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Abstract(s)
Aliarcobacter butzleri é um enteropatógeno emergente que tem sido considerado um grave perigo para a saúde humana. Nos últimos anos, vários isolados resistentes a antibióticos, comummente utilizados no tratamento de infeções por Al. butzleri, nomeadamente a macrólidos, têm sido identificados. Contudo, os mecanismos de resistência associados ainda não se encontram completamente elucidados. Para além disso, tem-se verificado que isolados com altos níveis de resistência aos macrólidos não são frequentemente encontrados no meio ambiente. Assim, os objetivos deste trabalho focaram-se na avaliação dos potenciais mecanismos que podem estar associados à resistência a macrólidos e os efeitos que esta aquisição de resistência pode ter na robustez da bactéria e na sua suscetibilidade a outros antibióticos. Para tal, procedeu-se a: i) avaliação da contribuição de mutações espontâneas na aquisição de resistência à eritromicina; ii) indução, in vitro, de elevados níveis de resistência ao referido antibiótico, a fim de identificar os mecanismos de resistência envolvidos; iii) avaliação do impacto da aquisição de resistência na suscetibilidade e robustez de Al. butzleri. Inicialmente, a fim de perceber de que forma a alteração da expressão das bombas de efluxo afetava o aparecimento de mutações espontâneas, foi determinada a frequência de mutação da estirpe nativa e duas estirpes mutantes para o gene areB da bomba de efluxo AreABC e respetivo repressor transcricional areR. Com base nos resultados é possível especular que o aumento da expressão da bomba de efluxo AreABC origina um aumento da frequência com que ocorrem mutações espontâneas nesta espécie. Seguidamente, foi determinada a taxa de mutação espontânea da estirpe de Al. butzleri DQ40A1, através do ensaio de flutuação, o que permitiu classificar a estirpe nativa como fracamente hipermutável. Nos ensaios de indução de resistência à eritromicina, in vitro, foi possível efetuar a indução de elevados níveis de resistência a este antibiótico, verificando-se aumentos de 512× na concentração mínima inibitória. Ao avaliar a suscetibilidade de estirpes provenientes dos ensaios de flutuação (cinco mutantes), e de evolução (evolução 8 e 9), na ausência de eritromicina, não se verificaram alterações significativas no perfil de crescimento das estirpes em estudo, à exceção da estirpe resistente da evolução 8 que apresentou uma diminuição da taxa de crescimento. Em relação ao perfil de crescimento na presença de eritromicina foi observado que as estirpes altamente resistentes da evolução 8 e 9 e a estirpe controlo do solvente da evolução 9 apresentavam crescimento na presença das concentrações mais elevadas de eritromicina. Além disso, as estirpes em estudo apresentaram uma diminuição da suscetibilidade à ciprofloxacina e à tetraciclina, contudo não foi verificada qualquer alteração em relação à resistência a compostos indutores de stress oxidativo. Avaliando a expressão fenotípica das bombas de efluxo, observou-se que tanto as estirpes provenientes do ensaio de flutuação com as estirpes com elevados níveis de resistência induzida in vitro demonstraram uma diminuição da capacidade de acumulação de brometo de etídio. No caso da estirpe altamente resistente da evolução 8, este comportamento está correlacionado com o aumento da expressão relativa do gene areB, em cerca de 50 vezes quando comparado com a estirpe nativa, o que é explicado pela mutação observada no gene que codifica para o repressor transcricional do sistema de efluxo correspondente (AreABC). Em relação à avaliação da robustez, de forma geral, as estirpes em estudo demonstraram menor motilidade, menor capacidade de formação de biofilme e não apresentaram diferenças significativas na sobrevivência em modelos alimentares, quando comparados com a estirpe nativa. Assim, os resultados obtidos sugerem que os sistemas de efluxo podem ter um papel determinante na aquisição de resistência à eritromicina e que alterações na suscetibilidade das estirpes a este agente antimicrobiano podem ter um papel na virulência e na robustez da bactéria.
Aliarcobacter butzleri is an emerging enteropathogen that has been considered a serious hazard to human health. In the past few years, several isolates resistant to antibiotics, which are commonly used to treat infections by Al. butzleri, namely to macrolides, have been reported. However, the associated resistance mechanisms have not yet been fully elucidated. Furthermore, it has been noticed that isolates with high resistance levels to macrolides are not frequently found in the environment. Thus, the aims of this work were focused on evaluating the potential mechanisms that may be associated with macrolide resistance and the effects that this acquisition of resistance may have on the robustness of the bacterium and on its susceptibility to other antibiotics. For this purpose, we proceeded with the i) assessment of the contribution of spontaneous mutations in the acquisition of resistance to erythromycin; ii) in vitro induction, of high levels of resistance to this antibiotic, to identify the resistance mechanisms involved; iii) and the evaluation of the impact of the resistance acquisition on the susceptibility and robustness of Al. butzleri. Initially, to understand how the alteration in the expression of the efflux pumps affected the emergence of spontaneous mutations, the mutation frequency of the native strain and two mutant strains, for the areB gene of the AreABC efflux pump and its transcriptional repressor areR, has been determined. Based on the results, it can be speculated that an increased expression of the AreABC efflux pump leads to an increase in the frequency with which spontaneous mutations occur in this species. Next, the spontaneous mutation rate of Al. butzleri strain DQ40A1 was determined using the fluctuation assay, which allowed the native strain to be classified as weakly hypermutable. In the assays of in vitro induction of erythromycin resistance, it was possible to induce high levels of resistance to this antibiotic, with increases of 512× of the minimum inhibitory concentration. Evaluating the susceptibility of strains from the fluctuation (five mutants), and evolution (evolution 8 and 9) assays in the absence of erythromycin, there were no significant changes in the growth profile of the strains under study, except the resistant strain of evolution 8 that showed a decreased growth rate. Regarding the growth profile in the presence of erythromycin, it was observed that the highly resistant strains of evolution 8 and 9 and the solvent control strain of evolution 9 exhibited growth in the presence of the highest concentrations of erythromycin. In addition, the strains under study showed decreased susceptibility to ciprofloxacin and tetracycline; however, no changes were observed concerning the resistance to oxidative stress inducing compounds. By assessing the phenotypic expression of efflux pumps, it was observed that strains from the fluctuation assay and strains with high levels of in vitro induced resistance showed a decreased ability to accumulate ethidium bromide. In the case of the highly resistant strain of evolution 8, this behavior correlates with an increase in the areB gene relative expression by around 50-fold when compared to the native strain, which is explained by the mutation observed in the gene encoding for the transcriptional repressor of the corresponding efflux system (AreABC). Regarding robustness evaluation, overall, the strains under study demonstrated lower motility, decreased biofilm-forming ability, and showed no significant differences in survival in food models when compared to the native strain. Thus, the results obtained suggest that efflux systems may play a determining role in the acquisition of resistance to erythromycin and that changes in the susceptibility of strains to this antimicrobial agent might play a pivotal role in bacterial virulence and robustness.
Aliarcobacter butzleri is an emerging enteropathogen that has been considered a serious hazard to human health. In the past few years, several isolates resistant to antibiotics, which are commonly used to treat infections by Al. butzleri, namely to macrolides, have been reported. However, the associated resistance mechanisms have not yet been fully elucidated. Furthermore, it has been noticed that isolates with high resistance levels to macrolides are not frequently found in the environment. Thus, the aims of this work were focused on evaluating the potential mechanisms that may be associated with macrolide resistance and the effects that this acquisition of resistance may have on the robustness of the bacterium and on its susceptibility to other antibiotics. For this purpose, we proceeded with the i) assessment of the contribution of spontaneous mutations in the acquisition of resistance to erythromycin; ii) in vitro induction, of high levels of resistance to this antibiotic, to identify the resistance mechanisms involved; iii) and the evaluation of the impact of the resistance acquisition on the susceptibility and robustness of Al. butzleri. Initially, to understand how the alteration in the expression of the efflux pumps affected the emergence of spontaneous mutations, the mutation frequency of the native strain and two mutant strains, for the areB gene of the AreABC efflux pump and its transcriptional repressor areR, has been determined. Based on the results, it can be speculated that an increased expression of the AreABC efflux pump leads to an increase in the frequency with which spontaneous mutations occur in this species. Next, the spontaneous mutation rate of Al. butzleri strain DQ40A1 was determined using the fluctuation assay, which allowed the native strain to be classified as weakly hypermutable. In the assays of in vitro induction of erythromycin resistance, it was possible to induce high levels of resistance to this antibiotic, with increases of 512× of the minimum inhibitory concentration. Evaluating the susceptibility of strains from the fluctuation (five mutants), and evolution (evolution 8 and 9) assays in the absence of erythromycin, there were no significant changes in the growth profile of the strains under study, except the resistant strain of evolution 8 that showed a decreased growth rate. Regarding the growth profile in the presence of erythromycin, it was observed that the highly resistant strains of evolution 8 and 9 and the solvent control strain of evolution 9 exhibited growth in the presence of the highest concentrations of erythromycin. In addition, the strains under study showed decreased susceptibility to ciprofloxacin and tetracycline; however, no changes were observed concerning the resistance to oxidative stress inducing compounds. By assessing the phenotypic expression of efflux pumps, it was observed that strains from the fluctuation assay and strains with high levels of in vitro induced resistance showed a decreased ability to accumulate ethidium bromide. In the case of the highly resistant strain of evolution 8, this behavior correlates with an increase in the areB gene relative expression by around 50-fold when compared to the native strain, which is explained by the mutation observed in the gene encoding for the transcriptional repressor of the corresponding efflux system (AreABC). Regarding robustness evaluation, overall, the strains under study demonstrated lower motility, decreased biofilm-forming ability, and showed no significant differences in survival in food models when compared to the native strain. Thus, the results obtained suggest that efflux systems may play a determining role in the acquisition of resistance to erythromycin and that changes in the susceptibility of strains to this antimicrobial agent might play a pivotal role in bacterial virulence and robustness.
Description
Keywords
Aliarcobacter Butzleri Bombas de Efluxo Eritromicina Resistência Robustez Suscetibilidade