Study of molecular and cellular pathogenicity mechanisms of Arcobacter species

Media type: E-Book

Title: Study of molecular and cellular pathogenicity mechanisms of Arcobacter species

Contributor: Ferreira, Susana Margarida Paraíso [VerfasserIn]

imprint: [Erscheinungsort nicht ermittelbar]: [Verlag nicht ermittelbar], 2017

Language: English

Identifier:

Origination:

University thesis: Dissertation, 2017

Footnote:

Description: Arcobacter is a genus of Gram-negative, spiral-shaped bacteria in the Epsilonproteobacteria class. It was first proposed in 1991 and was included in the Campylobacteraceae family together with the genera Campylobacter and Sulfurospirillum. There are currently 18 species described, among which A. butzleri, A. cryaerophilus and A. skirrowii are known to be human and animal pathogens. Arcobacter genus shows an unusually wide range of habitats, having been isolated from food, water, food processing and handling facilities, diverse environmental, animal and human samples. In fact, consumption of Arcobacter-contaminated food or water is regarded as the most probable cause of infection by this bacterium. Therefore it is important to evaluate its distribution in food or food processing environment, together with the assessment of its genetic variability and other phenotypic and genotypic features associated with virulence potential, to be able to understand the persistence mechanism of Arcobacter in the food chain. Thus, this study intend to evaluate the presence, genetic diversity, and virulence characteristics (namely antibiotic resistance, biofilm-forming capacity and detection of putative virulence genes) of A. butzleri strains isolated from poultry and from the environment of a Portuguese slaughterhouse. A. butzleri isolates revealed a high genetic diversity, with all isolates showing to be susceptible to gentamicin, in contrast to 55.8% that were resistant to ciprofloxacin. The latter resistance was associated with the presence of a cytosine to thymine transition in the quinolone resistance determining region of the gyrA gene. Among selected isolates, 72.2 % presented biofilm-forming ability and in all strains putative virulence genes were detected. These results highlight the relevance of A. butzleri relevance as food-borne pathogen. Taking into account that Arcobacter is potentially transmitted through contaminated food and is resistant to common antimicrobials, it is important to develop alternative control strategies that could be both effective and safe for human consumption. Therefore, the antimicrobial properties of resveratrol against A. butzleri and A. cryaerophilus were studied. Resveratrol exhibited a bacteriostatic or bactericidal activity dependent on cellular growth phase and resveratrol concentration, leading to both DNA content and metabolic activity reduction on Arcobacter cells. Resveratrol also showed the ability to act as an efflux pump inhibitor, and to induce cellular damage. Thus, resveratrol showed anti-Arcobacter activity, with the results obtained suggesting that this compound inhibits this microorganism through different pathways, which together with resveratrol beneficial properties described for human health may encourage its use as a food preservative. Some of the species of Arcobacter genus have been associated with gastrointestinal disease in humans, however there were a lack of studies evaluating its prevalence in Portugal, with the same happening for non-Campylobacter jejuni/coli species. Therefore, the frequency of Arcobacter and Campylobacter species in faeces from patients with diarrhoea in Portugal was assessed using a molecular approach. Concerning, Arcobacter and Campylobacter prevalence and distribution, 298 diarrhoeal samples from Portuguese patients were analysed, 1.3% of the samples were positive for A. butzleri and 0.3 % for A. cryaerophilus. Campylobacter species were found in 31.9 % of diarrhoeic faeces samples, with C. jejuni and C. concisus being the most prevalent species of this genus (13.7 % and 8.0 %, respectively). In this cohort of samples, A. butzleri was the fourth most frequent species. These results evidence the importance of Arcobacter and Campylobacter species as aetiological agents of acute gastroenteritis among Portuguese patients, affecting particularly the paediatric age group. Although A. butzleri has been implicated in human diseases, much of its pathogenesis and virulence factors remain unclear. Thus, A. butzleri virulence potential was also investigated, through the characterization of genotypic and pathogenic properties of human and non- human isolates. The isolates showed to be susceptible to tetracyclines and aminoglycosides, however displaying high resistance to quinolones. A. butzleri demonstrated a weak haemolytic activity and the ability to form biofilms in polystyrene surfaces. Adhesion levels similar to Salmonella Typhimurium were found for A. butzleri on Caco-2 cells, with pre- existing inflammation showing no significant effect on its adherence ability, yet invasion ability showed to vary among the isolates. A. butzleri was able of intracellular survival in Caco-2 cells and to induce a significant up-regulation of interleukin-8 secretion, as well as to promote structural cell disturbance. These data brings new insights to A. butzleri virulence and highlights its pathogenic potential. Overall in this work, the prevalence of Arcobacter species both in human and food-related samples was evaluated, contributing to understanding the epidemiology of Arcobacter in Portugal. The survival and persistence of this organism in the environment was highlighted due to its ability to form biofilms. Its relevance as a human pathogen was underlined by the resistance to antimicrobials, the presence of several putative virulence genes, along with its adherence, invasion, intracellular survival abilities and induction of proinflammatory cytokine secretion in intestinal epithelial cells. Finally, resveratrol was tested as an alternative to control the growth of Arcobacter. This work provided new insights on the epidemiology and pathogenicity of Arcobacter, and also identified a natural compound with anti-Arcobacter activity, which may contribute for future developments of new control approaches. ; O género Arcobacter foi proposto em 1991 e faz parte da família Campylobacteraceae juntamente com os géneros Campylobacter e Sulfurospirillum, compreendendo atualmente 18 espécies. Entre as espécies reconhecidas, Arcobacter butzleri, Arcobacter cryaerophilus e Arcobacter skirrowii têm sido associados a doença em humanos e animais. O género Arcobacter está amplamente distribuído, tendo sido isoladas espécies em alimentos, água, instalações de processamento e manipulação de alimentos, diversas amostras ambientais, animais e amostras humanas. Em produtos de origem animal, este microrganismo, tem sido isolado com maior prevalência em frangos, seguido de porco e vaca. O consumo de alimentos ou água contaminados com Arcobacter é considerado como a via de transmissão mais provável. Assim, o conhecimento sobre a distribuição de Arcobacter em alimentos ou ambiente de processamento alimentar, juntamente com a avaliação da variabilidade genética e outras características fenotípicas e genotípicas associadas ao seu potencial de virulência, pode ajudar na compreensão do mecanismo de persistência de Arcobacter na cadeia alimentar e contribuir para o seu controlo. Devido à ausência de estudos prévios sobre a prevalência de Arcobacter em Portugal para humanos ou relativos ao sector alimentar, procedeu-se neste trabalho ao seu isolamento a partir de amostras recolhidas de três bandos de frangos e de superfícies da linha de processamento do matadouro. A. butzleri foi isolado e identificado em todas as amostras recolhidas, com exceção das amostras correspondentes ao conteúdo cecal. Quarenta e três isolados de A. butzleri foram caracterizados por eletroforese em gel de campo pulsado, utilizando as enzimas de restrição Smal e Sacll, revelando uma elevada diversidade genética, com os 43 isolados pertencendo a 32 padrões distintos. No entanto, foram identificados genótipos comuns entre diferentes amostras, indicando a possibilidade de ocorrência de contaminação cruzada durante o processo de abate. Verificou-se ainda neste estudo que os isolados de A. butzleri apresentaram elevados níveis de resistência aos antibióticos em estudo, com todos os isolados a serem resistentes a pelo menos três antibióticos dos nove testados. Enquanto os 43 isolados demonstraram ser suscetíveis à gentamicina e 2,3 % resistentes ao cloranfenicol, 55,8 % das estirpes foram resistentes à ciprofloxacina. Genotipicamente a resistência à ciprofloxacina foi associada a uma transição de citosina para timina na região determinante de resistência às quinolonas do gene gyrA, como previamente descrito. Entre 36 isolados selecionados, 72,2 % apresentaram capacidade de formação de biofilme, o que poderá em parte estar associado à sobrevivência de A. butzleri em ambiente de matadouro, provavelmente por favorecer a dispersão e contaminação cruzada ao longo da linha de processamento. A deteção de genes putativos de virulência por PCR demonstrou a presença dos genes cadF, ciaB, cj1349, mviN, pldA e tlyA em todos os isolados, com uma deteção variável dos genes hecA (75 %), hecB (89 %) e irgA (42 %). Como neste estudo se encontrou uma elevada pre...

Access State: Open Access

Search in field:

Recently searched for: