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Authors
Abstract(s)
O género Arcobacter foi proposto pela primeira vez em 1991 por Vandamme e, até à data,
compreende 29 espécies reconhecidas, incluindo Arcobacter butzleri. Esta espécie é
considerada o quarto organismo Campylobacter-like mais frequentemente encontrado em
amostras de fezes diarreicas no homem, sendo responsável por causar cólicas abdominais,
náusea e diarreia. De facto, foi incluída na lista de microrganismos considerados um sério risco
para a saúde humana pela Comissão Internacional de Especificações Microbiológicas de
Alimentos. Arcobacter spp. tem demonstrado um aumento da resistência a vários
antimicrobianos, que podem estar associados a diversos mecanismos, um dos quais através das
bombas de efluxo. As bombas de efluxo são estruturas proteicas que representam importantes
elementos pertencentes ao repertório microbiano das bactérias e são consideradas responsáveis
pela resistência a muitas substâncias. Em bactérias de Gram-negativo, as bombas de efluxo da
família resistance-nodulation-division cell (RND) têm um papel relevante na resistência a
múltiplos fármacos. Vários genes que codificam para este tipo de sistema foram identificados
no genoma de A. butzleri. Assim, o objetivo deste trabalho foi avaliar o efeito de mutantes
para a bomba de efluxo do tipo RND (AreDEF) na resistência e virulência de A. butzleri. Como
tal, foram construídos mutantes, usando duas estirpes diferentes de A. butzleri, DQ40A1 e
AB28/11, por deleção do gene areE do operão da bomba de efluxo. Em seguida, foram testados
os efeitos desta deleção na resistência a antimicrobianos, crescimento, robustez bacteriana,
motilidade, hidrofobicidade e sobrevivência sob condições adversas, como stress oxidativo,
osmótico, biliar e resistência na presença de clorexidina. Quando comparados os mutantes com
as estirpes parentais correspondentes, o mutante AB28/11?areE apresentou um aumento na
suscetibilidade a cefalosporinas, aminoglicosídeos, tetraciclina, macrólidos, levofloxacina,
colato de sódio e cloreto de benzalcónio; no entanto, este fenótipo não foi observado para o
mutante DQ40A1?areE. Não foram observadas diferenças no crescimento bacteriano entre as
estirpes parentais e mutantes; contudo, A. butzleri AB28/11?areE apresentou uma redução na
robustez quando comparada com a estirpe parental. Ambas os mutantes mostraram alterações
no valor da hidrofobicidade, porém apenas AB28/11?areE apresentou uma motilidade
diminuída. Além disso, A. butzleri AB28/11?areE exibiu um aumento na suscetibilidade às
condições de stress testadas quando comparada com a correspondente estirpe parental, mas
A. butzleri DQ40A1?areE mostrou alteração apenas em relação ao stress biliar. No ensaio de
sobrevivência à clorexidina, verificou-se uma tendência para o aumento da suscetibilidade para
ambos os mutantes em estudo. Em conclusão, este trabalho mostrou um potencial papel da
bomba de efluxo AreDEF na resistência e virulência de Arcobacter butzleri, no entanto mais
estudos serão necessários.
The Arcobacter genus was first proposed in 1991 by Vandamme, and to date comprises 29 recognized species, including Arcobacter butzleri. This species is considered the fourth most frequent Campylobacter-like organism found in human diarrheic stool samples, being responsible for causing abdominal cramps, nausea and diarrhea. In fact, it was included on the list of microorganisms considered a serious hazard to human health by the International Commission on Microbiological Specifications for Foods. Arcobacter spp. is showing an increased resistance to various antimicrobials, which can be associated with several mechanisms, such as efflux pumps. Efflux pumps are protein structures representing important elements belonging to the microbial repertoire of bacteria and are considered responsible for the resistance to many substances. In Gram negative bacteria, efflux pumps from the resistance nodulation cell division (RND) family have a relevant role for multidrug resistance. Various genes coding for this type of systems were identified in the genome of A. butzleri, among them the complete operon AreDEF. The aim of this study was to examine the effect of areE mutants in resistance and virulence of A. butzleri. So, to evaluate the role of AreDEF, we constructed mutants, using two different A. butzleri strains, DQ40A1 and AB28/11, by deletion of the gene areE from the efflux pump operon. Next, the effect on resistance to antimicrobials, growth, strain fitness, motility, hydrophobicity, and survival under adverse conditions, such as oxidative stress, osmotic, bile and survival to chlorhexidine were tested. When we compared mutants with the correspondent wild-type strains, AB28/11?areE displayed an increase in the susceptibility to cephalosporins, aminoglycosides, tetracycline, macrolides, levofloxacin, sodium cholate and benzalkonium chloride; nonetheless the same profile was not observed for A. butzleriDQ40A1?areE. No differences in growth kinetics were recorded between parental and mutant strains; however, the mutant A. butzleri AB28/11?areE showed a reduction in the fitness cost when compared with the parental wild-type strain. Both mutants had modification in the value of hydrophobicity, however only AB28/11?areE had a decreased motility. In addition, A. butzleri AB28/11?areE exhibited higher susceptibility to all stresses when compared to its correspondent parental strain, but A. butzleri DQ40A1?areE showed alteration only concerning bile stress. In the chlorhexidine assay, a tendency for increased susceptibility was observed for both strains, although more studies are needed. In conclusion, the results obtained point to a potential role of the efflux pump AreDEF in the resistance and virulence of Arcobacter butzleri.
The Arcobacter genus was first proposed in 1991 by Vandamme, and to date comprises 29 recognized species, including Arcobacter butzleri. This species is considered the fourth most frequent Campylobacter-like organism found in human diarrheic stool samples, being responsible for causing abdominal cramps, nausea and diarrhea. In fact, it was included on the list of microorganisms considered a serious hazard to human health by the International Commission on Microbiological Specifications for Foods. Arcobacter spp. is showing an increased resistance to various antimicrobials, which can be associated with several mechanisms, such as efflux pumps. Efflux pumps are protein structures representing important elements belonging to the microbial repertoire of bacteria and are considered responsible for the resistance to many substances. In Gram negative bacteria, efflux pumps from the resistance nodulation cell division (RND) family have a relevant role for multidrug resistance. Various genes coding for this type of systems were identified in the genome of A. butzleri, among them the complete operon AreDEF. The aim of this study was to examine the effect of areE mutants in resistance and virulence of A. butzleri. So, to evaluate the role of AreDEF, we constructed mutants, using two different A. butzleri strains, DQ40A1 and AB28/11, by deletion of the gene areE from the efflux pump operon. Next, the effect on resistance to antimicrobials, growth, strain fitness, motility, hydrophobicity, and survival under adverse conditions, such as oxidative stress, osmotic, bile and survival to chlorhexidine were tested. When we compared mutants with the correspondent wild-type strains, AB28/11?areE displayed an increase in the susceptibility to cephalosporins, aminoglycosides, tetracycline, macrolides, levofloxacin, sodium cholate and benzalkonium chloride; nonetheless the same profile was not observed for A. butzleriDQ40A1?areE. No differences in growth kinetics were recorded between parental and mutant strains; however, the mutant A. butzleri AB28/11?areE showed a reduction in the fitness cost when compared with the parental wild-type strain. Both mutants had modification in the value of hydrophobicity, however only AB28/11?areE had a decreased motility. In addition, A. butzleri AB28/11?areE exhibited higher susceptibility to all stresses when compared to its correspondent parental strain, but A. butzleri DQ40A1?areE showed alteration only concerning bile stress. In the chlorhexidine assay, a tendency for increased susceptibility was observed for both strains, although more studies are needed. In conclusion, the results obtained point to a potential role of the efflux pump AreDEF in the resistance and virulence of Arcobacter butzleri.
Description
Keywords
Arcobacter Butzleri Bombas de Efluxo Rnd Resistência Virulência