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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, F.C.Arcobacter 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.
- Virulence and antibiotic resistance plasticity of Arcobacter butzleri: Insights on the genomic diversity of an emerging human pathogenPublication . Isidro, Joana; Ferreira, Susana; Pinto, Miguel; Domingues, F. C.; Oleastro, Mónica; Gomes, João Paulo; Borges, VítorArcobacter butzleri is a foodborne emerging human pathogen, frequently displaying a multidrug resistant character. Still, the lack of comprehensive genome-scale comparative analysis has limited our knowledge on A. butzleri diversification and pathogenicity. Here, we performed a deep genome analysis of A. butzleri focused on decoding its core- and pan-genome diversity and specific genetic traits underlying its pathogenic potential and diverse ecology. A. butzleri (genome size 2.07-2.58 Mbp) revealed a large open pan-genome with 7474 genes (about 50% being singletons) and a small but diverse core-genome with 1165 genes. It presents a plastic virulome (including newly identified determinants), marked by the differential presence of multiple adaptation-related virulence factors, such as the urease cluster ureD(AB)CEFG (phenotypically confirmed), the hypervariable hemagglutinin-encoding hecA, a type I secretion system (T1SS) harboring another agglutinin and a novel VirB/D4 T4SS likely linked to interbacterial competition and cytotoxicity. In addition, A. butzleri harbors a large repertoire of efflux pumps (EPs) and other antibiotic resistant determinants. We unprecedentedly describe a genetic mechanism of A. butzleri macrolides resistance, (inactivation of a TetR repressor likely regulating an EP). Fluoroquinolones resistance correlated with Thr-85-Ile in GyrA and ampicillin resistance was linked to an OXA-15-like β-lactamase. Remarkably, by decoding the polymorphism pattern of the main antigen PorA, we show that A. butzleri is able to exchange porA as a whole and/or hypervariable epitope-encoding regions separately, leading to a multitude of chimeric PorA presentations that can impact pathogen-host interaction during infection. Ultimately, our unprecedented screening of short sequence repeats indicates that phase variation likely modulates A. butzleri key adaptive functions. In summary, this study constitutes a turning point on A. butzleri comparative genomics revealing that this human gastrointestinal pathogen is equipped with vast and diverse virulence and antibiotic resistance arsenals that open a multitude of phenotypic fingerprints for environmental/host adaptation and pathogenicity.
- Current insights on Arcobacter butzleri in food chainPublication . Ferreira, Susana; Oleastro, Mónica; Domingues, F.C.Arcobacter 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 etiology. 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 toward 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.
- Genotypic and phenotypic features of Arcobacter butzleri pathogenicityPublication . Ferreira, Susana; Queiroz, João; Oleastro, Mónica; Domingues, F.C.Even though Arcobacter butzleri has been implicated in some human disease as diarrhoea and bacteraemia, much of its pathogenesis and virulence factors remain unclear. In this work we have compared pathogenic and genotypic properties of six A. butzleri isolates from human and non-human sources. The tested isolates showed to be susceptible to tetracyclines and aminoglycosides, however non-human isolates were all resistant to quinolones. The ability to form biofilms was variable among the tested strains, and all of them showed a weak haemolytic activity. The presence of nine putative virulence genes was determined, with cadF, ciaB, cj1349, mviN, pldA, tlyA being detected in all strains, while irgA (3/6), hecA (5/6), hecB (4/6) were detected only in some strains. High levels of adhesion were observed for A. butzleri on Caco-2 cells, with pre-existing inflammation showing no significant effect on the adherence ability; yet variable levels of invasion were observed. A. butzleri isolates were able to survive intracellularly in Caco-2 cells and to induce a significant up-regulation of interleukin-8 secretion and structural cell rearrangements. These data brings new insights on A. butzleri virulence and highlights its pathogenic potential.
- Genetic diversity, antibiotic resistance and biofilm-forming ability of Arcobacter butzleri isolated from poultry and environment from a Portuguese slaughterhousePublication . Ferreira, Susana; Fraqueza, Maria J.; Queiroz, João; Domingues, F.C.; 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 biofilm-forming 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.