Iberian Wolf as a Reservoir of Extended-Spectrum β-Lactamase-Producing Escherichia coli of the TEM,SHV,and CTX-M Groups

Introduction

β-lactams are among the most clinically important antimicrobial agents in both human and veterinary medicine. However, resistance to these antimicrobials has been increasingly observed in different microorganisms.^9,16,17 The mechanisms of β-lactam resistance include, among others, inactivation of the drugs by β-lactamases. The genes encoding these enzymes often coexist with other antimicrobial resistance determinants and can also be associated with transposons/integrons, increasing the dissemination of the resistance determinants among other bacterial species.^11,13 Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli strains have been isolated from farm and wild animals, food, and environmental samples in different countries,^9,17 and have caused a main apprehension due to the frequent implication of these resistant microorganisms in human infections.^1,15 However, studies concerning the incidence of ESBL in wild animals are limited. The flow of resistant microorganisms and resistant genes from livestock and humans to wildlife, or vice versa, remains poorly understood, despite the fact that wild animals may act as reservoirs of resistant genetic elements that could be spread across the environment. ¹⁷

This is the first study of ESBL-producing E. coli isolates performed in Iberian wolf, an endangered species. Due to their predatory and wild nature, these animals may serve as important indicators of environmental contamination with antimicrobial-resistant bacteria.

Material and Methods

Results

ESBL-producing E. coli isolates were recovered from 13 of the 237 (5.5%) fecal samples of Iberian wolf. Two isolates per sample were initially recovered, although only one isolate per sample was further studied, unless they showed different antimicrobial resistance profiles. Fifteen ESBL-producing E. coli isolates were completely characterized (Table 1). All isolates showed a positive screening test for ESBL production, and the following variants were identified: five isolates as bla_CTX-M-1, four isolates as bla_CTX-M-14a, two isolates as bla_CTX-M-14b, one isolate as bla_TEM-52, and three isolates as bla_SHV-12 (Table 1). The ISEcp1 sequence was found upstream of the bla_CTX-M- genes in six isolates, and the orf477 sequence was detected downstream in five of these genes.

The tet(A), tet(B), aadA, strA-strB, different combinations of sul genes, aac(3)-II, and cmlA genes, were observed among our E. coli isolates (Table 1). The presence of class 1 integrons was confirmed in three isolates containing the gene cassette array: dfrA1+aadA1 (Table 1). Furthermore, amino acid changes in GyrA and ParC were also detected in quinolone-resistant isolates (Table 1).

Different plasmid replicon types were identified among our ESBL-producing E. coli isolates (Table 1). Most of bla_CTX-M-14a-positive isolates carried the IncK replicon, and all bla_CTX-M-1 and bla_SHV-12 isolates carried the IncI1 or IncN replicon types. Thirteen different patterns were identified by PFGE analysis among the 15 ESBL-producing isolates (with more than six bands of difference among them) (Table 1).

Five of the ESBL-positive isolates were ascribed to the phylogenetic group A, five isolates to B1, and another five isolates to phylogroup D. Concerning the genes encoding virulence factors, fimA and aer were detected in 14 and 9 isolates, respectively (Table 1).

Discussion

This is the first time, to our knowledge, that CTX-M-producing E. coli isolates have been detected in the Iberian wolf. Acquisition of ESBL-producing E. coli isolates by this species could be explained through the predatory behavior of these animals. Travelling large distances could expose this species to food remains or fecal material from farm animals or even from humans who carry resistant strains. Additionally, the Iberian wolf might be contaminated through the food chain, as the presence of ESBL-producing E. coli strains has been previously detected in one of his primary preys, wild boars. ¹⁶

The CTX-M-1- and CTX-M-14-type β-lactamases have been frequently detected in E. coli isolates implicated in human infections and in some animal species, in Portugal and Spain.^11,13 Similarly, the TEM-52 and SHV-12 type β-lactamase has already been reported in hospitals, and is a frequent type among humans and animals.^7,12,13,21 The insertion sequences ISEcp1 and orf477 detected upstream and downstream, respectively, of the CTX-M β-lactamase genes have been previously reported.^8,17 The association of insertion sequences upstream and downstream of the CTX-M β-lactamase genes may be involved in their dissemination and expression processes. Additionally, the CTX-M and SHV-12 β-lactamase genes have been previously found to be included in IncK, IncI1, or IncF plasmids in E. coli isolates of different countries and origins.^6,14,23

The genes contained in the variable region of the class 1 integrons are associated with trimethoprim and streptomycin resistance. A similar structure has been previously reported in human and animal E. coli isolates.^11,13 This prevalence of integrons is a cause of concern and even if they do not have the capacity of genetic mobilization, they can be disseminated through plasmids or transposons to other bacteria.

PFGE analysis revealed a high clonal diversity of ESBL-producing E. coli isolates. This indicates that the emergence of resistance is probably due to the transference of mobile genetic elements that included the ESBL genes as opposed to the dissemination of a specific clone.

Since the B2 and D phylogenetic groups are associated with virulent strains, the higher prevalence of A and B1 phylogroups compared with D phylogroup among our isolates could be explained by their fecal origin.^5,17

This study highlights that ESBLs are found in microorganisms of ecosystems other than those closely related to humans or containing obvious antimicrobial resistance selection pressures. In addition, the presence of resistant genes in integrons and the existence of virulence determinants were shown. The Iberian wolf can act as a reservoir of resistance genes and since they travel large distances, they could spread resistant bacteria, and their resistant genes, throughout the environment. Monitoring ESBL-producing E. coli strains in wild animals should be continued in order to evaluate their evolution and dissemination.