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- Occurrence, genetic diversity and antibiotic resistance of Arcobacter sp. in a dairy plantPublication . Ferreira, Susana; Oleastro, Mónica; Domingues, F.C.Aims The aim of this study was to evaluate the occurrence, diversity and resistance to antibiotics of Arcobacter sp. in a dairy plant samples. Methods and Results A total of 75 samples from dairy plant surfaces and materials and several food products collected in different steps of the cheese production process were analysed by culture, under aerobic and microaerobic atmospheric conditions, and by enrichment molecular detection. Isolates were identified and genotyped by ERIC‐PCR, and their susceptibility to nine antibiotics was evaluated by agar dilution. Global prevalence of Arcobacter sp. was 42·7%, where 20 of the 42 food samples analysed were positive for A. butzleri by both culture and molecular detection, one for A. marinus by culture and one for A. cryaerophilus by molecular detection only; 10 of the 30 analysed materials and plant surfaces were positive for A. butzleri. All A. butzleri isolates were resistant to nalidixic acid and showed high resistance rates to ampicillin (56·2%) and cefotaxime (97·9%), being all strains susceptible to gentamicin and erythromycin. Conclusions Contamination of dairy plant environment with A. butzleri and its progression along cheese production process were observed, however, the cheese ripening process may have a relevant role in the reduction of the contamination. Significance and Impact of the Study This study showed the presence of Arcobacter sp. in a dairy plant, displaying its high prevalence and genetic diversity and highlighting its high resistance rates. The data obtained could contribute to further acknowledge the Arcobacter food contamination as a potential health hazard.
- Molecular diagnosis of Arcobacter and Campylobacter in diarrhoeal samples among Portuguese patientsPublication . Ferreira, Susana; Júlio, Cláudia; Queiroz, João; Domingues, F.C.; 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.
- A meta-analytic perspective on Arcobacter spp. antibiotic resistancePublication . Ferreira, Susana; Luís, Ângelo; Oleastro, Mónica; Pereira, L.; Domingues, F.C.Objectives: Over the years, an increased prevalence of resistant strains of Arcobacter has been observed, which may be due to Arcobacter exposure to antibiotics used both in animal production and human medicine. A systematic review was performed with the objective of summarising the results of the rates of antimicrobial resistance of Arcobacter isolates. Methods: The systematic review was performed according to PRISMA (Preferred Reported Items for Systematic Reviews and Meta-Analysis) recommendations, followed by meta-analysis. Results: It was observed that the resistance rate ranged between 69.3-99.2% for penicillins and 30.5-97.4% for cephalosporins. The overall percentage of resistance to fluoroquinolones ranged from 4.3% to 14.0%, with the highest resistance percentage observed for levofloxacin. Resistance rates ranged between 10.7-39.8% for macrolides, 1.8-12.9% for aminoglycosides and 0.8-7.1% for tetracyclines. Conclusions: These results show that Arcobacter spp. present resistance to various antibiotics commonly used and advocate further studies of the associated resistance mechanisms.
- Resveratrol against Arcobacter butzleri and Arcobacter cryaerophilus: Activity and effect on cellular functionsPublication . Ferreira, Susana; Silva, Filomena; Queiroz, João; Oleastro, Mónica; Domingues, F.C.The frequent isolation of Arcobacter butzleri and Arcobacter cryaerophilus from food samples makes it imperative to search for potential compounds able to inhibit the development of these bacteria. Taking this into consideration, this study focuses on the antimicrobial activity of resveratrol and its mechanism of action against A. butzleri and A. cryaerophilus. The activity of resveratrol was assessed by a microdilution method and time-kill curves. Resveratrol effect on cellular functions was assessed by flow cytometry evaluating intracellular DNA content and metabolic activity. Ethidium bromide (EtBr) accumulation in the presence of resveratrol was also evaluated, as well as the susceptibility to resveratrol in the presence of phenylalanine-arginine β-naphthylamide (PAβN). Scanning electron microscopy (SEM) was used to further evaluate cell damage caused by resveratrol. Resveratrol presented MIC values of 100 and 50μg/mL to A. butzleri and A. cryaerophilus, respectively. Based on the time-kill curves, resveratrol exhibited bactericidal activity, leading to a ≥3log10CFU/mL reduction of initial inoculums, for A. butzleri exponential phase cells incubated for 6h with 1× MIC or with 2× MIC after 24h for stationary phase cells. For A. cryaerophilus cells in exponential growth phase, 99.9% killing was achieved after 24h incubation with 2× MIC, whereas, for stationary phase cells, bactericidal activity was only detected after incubation with 4× MIC. Incubation with resveratrol led to a decrease in both intracellular DNA content and metabolic activity. An increase in the accumulation of EtBr was observed in the presence of resveratrol, and the efflux pump inhibitor PAβN reduced the MIC of resveratrol. SEM analysis revealed disintegration of A. butzleri cells treated with resveratrol, whereas no morphological alteration was observed for A. cryaerophilus cells. Resveratrol has a good anti-Arcobacter activity, and the results obtained suggest that this compound could act through several different mechanisms in the inhibition of this microorganism. The results encourage the use of this compound for the development of potential strategies to control Arcobacter in food products.
- 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.
- Insights in the pathogenesis and resistance ofArcobacter: A reviewPublication . Ferreira, Susana; Queiroz, João; Oleastro, Mónica; Domingues, F.C.Arcobacter genus currently comprises 18 recognized species, among which Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii have been associated with human and animal disease. Although these organisms, with special emphasis A. butzleri, are emerging as clinical pathogens, several aspects of their epidemiology and virulence are only starting to be clarified. In vitro human and animal cell culture assays have been used to show that several Arcobacter species can adhere to and invade eukaryotic cells, induce an immune response and produce toxins that damage host cells. In addition, data from genome sequencing highlighted several potential markers that may be helpful candidates for the study and understanding of these mechanisms; however, more work is necessary to clarify the molecular mechanisms involved in Arcobacter virulence. Arcobacter can be considered a relatively robust organism showing to be able to survive in adverse conditions, as the ones imposed by food processing and storage. Moreover, these bacteria have shown increased antibiotic resistance, along with high multidrug resistance. In this review, we seek to update the state-of-the-art concerning Arcobacter distribution, its interaction with the host, the trends of antibiotic resistance, its ability to survive, and finally the use of natural antimicrobials for control of Arcobacter.
- Arcobacter spp. at retail food from Portugal: Prevalence, genotyping and antibiotics resistancePublication . Martins, Sofia Vicente; Oleastro, Mónica; Domingues, F.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.
- 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.