Browsing by Author "Domingues, Fernanda"
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- Anti-Helicobacter pylori and urease inhibitory activities of resveratrol and red winePublication . Paulo, Luísa; Oleastro, Mónica; Gallardo, Eugenia; Queiroz, João António; Domingues, FernandaThere is considerable interest in alternative approaches for the eradication of Helicobacter pylori using biologically active compounds including antioxidants from a wide range of natural sources. In this work we have investigated the antibacterial properties of resveratrol towards different H. pylori strains. In addition we studied the inhibition of H. pylori urease by resveratrol and red wine. In those assays, resveratrol inhibited the growth of all the 17 H. pylori strains tested, with inhibition diameters ranging from 16 to 28 mm and minimum inhibitory concentration values varying from 25 to 100 mu g/mL, confirming its antimicrobial properties. Moreover, resveratrol and red wines showed an inhibitory effect on H. pylori urease activity, which is considered a virulence factor of this organism and essential for colonization and establishment of the infection. Further kinetic analysis revealed that inhibition occurred in a non-competitive and concentration-dependent manner. Overall, the results suggest that resveratrol and red wine may have potential for new therapy schemes that include natural products as an alternative therapeutic approach.
- 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.
- 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.
- Insights into macrolide resistance in Arcobacter butzleri: potential resistance mechanisms and impact on bacterial fitness and virulencePublication . Couto, Francisca; Martins, Inês; Vale, Filipa; Domingues, Fernanda; Oleastro, Mónica; Ferreira, SusanaBackground: Macrolides are recommended for treating the emerging enteropathogen Arcobacter butzleri; nonetheless, this bacterium often exhibits highly variable resistance rates, and the mechanisms behind this resistance phenotype remain largely unexplored. Objectives: To understand the phenotypic and genotypic consequences associated with the acquisition of erythromycin resistance in A. butzleri, as well as the effects on the fitness of this species. Methods: Resistant strains resulting from spontaneous mutations and adaptive laboratory evolution under increasing erythromycin concentrations were examined regarding their cross-resistance and collateral susceptibility profiles. Genetic causes of phenotypic antibiotic resistance were analysed by sequencing and bioinformatics, with functional correlation through ethidium bromide accumulation assays. Growth profiles in the presence and absence of erythromycin, motility and biofilm formation abilities were assessed to detect potential changes in fitness and virulence. Results: Clones from spontaneous mutation rate evolution demonstrated decreased susceptibility to erythromycin and other classes of antibiotics, associated with mutations in the transcriptional repressor areR, causing overexpression of the AreABC efflux pump. In turn, WGS analysis of the evolved strain showed additional mutations in the ribosomal proteins L4 and L22 and in the areR gene. Furthermore, the acquisition of macrolide resistance altered A. butzleri virulence and entailed a high biological cost. Conclusions: The findings of this study have proved that efflux activity contributes synergistically with mutations in the ribosomal proteins L4 and L22 to A. butzleri's high-level macrolide resistance. The results further suggest an impact on the bacterial physiology and virulence, with the increased fitness cost justifying the low worldwide prevalence of high-level resistant circulating strains.
- Molecular diagnosis of Arcobacter and Campylobacter in diarrhoeal samples amongPublication . Ferreira, Susana; Júlio, Cláudia; Queiroz, João; Domingues, Fernanda; Oleastro, MónicaThe present study was conducted to investigate the prevalence and diversity of Arcobacter and Campylobacter spp. in 298 stool samples of patients with diarrhoea, collected from 22 Portuguese hospitals, between September and November 2012. Detection of Arcobacter and Campylobacter spp. was performed using molecular-based detection techniques, such as real-time fluorescence resonance energy transfer PCR, species-specific PCR, and sequencing of amplified PCR products. Overall, 1.3% of the samples were positive for Arcobacter butzleri and 0.3% for Arcobacter cryaerophilus. Campylobacter spp. were found in 31.9% of diarrhoeic faeces. Campylobacter jejuni and Campylobacter concisus were the most prevalent species (13.7% and 8.0%, respectively). The prevalence of Arcobacter and Campylobacter spp. was significantly different between children and adults (39.7% versus 22.8%, P = 0.003). We underline the high prevalence of these pathogens in diarrhoeal samples among Portuguese patients, with particular relevance in the paediatric age group.