1 Service de bactériologie-virologie, Hôpital de Bicêtre, Assistance publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, 94275 Le Kremlin-Bicêtre, France; 2 Department of Microbiology, Istanbul Medical Faculty, Capa, Istanbul, Turkey
Keywords: extended-spectrum ß-lactamases, ESBLs, K. pneumoniae, resistance, CTX-M-15, Turkey
Sir,
At the beginning of the 1990s, extended-spectrum ß-lactamase (ESBL)-producing Gram-negative bacteria, exhibiting a higher level of resistance to cefotaxime than to ceftazidime, were described in Germany (1990), France and Argentina (1992).1 These ESBLs were named CTX-M type ß-lactamases, owing to their high level of activity against cefotaxime.1 Such Ambler class A ß-lactamases have <40% identity with the ß-lactamases of the TEM and SHV series.1 Twenty-nine different genes encoding CTX-Ms have been characterized so far. They have been identified mostly from Enterobacteriaceae isolated from various geographical areas, mainly from South America, Europe and East Asia.1 Unlike most CTX-Ms, CTX-M-15, CTX-M-16 and CTX-M-19 hydrolyse ceftazidime at a higher rate than cefotaxime.2
We report here the first detection of a clinical isolate from Turkey that expresses CTX-M-15.
In September 2001, a multiresistant isolate of Klebsiella pneumoniae (KP7881) was isolated from a urine sample of a patient hospitalized in the surgical unit of the Istanbul Faculty Hospital. This isolate was resistant to all ß-lactams except imipenem. MICs of ß-lactams were determined by an agar dilution method (Table 1).2 A ceftazidime/co-amoxiclav synergy test was slightly positive, whereas a cefotaxime/co-amoxiclav synergy test remained negative. The isoelectric points (pI)2 of culture extracts of this isolate were 5.6, 6.1, 7.4, 8.2 and 8.6. Plasmid DNA was isolated by the Kieser procedure3 and transformed into Escherichia coli DH10B by electroporation as described.2 Four antibiotic resistance phenotypes of transformants were obtained: three gave different ESBL phenotypes and another exhibited resistance to penicillins and co-amoxiclav. The MICs of ß-lactams for the four transformants and the parental isolate are shown in Table 1. Transformant 1 was resistant to cefotaxime and of intermediate susceptibility to ceftazidime. It was also resistant to kanamycin, tobramycin, amikacin, netilmicin, spectinomycin, tetracycline, sulphonamides, trimethoprim and chloramphenicol. PCR amplification, performed with DNA of transformant 1 as template using blaCTX-M-specific primers,2 was positive, and sequencing of the PCR product identified the blaCTX-M-15 gene. Using PCR experiments, an insertion sequence ISEcp1 was identified upstream of the 5' end of this gene, with primers hybridizing to the ends of this insertion sequence.2 The pI value of this ESBL was 8.6, and the blaCTX-M-15 gene was located on a 60 kb plasmid. Two other transformants (transformants 2 and 3) exhibited an ESBL phenotype with a higher resistance to ceftazidime than to cefotaxime. Using primers specific for the SHV gene,2 blaSHV-12 (pI 8.2) was identified in transformant 2 located on a 70 kb plasmid, and in transformant 3 on a 190 kb plasmid. In addition, in transformant 3, three other ß-lactamase genes were identified [blaTEM-2 (pI 5.4), blaOXA-1 (pI 7.4) and blaOXA-10 (pI 6.1)] also using primers specific for the TEM gene,2 the OXA-1 gene2 and the OXA-10 gene.2 Transformant 4, exhibiting a restricted spectrum ß-lactamase phenotype, encoded two ß-lactamases that corresponded to TEM-2 and OXA-10, and was located on a 140 kb plasmid.
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ß-Lactamase CTX-M-15, which differs from CTX-M-3 by a single amino acid substitution Asp-240 to Gly, was identified first in India2 and in Japan (GenBank accession no. AY013478), and was subsequently detected in Canada, Russia, Bulgaria, Poland5 and France.6 Its report now in Turkey suggests that the blaCTX-M-15 gene may be highly prevalent worldwide. Moreover, as found previously in India2 and Poland,5 K. pneumoniae KP7881 also possesses ISEcp1 located upstream of the blaCTX-M-15 gene. The ISEcp1 element was located at exactly the same position upstream of blaCTX-M-15 as found in Indian isolates, but closer to the blaCTX-M-15 gene, as opposed to what was reported from Polish isolates.5 Thus, an identical blaCTX-M-15 gene may be spreading in Poland and Turkey but located on different genetic backgrounds. The ability of the ISEcp1 element to mobilize and promote expression of ß-lactamase genes may explain, in part, the current spread of the blaCTX-M-15 gene.
Acknowledgements
This work was funded by a grant from the Ministère de lEducation Nationale et de la Recherche (UPRES-EA3539), Université Paris XI, France.
Footnotes
* Corresponding author. Tel: +33-1-45-21-36-32; Fax: +33-1-45-21-63-40; E-mail: nordmann.patrice{at}bct.ap-hop-paris.fr
References
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2 . Karim, A., Poirel, L., Nagarajan, S. et al. (2001). Plasmidmediated extended-spectrum ß-lactamase (CTX-M-3 like) from India and gene association with insertion sequence ISEcp1. FEMS Microbiology Letters 201, 23741.[CrossRef][ISI][Medline]
3 . Kieser, T. (1984). Factors affecting the isolation of CCC DNA from Streptomyces lividans and Escherichia coli. Plasmid 12, 1936.[ISI][Medline]
4 . Hernandez-Alles, S., Albert, S., Alvarez, D. et al. (1999). Porin expression in clinical isolates of Klebsiella pneumoniae. Microbiology 145, 6739.[Abstract]
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