Genetic Detection of Extended-Spectrum β-Lactamase-Containing Escherichia coli Isolates from Birds of Prey from Serra da Estrela Natural Reserve in Portugal

The high and sometimes excessive use of antibiotics is directly related to the great spread and development of bacterial antibiotic resistance, a problem in public health nowadays. Thus, many studies have been done in order to understand the mechanisms of bacterial drug resistance, as many multidrug-resistant bacterial strains have been detected in domestic animals and humans (12). Recent studies from our research group have focused on wild animals, trying to understand how they acquire resistance to antibiotics without direct contact (11, 17, 20). One of the most clinically relevant antimicrobial resistance mechanisms is constituted by extended-spectrum β-lactamases (ESBLs) (3, 16) harbored by Enterobacteriaceae, such as Escherichia coli, commensals of the gastrointestinal tract of animals and humans, serving as a reservoir for resistances, namely, to β-lactam antibiotics (1, 15). Most ESBLs are derivatives of TEM or SHV enzymes, but a new family of plasmid-mediated ESBLs, CTX-M, has also been reported worldwide in E. coli and other Enterobacteriaceae. Different variants of the β-lactamase TEM, as in the case of TEM-1, are often reported in cases of plasmid-mediated β-lactamase resistance (2, 5, 8, 14, 21, 22). In this study, fecal samples from wild birds of prey at the Serra da Estrela Natural Reserve were analyzed in order to detect ESBL-containing E. coli isolates and characterize the type of ESBL-encoding genes and other associated resistance genes and the corresponding phylogenetic groups.

One hundred nineteen fecal samples from birds of prey were recovered from April to July 2008 and studied for the presence of ESBL-containing E. coli strains (Table 1). All the fecal samples were collected individually from each animal and obtained in collaboration with CERVAS (Center of Ecology, Collecting, Welcome and Handling of Wild Animals). This Center receives injured animals found in its natural environments, is located at the Serra da Estrela Natural Reserve, and belongs to the Portuguese Institute of the Nature Management. None of the animals had been previously fed by humans or had received antibiotics. Each bird that arrives at CERVAS is placed in an individual cage to be treated and released back into the environment. Sampling was performed by recovering the fecal material in the cloaca by using a sterile swab. They were immediately transferred to peptone water and manipulated during the first 24 h of arrival to the laboratory. Samples were seeded in Levine agar supplemented with cefotaxime (2 mg/liter), and colonies with typical E. coli morphology were selected, identified by classical biochemical methods and by the API 20E system (bioMérieux, La Balme Les Grottes, France), and further studied. Susceptibility to 16 antibiotics (ampicillin, amoxicillin plus clavulanic acid [AMC], cefoxitin, cefotaxime [CTX], ceftazidime [CAZ], aztreonam [ATM], imipenem, gentamicin, amikacin, tobramycin, streptomycin, nalidixic acid, ciprofloxacin, sulfamethoxazole-trimethoprim, tetracycline, and chloramphenicol) was tested by the disc diffusion method (7) for all recovered E. coli isolates. E. coli ATCC 25922 was used as a quality control strain. The double-disc diffusion test (CTX, CAZ, and ATM in the presence or absence of AMC) was performed to detect ESBL production (7).

The presence of genes encoding TEM, SHV, OXA, CTX-M, and CMY type β-lactamases was studied by specific PCRs (17). All obtained amplicons were sequenced on both strands, and sequences were compared with those included in the GenBank database and at the Lahey Clinic website (http://www.lahey.org/Studies/) to identify the β-lactamase genes.

Non-β-lactamase genes [tetA/tetB, aadA, aac(3)-II/aac(3)-IV, aac(6′), cmlA, and sul1/sul2/sul3, associated with tetracycline, streptomycin, gentamicin, amikacin, chloramphenicol, and trimethoprim-sulfamethoxazole resistance, respectively] were also studied by PCR (17). The presence of the genes intI1 and intI2, encoding class 1 and 2 integrases, respectively, was studied by PCR. Positive and negative controls from the bacterial collection of the University of Trás-os-Montes and Alto Douro were used in all assays. The ESBL-positive isolates were classified into one of the four main phylogenetic groups, A, B1, B2, and D, by PCR as described previously based on the presence or absence of the chuA, yjaA, or tspE4.C2 gene (6).

E. coli isolates were detected in Levine-CTX plates for 32 of the 119 samples (26.9%) from wild birds of prey at the Serra da Estrela Natural Reserve (Table 1). All 32 isolates recovered in this survey resulted in a positive ESBL screening test, which also indicated a resistant phenotype to CTX and/or CAZ. The β-lactamase genes detected in these isolates were as follows: bla_CTX-M-1 (n = 13), bla_CTX-M-1 plus bla_TEM-1b (n = 8), bla_CTX-M-1 plus bla_TEM-1d (n = 6), bla_CTX-M-1 plus bla_TEM-20 (n = 1), bla_SHV-5 plus bla_TEM-1b (n = 2), bla_SHV-5 (n = 1), and bla_TEM-20 (n = 1) (Table 2). The bla_CTX-M-1 gene was found in most of our ESBL-positive E. coli isolates (n = 28); this ESBL was also reported in studies of healthy poultry (13) and by our research group in healthy pets (9) and wild animals (10, 17). It is interesting to underline that approximately half of the bla_CTX-M-1-containing E. coli isolates also harbored a variant of a bla_TEM gene, concretely bla_TEM-1b, bla_TEM-1d, or bla_TEM-20. The bla_TEM-1b gene is the most frequent variant found in β-lactam-resistant E. coli isolates of food, humans, and healthy animals or in wild boars in other studies (4, 18). The bla_TEM-1d genetic variant is relatively infrequent, although it has been detected in some other studies (19). Two ESBL-containing E. coli isolates were positive for the presence of bla_TEM-20, and another three isolates carried the bla_SHV-5 gene. Most ESBL-positive E. coli isolates of this study were classified into the A or B1 phylogenetic group (72%), although nine isolates which harbored bla_CTX-M-1 with or without bla_TEM-1b were included in the B2 phylogenetic group. The predominance of CTX-M-1-producing isolates belonging to phylogenetic group B2 is of great concern, as, in fact, this phylogenetic group often carries virulence determinants that are less frequently present in other phylogenetic groups. Similar results were found in wild boars by our research group (18).

The common phenotype of resistance to multiple antibiotics among our ESBL-producing isolates is probably due to the coexistence of bla_CTX-M-1 with other antibiotic resistance genes in the same plasmid, contributing to maintain ESBL-producing populations under different antibiotic selective pressures. With this study, it was possible to detect and characterize ESBL-producing E. coli isolates from birds of prey at the Serra da Estrela Natural Reserve in Portugal, with two types of ESBLs detected as CTX-M-1 and SHV-5. However, we cannot exclude the possibility that these wild animals had been exposed to fecal material of farm animals or even that of humans. These facts might be involved in the acquisition and dissemination of antibiotic-resistant bacteria even in the absence of direct antibiotic pressure and might explain the presence of ESBL-positive E. coli isolates. However, more studies should be performed to better understand the role of these animals in the spread of this type of resistance.

• American Society for Microbiology