Browsing by Author "Ferreira, Susana"
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- Arcobacter butzleri Ciprofloxacin Resistance: Point Mutations in DNA Gyrase A and Role on Fitness CostPublication . Ferreira, Susana; Correia, Daniela R; Oleastro, Mónica; Domingues, Fernanda CArcobacter butzleri is a widely distributed emerging pathogen resistant to various classes of antimicrobial agents, namely fluoroquinolones. A. butzleri resistance to fluoroquinolones is conferred by point mutations at the antibiotic target. The aim of this study was to evaluate mutations at gyrA associated with ciprofloxacin resistance and evaluate whether acquisition of resistance impacts on fitness and stress tolerance of A. butzleri. A. butzleri ciprofloxacin mutants were generated by laboratory induction. Identification of mutations associated with ciprofloxacin resistance was performed by gyrA sequencing. Growth kinetics, cost of fitness, biofilm formation ability, and stress tolerance were assessed. Two amino acid substitutions in the quinolone resistance-determining region of GyrA were identified in the mutant strains, one previously described (Thr-85-Ile) and a new substitution (Asp-89-Tyr). No differences in growth kinetics were recorded between parental and mutant strains; however, fitness cost was variable, according to the genetic background of the strains, and independently of ciprofloxacin resistance. Overall, the ciprofloxacin resistance development did not significantly affect stress tolerance, motility, or biofilm-forming ability. In conclusion, acquisition of ciprofloxacin resistance in A. butzleri is associated with mutations in gyrA and is likely well compensated, with cost of fitness reflecting the diversity in genetic background of this bacterium.
- Arcobacter spp. at retail food from Portugal: Prevalence, genotyping and antibiotics resistancePublication . Vicente-Martins, Sofia; Oleastro, Mónica; Domingues, Fernanda C.; Ferreira, SusanaArcobacter genus is considered a foodborne emergent pathogen able to cause disease among humans and animals. The wide distribution and high prevalence of Arcobacter in food is becoming a concern, since consumption of contaminated food and water is considered the most probable route of Arcobacter transmission to humans. In this work, ready-to-eat packaged vegetables, poultry, pork, beef and fish meat were purchased from several retail markets and supermarkets and tested for the presence of Arcobacter spp. Suspected colonies were isolated and identified, characterized through ERIC-PCR and resistance to nine antibiotics commonly used to treat infections by this microorganism was assessed; in parallel, Arcobacter spp. was directly detected on enrichment broth by multiplex PCR. The results show a high prevalence of Arcobacter spp. among retail food, with an overall prevalence of 60.5% obtained by both molecular and culture detection. A. butzleri was the most frequently isolated species (58.5%) followed by A. cryaerophilus (35.8%) and A. skirrowii (5.7%). A high genetic diversity among the isolates was observed, despite the detection of a possible cross-contamination between food samples. Also, a high rate of multidrug resistance among the isolates (85.7%) was observed. Taken together, our results suggest that the consumption of Arcobacter-contaminated food products is of public health concern.
- Characterization of Arcobacter butzleri isolates from poultry and slaughterhouse EnvironmentPublication . Ferreira, Susana; Fraqueza, Maria João; Queiroz, João; Domingues, Fernanda; Oleastro, MónicaThe genus Arcobacter is an emerging pathogen associated with several clinical symptoms, namely diarrhoea, abdominal pain or bacteraemia. This genus is widely distributed and has been isolated from environmental, animal, food and human samples, being poultry considered the major reservoir. In this study, forty three Arcobacter butzleri strains were isolated from poultry of three flocks from different farms and environment samples at a Portuguese slaughterhouse, also three reference strains were included. All isolates were confirmed at species level by multiplex PCR and genomic DNA fingerprints of all isolates were determined using Pulsed Field Gel Electrophoresis (PFGE) after enzymatic digestion with SmaI and the isolates with undistinguishable SmaI patterns were further analyzed with a second restriction enzyme, SacII. Phenotypic resistance profiles to nine antibiotics were assessed by broth microdilution method. Biofilm formation assays were performed in the 36 out of the 43 A. butzleri isolates, either presenting different pulsetypes or similar PFGE patterns but different origin or even different resistance patterns. PFGE patterns obtained using restriction enzymes SmaI and SacII revealed genetic diversity, with 32 distinct PFGE patterns. A high percentage of A. butzleri isolates was found to be resistant, recording four different resistance profiles. Twenty four of the 43 isolates presented a phenotypic resistance to ciprofloxacin, in contrast to the great susceptibility against gentamicin and chloramphenicol. Among the 36 selected A. butzleri isolates, 13.9% were categorized as moderately adherent, while 58.3% were defined as weakly adherent. Overall, the results showed a high degree of genetic heterogeneity among A. butzleri isolates, along with high levels of resistance to several antibiotics. Biofilm formation ability of A. butzleri can possibly favour dispersion and cross-contamination along the slaughterhouse processing line. These findings may represent a contribution to get insight the survival and even persistence mechanisms of this organism in the environment and on its relevance as a potential hazard for foodborne infections.
- Characterization of AreABC, an RND-Type Efflux System Involved in Antimicrobial Resistance of Aliarcobacter butzleriPublication . Ferreira, Susana; Silva, Ana L.; Tomás, Joana; Mateus, Cristiana; Domingues, Fernanda; Oleastro, MónicaAliarcobacter butzleri is an emergent enteropathogen for which resistance to several classes of antimicrobial agents has been described, although the underlying mechanisms have been poorly addressed. We aimed to evaluate the contribution of the resistance-nodulation-division-type (RND) efflux system, AreABC, to drug resistance in A. butzleri. A. butzleri strains were first tested against several antimicrobials with and without an efflux pump inhibitor. Then, erythromycin-resistant strains were screened for the presence of a premature stop codon in a putative transcriptional regulator of the AreABC system, areR. Lastly, antimicrobial susceptibility and ethidium bromide (EtBr) accumulation were evaluated using an areB knockout strain and a strain overexpressing the AreABC system through areR truncation. The presence of the efflux pump inhibitor resulted in increased susceptibility to most of the antimicrobials tested. A correlation between erythromycin resistance and the presence of premature stop codons in areR was observed. The truncation of areR resulted in increased expression of the AreABC system and decreased susceptibility to various antimicrobials. In contrast, areB inactivation resulted in increased susceptibility and a higher intracellular accumulation of EtBr. In conclusion, the AreABC efflux pump plays a role in the resistance of A. butzleri to multiple drugs and is regulated by a putative transcriptional repressor, areR. Our results support the importance of efflux pumps in this bacterium's resistance to major classes of antibiotics and other antimicrobials.
- Current insights on Arcobacter butzleri in food chainPublication . Ferreira, Susana; Oleastro, Mónica; Domingues, FernandaArcobacter butzleri is an emerging pathogen with a global distribution, frequently found in the food chain. Its wide spread and association with human illness may point this bacterium as a possible causative agent of foodborne diseases usually associated with unknown aetiology. Thus, the true pathogenic potential of A. butzleri and the associated risk of disease development must be clarified. Since its dispersion throughout the food chain is clear, this points towards a potential public health problem due to contaminated food and water, which is strengthened by the ability of this microorganism to survive in food products and water and by its resistance to stress created during food storage and processing.
- Effect of Atmospheric Conditions on Pathogenic Phenotypes of Arcobacter butzleriPublication . Martins, Rodrigo; Mateus, Cristiana; Domingues, Fernanda; Bücker, Roland; Oleastro, Mónica; Ferreira, SusanaArcobacter butzleri is an emergent gram-negative enteropathogenic bacterium widespread in different environments and hosts. During the colonization of the gastrointestinal tract, bacteria face a variety of environmental conditions to successfully establish infection in a new host. One of these challenges is the fluctuation of oxygen concentrations encountered not only throughout the host gastrointestinal tract and defences but also in the food industry. Oxygen fluctuations can lead to modulations in the virulence of the bacterium and possibly increase its pathogenic potential. In this sense, eight human isolates of A. butzleri were studied to evaluate the effects of microaerobic and aerobic atmospheric conditions in stressful host conditions, such as oxidative stress, acid survival, and human serum survival. In addition, the effects on the modulation of virulence traits, such as haemolytic activity, bacterial motility, biofilm formation ability, and adhesion and invasion of the Caco-2 cell line, were also investigated. Overall, aerobic conditions negatively affected the susceptibility to oxygen reactive species and biofilm formation ability but improved the isolates’ haemolytic ability and motility while other traits showed an isolate-dependent response. In summary, this work demonstrates for the first time that oxygen levels can modulate the potential pathogenicity of A. butzleri, although the response to stressful conditions was very heterogeneous among different strains.
- Evaluation of Bile Salts on the Survival and Modulation of Virulence of Aliarcobacter butzleriPublication . Mateus, Cristiana; Maia, Cláudio J.; Domingues, Fernanda; Bücker, Roland; Oleastro, Mónica; Ferreira, SusanaAliarcobacter 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.
- Exploring flagellar contributions to motility and virulence in Arcobacter butzleriPublication . Santos, Raquel; Mateus, Cristiana; Oleastro, Mónica; Ferreira, SusanaFlagella is a well-known bacterial structure crucial for motility, which also plays pivotal roles in pathogenesis. Arcobacter butzleri, an enteropathogen, possesses a distinctive polar flagellum whose functional aspects remain largely unexplored. Upon investigating the factors influencing A. butzleri motility, we uncovered that environmental conditions like temperature, oxygen levels, and nutrient availability play a significant role. Furthermore, compounds that are found in human gut, such as short-chain fatty acids, mucins and bile salts, have a role in modulating the motility, and in turn, the pathogenicity of A. butzleri. Further investigation demonstrated that A. butzleri ΔflaA mutant showed a reduction in motility with a close to null average velocity, as well as a reduction on biofilm formation. In addition, compared with the wild-type, the ΔflaA mutant showed a decreased ability to invade Caco-2 cells and to adhere to mucins. Taken together, our findings support the role of environmental conditions and gut host associated compounds influencing key physiological aspects of the gastrointestinal pathogen A. butzleri, such as motility, and support the role of the flagellum on bacterial virulence.
- Genetic diversity and antibiotic resistance of Arcobacter butzleri isolated from poultry and slaughterhouse environment in PortugalPublication . Ferreira, Susana; Fraqueza, Maria João; Queiroz, João; Domingues, Fernanda; Oleastro, MónicaArcobacter is considered an emerging enteric pathogen, commonly associated with diarrhea, abdominal pain and in some cases with bacteriemia. This genus is widely distributed, with fteen species identi ed to date, of which the most common is Arcobacter butzleri. Arcobacter spp. has been isolated from environmental, animal, food and human samples, but poultry is considered its main reservoir. The extended use of antibiotics for disease control in modern food animal production, leads to a spread of resistant pathogenic bacteria, and Arcobacter spp. is no exception to this rule. In this study, 43 A. butzleri isolates were obtained from poultry and environment samples at a Portuguese slaughterhouse, also three reference strains were included. All isolates were con rmed at species level by multiplex PCR; genomic DNA ngerprints of all isolates were determined using Pulsed Field Gel Electrophoresis (PFGE) after enzymatic digestion with SmaI. Resistance pro les to nine antibiotics were assessed by broth microdilution method. Fifteen unique and 11 common PFGE ngerprints were identi ed among the 43 Arcobacter isolates studied, generating a total of 26 di erent PFGE ngerprints. This data demonstrates the high genetic diversity observed among Arcobacter isolates. Concerning the antibiotic susceptibility, all isolates tested were susceptible to gentamycin and one strain presented resistance to chloramphenicol. In contrast, 24 of the 43 isolates (55.8%) were resistant to cipro oxacin. All the studied isolates presented resistance to multiple antibiotics simultaneously, especially to ampicillin, vancomycin, trimethoprim, piperacillin, cefoperazone and amoxicillin. The results showed that A. butzleri isolated in Portugal presents a high genetic diversity, but also show high levels of resistance to several antimicrobial agents, this fact could represent a potential health hazard for humans through food chain contamination.
- Genetic diversity, antibiotic resistance and biofilm-forming ability of Arcobacter butzleri isolated from poultry and environment from a Portuguese slaughterhousePublication . Ferreira, Susana; Fraqueza, Maria João; Queiroz, João; Domingues, Fernanda; Oleastro, MónicaThe genus Arcobacter is an emerging pathogen associated with several clinical symptoms. This genus is widely distributed and has been isolated from environmental, animal, food and human samples, where poultry is considered the major source. In this study, forty three Arcobacter butzleri strains isolated from poultry and environment of a Portuguese slaughterhouse, were characterized by pulsed field gel electrophoresis (PFGE) and assessed for antimicrobial susceptibility and ability to form biofilms. PFGE patterns obtained using restriction enzymes SmaI and SacII revealed high genetic diversity, with 32 distinct PFGE patterns. Most of A. butzleri isolates presented multiple antimicrobial resistance, exhibiting four different resistance profiles. All 43 isolates were susceptible to gentamicin and 2.3% were resistant to chloramphenicol, in contrast to twenty four (55.8%) that were resistant to ciprofloxacin. Among 36 selected isolates, 26 strains presented biofilmforming ability, which was dependent on the atmosphere and initial inoculum density. Overall, the results showed that A. butzleri displays a high genetic diversity, and presents resistance to several antibiotics, which together with its biofilm formation ability may represent a potential hazard for foodborne infections and a considerable risk for human health.