Detection of Escherichia coli harbouring extended-spectrum -lactamases of the CTX-M, TEM and SHV classes in faecal samples of wild animals in Portugal

Abstract

Sir, Production of extended-spectrum β-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) β-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 Author Webpage, in order to determine the specific type of β-lactamase gene. The β-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. Characteristics of the nine ESBL-harbouring E. coli detected in faecal samples of nine wild animals in this study aGEN, gentamicin; TOB, tobramycin; CIP, ciprofloxacin; NAL, nalidixic acid; STR, streptomycin; SXT, sulfamethoxazole/trimethoprim; TET, tetracycline. bSequences of gyrA and parC genes were studied in our isolates and compared with those included in GenBank with accession numbers X06373 for gyrA and M58408 with the modification included