Genetic relatedness between human and animal polymorphic blaTEM genes strengthens zoonotic potential among uropathogenic Escherichia coli strains

Manuela Caniça1,*, Ricardo Dias1, José Duarte Correia2 and Constança Pomba2

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-39->Lys, 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.


View this table:
[in this window]
[in a new window]
 
Table 1. Nucleotides at selected positions in parental blaTEM-1A, blaTEM-1B, blaTEM-1C, blaTEM-2 genes and the 17 blaTEM genes in animal isolates, as determined by PCR-RFLP and nucleotide sequencing

 
PCR-RFLP analysis discriminated the blaTEM genes into three restriction linkage groups (according to polymorphic sequence variations) (Table 1): the ‘TEM-1A type’ (n=1), the ‘TEM-1B type’ (n=14) and the ‘TEM-1C type’ (n=2). Nucleotide sequencing of blaTEM genes representative of each group confirmed this classification. The linkage groups of these blaTEM genes from animal sources were identical to those of the corresponding parental blaTEM genes from human specimens.13 All linkage groups had C32 in the promoter region, indicating a weak promoter.1 However, some of the strains presented resistance phenotypes (with MICs of amoxicillin 1024–>2048 mg/L, cefalothin 16–>32 mg/L and mecillinam 0.5–>256 mg/L, data not shown). This may be due to enzyme hyperproduction as a result of the gene being on multicopy plasmids, tandem-genes in a single plasmid and/or reduced permeability of outer cell membrane to antibiotics.

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

1 . Goussard, S. & Courvalin, P. (1991). Sequence of the genes blaT-1B and blaT-2. Gene 102, 71–3.[CrossRef][ISI][Medline]

2 . Caniça, M. M. M., Chang, Y. L., Krishnamoorthy, R. et al. (1997). Molecular diversity and evolution of blaTEM genes encoding ß-lactamases resistant to clavulanic acid in clinical E. coli. Journal of Molecular Evolution 44, 57–65.[ISI][Medline]

3 . Leflon-Guibout, V., Speldooren, V., Heym, B. et al. (2000). Epidemiological survey of amoxicillin-clavulanate resistance and corresponding molecular mechanisms in Escherichia coli isolates in France: new genetic features of blaTEM genes. Antimicrobial Agents and Chemotherapy 44, 2709–14.[Abstract/Free Full Text]

4 . Pomba-Féria, C. & Caniça, M. (2003). A novel sequence framework (blaTEM-1G) encoding the parental TEM-1 ß-lactamase. FEMS Microbiology Letters 220, 177–80.[CrossRef][ISI][Medline]

5 . Féria, C., Machado, J., Duarte Correia, J. et al. (2001). Virulence genes and P fimbriae PapA subunit diversity in canine and feline uropathogenic Escherichia coli. Veterinary Microbiology 82, 81–9.[CrossRef][ISI][Medline]

6 . Johnson, J. R., O'Bryan, T. T., Low, D. A. et al. (2000). Evidence of commonality between canine and human extraintestinal pathogenic Escherichia coli strains that express papG allele III. Infection and Immunity 68, 3327–36.[Abstract/Free Full Text]





This Article
Extract
FREE Full Text (PDF)
All Versions of this Article:
54/1/284    most recent
dkh307v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Disclaimer
Request Permissions
Google Scholar
Articles by Caniça, M.
Articles by Pomba, C.
PubMed
PubMed Citation
Articles by Caniça, M.
Articles by Pomba, C.