Antibiotic resistance in gastrointestinal bacteria isolated from the Iberian lynx and Iberian wolf: species conservation and public health concerns
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2015-05-21
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Enterococcus spp. e Escherichia coli são bactérias comensais, habitantes regulares do trato gastrointestinal de humanos e animais, incluindo os animais selvagens. Estas espécies
bacterianas são capazes de adquirir e disseminar genes de resistência a antibióticos pelo
meio-ambiente, representando um problema grave em medicina humana e veterinária. Esta
tese teve como objetivo avaliar o potencial de duas espécies animais ameaçadas de extinção,
o Lince Ibérico e o Lobo Ibérico, como reservatórios de estirpes comensais de Escherichia
coli e Enterococcus spp. que abrigam genes de resistência e de virulência.
No capítulo 1 determinou-se a prevalência de Enterococcus spp. resistentes à
vancomicina (“Vancomycin-Resistant Enterococci”, VRE) em amostras fecais de Lobo
Ibérico (2,3%) e de Lince Ibérico (2%). No Lince Ibérico não foram detectados isolados com
mecanismos de resistência adquirida à vancomicina. No Lobo Ibérico, um dos dois isolados E.
faecium contendo o gene vanA pertencia ao tipo de sequência ST18 (incluído na linhagem
clonal CC17), enquanto o segundo foi inserido num novo tipo de sequência, incluído na base
de dados MLST e registado como ST573. Ambos os isolados vanA apresentaram resistência à
tetraciclina e eritromicina [com os genes erm(B) e tet(L)] e um exibiu ainda resistência à
ampicilina e à canamicina [com os genes erm(B), tet(M) e aph(3')-III ].
No capítulo 2, caracterizou-se a diversidade dos isolados de E. coli produtores de β-
lactamases de espectro alargado (“Extended-spectrum β-lactamase”, ESBL), recuperados na
microbiota fecal de Lince Ibérico e de Lobo Ibérico. Pela primeira vez em espécimes de
Lince Ibérico em cativeiro (9%) e em Lobo Ibérico (5,5%) foram detectados isolados de E.
coli produtores de ESBL do tipo TEM-52, SHV-12, CTX-M-1, e/ou CTX-M-14. Neste
estudo detectou-se ainda a ocorrência de isolados de E. coli multirresistentes, provenientes de
diferentes linhagens clonais, incluindo a presença de ESBL, genes de resistência em integrões
e genes codificadores de factores de virulência.
No capítulo 3, estudou-se a incidência de Enterococcus spp. e E. coli resistentes a
diferentes antibióticos, incluindo a presença de genes de resistência em integrões e de genes
codificadores de factores de virulência. Observou-se uma percentagem elevada de isolados
resistentes à tetraciclina, eritromicina, estreptomicina, ácido nalidíxico e sulfametoxazol-trimetoprim. A maioria dos isolados de E. coli provenientes de Lince Ibérico possuíam pelo
menos um dos genes de virulência estudados e a distribuição filogenética revelou uma
percentagem elevada de isolados pertencentes aos grupos filogenéticos B2 e D (37% e 11%,
respectivamente).
Por último, no capítulo 4, estudaram-se as alterações nos proteomas subcelulares da
estirpe E. coli produtora de ESBL WA57, originária do Lobo Ibérico, durante a exposição
desta estirpe bacteriana à cefotaxima. Este estudo identificou 40 proteínas com expressão
diferencial na sua maioria relacionadas com a biossíntese de proteínas, a resposta ao stress, a
glicólise, o transporte e o metabolismo energético. A análise da interação proteína-proteína
mostrou um aumento da expressão de proteínas associadas a processos biológicos que
envolvem o metabolismo do DNA, o catabolismo de aminoácidos, a resposta ao
desdobramento de proteínas e o transporte. As proteínas sub-expressas mostraram estar
predominantemente relacionadas com a aminoacilação do tRNA e com processos
metabólicos de aminoácidos. Na fracção periplasmática identificou-se a sub-expressão da
enzima CTX-M-14.
Neste trabalho verificou-se que bactérias multirresistentes alcançaram espécies
animais raras e não-sinantrópicas como o Lince Ibérico e o Lobo Ibérico. Os mesmos genes
encontrados em bactérias de origem ambiental ou humana codificam os mecanismos de
resistência a antibióticos encontrados nestes estudos, o que indica a circulação de bactérias e
genes de resistência entre animais, ambiente e os ecossistemas humanos. Além disso, o Lince
Ibérico e o Lobo Ibérico, devido aos hábitos predatórios e ao vasto alcance do habitat, podem
disseminar estas bactérias resistentes, e/ou genes de resistência, por diferentes ecossistemas.
Por último, no futuro, o aumento do risco de infecção e falha terapêutica devido à presença
de genes de virulência/resistência pode representar um problema grave para os programas de conservação destas espécies.
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 β-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.
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 β-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.
Descrição
Tese de Doutoramento em Genética Molecular Comparativa e Tecnológica
Palavras-chave
Resistência a antibióticos , Microorganismo , Animais selvagens , Genómica , Proteómica