Caracterização de genes de resistência a carbapenemos em estirpes bacterianas isoladas de animais selvagens
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2008
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O uso excessivo de antibióticos na medicina veterinária, na medicina humana, aquacultura e na agricultura tem contribuído para o aparecimento e disseminação de microrganismos multirresistentes. Os ß-lactâmicos estão entre os antibióticos mais utilizados, tanto na terapêutica humana como na veterinária, nomeadamente no tratamento de infecções bacterianas dada a sua elevada eficácia terapêutica e a sua baixa toxicidade para as células animais. De entre os ß-lactâmicos, os carbapenemos são considerados os antibióticos mais eficazes. A produção de carbapenemases constitui um dos mecanismos de resistência emergente aos carbapenemos. Dos quatrocentos e cinquenta e seis isolados (Gram negativo) de animais selvagens de diferentes nichos ecológicos, obtidos durante os dois períodos de amostragem: (i) Março a Junho de 2007 e (ii) Setembro a Novembro de 2007, no CRATAS projecto do NEPA sediado na UTAD. Foram seleccionadas vinte estirpes (durante os 7 meses) para serem estudadas devido ao facto de apresentarem pelo menos uma das seguintes características: resistentes aos carbapenemos e/ou todos os isolados pertencentes ao género Aeromonas. A identificação dos isolados foi realizada por biotipificação numérica e por sequenciação dos genes 16S rDNA e gyrB tendo sido identificados os géneros Aeromonas (n=16), Klebsiella (n=1), Pseudomonas (n=1), Shewanella (n=1), e uma espécie não descrita do género Acinetobacter (n=1).
Determinou-se a susceptibilidade destas bactérias a vários grupos de antibióticos, pelo método de difusão em disco. Relativamente aos ß-lactâmicos e em particular aos carbapenemos estudou-se os mecanismos de resistência associados aos fenótipos observados. De um modo geral verificou-se multiresistência das estirpes isoladas aos antibióticos testados, permitindo os resultados obtidos inferir sobre o facto de apesar de existir uma baixa pressão selectiva a que as estirpes ambientais estão submetidas, estas podem ser reservatórios de genes de resistência. De salientar o facto de quatro dos isolados apresentarem resistência aos carbapenemos (imipenemo), antibióticos usados apenas para uso clínico em ambiente hospitalar, o que justificou o estudo dos mecanismos de resistência subjacentes ao fenótipo observado. Na detecção de genes de ß-lactamases, nomeadamente ß-lactamases de espectro alargado (ESBLs) e carbapenemases (serina carbapenemases e metalo-ß-lactamases) esta foi verificada em dois dos isolados MDC 1277 (tipo-OXA) e MDC 1280 (tipo-OXA, tipo-TEM e tipo-SHV). O teste de difusão em agar Mueller-Hinton com tiras (E-test) e o teste de sinergismo do duplo disco foram usados para a triagem dos isolados relativamente a presença de ß-lactamases de espectro alargado, observando-se que apenas o isolado MDC 1280 apresentou teste fenotípico positivo, em ambos os testes. Nos dezasseis isolados do género Aeromonas as metalo-β-lactamases tipo-IMP e tipo-VIM não foram detectadas. Verificou-se a presença de CphA em sete dos isolados identificados como Aeromonas. Em quatro isolados blaCphA positivos foi detectado também o gene blaOXA. De referir que estes isolados não apresentavam um fenótipo de resistência aos carbapenemos.
The excessive use of antibiotics in veterinary medicine, in human medicine, aquaculture and agriculture has contributed to the emergence and spread of multi-resistant organisms. The ß-lactam antibiotics are among the most widely used both in human and in veterinary therapies, particularly in the treatment of bacterial infections because of their high therapeutic efficacy and generally low toxicity to animal cells. Among the ß-lactams, the carbapenems are considered the most effective antibiotics. The production of carbapenemases is one of the emerging mechanisms of resistance to carbapenems. Of four hundred and fifty-six isolates (Gram negative) from wild animals of different ecological niches, obtained during the two periods: (i) March to June 2007 and (ii) in September to November 2007, in the CRATAS (NEPA) project of UTAD, twenty were selected (during the 7 months) to be studied since they possessed at least one of the following characteristics: resistance to carbapenems and / or all isolates included in the genus Aeromonas. The identification of the isolates was performed by numerical bio-typing and by sequencing the 16S rRNA gene and the gene gyrB. The isolates included those from genera Aeromonas (n = 16), Klebsiella (n = 1), Pseudomonas (n = 1), Shewanella (n = 1), and a previously un-described species of the genus Acinetobacter (n = 1). The susceptibility of the bacteria to various groups of antibiotics was determined using the disk diffusion method. The mechanisms of resistance to ß-lactams and in particular to carbapenems associated with the phenotypes observed were studied. In general multi-resistance was observed for the isolated strains. These results infer that there is a low selection pressure and that these environmental strains may be reservoirs of resistant genes. We must emphasize the fact that 25% of the isolates were resistant to carbapenems, antibiotics used only for clinical use in hospitals, which justified the study of mechanisms underlying the resistance phenotype observed. Genes of ß-lactamases were only found in two strains of MDC 1277 (oxa-type) and MDC 1280 (oxa-type, TEM-type and SHV-type). The test on Mueller-Hinton agar with strips (E-test) and the test for synergism of the double disc were used to assess the presence of ß-lactamases of extended spectrum, noting that only the isolate MDC 1280 was positive in both tests. In sixteen of the isolates belonging to genus Aeromonas the metallo-β-lactamases IMP and VIM were not detected. By sequencing the CphA gene was found in seven of these isolates. In four that were blaCphA positive the gene blaOXA-type was also detected the gene. It should be noted that these isolates did not have a phenotype of resistance to carbapenems.
The excessive use of antibiotics in veterinary medicine, in human medicine, aquaculture and agriculture has contributed to the emergence and spread of multi-resistant organisms. The ß-lactam antibiotics are among the most widely used both in human and in veterinary therapies, particularly in the treatment of bacterial infections because of their high therapeutic efficacy and generally low toxicity to animal cells. Among the ß-lactams, the carbapenems are considered the most effective antibiotics. The production of carbapenemases is one of the emerging mechanisms of resistance to carbapenems. Of four hundred and fifty-six isolates (Gram negative) from wild animals of different ecological niches, obtained during the two periods: (i) March to June 2007 and (ii) in September to November 2007, in the CRATAS (NEPA) project of UTAD, twenty were selected (during the 7 months) to be studied since they possessed at least one of the following characteristics: resistance to carbapenems and / or all isolates included in the genus Aeromonas. The identification of the isolates was performed by numerical bio-typing and by sequencing the 16S rRNA gene and the gene gyrB. The isolates included those from genera Aeromonas (n = 16), Klebsiella (n = 1), Pseudomonas (n = 1), Shewanella (n = 1), and a previously un-described species of the genus Acinetobacter (n = 1). The susceptibility of the bacteria to various groups of antibiotics was determined using the disk diffusion method. The mechanisms of resistance to ß-lactams and in particular to carbapenems associated with the phenotypes observed were studied. In general multi-resistance was observed for the isolated strains. These results infer that there is a low selection pressure and that these environmental strains may be reservoirs of resistant genes. We must emphasize the fact that 25% of the isolates were resistant to carbapenems, antibiotics used only for clinical use in hospitals, which justified the study of mechanisms underlying the resistance phenotype observed. Genes of ß-lactamases were only found in two strains of MDC 1277 (oxa-type) and MDC 1280 (oxa-type, TEM-type and SHV-type). The test on Mueller-Hinton agar with strips (E-test) and the test for synergism of the double disc were used to assess the presence of ß-lactamases of extended spectrum, noting that only the isolate MDC 1280 was positive in both tests. In sixteen of the isolates belonging to genus Aeromonas the metallo-β-lactamases IMP and VIM were not detected. By sequencing the CphA gene was found in seven of these isolates. In four that were blaCphA positive the gene blaOXA-type was also detected the gene. It should be noted that these isolates did not have a phenotype of resistance to carbapenems.
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Dissertação de Mestrado em Biologia Clínica Laboratorial