX-M-15 ß-lactamase was found in several enterobacterial isolates from India in 1999 (4). Worldwide spread of CTX-M-15 ß-lactamases is now well documented (1-5, 7, 8, 12). CTX-M ß-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 ß-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).


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  		TABLE 1. MICs of ß-lactam antibiotics alone or in association with ß-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.


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T. Conceição
A. Brízio
A. Duarte*
Laboratório de Microbiologia
Faculty of Pharmacy
Av. Forças Armadas 1649-049
Lisbon, Portugal

L. M. Lito
J. Melo Cristino
M. J. Salgado
Faculty of Medicine
Hospital Santa Maria
Lisbon, Portugal

* Phone: 351 21 79 46440,
Fax: 351 21 7986055,
E-mail: aduarte{at}ff.ul.pt

Antimicrobial Agents and Chemotherapy, January 2005, p. 477-478, Vol. 49, No. 1
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.1.477-478.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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