Julie Baré

Occurrence of Putative Virulence Genes in Arcobacter Species Isolated from Humans and Animals

ABSTRACT Interest in arcobacters in veterinary and human public health has increased since the first report of the isolation of arcobacters from food of animal origin. Since then, studies worldwide have reported the occurrence of arcobacters on food and in food production animals and have highlighted possible transmission, especially of Arcobacter butzleri, to the human population. In humans, arcobacters are associated with enteritis and septicemia. To assess their clinical relevance for humans and animals, evaluation of potential virulence factors is required. However, up to now, little has been known about the mechanisms of pathogenicity. Because of their close phylogenetic affiliation to the food-borne pathogen Campylobacter and their similar clinical manifestations, the presence of nine putative Campylobacter virulence genes (cadF, ciaB, cj1349, hecA, hecB, irgA, mviN, pldA, and tlyA) previously identified in the recent Arcobacter butzleri ATCC 49616 genome sequence was determined in a large set of human and animal Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii strains after the development of rapid and accurate PCR assays and confirmed by sequencing and dot blot hybridization.

Dogs as carriers of the emerging pathogen Arcobacter.

Dogs and cats living in a household have previously been identified as a risk factor for human infection with Campylobacter and Helicobacter. In this study, carried out between July 2006 to September 2007, feces and oral swabs from 267 dogs and 61 cats were examined for the presence of the emerging pathogen Arcobacter. Isolates, obtained by an Arcobacter selective isolation procedure, were identified with an Arcobacter species-specific multiplex-PCR and characterized by modified enterobacterial repetitive intergenic concensus PCR. No arcobacters were isolated from cats. Five dogs excreted arcobacters in the feces and two other dogs carried arcobacters in the mouth. In the follow-up, one dog excreted the same Arcobacter butzleri strain for at least 1 week. Six dogs carried each an unique A. cryaerophilus strain although three of them lived in the same family. Therefore, beside the consumption of food and water, contact with dogs is another potential source of Arcobacter infection.

Microscopic and Molecular Studies of the Diversity of Free-Living Protozoa in Meat-Cutting Plants

ABSTRACT The diversity of free-living protozoa in five meat-cutting plants was determined. Light microscopy after enrichment culturing was combined with sequencing of PCR-amplified, denaturing gradient gel electrophoresis (DGGE)-separated 18S rRNA gene fragments, which was used as a fast screening method. The general results of the survey showed that a protozoan community of amoebae, ciliates, and flagellates was present in all of the plants. Protozoa were detected mainly in floor drains, in standing water on the floor, on soiled bars of cutting tables, on plastic pallets, and in out-of-use hot water knife sanitizers, but they were also detected on surfaces which come into direct contact with meat, such as conveyer belts, working surfaces of cutting tables, and needles of a meat tenderizer. After 7 days of incubation at refrigerator temperature, protozoa were detected in about one-half of the enrichment cultures. Based on microscopic observations, 61 morphospecies were found, and Bodo saltans, Bodo spp., Epistylis spp., Glaucoma scintillans, Petalomonas spp., Prodiscophrya collini, and Vannella sp. were the most frequently encountered identified organisms. Sequencing of DGGE bands resulted in identification of a total of 49 phylotypes, including representatives of the Amoebozoa, Chromalveolata, Excavata, Opisthokonta, and Rhizaria. Sequences of small heterotrophic flagellates were affiliated mainly with the Alveolata (Apicomplexa), Stramenopiles (Chrysophyceae), and Rhizaria (Cercozoa). This survey showed that there is high protozoan species richness in meat-cutting plants and that the species included species related to known hosts of food-borne pathogens.

Influence of temperature, oxygen and bacterial strain identity on the association of Campylobacter jejuni with Acanthamoeba castellanii.

Campylobacteriosis is the most frequently reported foodborne disease in the industrialized world, mainly through consumption of contaminated chicken meat. To date, no information is available on the primary infection sources of poultry. In this study, the ability of five Campylobacter jejuni strains with different invasion potential towards Caco-2 cells to survive and replicate in the protozoan Acanthamoeba castellanii was tested under simulated in situ conditions (i.e. chicken broiler houses). Results indicate that environmental conditions play a crucial role in C. jejuni-A. castellanii interactions. Co-culture in general did not result in an increase of either bacteria or amoebae. However, co-culture with Acanthamoeba did result in a delayed decline and an increased long-term survival of Campylobacter. Bacterial strain-specific effects were observed, with higher survival rates for low-invasive strains. The presence of C. jejuni in general did not affect A. castellanii viability, except at 37 °C under microaerobic conditions, where the presence of the reference and low-invasive Campylobacter strains resulted in a significant decline in amoebal viability. Confocal laser scanning microscopy revealed that intra-amoebal campylobacters were not always colocated with acidic organelles, suggesting potential bacterial interference with digestive processes. As Acanthamoeba enhances the persistence of C. jejuni, the presence of the amoeba in broiler house environments may have important implications for the ecology and epidemiology of this food pathogen.

Diversity and Habitat Specificity of Free-Living Protozoa in Commercial Poultry Houses

ABSTRACT Despite stringent biosecurity measures, infections by bacterial food pathogens such as Campylobacter are a recurrent problem in industrial poultry houses. As the main transmission route remains unclear, persistence of these infections has been linked to bacterial survival and possibly multiplication within protozoan vectors. To date, however, virtually no information is available on the diversity and occurrence of free-living protozoa in these environments. Using a combination of microscopic analyses of enrichment cultures and molecular methods (denaturing gradient gel electrophoresis [DGGE]) on natural samples, we show that, despite strict hygiene management, free-living protozoa are common and widespread throughout a 6-week rearing period in both water and dry samples from commercial poultry houses. Protozoan communities were highly diverse (over 90 morphotaxa and 22 unique phylotypes from sequenced bands) and included several facultative pathogens and known bacterial vectors. Water samples were consistently more diverse than dry ones and harbored different communities, mainly dominated by flagellates. The morphology-based and molecular methods yielded markedly different results: amoebic and, to a lesser degree, ciliate diversity was seriously underestimated in the DGGE analyses, while some flagellate groups were not found in the microscopic analyses. Some recommendations for improving biosecurity measures in commercial poultry houses are suggested.

Interactions of Foodborne Pathogens with Free-living Protozoa: Potential Consequences for Food Safety

Free-living protozoa (FLP) are ubiquitous in natural ecosystems where they play an important role in the reduction of bacterial biomass and the regeneration of nutrients. However, it has been shown that some species such as Acanthamoeba castellanii, Acanthamoeba polyphaga, and Tetrahymena pyriformis can act as hosts of pathogenic bacteria. There is a growing concern that FLP might contribute to the maintenance of bacterial pathogens in the environment. In addition to survival and/or replication of bacterial pathogens in FLP, resistance to antimicrobial agents and increased virulence of bacteria after passage through protozoa have been reported. This review presents an overview of FLP in food-associated environments and on foods, and discusses bacterial interactions with FLP, with focus on the foodborne pathogens Campylobacter jejuni, Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes. The consequences of these microbial interactions to food safety are evaluated.

Occurrence and diversity of free-living protozoa on butterhead lettuce.

The occurrence and diversity of free-living protozoa (FLP) on butterhead lettuce (Lactuca sativa L.) was investigated using four different sampling techniques (washing, swabbing, homogenization, and excising). FLP were recovered from all leaf samples (n=64), and cultures were FLP-positive after 1 week. Identification of FLP was performed by light microscopy and sequencing of denaturing gradient gel electrophoresis (DGGE)-separated 18S rRNA gene fragments. Bodo saltans, Spumella (-like) spp. and Cercozoa were the most common heterotrophic nanoflagellates. Amoebae belonged mainly to the Vannellida and Tubulinida. Colpoda steinii and Cyclidium glaucoma were the most common ciliates. The total number of FLP on middle leaves estimated by the Most Probable Number method ranged from 9.3 × 10(2)MPN/g to 2.4 × 10(5)MPN/g leaf, with flagellates (92 MPN/g to 2.4 ×10(5)MPN/g) being more abundant than amoebae (<3 MPN/g to 9.3 × 10(3)MPN/g) and ciliates (<3 MPN/g to 9.3 × 10(2)MPN/g). Washing or rinsing leaves followed by spin-drying in a household salad spinner reduced the protozoan number with maximum one log unit. Our survey shows that FLP on lettuce leaves are a common and diverse but largely unexplored group of microorganisms.

Contamination of freshly slaughtered pig carcasses with enteropathogenic Yersinia spp.: Distribution, quantification and identification of risk factors

A cross-sectional survey was undertaken to determine the overall prevalence of enteropathogenic Yersinia spp. in the tonsils, feces and on carcasses of pigs at slaughter. Moreover, factors associated with Yersinia contamination of freshly eviscerated pig carcasses were studied. Yersinia enterocolitica serotype O:3 was isolated from the tonsils and feces of 55.3% and 25.6% of pigs, and Y. pseudotuberculosis from 1.4% and 0.6%, respectively. The pathogens were also recovered from 39.7% of carcass surfaces post-evisceration. The highest prevalence was found at the mandibular region (28.9%), followed by the sternal region (16.4%), pelvic duct (7.8%), and split surface near the sacral vertebrae (6.9%). Regarding the quantification of the pathogen, the median concentration of pathogenic Y. enterocolitica was 4.14l og10 CFU/g in tonsils with countable numbers (n=143) and 2.80 log10 CFU/g for fecal samples with countable numbers (n=26). The quantitative load on the carcass surface was generally low as the majority of the carcass samples (97.0%) had Yersinia concentrations below the detection limit of enumeration (<1.30 log10 CFU/100 cm(2)). The initial presence of Y. enterocolitica in the tonsils and/or feces was significantly associated with carcass contamination at all sampled areas. Other risk factors for carcass contamination are the splitting of the head together with the carcass, and incision of the tonsils during removal of the pluck. Small adaptations in slaughter practices and the training of slaughterhouse personnel to respect basic hygienic instructions may diminish carcass contamination with enteropathogenic Yersinia.

Campylobacter carcass contamination throughout the slaughter process of Campylobacter-positive broiler batches.

Campylobacter contamination on broiler carcasses of Campylobacter colonized flocks was quantified at seven sampling sites throughout the slaughter process. For this purpose, in four slaughterhouses samples were collected from twelve Campylobacter positive batches. Broilers from all visits carried high numbers of campylobacters in their caeca (≥7.9log10cfu/g). Campylobacter counts on feathers (up to 6.8log10cfu/g), positively associated with the breast skin contamination of incoming birds and carcasses after plucking, were identified as an additional source of carcass contamination. A high variability in Campylobacter carcass contamination on breast skin samples within batches and between batches in the same slaughterhouse and between slaughterhouses was observed. In slaughterhouses A, B, C and D Campylobacter counts exceeded a limit of 1000cfu/g on 50%, 56%, 78% and 11% of carcasses after chilling, respectively. This finding indicates that certain slaughterhouses are able to better control Campylobacter contamination than others. Overall, the present study focuses on the descriptive analysis of Campylobacter counts in different slaughterhouses, different batches within a slaughterhouse and within a batch at several sampling locations.

Influence of isolation methods on the occurrence of plasmid-carrying Yersinia enterocolitica serotype O:3 in slaughter pig tonsils, faeces and carcass surface swabs

Yersinia enterocolitica is an important foodborne pathogen that is primarily transmitted to humans through the consumption of contaminated pork. Different matrices of pigs at slaughter were tested for the presence of human pathogenic types of Y. enterocolitica using direct plating, selective enrichment, and cold enrichment. Y. enterocolitica serotype O:3 was isolated from the tonsils and faeces of 55.3% and 25.6% of pigs, respectively. The pathogen was also recovered from 15.0% of swab samples taken from the carcass surface post-evisceration. Tonsils positive by direct plating revealed an average concentration of 3.99 log10 Y. enterocolitica per gram, whereas the majority of positive faecal and carcass samples were contaminated below the detection limit of enumeration. The relative sensitivity of the methods to recover pathogenic Y. enterocolitica varied among the different matrices. Nevertheless, cold enrichment was significantly more efficient than direct plating and selective enrichment for all three sample matrices. From the 2082 recovered Y. enterocolitica isolates, 1742 (83.7%) harboured the virulence plasmid. Isolates obtained from faeces were more likely to contain the virulence plasmid than isolates from tonsils and carcass swabs. To obtain reliable results regarding the presence of plasmid-carrying Y. enterocolitica isolates, sensitive isolation methods should be combined with testing of a sufficient number of isolates.