1 Sera and Vaccines Central Research Laboratory, ul. Cheßmska 30/34, 00-725 Warsaw, Poland; 2 Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique-Hôpitaux de Paris, Faculté de Médecine Paris-Sud, 78 rue du Général Leclerc, 94275 Le Kremlin-Bicêtre, France
Received 17 May 2002; returned 7 June 2002; revised 12 June 2002; accepted 14 June 2002
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Abstract |
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Keywords: lactamases, ceftazidime, Enterobacteriaceae
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Introduction |
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Materials and methods |
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Three clinical isolates analysed here were identified during the study of cefotaxime-resistant ESBL-producing Enterobacteriaceae strains in Polish hospitals.7 Apart from the high-level resistance to cefotaxime, they also demonstrated resistance to ceftazidime in routine susceptibility testing. Escherichia coli WR 3551/98 was recovered in May 1998 from the post-operative wound of a patient in the intensive care unit (ICU) of a hospital in Wrocßaw, and two Serratia marcescens isolates, BB 1758 and BB 1763, were cultured in February and April 2000 from intubation tubes of two patients in the ICU of a hospital in Bielsko-Biaßa. Species identification was performed with the ATB ID32E test (bioMérieux, Marcy lÉtoile, France), and ESBL expression was determined by the double disc synergy (DDS) test.8
Resistance transfer
The cefotaxime-resistance transfer experiment was performed as described previously,9 with E. coli A15 resistant to rifampicin as the recipient strain.
PCR detection of blaCTX-M genes
Total DNAs of clinical isolates were used in PCR with primers specific for the blaCTX-M-1 and blaCTX-M-3 genes.9 The entire coding regions of the genes were amplified with primers ALA2, which is complementary to the 5' end of the blaCTX-M-1 and blaCTX-M-3 coding regions, and P2D,9 which anneals downstream of the genes (Table 1). The reaction conditions were as reported previously.9
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PCR amplicons containing the blaCTX-M coding regions were sequenced using primers ALA2, A, P1A, P2D, P2A and P2B (Table 1).7 To sequence the 5'-adjacent regions of blaCTX-M genes, the gene-carrying plasmids were purified with the Qiagen Plasmid Midi Kit (Qiagen, Hilden, Germany) and used in reactions with primers ALA3 and ALA4 (Table 1). ALA3 anneals close to the 5' end inside the coding region and is directed upstream of the gene, whereas ALA4 anneals far upstream of the gene and is directed towards it. Sequencing was performed with an ABI PRISM 310 automatic sequencer (PE Biosystems, Foster City, CA, USA).
Typing by randomly amplified polymorphic DNA analysis and plasmid fingerprinting
Total DNA preparations of clinical isolates were used for randomly amplified polymorphic DNA (RAPD) typing, which was performed as described previously.7 Plasmid DNA was subjected to the fingerprinting analysis that was carried out using the PstI restriction enzyme (MBI Fermentas, Vilnius, Lithuania), as reported previously.7 Representative isolates identified as CTX-M-3 producers in the previous studies7 were included in the analyses.
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Results |
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E. coli WR 3551/98, and S. marcescens BB 1758 and BB 1763 isolates were subjected to conjugation, in which cefotaxime was used for selection of recombinant strains. Only E. coli WR 3551/98 produced transconjugants, and the resistance transfer efficiency was relatively high (104 recombinants per donor cell).
ESBL gene identification by PCR and sequencing
For all the clinical isolates, PCR with primers ALA2 and P2D yielded products of the expected size of 1 kb, which contained the entire blaCTX-M gene coding regions. Sequencing revealed that the three coding regions were identical to each other and differed only by a single nucleotide substitution (A-725
G) from the coding region of the blaCTX-M-3 gene.9 This difference determines the aspartate to glycine substitution in position 240 (Asp-240
Gly) of the deduced protein sequence. The nucleotide sequence was identical to coding regions of blaCTX-M-15 genes from India5 and from Japan (GenBank accession no. AY013478).2
Sequencing the upstream regions of the blaCTX-M-15 and blaCTX-M-3 genes
Plasmid DNA from the E. coli WR 3551/98 transconjugant was used for sequencing the entire blaCTX-M-15 gene, together with its 5'-adjacent region. The blaCTX-M-3 gene from Citrobacter freundii 2526/96, in which the gene was originally identified,9 was included in the analysis. The 373 bp DNA sequences located upstream of the blaCTX-M-15 and blaCTX-M-3 coding regions differed only at a single position, 17, with regard to the start of the coding region. Similar to CTX-M-15-producing isolates from India,5 the insertion sequence ISEcp110 was identified in the upstream regions of the genes. However, in Polish isolates it was located further upstream of the genes (by 80 bp) than in the Indian isolates.5
Epidemiological analysis of CTX-M-15-producing isolates
The CTX-M-15-producing E. coli WR 3551/98 and S. marcescens BB 1758 and BB 1763 isolates were typed by RAPD along with representatives of all RAPD types of CTX-M-3-producing isolates of these species that had been identified in Poland.7 E. coli WR 3551/98 was compared with 21 E. coli isolates collected during 19962000 in 12 hospitals, including the hospitals in Wrocßaw and Bielsko-Biaßa, and it represented a unique RAPD type. The S. marcescens isolates BB 1758 and BB 1763 were typed together with 12 isolates of this species, which were identified in seven hospitals between 1996 and 2000. The two CTX-M-15-producing isolates were indistinguishable from each other, but were different from the CTX-M-3-producing S. marcescens strains.
Plasmid DNA of the transconjugant of E. coli 3551/98 was subjected to fingerprinting analysis together with 32 plasmid variants identified in CTX-M-3-producing Enterobacteriaceae in Poland.7 The blaCTX-M-15 gene-carrying plasmid of the E. coli WR 3551/98 isolate turned out to belong to the widespread family of plasmids with blaCTX-M-3 genes (family A), and was very similar to its most prevalent variant (A1) (Figure 1).
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Discussion |
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CTX-M-15 has been identified in three geographically distant countries: India,5 Japan (GenBank accession no. AY013478)2 and Poland. The span of the CTX-M-15 distribution parallels that of CTX-M-3, which, among others, has been reported in Poland7,9 and France.4 Although plasmids carrying the blaCTX-M-3 gene from Polish and French isolates were found to be similar to each other,4 the repeated incidence of the same CTX-M variants in different countries should rather be attributed to independent selection. Although the blaCTX-M-3 and blaCTX-M-15 genes from Polish isolates and blaCTX-M-15 genes from Indian isolates5 were flanked by the same ISEcp1 transposable element on their 5' side, it was inserted in different locations in the isolates from the two countries. ISEcp1 was also identified in the vicinity of plasmidic blaCMY-4 and many other blaCTX-M genes, and it was proposed that this insertion sequence mobilizes various ß-lactamase genes to transfer between different replicons.4,5,10 It is possible that chromosomal ß-lactamase genes, which are probably the ancestors of blaCTX-M-3 and blaCTX-M-15, might be independently mobilized in different strains of an as yet unknown host species, which could trigger the parallel dissemination of these genes in local Enterobacteriaceae populations.
The nucleotide sequence of the blaCTX-M-15 gene described here will appear in the DDBJ/EMBL/GenBank database under the accession no. AJ310929, designated as blaCTX-M-11.
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Acknowledgements |
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Footnotes |
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References |
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