Antibiotic resistance in gastrointestinal bacteria isolated from the Iberian lynx and Iberian wolf: species conservation and public health concerns

Resumen

Enterococcus spp. and Escherichia coli are common commensal inhabitants of the gastrointestinal tracts of human and animals, including wild animals, which can easily acquire and transfer resistance genes. Antibiotic resistance in wildlife across the environment presents a critical threat to both human and animal health. The main objective of this thesis was to assess the potential of two endangered animal species native to the Iberian Peninsula, Iberian lynx and Iberian wolf, as reservoirs of Escherichia coli and Enterococcus spp. commensal strains harbouring antibiotic resistance and virulence genes. In Chapter 1 the prevalence of Vancomycin-Resistant Enterococci (VRE) in faecal samples of Iberian wolf (2.3%) and Iberian lynx (2%) was detected. Strains with an acquired mechanism of resistance to vancomycin were not detected among Iberian lynx. In Iberian wolf, one of the two vanA-containing E. faecium isolates detected belonged to the sequence type ST18 (included in the clonal lineage CC17), while the other one was assigned to a new sequence type, that was included in the MLST database and registered as ST573. vanAcontaining isolates showed tetracycline and erythromycin resistance [with erm(B) and tet(L) genes] and one isolate also exhibited ampicillin and kanamycin resistance [with erm(B), tet(M) and aph(3')-III genes]. In Chapter 2, the diversity of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli isolates recovered within the faecal microbiota of Iberian lynx and Iberian wolf was characterized. For the first time among captive specimens of Iberian lynx and the Iberian wolf it were detected ESBL-producing E. coli isolates of the TEM-52, SHV-12, CTX-M-1, and/or CTX-M-14-type (9% and 5.5%, respectively). The occurrence of unrelated multiresistant E. coli within the faecal microbiota of captive specimens of Iberian lynx and the Iberian wolf including the presence of ESBLs, resistance genes in integrons and virulence determinants was shown in this study. In Chapter 3, the incidence of resistant enterococci and E. coli isolates in the faecal microbiota of free-range Iberian lynx, Iberian lynx specimens from the captive breeding program, and Iberian wolf, including the presence of resistant genes in integrons, and virulence determinants was studied. A high frequency of resistance for tetracycline, erythromycin, streptomycin, nalidixic acid and sulfamethoxazol-trimethoprim was detected. Furthermore, the majority of the Iberian lynx E. coli isolates harboured at least one of the studied virulence genes and the phylogenetic distribution was unexpected with a high percentage of strains being ascribed to phylogenetic groups B2 and D (37% and 11%, respectively). Lastly, in Chapter 4, changes in the subcellular proteomes of the cefotaxime-exposed Iberian wolf ESBL-producing E. coli strain WA57 was analysed. This study identified 40 differentially expressed proteins mostly related to protein biosynthesis, the stress response, glycolysis, transport, and energy metabolism. Protein-protein interaction analysis of these proteins showed an upregulation of proteins associated with biological processes involving DNA metabolism, cellular amino acid catabolism, responses to unfolded protein, and transport, while the down-regulated proteins predominantly involved tRNA aminoacylation and amino acid metabolic processes. Surprisingly, the ESBL enzyme CTX-M-14 was identified as downregulated in the periplasmic fraction what requires further research. This thesis showed that multiresistant bacteria have reached species as rare and completely non-synanthropic as the free-range Iberian lynx and the Iberian wolf. The same genes found in bacteria from environment and human origins encode the antimicrobial resistance mechanisms found in these studies, indicating the possible circulation of bacteria and resistance genes between animal, environment and human ecosystems. Moreover, Iberian lynx and Iberian wolf, through their predatory and travelling habits, could spread these resistant bacteria, and/or their resistance genes, throughout the environment. Lastly, in the future increased risk of infection and therapeutic failure due to virulence/resistance genes might represent a serious setback for these species conservation programs.