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Unravelling the virulence potential of Aliarcobacter butzleri
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Evaluation of Bile Salts on the Survival and Modulation of Virulence of Aliarcobacter butzleri
Publication . Mateus, Cristiana; Maia, Cláudio J.; Domingues, Fernanda; Bücker, Roland; Oleastro, Mónica; Ferreira, Susana
Aliarcobacter butzleri is a Gram-negative bacterium associated with infections of the gastrointestinal tract and widely distributed in various environments. For successful infection, A. butzleri should be able to tolerate various stresses during gastrointestinal passage, such as bile. Bile represents an antimicrobial host barrier that acts against external noxious agents and consists of a variety of bile salts. The intestinal bile salts act as detergents involved in the antimicrobial host defense; although, on the bacterial side, they could also serve as a signal to activate virulence mechanisms. The aim of this work was to understand the effects of bile salts on the survival and virulence of A. butzleri. In our study, A. butzleri was able to survive in the presence of human physiological concentrations of bile salts. Regarding the virulence features, an increase in cellular hydrophobicity, a decrease in motility and expression of flaA gene, as well as an increase in biofilm formation with a concomitant change in the type of biofilm structure were observed in the presence of sub-inhibitory concentration of bile salts. Concerning adhesion and invasion ability, no significant difference was observed. Overall, the results demonstrated that A. butzleri is able to survive in physiological concentrations of bile salts and that exposure to bile salts could change its virulence mechanisms.
RND Efflux Systems Contribute to Resistance and Virulence of Aliarcobacter butzleri
Publication . Mateus, Cristiana; Nunes, Ana Rita; Oleastro, Mónica; Domingues, Fernanda; Ferreira, Susana
Aliarcobacter butzleri is an emergent enteropathogen that can be found in a range of environments. This bacterium presents a vast repertoire of efflux pumps, such as the ones belonging to the resistance nodulation cell division family, which may be associated with bacterial resistance, as well as virulence. Thus, this work aimed to evaluate the contribution of three RND efflux systems, AreABC, AreDEF and AreGHI, in the resistance and virulence of A. butzleri. Mutant strains were constructed by inactivation of the gene that encodes the inner membrane protein of these systems. The bacterial resistance profile of parental and mutant strains to several antimicrobials was assessed, as was the intracellular accumulation of the ethidium bromide dye. Regarding bacterial virulence, the role of these three efflux pumps on growth, strain fitness, motility, biofilm formation ability, survival in adverse conditions (oxidative stress and bile salts) and human serum and in vitro adhesion and invasion to Caco-2 cells was evaluated. We observed that the mutants from the three efflux pumps were more susceptible to several classes of antimicrobials than the parental strain and presented an increase in the accumulation of ethidium bromide, indicating a potential role of the efflux pumps in the extrusion of antimicrobials. The mutant strains had no bacterial growth defects; nonetheless, they presented a reduction in relative fitness. For the three mutants, an increase in the susceptibility to oxidative stress was observed, while only the mutant for AreGHI efflux pump showed a relevant role in bile stress survival. All the mutant strains showed an impairment in biofilm formation ability, were more susceptible to human serum and were less adherent to intestinal epithelial cells. Overall, the results support the contribution of the efflux pumps AreABC, AreDEF and AreGHI of A. butzleri to antimicrobial resistance, as well as to bacterial virulence.
Natural Transformation as a Mechanism of Horizontal Gene Transfer in Aliarcobacter butzleri
Publication . Bonifácio, Marina; Mateus, Cristiana; Alves, Ana R.; Maldonado, Emanuel; Duarte, Ana P.; Domingues, Fernanda; Oleastro, Mónica; Ferreira, Susana
Aliarcobacter butzleri is an emergent enteropathogen, showing high genetic diversity, which likely contributes to its adaptive capacity to different environments. Whether natural transformation can be a mechanism that generates genetic diversity in A. butzleri is still unknown. In the present study, we aimed to establish if A. butzleri is naturally competent for transformation and to investigate the factors influencing this process. Two different transformation procedures were tested using exogenous and isogenic DNA containing antibiotic resistance markers, and different external conditions influencing the process were evaluated. The highest number of transformable A. butzleri strains were obtained with the agar transformation method when compared to the biphasic system (65% versus 47%). A. butzleri was able to uptake isogenic chromosomal DNA at different growth phases, and the competence state was maintained from the exponential to the stationary phases. Overall, the optimal conditions for transformation with the biphasic system were the use of 1 μg of isogenic DNA and incubation at 30 °C under a microaerobic atmosphere, resulting in a transformation frequency ~8 × 10-6 transformants/CFU. We also observed that A. butzleri favored the transformation with the genetic material of its own strain/species, with the DNA incorporation process occurring promptly after the addition of genomic material. In addition, we observed that A. butzleri strains could exchange genetic material in co-culture assays. The presence of homologs of well-known genes involved in the competence in the A. butzleri genome corroborates the natural competence of this species. In conclusion, our results show that A. butzleri is a naturally transformable species, suggesting that horizontal gene transfer mediated by natural transformation is one of the processes contributing to its genetic diversity. In addition, natural transformation can be used as a tool for genetic studies of this species.
The influence of short-chain fatty acids on the survival and virulence of Arcobacter butzleri
Publication . Fonseca, Inês M.; Mateus, Cristiana; Vieira, Alexandre; Domingues, Fernanda; Manageiro, Vera; Oleastro, Mónica; Ferreira, Susana
Aims: Arcobacter butzleri, a widespread bacterium linked to gastrointestinal disease, can bypass host colonization resistance mechanisms; however, its response to short-chain fatty acids (SCFAs) remains poorly understood. This study investigated the impact of SCFAs on A. butzleri ’s survival and virulence.
Methods and results: Eight A. butzleri isolates were assessed under varying concentrations of individual SCFAs and mixtures (m-SCFAs). Higher SCFAs concentrations inhibited bacterial growth in a strain-dependent manner. Transcript analysis of putative virulence genes revealed upregula- tion of ciaB and flaA across most m-SCFAs concentrations, while luxS expression increased at 90 mM. SCFAs generally reduced bacterial motility, with sodium propionate reducing motility but enhancing biofilm-forming ability in the model strain. Additionally, SCFAs exposure decreased the ability of A. butzleri to adhere to and invade the Caco-2 intestinal epithelial cell line. Whole-genome sequencing of the eight A. butzleri isolates revealed extensive genetic diversity, particularly in virulence- and stress-associated genes, although consistent genot ype/phenot ype correlations were not observed.
Conclusions: Altogether, these findings demonstrate that SCFAs modulate A. butzleri survival and virulence, providing novel insights into their significance in shaping pathogen behaviour and host-pathogen interactions.
The dual action of probiotic lactobacilli in suppressing virulence and survival of Arcobacter butzleri
Publication . Vieira, Alexandre; Mateus, Cristiana; Fonseca, Inês M.; Domingues, Fernanda; Oleastro, Mónica; Ferreira, Susana
Arcobacter butzleri is a widely distributed foodborne and waterborne pathogen, increasingly recognized as an emerging enteropathogen. Understanding its survival mechanisms and interactions with probiotics is crucial for developing targeted intervention strategies. A. butzleri must withstand various hostile conditions to successfully colonize the gastrointestinal tract, including inhibition by probiotics, such as Limosilactobacillus reuteri, Lactobacillus acidophilus and Lactiplantibacillus plantarum. Thus, this study aimed to assess the survival of A. butzleri under acidic conditions and determine its minimum inhibitory concentration (MIC) for bile salts. Additionally, the antimicrobial potential of the lactobacilli strains was evaluated by analysing the effects of their culture-free supernatant (CFS) on A. butzleri growth, coculture interactions, and biofilm formation. The influence of lactobacilli on A. butzleri was further investigated through competition, displacement and exclusion assays using Caco-2 cell models. The results indicate that lactobacilli strains exhibit tolerance to acidic environments and physiological bile salt concentrations, whereas A. butzleri was more susceptible to acidic stress. The antagonistic effect of lactobacilli was evidenced by growth inhibition of A. butzleri in the presence of CFS or during coculture. However, CFS from certain lactobacilli strains was found to enhance biofilm formation, highlighting potential consequences. Furthermore, while lactobacilli did not demonstrate significant ntagonistic effects in competition assays, they effectively displaced and excluded A. butzleri in the Caco-2 infection model. Overall, these findings suggest that probiotic lactobacilli can inhibit A. butzleri growth, yet their impact on its virulence remains uncertain. This underscores the need for strain-specific probiotic selection to effectively target this pathogen and emphasizes that not every probiotic contribute to the prevention of A. butzleri infections.
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Fundação para a Ciência e a Tecnologia
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UI/BD/151023/2021