JAC Advance Access originally published online on October 5, 2006
Journal of Antimicrobial Chemotherapy 2006 58(6):1311-1312; doi:10.1093/jac/dkl415
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Correspondence |
Detection of Escherichia coli harbouring extended-spectrum ß-lactamases of the CTX-M, TEM and SHV classes in faecal samples of wild animals in Portugal
1 Departamento de Ciências Veterinárias, Universidade de Trás-os-Montes e Alto Douro Vila Real, Portugal 2 Area de Bioquímica y Biología Molecular, Universidad de La Rioja Madre de Dios 51, Logroño, Spain 3 Centro de Estudos de Ciência Animal e Veterinária Vila Real, Portugal
*Corresponding author. Tel: +34-941299750; Fax: +34-941299721; E-mail: carmen.torres{at}daa.unirioja.es
Keywords: CTX-M-14 , CTX-M-1 , TEM-52 , SHV-12
Sir,
Production of extended-spectrum ß-lactamases (ESBLs) by Enterobacteriaceae, specifically by Escherichia coli, has caused a major concern in several countries, being frequently implicated in human infections. In the last few years, ESBL-containing E. coli strains have also been found and reported in healthy animals.1,2 In Portugal there are few studies on ESBL-producing E. coli strains; two cases have been reported in humans3,4 and one case in pets.5 To our knowledge, there are no previous reports of ESBL-containing E. coli strains isolated from wild animals, either in Portugal or in other countries.
A total of 72 faecal samples of wild animals were recovered from Natural Parks located in the north or centre of Portugal during the period 2003–2004 (14 birds of prey, 10 owls, 7 foxes, 6 wild rabbits, 5 genets, 4 forest wildcats, 3 storks, 3 deer, 3 otters, 2 wolves, 2 mouflons, 1 badger, 1 partridge, 1 hedgehog, 1 pigeon, 1 ferret, 1 quail, 1 wild boar, 1 salamander, 1 snake, 1 winter wren, 1 jay, 1 magpie and 1 Mediterranean turtle). The samples were seeded on Levine agar plates supplemented with 2 mg/L cefotaxime (Levine-CTX plates) and incubated for 24 h at 37°C. Samples were also seeded on non-supplemented medium (Levine plates) to control faecal E. coli colonization of animals. E. coli isolates were identified by classical biochemical methods. Susceptibility to 16 antibiotics (ampicillin, amoxicillin + clavulanic acid, cefoxitin, ceftazidime, cefotaxime, aztreonam, imipenem, gentamicin, amikacin, tobramycin, streptomycin, nalidixic acid, ciprofloxacin, sulfamethoxazole/trimethoprim, tetracycline and chloramphenicol) was tested by the disc diffusion method (CLSI criteria, formerly NCCLS) in E. coli isolates recovered from Levine-CTX plates. Broad-spectrum cephalosporin-resistant isolates were selected for further studies (one isolate per sample). Screening for the detection of ESBLs was carried out by the double disc diffusion test (using cefotaxime, ceftazidime and amoxicillin + clavulanic acid discs) (CLSI criteria).
E. coli isolates were detected in Levine plates from 56 of the 72 animal faecal samples (78%), and in Levine-CTX plates from 9 of these faecal samples. One E. coli isolate from Levine-CTX plates was selected from each of the faecal samples. All of the nine isolates showed an intermediate susceptibility or resistant phenotype to cefotaxime and/or ceftazidime, and a positive screening test for ESBLs was obtained in all of them.
The presence of genes encoding TEM (F: 5'-TTCTTGAAGACGAAAGGGC-3'; R: 5'-ACGCTCAGTGGAACGAAAAC-3', amplicon size 1150 bp), SHV (F: 5'-CACTCAAGGATGTATTGTG-3'; R: 5'-TTAGCGTTGCCAGTGCTCG-3'; amplicon size 885 bp), OXA-1 (F: 5'-ACACAATACATATCAACTTCGC-3'; R: 5'-AGTGTGTTTAGAATGGTGATC-3'; amplicon size 813 bp), OXA-2 (F: 5'-TTCAAGCCAAAGGCACGATAG-3'; R: 5'-TCCGAGTTGACTGCCGGGTTG-3', amplicon size 702 bp), OXA-10 (F: 5'-CGTGCTTTGTAAAAGTAGCAG-3'; R: 5'-CATGATTTTGGTGGGAATGG-3'; amplicon size: 651 bp), CTX-M-9 group (F: 5'-GTGACAAAGAGAGTGCAACGG-3'; R: 5'-ATGATTCTCGCCGCTGAAGCC-3'; amplicon size: 857 bp), CTX-M-3 group (F: 5'-GTTACAATGTGTGAGAAGCAG-3'; R: 5'-CCGTTTCCGCTATTACAAAC-3'; amplicon size: 800 bp) and CMY-type (F:, 5'-GATTCCTTGGACTCTTCAG-3'; R: 5'-TAAAACCAGGTTCCCAGATAGC-3'; amplicon size: 1800 bp) ß-lactamases was analysed by PCR and sequencing on both strands. The same set of primers was used for PCR analysis and for sequencing purposes. DNA and the deduced amino acid sequences were compared with those included in the GenBank database as well as with those deposited at the website http://www.lahey.org/Studies/, in order to determine the specific type of ß-lactamase gene. The ß-lactamase genes detected in these nine isolates were as follows (number of isolates): blaTEM-52 (3); blaTEM-52 + blaCTX-M-14 (1); blaCTX-M-14 + blaTEM-1 (2); blaCTX-M-1 + blaTEM-1 (1); and blaSHV-12 (1) (Table 1). The remaining isolate (E. coli 52) harboured the blaCTX-M-14 gene and a blaTEM amplicon was also detected by PCR in this isolate. Sequencing of this blaTEM amplicon indicated the presence of more than one blaTEM gene and future studies will clarify the specific genes included. The genetic environment of blaCTX-M genes was studied by PCR and sequencing in all blaCTX-M-containing isolates using primers previously reported.6 The presence of ISEcp1 was demonstrated upstream of blaCTX-M-14 and blaCTX-M-1 genes in all five isolates that contained those genes. In addition, IS903 and orf477 were detected downstream of the blaCTX-M-14 and blaCTX-M-1 genes, respectively.
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It is interesting to underline the high percentage of wild animals colonized by ESBL-containing E. coli (16% of those animals in which E. coli was recovered in Levine plates), mainly among birds of prey (5 of 14 animals tested, 36%). This type of animal can live in closer contact or even can eat other animals that are in closer contact with humans, and this might explain, at least in part, the obtained results. Other animals in which ESBLs were detected were deer (2 of 3 animals), fox (1 of 7 animals) and owls (1 of 10 animals). Another interesting aspect is the detection of two ESBL genes in the same E. coli isolate of one bird of prey.
The presence of other resistance genes [tet(A)/tet(B), aadA, sul1/sul2/sul3, and qnr, associated with tetracycline, streptomycin and spectinomycin, sulphonamide, and quinolone resistance, respectively], as well as the amino acid changes in GyrA and ParC proteins were also analysed among our isolates by PCR and sequencing. A variety of resistance genes [tet(A), tet(B), aadA, sul1, sul2 and sul3, but not the qnr gene], as well as amino acid changes in GyrA and ParC were observed among our ESBL-producing E. coli isolates (Table 1).
This is the first time, to our knowledge, that ESBL-producing E. coli isolates have been detected in wild animals. It is of interest to underline the wide variety of ESBLs detected in this study. The high proportion of animals colonized by E. coli with ESBLs of different classes detected in this study is worrisome, and more studies should be performed in the future with this class of animals to confirm the wide dissemination of this type of resistance.
Transparency declarations
None to declare.
Acknowledgements
We thank the Veterinary Hospital Montenegro of Porto and the CRATAS (Centre of Collecting, Welcome and Handling of Wild Animals) for their contribution to the collection of samples. This work has been supported in part by Acções Integradas Luso-Espanholas (E-110/06, HP2005-0052).
References
1
Briñas L, Moreno MA, Teshager T, et al. (2005) Monitoring and characterization of extended-spectrum ß-lactamases in Escherichia coli strains from healthy and sick animals in Spain in 2003. Antimicrob Agents Chemother 49:1262–4.
2
Carattoli AS, Lovari A, Franco G, et al. (2005) Extended-spectrum ß-lactamases in Escherichia coli isolated from dogs and cats in Rome, Italy, from 2001 to 2003. Antimicrob Agents Chemother 49:833–5.
3 Machado E, Coque TM, 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. Clin Microbiol Infect 50:1608–9.
4
Mendonça N, Louro D, Castro AP, et al. (2006) CTX-M-15, OXA-30 and TEM-1-producing Escherichia coli in two Portuguese regions. J Antimicrob Chemother 57:1014–6.
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Costa D, Poeta P, Briñas L, et al. (2004) Detection of CTX-M-1 and TEM-52 ß-lactamases in Escherichia coli strains from healthy pets in Portugal. J Antimicrob Chemother 54:960–1.
6
Eckert C, Gautier V, Arlet G. (2006) DNA sequence analysis of the genetic environment of various blaCTX-M genes. J Antimicrob Chemother 57:14–23.
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