Ceftazidime-hydrolysing CTX-M-15 extended-spectrum ß-lactamase (ESBL) in Poland

Anna Baraniak1, Janusz Fiett1, Waleria Hryniewicz1, Patrice Nordmann2 and Marek Gniadkowski1,*

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The CTX-M-15 extended-spectrum ß-lactamase (ESBL) was recently identified in Enterobacteriaceae isolates in India, and demonstrated significant hydrolytic activity against ceftazidime, in contrast to the majority of CTX-M enzymes. CTX-M-15 differs from CTX-M-3, which is one of the most prevalent ESBLs in Poland, by only a single amino acid change (Asp-240->Gly). Three cefotaxime- and ceftazidime-resistant Enterobacteriaceae isolates, recovered during 1998–2000 in two Polish hospitals, were found to produce CTX-M-15. Similar to those from India, the isolates contained the ISEcp1 insertion sequence located upstream of the blaCTX-M-15 gene, which has been recently demonstrated to mobilize 3'-adjacent genes to transfer between DNA replicons. However, its different position with respect to the ß-lactamase gene indicated the independent selection of the ESBL gene in the two countries.

Keywords: lactamases, ceftazidime, Enterobacteriaceae


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
CTX-M ß-lactamases constitute one of the most rapidly growing extended-spectrum ß-lactamase (ESBL) families. Over the last decade they have been identified in numerous countries of Africa, South America, Asia and Europe.1,2 Four evolutionary groups of CTX-M enzymes have been discerned based on the comparison of their sequences, and these are groups of CTX-M-1, CTX-M-2, CTX-M-8 and CTX-M-9.2,3 CTX-Ms are closely related to chromosomally encoded ß-lactamases in the genus Kluyvera, and the enzymes identified in Kluyvera ascorbata (GenBank accession nos AJ251722 and AJ272538) seem to be the direct evolutionary ancestors of CTX-M-2 group members.4 Despite their structural diversity, most CTX-M ß-lactamases have similar hydrolysis profiles, which include cefotaxime and ceftriaxone, but not ceftazidime.1 Recently however, three novel CTX-M variants, CTX-M-15, -16 and -19, were found to efficiently hydrolyse ceftazidime as well.3,5,6 In this article we report the selection of CTX-M-15-producing Enterobacteriaceae in Poland.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Clinical isolates

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 l’Étoile, 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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Table 1.  Primers used in the study for PCR amplification and sequencing of blaCTX-M genes
 
DNA sequencing

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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Resistance transfer

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 (~10–4 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 1996–2000 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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Figure 1. Part of the plasmid fingerprinting analysis performed with the PstI restriction enzyme. M, DNA molecular weight markers (GeneRuler 100 bp DNA Ladder Plus; MBI Fermentas); *, the blaCTX-M-15 gene-carrying plasmid purified from the transconjugant of E. coli WR 3551/98. The remaining lanes contain plasmids purified from different CTX-M-3-producing isolates identified in the previous work;7 A1, an example of a plasmid with the fingerprint described as A1.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Data presented in this work document the selection of CTX-M-15 ESBL-producing enterobacterial isolates in Poland. The E. coli and two clonally related S. marcescens isolates were identified in an interval of 2 years in two geographically distant hospitals. The blaCTX-M-15 gene coding region differs from blaCTX-M-3 by only a single nucleotide substitution; moreover, the blaCTX-M-15 and blaCTX-M-3 genes compared in this work were located within the same wider sequence context. At least in the E. coli isolate from Wrocßaw, the blaCTX-M-15 gene was present in a plasmid that was very similar to the widespread blaCTX-M-3-carrying plasmid in Poland.7 These data indicated that the blaCTX-M-15 genes analysed here most likely evolved directly from plasmid-located blaCTX-M-3 genes. The emergence of blaCTX-M-15 genes in microbial populations in the two hospitals was probably due to independent selection, because in both institutions organisms producing CTX-M-3 were common at the time.7 The CTX-M-15-expressing isolates were not related to any CTX-M-3-producing isolates of their species identified previously in Poland; however, this observation is not surprising considering the clonal diversity of CTX-M-3-producing populations in Polish hospitals.7 Out of many CTX-M variants that have been reported in recent years,2 CTX-M-15 belongs to a specific group of these enzymes, which are characterized by the increased ceftazidime-hydrolysing activity. In the case of CTX-M-19 this activity has been attributed to the Pro-167->Ser mutation6 and in CTX-M-15 and CTX-M-16 it is due to the Asp-240->Gly substitution.3,5 What is noteworthy is that CTX-M-15 and CTX-M-16 belong to two different groups within the CTX-M family,2,3 which may have originated from different ancestral ß-lactamases. It is possible that the repetitive selection of ceftazidime-hydrolysing variants of CTX-M enzymes is a result of the ceftazidime pressure that is exerted on Enterobacteriaceae populations in many hospitals over the world.

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.


    Acknowledgements
 
We would like to thank Anna Przondo-Mordarska, Beata MÃczyáska and Hildegarda Maílanka for collecting clinical isolates, Agnieszka Mrówka for her technical assistance, and Barbara S. Ink and Ewa Sadowy for critical reading of the manuscript.


    Footnotes
 
* Corresponding author. Fax: +48-22-841-29-49; E-mail: gniadkow{at}cls.edu.pl Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Tzouvelekis, L. S., Tzelepi, E., Tassios, P. T. & Legakis, N. J. (2000). CTX-M-type ß-lactamases: an emerging group of extended spectrum enzymes. International Journal of Antimicrobial Agents 14, 137–42.[ISI][Medline]

2 . Bush, K. & Jacoby, G. A. (2002). Amino acid sequences for TEM, SHV and OXA extended-spectrum and inhibitor resistant ß-lactamases. Published by the Lahey Clinic. [Online.] http://www.lahey.org/studies/webt.htm (date last accessed 10 May 2002).

3 . Bonnet, R., Dutour, C., Sampaio, J. L. M., Chanal, C., Sirot, D., Labia, R. et al. (2001). Novel cefotaximase (CTX-M-16) with increased catalytic efficiency due to substitution Asp240ÆGly. Antimicrobial Agents and Chemotherapy 45, 2269–75.[Abstract/Free Full Text]

4 . Dutour, C., Bonnet, R., Marchandin, H., Boyer, M., Chanal, C., Sirot, D. et al. (2002). CTX-M-1, CTX-M-3, and CTX-M-14 ß-lactamases from Enterobacteriaceae in France. Antimicrobial Agents and Chemotherapy 46, 534–7.[Abstract/Free Full Text]

5 . Karim, A., Poirel, L., Nagarajan, S. & Nordmann, P. (2001). Plasmid-mediated extended-spectrum ß-lactamase (CTX-M-3 like) from India and gene association with insertion sequence ISEcp1. FEMS Microbiology Letters 201, 237–41.[ISI][Medline]

6 . Poirel, L., Naas, T., Le Thomas, I., Karim, A., Bingen, E. & Nordmann, P. (2001). CTX-M-type extended-spectrum ß-lactamase that hydrolyzes ceftazidime through a single amino acid substitution in the omega loop. Antimicrobial Agents and Chemotherapy 45, 3355–61.[Abstract/Free Full Text]

7 . Baraniak, A., Fiett, J., Sulikowska, A., Hryniewicz, W. & Gniadkowski, M. (2002). Countrywide spread of CTX-M-3 extended-spectrum ß-lactamase (ESBL)-producing microorganisms of the family Enterobacteriaceae in Poland. Antimicrobial Agents and Chemotherapy 46, 151–9.[Abstract/Free Full Text]

8 . Jarlier, V., Nicolas, M., Fournier, G. & Philippon, A. (1988). Extended broad-spectrum ß-lactamases conferring transferable resistance to newer ß-lactam agents in Enterobacteriaceae: hospital prevalence and susceptibility patterns. Reviews of Infectious Diseases 10, 867–78.[ISI][Medline]

9 . Gniadkowski, M., Schneider, I., Paßucha, A., Jungwirth, R., Mikiewicz, B. & Bauernfeind, A. (1998). Cefotaxime-resistant Enterobacteriaceae isolates from a hospital in Warsaw, Poland: identification of a new CTX-M-3 cefotaxime-hydrolyzing ß-lactamase that is closely related to the CTX-M-1/MEN-1 enzyme. Antimicrobial Agents and Chemotherapy 42, 827–32.[Abstract/Free Full Text]

10 . Stapleton, P. D. (1999). Novel insertion sequence, ISEcp1, mobilizes the plasmid-mediated class C ß-lactamase-coding gene, blaCMY-4. In Program and Abstracts of the Thirty-ninth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 1999. Abstract 1457, p. 132. American Society for Microbiology, Washington, DC, USA.