It is now widely accepted that surface contamination plays an important role in the transmission of both respiratory and gastrointestinal infections in the domestic environment and community setting. The efficiency of transfer of a pathogen to the hand from a fomite is important in modeling transmission in microbial risk assessment models. The objective of this study was to use published literature to assess the role of fomites and hands in disease transmission, and to conduct fomite-to-finger transfer studies from various porous and nonporous fomites under different relative humidity condition using non-pathogenic strains of Escherichia coli, Staphylococcus aureus, MS2 coliphage, Bacillus thuringiensis spores, and poliovirus 1; to evaluate the persistence of bacteria and viruses on surfaces; to examine bacteria and virus transfer from treated surfaces; and to conduct a foodborne quantitative microbial risk assessment using Campylobacter jejuni from the data obtained in these studies. It was found that numerous factors influence the transfer efficiency of microorganisms, with moisture being the most important, with greater transfer under humid conditions. Other factors influencing transfer include drying time, contact time, pressure, friction, type of material, and porosity of the fomite. Percent transfer was greater under high relative humidity for both porous and nonporous surfaces. Most organisms on average had greater transfer under high relative humidity (40 - 65%) compared to low relative humidity (15 - 32%). Relative humidity and fomite type influenced the survival of all studied organisms; survival was greater on nonporous surfaces than those for porous surfaces. Test organisms were reduced up to 99.997% on the fomites after the surfaces were wiped with a disinfectant wipe. Microbial fomite-to-finger transfer from disinfectant wipe-treated surfaces were, lower than from non-treated surfaces. The disinfectant-wipe intervention reduced the risk of Campylobacter infection, illness, and death by 2 to 3 orders on all fomites. The disinfectant-wipe intervention reduced the annual risk of illness below the reported national average of diagnosed Campylobacteriosis cases 1.3E-04. This risk assessment demonstrates that the use of disinfectant wipes to decontaminate surface areas after chicken preparation reduces the risk of C. jejuni infections up to 99.2%.

Chez les bactéries à chromosome circulaire, la réplication peut engendrer des dimères que le système de recombinaison site-spécifique dif/Xer résout en monomères afin que la ségrégation des chromosomes fils et la division cellulaire se fassent normalement. Ses composants sont une ou deux tyrosines recombinases de type Xer qui agissent à un site de recombinaison spécifique, dif, avec l’aide de la translocase FtsK qui mobilise l’ADN au septum avant la recombinaison. Ce système a été d’abord identifié et largement caractérisé chez Escherichia coli mais il a également été caractérisé chez de nombreuses bactéries à Gram négatif et positif avec des variantes telles que les systèmes à une seule recombinase comme difSL/XerS chez Streptococcus sp et Lactococcus sp. Des études bio-informatiques ont suggéré l’existence d’autres systèmes à une seule recombinase chez un sous-groupe d’ε-protéobactéries pathogènes, dont Campylobacter jejuni et Helicobacter pylori. Les acteurs de ce nouveau système sont XerH et difH. Dans ce mémoire, les premières recherches in vitro sur ce système sont présentées. La caractérisation de la recombinase XerH de C. jejuni a été entamée à l’aide du séquençage de son gène et de tests de liaison et de clivage de l’ADN. Ces études ont montré que XerH pouvait se lier au site difSL de S. suis de manière non-coopérative : que XerH peut se lier à des demi-sites de difSL mais qu’elle ne pouvait, dans les conditions de l’étude effectuer de clivage sur difSL. Des recherches in silico ont aussi permis de faire des prédictions sur FtsK de C. jejuni. / DNA replication can form dimers in bacteria harboring a circular chromosome. The dif/Xer recombination system resolves monomers them so that chromosome segregation and cell division take place normally. This system is composed of one or two tyrosine recombinases that act at a specific recombination site, dif, with the help of the FtsK translocase that mobilises DNA to the septum before recombination. The Xer system has been first identified and widely characterized in Escherichia coli where XerC and XerD are the recombinases. The system has been found and studied in many other Gram negative and positive bacteria. A different form, carrying a single recombinase acting on an atypical site, has been identified in Streptococci and Lactococci, difSL/XerS. In silico studies suggested the existence of other single recombinase systems in a sub-group of pathogenic ε-proteobacteriasuch as Campylobacter jejuni and Helicobacter pylori. The components of this system were identified as XerH and difH. In this thesis, the first in vitro studies made on this system are presented. The characterization of the XerH recombinase of C. jejuni started with the sequencing of its gene and with the DNA binding and cleavage assays. These studies showed that XerH could bind difSL of S. suis non-cooperatively, that it could bind difSL half-sites and that it was unable to perform cleavage on difSL. Also, in silico comparisons permitted predictions on FtsK of C. jejuni.

Neonatal mortality in grey seals on the Isle of May breeding colony and in a rehabilitation centre were investigated by detailed systematic post-mortem examinations (n=59), on-site bacteriology and advanced molecular diagnostic techniques for specific pathogens. Causes of death on the breeding colony included starvation (30%), omphalitis-peritonitis (26%), septicaemia (22%), stillbirth (10%) and trauma (4%) and in the rehabilitation centre starvation (44%) and septicaemia (22%). Detailed key gross and histopathological findings and pathogens are described and include the first report of Listeria monocytogenes in any marine mammal. Phocid herpes virus 1 nucleic acids were detected in nasal swabs of 58% live, free-ranging grey seal pups (n=90) and 28% yearlings (n=19), suggesting recrudescence in the latter. Previously undetected in Scotland, phocid herpes virus 2 nucleic acids were identified only in yearlings (15%); sealpox was detected in a single live stranded grey seal pup and phocine distemper virus was not detected. Given their unique characteristics and potential for acting as sentinels of coastal marine health several pathogens of putative anthropogenic origin were investigated: Toxoplasma gondii, Neospora caninum, Salmonella spp. and Campylobacter spp. Toxoplasma gondii DNA was detected in 6% dead free-ranging grey seal pups (n=50) but N. caninum was not found. Salmonella (20%) and Campylobacter (50%) were isolated from rectal swabs of live and dead grey seal pups and Campylobacter was significantly associated with moderate to severe colitis implying pathogenicity. These findings imply a land-sea-land transfer of T. gondii and early exposure of pups to this parasite. Extensive genetic fingerprinting suggested an exchange of Salmonella between grey seal, cattle and human populations and that the Campylobacter isolates may share the same origin as human clinical isolates. This work provides a solid base line study of diseases present in grey seal pups and demonstrates that they are useful indicators of coastal marine microbial contamination.

Sterile ceramic plates and the surface of beef steaks were inoculated with the pathogenic microorganisms Listeria monocytogenes, Campylobacter jejuni, Escherichia coli and Salmonella typhimurium. Samples were also inoculated with nonpathogenic microorganisms Clostridium sporogenes ATCC 7955, Pseudomonas aeruginosa, and Bacillus stearothermophilus. Concentrations of organisms in the pure culture used to inoculate the samples were selected within the range of 106 to 108 colony forming units/ml (CFU/ml). Samples were treated with ultra-high temperature (UHT), and· the surviving organisms were recovered and counted. Meat samples were exposed to 1100°C for 22 seconds. Beef steaks inoculated with pathogenic microorganisms had low survival rates. The percent destruction ranged from 99.9 to 99.8. Sixteen percent of the spores from putrefactive anaerobe 3679 were destroyed. UHT was not found to be effective in destroying the spores of this organism. UHT destroyed 99.9 to 100 percent of the nonpathogenic microorganisms Pseudomonas and Bacillus stearothermophilus, respectively, inoculated on the surface of beef steaks prior to treatment. UHT pasteurization technology proved to be an effective method of controlling vegetative pathogens and vegetative spoilage organisms on meat surfaces.

Poultry meat consumption in New Zealand has been increasing since 1975 with the highest peak reported in 2006. The total poultry meat consumption was 36.5 kg per capita in the year ending September 2006. Consumption of contaminated food with raw poultry can lead to campylobacteriosis, which is a food-borne disease that causes gastroenteritis in humans and it is a major problem in New Zealand. There were 12,776 reported cases of campylobacteriosis in 2007, which accounts for 65.9% of the overall notified diseases. Campylobacteriosis can lead to Guillain-Barré syndrome in some patients, an autoimmune disorder of the peripheral nervous system. Campylobacteriosis is caused by consumption of either Campylobacter jejuni or Campylobacter coli. Campylobacter spp. have been found in commercially raised poultry being infected predominantly by C. jejuni. C. jejuni has been found associated with biofilms of other bacterial species in the watering supplies and plumbing systems of animal husbandry facilities and animalprocessing plants. A biofilm is an assemblage of microbial cells that is associated with a surface and the cells are enclosed in a matrix of polysaccharides, which provides a survival advantage to the bacteria in the film. In this study, the ability to form biofilm was measured in a laboratory assay using microtitre plates. C. jejuni strains in monoculture were shown to attach to the abiotic surface and form biofilms to various degrees, thus potentially enhancing their survivability in the poultry environment. C. jejuni was also shown to have the ability to attach and survive in mixed-microbial populations. Biofilm formation may play a role in the epidemiology of C. jejuni infections. Enterococcus faecalis and Staphylococcus simulans may play a role in the biofilm formation in the poultry environment as both of these microorganisms were able to form, and harbour C. jejuni in their biofilms. Pseudomonas aeruginosa seemed to inhibit biofilm formation and C. jejuni in the mixed-microbial population. Further studies are required to establish control measures against the formation of biofilms containing C. jejuni in poultry processing plants and farms in New Zealand to reduce the reservoir of contamination and thus reduce the incidence of campylobacteriosis.

The purpose of this dissertation is threefold: to determine the differences in apparent prevalence and the antimicrobial susceptibility of Campylobacter spp. between antimicrobial-free and conventional swine farms; secondly, to introduce an appropriate statistical model to compare the minimum inhibitory concentration distributions of Escherichia coli and Campylobacter spp. isolated from both farm types; and thirdly, to examine the potential herd level risk factors that may be associated with antimicrobial resistance of Campylobacter spp. and E. coli isolates from finishers on antimicrobial-free and conventional farming systems. In addition, a critical review of studies that have compared the levels and patterns of antimicrobial resistance among animals from antimicrobial-free and conventional farming practices was performed. Fecal samples from 15 pigs were collected from each of 35 antimicrobial-free and 60 conventional farms in the Midwestern U.S. Campylobacter spp. was isolated from 464 of 1,422 fecal samples, and each isolate was tested for susceptibility to 6 antimicrobials. The apparent prevalence of Campylobacter spp. isolates was approximately 33 percent on both conventional and antimicrobial-free farms. The proportion of antimicrobial resistance among Campylobacter was higher for three antimicrobials within conventional compared to antimicrobial-free farms. The susceptibilities of populations of bacteria to antimicrobial drugs were summarized as minimum inhibitory concentration (MIC) frequency distributions. The use of MIC values removed the subjectivity associated with the choice of breakpoints which define an isolate as susceptible or resistant. A discrete-time survival analysis model was introduced as the recommended statistical model when MICs are the outcome. A questionnaire was completed by each farm manager on biosecurity, preventive medication, vaccines, disease history, and production management. Multivariable population-averaged statistical models were used to determine the relationships among antimicrobial susceptibility patterns and potential herd-level risk factors. Controlling for herd type (antimicrobial-free versus conventional), each antimicrobial-bacterial species combination yielded unique combinations of risk factors; however, housing type, history of rhinitis, farm ventilation, and history of swine flu were significant in more than one model. A variety of herd-level practices were associated with the prevalence of antimicrobial resistance on swine farms. Further studies are encouraged when considering interventions for antimicrobial resistance on both antimicrobial-free and conventional farms.

Chez les bactéries à chromosome circulaire, la réplication peut engendrer des dimères que le système de recombinaison site-spécifique dif/Xer résout en monomères afin que la ségrégation des chromosomes fils et la division cellulaire se fassent normalement. Ses composants sont une ou deux tyrosines recombinases de type Xer qui agissent à un site de recombinaison spécifique, dif, avec l’aide de la translocase FtsK qui mobilise l’ADN au septum avant la recombinaison. Ce système a été d’abord identifié et largement caractérisé chez Escherichia coli mais il a également été caractérisé chez de nombreuses bactéries à Gram négatif et positif avec des variantes telles que les systèmes à une seule recombinase comme difSL/XerS chez Streptococcus sp et Lactococcus sp. Des études bio-informatiques ont suggéré l’existence d’autres systèmes à une seule recombinase chez un sous-groupe d’ε-protéobactéries pathogènes, dont Campylobacter jejuni et Helicobacter pylori. Les acteurs de ce nouveau système sont XerH et difH. Dans ce mémoire, les premières recherches in vitro sur ce système sont présentées. La caractérisation de la recombinase XerH de C. jejuni a été entamée à l’aide du séquençage de son gène et de tests de liaison et de clivage de l’ADN. Ces études ont montré que XerH pouvait se lier au site difSL de S. suis de manière non-coopérative : que XerH peut se lier à des demi-sites de difSL mais qu’elle ne pouvait, dans les conditions de l’étude effectuer de clivage sur difSL. Des recherches in silico ont aussi permis de faire des prédictions sur FtsK de C. jejuni. / DNA replication can form dimers in bacteria harboring a circular chromosome. The dif/Xer recombination system resolves monomers them so that chromosome segregation and cell division take place normally. This system is composed of one or two tyrosine recombinases that act at a specific recombination site, dif, with the help of the FtsK translocase that mobilises DNA to the septum before recombination. The Xer system has been first identified and widely characterized in Escherichia coli where XerC and XerD are the recombinases. The system has been found and studied in many other Gram negative and positive bacteria. A different form, carrying a single recombinase acting on an atypical site, has been identified in Streptococci and Lactococci, difSL/XerS. In silico studies suggested the existence of other single recombinase systems in a sub-group of pathogenic ε-proteobacteriasuch as Campylobacter jejuni and Helicobacter pylori. The components of this system were identified as XerH and difH. In this thesis, the first in vitro studies made on this system are presented. The characterization of the XerH recombinase of C. jejuni started with the sequencing of its gene and with the DNA binding and cleavage assays. These studies showed that XerH could bind difSL of S. suis non-cooperatively, that it could bind difSL half-sites and that it was unable to perform cleavage on difSL. Also, in silico comparisons permitted predictions on FtsK of C. jejuni.

In this project the factors influencing the stability and antimicrobial activity of allyl isothiocyanate (AITC) against Campylobacter jejuni, Salmonella or Listeria monocytogenes as well as factors that enhance sinigrin (glucosinolate in Oriental mustard) hydrolysis by these pathogens were investigated. The minimum inhibitory concentration (MIC) of AITC against 5 strains of each of Salmonella or L. monocytogenes, ranged from 60-100 ppm at 37 ºC. This was reduced to 10-40 ppm at 21 ºC and a further reduction to 5-10 ppm against strains of L. monocytogenes was observed at 4 ºC. This was attributed to greater stability of AITC as temperature was decreased. C. jejuni strains were more susceptible to AITC with MICs of 0.63-1.25 ppm and 2.5-5 ppm at 37 and 42 ºC, respectively. AITC was more inhibitory at ≤ 21 ºC against Salmonella with acidic pH or against L. monocytogenes with neutral pH. C. jejuni, Salmonella and L. monocytogenes strains and mixtures had the ability to degrade sinigrin to form inhibitory concentrations of AITC, and sinigrin hydrolysis was significantly enhanced by higher incubation temperature (21 ºC > 10 ºC > 4 ºC), the presence of 10 mM ferric or ferrous irons, and the presence of < 0.25% glucose. This project also investigated the antimicrobial activity of AITC or Oriental mustard extract alone or combined with ethylenediamine tetraacetic acid (EDTA), malic acid and acetic acid in edible antimicrobial coatings against C. jejuni and Salmonella on fresh, refrigerated, vacuum-packed chicken breasts or L. monocytogenes on refrigerated, cured roast chicken. Malic acid improved the antimicrobial activity of Oriental mustard extract against L. monocytogenes, while EDTA improved its activity against Salmonella. Incorporation of 25 to 50 µl/g AITC or 100 to 250 mg/g Oriental mustard extract in 0.5%κ-carrageenan/2%chitosan coatings, prepared using 1.5% malic or acetic acid, reduced L. monocytogenes on cooked, cured, vacuum-packed chicken slices 4.2 to > 7.0 log10 CFU/g, compared to uncoated chicken by 70 d at 4 ºC. In addition, 0.2%κ-carrageenan/2%chitosan coatings (prepared using a 1% acetic acid solution) containing 250 mg/g mustard extract or 50 µl/g AITC reduced Salmonella numbers on vacuum-packed chicken breasts 3.0 log10 CFU/g by 21 d at 4 ºC. Further, 0.2%κ-carrageenan/2%chitosan coatings containing 50 or 100 µl/g AITC reduced numbers of C. jejuni on fresh, vacuum-packed chicken breasts > 5.0 log10 CFU/g (C. jejuni cells were not detected) after 5 d storage at 4 ºC, while coatings containing 200 to 300 mg/g Oriental mustard extract or 25 µl/g AITC reduced C. jejuni numbers by 3.6 to 4.6 log10 CFU/g. Numbers of lactic acid and aerobic bacteria on poultry meat products were significantly reduced by the coatings. It is clear that κ-carrageenan/chitosan coatings containing either AITC, mustard extract alone or combined with EDTA, malic or acetic acid significantly reduced C. jejuni and Salmonella on fresh, refrigerated, vacuum-packed chicken breasts and L. monocytogenes on refrigerated, cured roast chicken, and consequently enhanced their safety.

Les maladies diarrhéiques demeurent une cause majeure de mortalité infantile dans les pays en développement (PED). Du fait de l'insuffisance des plateaux techniques, les diagnostics étiologiques sont rarement réalisés et les traitements sont alors probabilistes. A Madagascar les données sur les diarrhées sont souvent parcellaires et anciennes. Le Réseau de surveillance sentinelle développé par l'Institut Pasteur de Madagascar à partir de 2007 nous a permis d'étudier la distribution spatio-temporelle des consultations pour diarrhée. Mais cette surveillance syndromique n'est pas couplée systématiquement à une surveillance biologique. Pour étudier les agents étiologiques des diarrhées, nous avons réalisé une enquête cas-témoins menée en 2008-2009 en milieu communautaire, chez les enfants de moins de 5 ans dans 14 districts. Nous avons pu identifier au moins un pathogène chez plus de la moitié des enfants (55%), avec une prédominance des étiologies parasitaires (37,2% des diarrhées), suivies par les bactéries (15%) puis les virus (6,7% de rotavirus). Les parasites ont été les seules étiologies pour lesquelles une pathogénicité a pu être mise en évidence. Parmi les étiologies bactériennes, l'infection à Campylobacter a été la plus fréquente (9,5%). Pour analyser le rôle de Campylobacter et les effets des facteurs environnementaux dans la survenue des diarrhées infantiles, nous avons initié et coordonné depuis 2010 une étude de cohorte dynamique d'enfants inclus avant l'âge de 24 mois et suivis jusqu'à l'âge de 36 mois à Moramanga, site où la prévalence de Campylobacter a été la plus élevée au cours de l'étude de 2008 (20,6%). Une surveillance des diarrhées a été menée 2 fois par semaine et les portages asymptomatiques évalués à l'inclusion et tous les 2 mois. Une étude de portage familial a été mise en œuvre ainsi qu'un suivi coprologique bi-annuel de la population avicole, des points d'eaux collectifs et de l'eau de boisson des familles. La recherche de Campylobacter chez les volailles portait sur les écouvillonnages rectaux. De janvier 2010 à mai 2012, 508 enfants correspondant à 256 346 enfant-jour ont participé à l'étude. La prévalence globale d'isolement de Campylobacter a été de 9,3%. Plus de 2/5 des enfants (43,3%) ont eu au moins un épisode d'infection à Campylobacter au cours de leur suivi. Les taux d'incidence annuelle des diarrhées ainsi que des infections symptomatiques ont été faibles, respectivement de 0,7 épisode /enfant et de 5,8 épisodes/100 enfant pouvant s'expliquer par le faible niveau d'exposition environnementale des enfants. Nous avons pu étudier l'importance des facteurs liés à l'hôte comme l'âge. Le pic d'infection à Campylobacter se situe entre 18 à 29 mois, celui des diarrhées entre 6 à 11 mois puis diminue ensuite. La 1ère infection à Campylobacter a été toujours pathogène chez les plus jeunes. Elle se situe vers le 8ème mois de la vie pour 10% d'entre eux. Les réinfections se font à des distances différentes de l'événement initial en fonction de l'âge. Ce profil d'infection pourrait traduire une compétence immunitaire différente selon l'âge et/ou une immunité acquise au cours du temps suite aux expositions répétées des enfants. L'environnement pourrait avoir un effet indirect dans l'entretien d'une immunité protectrice s'exprimant par un taux élevé d'infection asymptomatique. Il apparaît nécessaire de poursuivre des études de cohorte dans des zones à plus fort risque de transmission avec des données immunologiques car la compréhension actuelle des interactions entre l'hôte, le Campylobacter et l'environnement ne permet pas d'expliquer la variabilité de l'expression clinique de l'infection.

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