Detection of CTX-M-1 and TEM-52 ß-lactamases in Escherichia coli strains from healthy pets in Portugal

Daniela Costa1, Patricia Poeta1, Laura Briñas2, Yolanda Sáenz2, Jorge Rodrigues1,3 and Carmen Torres2,*

1 Departamento de Ciências Veterinárias, Universidade de Trás-os-Montes e Alto Douro, Vila Real; 3 Centro de Estudos de Ciências Animais e Veterinárias, Vila Real, Portugal; 2 Area de Bioquímica y Biología Molecular, Universidad de La Rioja, Madre de Dios 51, 26006 Logroño, Spain

Keywords: extended-spectrum ß-lactamases , healthy animals , Enterobacteriaceae

Sir,

In recent years, the dissemination of Escherichia coli strains harbouring extended-spectrum ß-lactamases (ESBLs) in clinical settings has caused a great deal of concern. Most ESBLs are derived from the classical TEM-1, TEM-2 and SHV-1 enzymes by amino acid substitutions in their sequences.1 A new type of ESBL, CTX-M enzymes, is increasingly being reported among human clinical E. coli strains;2 although only two references exist about detection of this type of ß-lactamase in animal E. coli strains,3,4 they have never been reported in pets before. The first detection of a CTX-M-containing E. coli strain in Portugal was reported very recently.5 It was recovered from a healthy human. Until now such strains had not been reported from animals in Portugal. The objective of this work was to study the intestinal colonization by ESBL-containing E. coli strains in healthy pets in Portugal.

Faecal samples of 75 healthy pets (39 dogs and 36 cats) that had not received previous antibiotic treatment, were recovered in Portugal during 2003 and tested for the presence of ESBL-containing E. coli strains. Samples were seeded in Levine agar supplemented with cefotaxime (2 mg/L), and colonies with typical E. coli morphology were selected and identified by classical biochemical methods. Susceptibility testing for 17 antibiotics was carried out by agar dilution and disc diffusion methods and those E. coli strains that showed broad-spectrum cephalosporin (cefotaxime or ceftazidime) resistance were selected (one per animal), and studied further. The screening of ESBL production was also analysed by the double disc test (cefotaxime and ceftazidime with or without clavulanic acid). The presence of TEM, SHV, OXA, CTX-M, FOX and CMY ß-lactamase-encoding genes was studied by PCR and sequencing3,6 in all the broad-spectrum cephalosporin-resistant E. coli strains recovered. Mutations in the promoter–attenuator region of the chromosomal ampC gene were also studied by PCR and sequencing.3

Broad-spectrum cephalosporin-resistant E. coli strains were detected in five of the 75 faecal samples analysed (6.6%, four dogs and one cat). Four of the five strains, obtained from four unrelated dogs, gave positive ESBL screening test results and were resistant to cefotaxime and/or ceftazidime. The blaTEM-52b gene was identified in three of these four strains and the blaCTX-M-1 gene in the remaining one, these strains being negative for the other ß-lactamase genes tested by PCR. The characteristics of these E. coli strains are included in Table 1. The three blaTEM-52b-containing strains were also resistant to streptomycin, and two of them also to tetracycline. The blaCTX-M-1-containing strain was resistant to streptomycin–tetracycline–sulfamethoxazole–chloramphenicol and also harboured the intI gene, associated with type I integrons (detected by PCR).


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Table 1. Characteristics of the four ESBL-containing E. coli strains recovered from healthy pets in Portugal

 
The remaining broad-spectrum cephalosporin-resistant E. coli strain that was recovered from a healthy cat, showed a negative ESBL screening test and all PCR assays for TEM, SHV, OXA, CTX-M, FOX and CMY ß-lactamase-encoding genes were negative. Mutations at the –42, –18, –1 and +58 positions of the promoter–attenuator region of the chromosomal ampC gene were detected by PCR and sequencing. This strain showed a pattern of multi-resistance, which included nalidixic acid, ciprofloxacin, tetracycline, streptomycin, gentamicin, tobramycin, trimethoprim/sulfamethoxazole and chloramphenicol, and this strain also harboured the intI gene.

To our knowledge, this is the first time that ESBL-encoding genes have been detected in healthy pets and also the first time that CTX-M ß-lactamases have been detected in E. coli strains from animal origin in Portugal. More studies should be carried out in the future to track the evolution of this type of ß-lactamase in different environments.

Acknowledgements

We thank the veterinary Hospital Montenegro of Porto (Portugal) for providing us with the faecal samples for this study. This work has been supported in part by a grant from the Fondo de Investigaciones Sanitarias of Spain (FIS 01/973).

Footnotes

* Corresponding author. Tel: +34-941-299750; Fax: +34-941-299721; Email: carmen.torres{at}daa.unirioja.es

References

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4 . Shiraki, Y., Shibata, N., Doi, Y. et al. (2004). Escherichia coli producing CTX-M-2 ß-lactamase in cattle, Japan. Emerging Infectious Diseases 10, 68–75.

5 . Machado, E., Coque, T. M., Cantón, R. et al. (2004). Emergence of CTX-M ß-lactamase-producing Enterobacteriaceae in Portugal: report of an Escherichia coli isolate harbouring blaCTX-M-14. Clinical Microbiology and Infection 10, 755–7.[CrossRef][ISI][Medline]

6 . Coque, T. M., Oliver, A., Pérez-Díaz, J. C. et al. (2002). Genes encoding TEM-4, SHV-2 and CTX-M-10 extended-spectrum ß-lactamases are carried by multiple Klebsiella pneumoniae clones in a single hospital (Madrid, 1989 to 2000). Antimicrobial Agents and Chemotherapy 46, 500–10.[Abstract/Free Full Text]