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Authors
Abstract(s)
Aliarcobacter butzleri Ʃ um enteropatogƩneo emergente reconhecido como a quarta espƩcie mais
comum em amostras diarreicas entre os Campylobacter-like organisms, encontrando-se na lista
de microrganismos considerados um perigo grave para a saĆŗde humana, pela ComissĆ£o
Internacional em EspecificaƧƵes MicrobiolĆ³gicas para Alimentos. Este organismo patogĆ©nico Ć©
conhecido por apresentar mĆŗltipla resistĆŖncia a antibiĆ³ticos, podendo assim originar um
problema no tratamento de infeƧƵes causadas por esta bactĆ©ria. InformaĆ§Ć£o prĆ©via acerca desta
bactƩria sugere que a mesma contƩm diversos sistemas putativos de efluxo que permanecem por
caracterizar, e que poderĆ£o ter um papel relevante como mecanismos de resistĆŖncia a
antimicrobianos. Assim, com este trabalho pretendeu-se estudar o papel de cinco sistemas de
efluxo distintos, todos da famĆlia "Resistance nodulation cell division". Para tal, o estudo foi
iniciado com construĆ§Ć£o de mutantes de A. butzleri, utilizando uma estirpe naturalmente
transformĆ”vel(AB28/11), atravĆ©s da interrupĆ§Ć£o dos genes codificantes da proteĆna de membrana
interna responsĆ”vel pelo reconhecimento do substrato. Dos cinco sistemas em estudo sĆ³ foi
possĆvel gerar mutantes para trĆŖs, onde se procedeu Ć interrupĆ§Ć£o dos genes areB, areE e areG,
correspondentes Ć s bombas de efluxo AreABC, AreDEF e AreGHI, respetivamente. A construĆ§Ć£o
dos mutantes foi confirmada pela tĆ©cnica de āReverse Transcription-Polimerase Chain
Reactionā. Posteriormente, foram realizados uma sĆ©rie de testes para comparaĆ§Ć£o entre os
mutantes e a estirpe parental. Quanto ao crescimento bacteriano nĆ£o foram observadas diferenƧas
significativas entre estirpes. De seguida, foi avaliada a contribuiĆ§Ć£o de cada sistema de efluxo em
estudo para a resistĆŖncia a antibiĆ³ticos, sais biliares e biocidas, pela determinaĆ§Ć£o da
concentraĆ§Ć£o mĆnima inibitĆ³ria pelo mĆ©todo de microdiluiĆ§Ć£o. Os resultados mostram que: a
inativaĆ§Ć£o do gene areG teve um impacto na resistĆŖncia a cinco das seis classes de antibiĆ³ticos
testadas, bem como na resistĆŖncia a diversos biocidas; a inativaĆ§Ć£o do gene areB teve um impacto
na resistĆŖncia Ć s cefalosporinas, estreptomicina, fluoroquinolonas e macrĆ³lidos; o perfil
fenotĆpico de resistĆŖncia do mutante AB28/11?areE foi semelhante Ć estirpe parental. Para alĆ©m
disso, procedeu-se Ć avaliaĆ§Ć£o do papel do sistema de efluxo na acumulaĆ§Ć£o intracelular de
compostos, usando um mĆ©todo de fluorescĆŖncia, que avaliou a acumulaĆ§Ć£o de brometo de etĆdio,
nas estirpes parental e mutantes. Esta avaliaĆ§Ć£o demonstrou que a bomba AreABC apresenta um
papel importante na extrusĆ£o de compostos tĆ³xicos, visto que se observou uma maior acumulaĆ§Ć£o
de brometo de etĆdio do que a observada para a estirpe parental, demonstrando uma menor
atividade global dos sistemas de efluxo das cĆ©lulas. Em suma, com este trabalho foi possĆvel
caraterizar o papel dos sistemas de efluxo AreABC, AreDEF e AreGHI na resistĆŖncia de A. butzleri,
tendo-se destacado o potencial papel dos sistemas AreABC e AreGHI para a multirresistĆŖncia
desta bactƩria.
Aliarcobacter butzleri is an emerging enteropathogen recognized as the fourth most common species in diarrheal samples among the Campylobacter-like organisms, being on the list of microorganisms considered to be a serious hazard to human health according to the International Commission on Microbiological Specifications for Food. This pathogenic organism is known to be multidrug resistant, which may cause complications in the treatment of infections caused by this bacterium. Previous studies regarding this bacterium suggest that it contains several putative efflux systems that remain to be characterized, and these may have a relevant role as mechanisms of resistance to antimicrobials. This study aimed to evaluate the role of five different efflux systems from the Resistance nodulation cell division pump family. To this end, the study started with the construction of A. butzleri mutants, using a naturally transformable strain (AB28/11), by interrupting the genes coding for the inner membrane protein responsible for substrate recognition. From the five systems under study, it was only possible to generate mutants for thee of them, where the genes areB, areE and areG, from to the AreABC, AreDEF and AreGHI efflux pumps, respectively, were interrupted. The construction of the mutants was confirmed by the technique of reverse transcription-polymerase chain reaction. Subsequently, a series of tests were carried out to compare the mutants and the parental strain. As for bacterial growth, no significant differences were observed among strains. Then, the contribution of each efflux system under study for resistance to antibiotics, bile salts and biocides was evaluated, by determining the minimum inhibitory concentration through the microdilution method. Following this, the results showed that: inactivation of the areG gene had an impact on resistance to five of the six classes of antibiotics tested, as well as on the resistance to several biocides; the inactivation of the areB gene had an impact on resistance to cephalosporins, streptomycin, fluoroquinolones and macrolides; the resistance phenotype of the mutant AB28/11?areE was similar to the one of the parental strain. In addition, the role of the efflux system in the intracellular accumulation of compounds was evaluated using a fluorescence method, which evaluated the accumulation of ethidium bromide, in parental and mutant strains. This assay showed that the pump AreABC provides an important role in the extrusion of toxic compounds, since a higher accumulation than the observed for the parental strain was observed, showing a reduced global activity of the efflux systems. In sum, with this work we were able to characterize the role of the AreABC, AreDEF and AreGHI pumps in the resistance of A. butzleri, focusing on the potential role of the AreABC and AreGHI systems on multidrug resistance of this bacterium.
Aliarcobacter butzleri is an emerging enteropathogen recognized as the fourth most common species in diarrheal samples among the Campylobacter-like organisms, being on the list of microorganisms considered to be a serious hazard to human health according to the International Commission on Microbiological Specifications for Food. This pathogenic organism is known to be multidrug resistant, which may cause complications in the treatment of infections caused by this bacterium. Previous studies regarding this bacterium suggest that it contains several putative efflux systems that remain to be characterized, and these may have a relevant role as mechanisms of resistance to antimicrobials. This study aimed to evaluate the role of five different efflux systems from the Resistance nodulation cell division pump family. To this end, the study started with the construction of A. butzleri mutants, using a naturally transformable strain (AB28/11), by interrupting the genes coding for the inner membrane protein responsible for substrate recognition. From the five systems under study, it was only possible to generate mutants for thee of them, where the genes areB, areE and areG, from to the AreABC, AreDEF and AreGHI efflux pumps, respectively, were interrupted. The construction of the mutants was confirmed by the technique of reverse transcription-polymerase chain reaction. Subsequently, a series of tests were carried out to compare the mutants and the parental strain. As for bacterial growth, no significant differences were observed among strains. Then, the contribution of each efflux system under study for resistance to antibiotics, bile salts and biocides was evaluated, by determining the minimum inhibitory concentration through the microdilution method. Following this, the results showed that: inactivation of the areG gene had an impact on resistance to five of the six classes of antibiotics tested, as well as on the resistance to several biocides; the inactivation of the areB gene had an impact on resistance to cephalosporins, streptomycin, fluoroquinolones and macrolides; the resistance phenotype of the mutant AB28/11?areE was similar to the one of the parental strain. In addition, the role of the efflux system in the intracellular accumulation of compounds was evaluated using a fluorescence method, which evaluated the accumulation of ethidium bromide, in parental and mutant strains. This assay showed that the pump AreABC provides an important role in the extrusion of toxic compounds, since a higher accumulation than the observed for the parental strain was observed, showing a reduced global activity of the efflux systems. In sum, with this work we were able to characterize the role of the AreABC, AreDEF and AreGHI pumps in the resistance of A. butzleri, focusing on the potential role of the AreABC and AreGHI systems on multidrug resistance of this bacterium.
Description
Keywords
Aliarcobacter Butzleri AntibiĆ³ticos ResistĆŖncia Sistemas de Efluxo