Gilthead Seabream ( Sparus aurata ) as Carriers of SHV-12 and TEM-52 Extended-Spectrum Beta-Lactamases-Containing Escherichia coli Isolates

Introduction

E xtended-spectrum Beta-lactamases (ESBLs) are increasingly detected in Escherichia coli, and its emergence in commensal bacteria is of great concern as the encoding genes are often located on mobile elements and can be transferred to pathogenic bacteria. ESBL-producing E. coli isolates have appeared as serious nosocomial pathogens globally (Cantón et al., 2008), although they have been also found in other ecological niches such as healthy humans, animals (including wild animals), and foods (Briñas et al., 2002; Costa et al., 2007; Carattoli, 2008; Poeta et al., 2008), suggesting its wide distribution in different ecosystems.

The gilthead seabream (Sparus aurata) is a marine teleost fish that represents a key element of the coastal marine ecosystem. Sparids are also of great importance for fisheries being excellent food fish, with high commercial value. The purpose of our study was to analyze the carriage of ESBL-containing E. coli isolates in fecal samples from S. aurata, and also to characterize the integron content and the clonal diversity of recovered ESBL-positive isolates.

Materials and Methods

The presence of ESBL-containing E. coli strains was studied in 118 fecal samples of S. aurata (one sample per animal). Animals were captured in the Atlantic Ocean, in the West coast of Portugal (Peniche city) by sports fishing practitioners using hand line technique (September–November, 2007). Small intestines of each animal were collected, weighed, and transferred to sterile Stomacher bags. Peptone solution was added in a proportion of 1:9 and the mixtures were homogenized using a Stomacher. Samples were seeded in levine agar supplemented with cefotaxime (2 mg/L), and colonies with typical E. coli morphology were selected and identified by classical biochemical methods and by the API 20E system (BioMérieux). One E. coli isolate per ESBL-positive sample was selected for further study. Susceptibility to 16 antibiotics (ampicillin, amoxicillin + clavulanic acid, cefoxitin, cefotaxime, ceftazidime, aztreonam, imipenem, gentamicin, amikacin, tobramycin, streptomycin, nalidixic acid, ciprofloxacin, sulfamethoxazole/trimethoprim, tetracycline, and chloramphenicol) was tested by the disk-diffusion method in all recovered isolates, and ESBL-phenotypic detection was carried out by double-disk test (CLSI, 2011). The presence of genes encoding TEM, OXA, SHV, and CTX-M type beta-lactamases was studied by polymerase chain reaction (PCR) and sequencing. Nonbeta-lactamase genes (tetA/tetB in tetracycline-resistant isolates; aadA in streptomycin-resistant isolates; aac(3)-II/aac(3)-IV, aac(6′) in gentamicin-resistant isolates; cmlA in chloramphenicol resistant isolates; sul1/sul2/sul3 in trimethoprim/sulfamethoxazole resistant isolates) were also studied by PCR (Poeta et al., 2008). The presence of intI1 and intI2 genes, encoding class 1 and 2 integrases, respectively, was analyzed by PCR and the characterization of the variable region of class 1 integrons was performed by PCR and subsequent sequencing (Gonçalves et al., 2010). The clonal relationship among the strains was studied by pulsed-field gel electrophoresis (PFGE) using XbaI as restriction enzyme (Gonçalves et al., 2010). In addition, isolates were also classified into one of the four main phylogenetic groups (A, B1, B2, and D) by PCR (Clermont et al., 2000).

Results and Discussion

ESBL-containing E. coli isolates were detected in 5 of 118 tested samples (4.2%) and 5 isolates were obtained (1 per positive-sample) (Table 1). These isolates showed high minimum inhibitory concentration values to cefotaxime (128 μg/mL) and moderate resistance to ceftazidime (minimum inhibitory concentrations 1–16 μg/mL). The bla_{S HV-12} gene was detected in three isolates and bla _TEM-52 in the remaining two ESBL-positive isolates. Four unrelated PFGE patterns (A–D) were identified among these isolates and four isolates from two samples shared identical PFGE types (Table 1). The detection of two samples containing indistinguishable ESBL-positive E. coli isolates is of interest and could reflect the acquisition of these resistant isolates from a common source. Nevertheless, unrelated strains were detected in four of the five ESBL-positive animals, indicating that different clones can acquire the genes encoding ESBLs.

TEM-52 and SHV-12 enzymes, among others, were frequently detected in Enterobacteriaceae from different clinical settings in Portugal (Machado et al., 2007). TEM-52 and SHV-12 beta-lactamases have also been previously identified in E. coli isolates from different type of animals in different countries (Briñas et al., 2002; Costa et al., 2007; Carattoli, 2008; Poeta et al., 2008, Gonçalves et al., 2010).

A variety of resistance genes (cmlA, tetA, aadA, sul1, sul2, and sul3) were observed among our ESBL-producing E. coli isolates (Table 1). Four isolates harbored class 1 integrons with the following gene cassettes in their variable region: dfrA1 + aadA1 (1 isolate) and sat (streptothricin acetyltransferase gene) + psp (phosphoserine phosphatase) + aadA2 (three isolates). None of the isolates harbored class 2 integrons. The dfrA1 and aadA1/2 genes are associated with trimethoprim and streptomycin resistance, respectively, and similar structures have also been previously reported in animal E. coli isolates (Briñas et al., 2002; Poeta et al., 2008). Integrons carrying resistance gene cassettes can be mobilized by plasmids, spreading the antibiotic resistance phenomenon and facilitating the emergence of multidrug resistance. It seems that the type of class 1 integrons detected among our ESBL-positive isolates is common among food isolates in different countries (Sunde, 2005). Four of the ESBL-positive E. coli isolates of this study were classified into the phylogenetic group A and the other isolate in the B1 phylogenetic group. The A and B1 groups are more frequently associated with commensal strains (Clermont et al., 2000), as already reported in previous studies in food-animal ESBL-producing E. coli (Gonçalves et al., 2010). No B2 or D phylogenetic groups were identified among our strains. Strains belonging to B2 and D groups have higher probability of occurrence in clinical settings (Clermont et al., 2000) and are usually associated to extraintestinal infections.

In this study it was possible to detect and characterize ESBL-producing E. coli isolates in gilthead seabream in Portugal, with two types of ESBLs detected, TEM-52 and SHV-12. However, we cannot exclude the possibility that these wild animals had been exposed to fecal material of other animals or even of humans in their aquatic environment. Fecal exposure 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. Fish are considered a potential vehicle of food-borne bacterial infections what might be of importance in human public health. These results add to our knowledge about the occurrence of ESBL-positive bacteria in nonhospital-associated environments, which could have implications in human health because these resistant bacteria might be transferred to humans through the food chain. ESBL producers are expected to increase in the future, in both animals and humans, and more prudent use of antimicrobials, in general, may be necessary, as well as the implementation of international measures to control zoonotic pathogens and limit the global emergence of these resistance traits.