1 Antibiotic Resistance Unit, Centre of Bacteriology, National Institute of Health Dr. Ricardo Jorge, Av. Padre Cruz, 1649-016 Lisbon; 2 CIISA, Faculty of Veterinary Medicine, Universidade Técnica de Lisboa, Rua Prof. Cid dos Santos, 1300-477 Lisbon, Portugal
Keywords: ß-lactamases , E. coli , bla genes , animal
Sir,
TEM ß-lactamases are encoded by genes which have various coding region frameworks and types of promoter. Six blaTEM genes coding for TEM-1 ß-lactamase (blaTEM-1A, blaTEM-1B, blaTEM-1C, blaTEM-1D, blaTEM-1E and blaTEM-1F) and the blaTEM-2 gene coding for TEM-2 ß-lactamase have been found in isolates from humans.13 Recently, we reported a new sequence framework blaTEM-1G in an Escherichia coli strain of animal origin.4 These genes differ by silent mutations at any of 10 positions in their nucleotide sequences (32, 162, 175, 226, 346, 436, 604, 682, 913 and 925). The amino acid sequences of TEM-1 and TEM-2 ß-lactamases differ at one position (Gln-39Lys, respectively) due to a single base difference (C317A).1 We investigated the molecular diversity of blaTEM genes encoding TEM-1 enzymes (pI of 5.4) produced by amoxicillin-resistant uropathogenic E. coli strains isolated from dogs.
Seventeen amoxicillin-resistant uropathogenic E. coli strains (named in this study from Fmv1 to Fmv17) were randomly chosen from the collection of the Clinical Pathology Laboratory, Faculty of Veterinary Medicine, Portugal. The MICs of various ß-lactams were determined (Table 1) using a microdilution method with an inoculum of 5x105 cfu/mL. DNA amplification by PCR, analysis of restriction-fragment length polymorphism (RFLP) and nucleotide sequencing were used for genotyping as previously described.2 PCR-RFLP identifies all the mutations cited above, except that at position 913 (Table 1). PCR DNA fragments of 1092 bp from five of the 17 blaTEM genes were purified with Qiaquick spin columns (Qiagen, Hilden, Germany), and then sequenced on both strands with the primer pair FIN/DEB, described previously,2 using an ABI377 sequencer (Applied Biosystems, Foster City, CA, USA). This allowed detection of mutations at nucleotide position 913, and also confirmed PCR-RFLP results.
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Analysis of the molecular diversity of blaTEM genes encoding ß-lactamases contributes to our understanding of this mechanism of resistance in Enterobacteriaceae isolated from humans,2,3 or animals (this study). We demonstrated the genetic relatedness between blaTEM genes in pathogenic E. coli strains isolated from humans,2,3 and animal species (this study). This genetic proximity is in agreement with the known zoonotic potential of uropathogenic E. coli strains: these strains display PapA subunit diversity and share the same papG alleles coding for the Pap fimbriae adhesin, both of which are virulence factors.5,6
Acknowledgements
This work was supported by POCTI/CVT/36253/99 (FEDER) grant from Fundação para a Ciência e a Tecnologia, Lisbon, Portugal.
Footnotes
* Corresponding author. Tel/Fax: +351-217519246; Email: manuela.canica{at}insa.min-saude.pt
References
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