Genomic and proteomic study of antimicrobial resistance in Escherichia coli and Enterococcus spp. recovered from wild animals

Zusammenfassung

Genomics, proteomics and bioinformatics tools allow for more in-depth knowledge about the physiology and structure of bacteria and also about mechanisms involved in antimicrobial resistance. Antimicrobial resistance, evolving and spreading among bacteria, poses a serious and growing threat to public health. Though, resistance has also been detected in the absence of antimicrobial exposure, such as in bacteria from wildlife, raising a question about the mechanisms of occurrence and persistence of resistant strains under similar conditions, and the implications for resistance control strategies. In order to study antimicrobial resistance in Escherichia coli and Enterococcus spp., 151 faecal samples were collected from three wild animal species, seagulls (Larus cachinnans, 57), common buzzards (Buteo buteo, 42) and red foxes (Vulpes vulpes, 52). Seagull samples were recovered from the Berlengas nature reserve, those of common buzzards from the Peneda Gerês natural park and other rural conservation areas of Portugal and those of red foxes were collected in the north of Portugal during red fox hunts. A total of 111 E. coli (73.5%) and 135 enterococci (89.4%) were isolated from Levine and Slanetz-Bartley plates without antimicrobial agents. After isolation in selective plates supplemented with cefotaxime (CTX) and vancomycin, respectively, the occurrence of extended-spectrum-ß-lactamase (ESBL)-producing E. coli was 12.7% (seagulls, 19.3%; common buzzards, 15.2%; red foxes, 4%) and the prevalence of vancomycin-resistant enterococci (VRE) was 20.4% (seagulls, 10.5%; common buzzards, 36.4%; red foxes, 21.1%). All CTX-resistant E. coli isolates exhibited a resistance phenotype to cefotaxime and/or ceftazidime and had a positive screening test for ESBL production. The most predominant ß- lactamase genes were the blaTEM-52 and blaCTX-M-32 genes (34.8%) together with the blaCTX-M-1 (17.4%), blaSHV-12 (8.7%), blaOXA-1 and blaCTX-M-14a (4.3%) genes. Most of the ESBL and non-ESBL E. coli isolates were multiresistant, with high levels of resistance to tetracycline (57.5%), streptomycin (53%) and sulfamethoxazole/trimethoprim (35.1%), due to the presence of tet(A) and/or tet(B) genes (81.7%), aadA gene (45.1%) and different combinations of sul genes (97.8%), respectively. The most common virulence factor gene was fimA (63.3%) and the majority of the E. coli isolates belonged to phylogenetic groups A (39.1%) and B1 (27.3%). Regarding to the VRE and non-VRE isolates, E. faecium and E. faecalis were the most predominant enterococcal species. High levels of tetracycline and erythromycin resistance were detected and resistance gene profiles were diverse amongst these isolates; the most prevalent genotype was tet(M)+tet(L)+erm(B). Concerning virulence factors, the gelE, hyl, cpd, esp and agg genes were the most predominant. VRE isolates with acquired vancomycin resistance (vanA genotype) were found in 11.3% of the faecal samples analysed; and 9.1% contained VRE isolates with intrinsic vancomycin resistance (vanC1 genotype). The vanA-E. faecium isolates belonged to multilocus sequence types ST273 and ST262 (clonal complex CC17) and also to ST5. ESBL-E. coli and vanA-containing enterococci were also studied through a detailed proteomic approach consisting in two-dimensional gel electrophoresis (2-DE) followed by matrixassisted laser desorption/ionization time-of-flight-mass spectrometry (MALDI/TOF-MS). A total of 686 spots were excised from 2-DE gels of 3 ESBL-E. coli and 4 VRE strains from which 562 proteins were successfully identified (81.9%) and fully characterized through database interrogations. The proteins identified were associated to different biological functions such as glycolysis, transport, biosynthesis, antimicrobial resistance, among others. The results also indicated in E. coli and enterococci strains the presence of proteins involved in stress response, as chaperone proteins DnaK, WrbA, GroEL, among others. Additionally, the VanA protein (Mw: 37419,10938 / PI: 5,79), which prevents vancomycin/teicoplanin binding, was identified when studying the wholecell proteomic profile of vanA-containing enterococci strains. This approach showed that it is possible to evaluate protein profiles in resistant bacteria and in response to antimicrobial stress conditions to further understand the associated resistance mechanisms. Quantitative proteomic analyses on specific protein classes or subproteomes in ESBL-E. coli and VRE strains will provide new functional insights into antimicrobial resistance mechanisms facilitating the identification of diagnostic or prognostic disease markers and the discovery of therapeutic targets. This thesis discussed the prevalence and spread of antimicrobial resistance in indicator bacteria such as E. coli and enterococci in wild animals and showed that wildlife may be an important reservoir of antimicrobial-resistant bacteria and resistance genes. Moreover, this research showed the presence of strains carrying virulence factor genes and belonged to high-risk clonal complexes, which adds to the public health concerns that arise from the spread of ESBL-E. coli and VRE into wildlife.