The enteric pathogen Escherichia albertii represents an emerging food safety challenge. It has been mistakenly identified as Hafnia alvei, Shigella, or as a member of the Enteropathogenic E. coli (EPEC). Isolates of certain strains of the organism are known to possess genes encoding pathogenesis factors capable of inducing attaching/effacing (A/E) lesions, cytolethal distending toxin and a variant Shiga toxin. The pathogen has been isolated from infants and adults and has been identified as a causative agent from an outbreak of foodborne disease occurring in an industrialized nation. Recent reports have detailed the ability of this pathogen to survive on ground beef and to resist several classes of therapeutic antibiotics. The objectives of this study were to: (i) determine the efficacy of E. albertii isolates to survive lactic acid exposure as a function of solution pH, and (ii) verify its inactivation in ground beef according to the USDA recommendations for in-home preparation. Rifampicin resistant (RifR) isolates of E. albertii (ATCC 9194, 19982, 10457) were obtained according to published methods. Thermal resistance of parent and RifR isolates were compared in vitro at 55 °C to confirm no significant differences in tolerance to heat as a result of antibiotic resistance capacity. Tolerance to food grade lactic acid (Purac, Olathe, KS) (3.0% w/v) at differing pH levels (3.0, 4.0, 5.0, 7.0) was examined in physiological saline at 35 °C. Finally, ground beef patties (80% lean) inoculated with individual RifR isolates were cooked to internal temperatures of 62, 71, or 76 °C to determine resistance of different internal temperatures. Experiments comparing the in vitro tolerances of parent and RifR E. albertii isolates indicated no differences between parent and mutant with regards to heat and lactic acid challenge. E. albertii inactivation in lactic acid increased as the pH of the solution was decreased; maximum reduction at pH 3.0 was at 30 min and maximum reduction for pH 4.0 at 2.5 hours. Changes in populations of E. albertii at pH 5.0 were not different than that at pH 7.0. Cooking of beef to 62 °C internal temperature produced reductions of all RifR isolates to non-detectable levels.

Introduction: Emerging pathogens (EP) in cystic fibrosis (CF) include organisms that have infected individuals with CF for many years e.g. Burkholderia multivorans and Mycobacterium abscessus and more recently identified potential pathogens in CF e.g. Pneumocystis jirovecii and Pandoraea spp. The clinical implications of infection with these organisms are emerging but much remains unknown. Current evidence suggests that infection with some EP is associated with a worse prognosis. This thesis aimed to investigate the epidemiology, prevalence and clinical impact of EP in adults with CF.Methods: (1) The prevalence of P. jirovecii was determined in adults attending Manchester Adult Cystic Fibrosis Centre (MACFC) who were clinically stable versus those experiencing an acute pulmonary exacerbation (PEx). (2) The prevalence of M. abscessus at MACFC was determined, isolates of M. abscessus were strain typed, and cross infection risk was assessed. The clinical impact of Gram-negative EP was assessed by: (3) assessing their prevalence and determining if any patients attending MACFC harboured identical strains and had opportunities for cross infection to occur, and by (4) following these patients longitudinally and comparing outcome with age, gender and FEV1 matched Pseudomonas aeruginosa infected controls. Results: (1) P. jirovecii was detected via sputum PCR in 10 (4.4%) of 226 samples tested from 111 patients. P. jirovecii was more likely to be detected in samples taken from an acute pulmonary exacerbation compared with samples taken from stable patient visits (7 (9.2%) of 76 exacerbations samples versus 3 (2%) of 150 stable visit samples, p = 0.033). (2) Prevalence of M. abscessus was stable at ≤3.6% from 2010 to 2015. 21 patients (91.3%) with a positive culture for M. abscessus since 2010 were infected with M. abscessus subsp abscessus. 2 clusters of 7 and 6 patients harboured strains with identical variable number tandem repeat profiles and some of these patients had opportunities for cross infection to occur. 28.6% of patients developed M. abscessus pulmonary disease, 38.1% were persistently culture positive with no related pulmonary disease, and 33.3% spontaneously cleared M. abscessus from their sputum. (3) Prevalence of Gram-negative EP ranged from 1.9% (Ralstonia spp.) to 6.2% (B. multivorans). Small numbers of patients shared strains of B. multivorans; Stenotrophomonas S. maltophilia and Achromobacter; Ralstonia and Pandoraea species. Epidemiological connections consistent with possible cross infection were found in patients infected with Pandoraea and Ralstonia species. (4) Patients with B. multivorans; S. maltophilia; Ralstonia spp. and Pandoraea spp. had higher antibiotic requirements than P. aeruginosa infected matched controls. B. multivorans; Achromobacter spp.; Ralstonia spp. and Pandoraea spp patients had median FEV1 (% predicted) values ≥10% (absolute) lower than the overall median FEV1.Conclusion: Prevalence of all EP investigated at MACFC was low. P. jirovecii was approximately 5 times more likely to be detected in patients with acute PEx compared with stable patients suggesting it may be a cause of PEx. Results suggest that some patients attending MACFC may have acquired infection with M. abscessus subsp abscessus, Pandoraea spp. or Ralstonia spp. through cross infection. Patient numbers are too small to establish this with certainty and a common environmental source is possible. Gram-negative EP other than Achromobacter spp. were associated with higher acute antibiotic requirements than P. aeruginosa matched controls suggesting these EP are associated with an increased risk of PEx. The fact that many Gram-negative EP were associated with lower median lung function may indicate that these EP cause accelerated lung function decline or that patients with more advanced disease are at most risk of acquiring EP.

Les Henipavirus sont des paramyxovirus zoonotiques émergents hautement pathogènes. Ils sont capables d’infecter un large spectre d’hôtes incluant notamment la chauve-souris frugivore (réservoir naturel), le porc et l’homme. Etant donné leur très grande dangerosité et en l’absence de traitements curatifs ou prophylactiques efficaces, ces virus doivent être manipulés dans un laboratoire de classe P4. Dans une première partie, nous étudions l’effet de composés glyco-amino-glycanes sur l’infection par les Henipavirus ainsi que leur potentielle application en tant que traitement. Dans une seconde partie, nous nous attachons à comprendre les interactions entre le système immunitaire de l’hôte et le virus. Afin de mieux comprendre ces interactions, nous avons utilisé une approche basée sur l’utilisation de souris déficientes pour certaines voies de l’immunité. En effet, bien que les récepteurs cellulaires au virus (EFN B2 et B3) soient fonctionnels chez la souris, celle-ci est résistante à l’infection par voie intrapéritonéale. Nous avons analysé la susceptibilité au virus Nipah (NiV) de souris privées de différentes voies du système immunitaire inné et adaptatif. Les résultats obtenus permettent d’envisager certaines lignées de ces souris comme nouveaux modèles animaux pour l’étude de l’immunopathogénèse du NiV. Cette étude suggère aussi que le système interféron de type I joue un rôle crucial dans la limitation de la propagation virale vers le cerveau et que les lymphocytes T sont nécessaires à la complète élimination du virus. Les macrophages jouent, quant à eux, un rôle central et indispensable, à l’interface entre système inné et adaptatif. Enfin, nous abordons les prémices d’un projet visant à identifier les différences d’interactions au niveau moléculaire entre les protéines non-structurales du virus et les protéines du système immunitaire inné chez l’Homme et la souris afin de voir s’il se dégage des différences d’interactions pouvant expliquer les différences de pathogénie. Ces travaux ont donc permis d’identifier de nouveaux modèles animaux et de mieux caractériser les interactions entre le pathogène et le système immunitaire de l’hôte, de l’échelle moléculaire à l’échelle de l’organisme entier. Néanmoins, les mécanismes précis de ces interactions restent à élucider et permettront certainement de mieux comprendre la grande diversité de pathogénie des Henipavirus. / Henipaviruses are highly pathogenic emerging zoonotic paramyxoviruses. They can infect a broad spectrum of mammals including flying foxes (Pteropus fruit bats), its reservoir, pigs and humans. As there are neither therapeutic drugs nor efficient prophylactic treatment towards these highly lethal viruses, they have to be manipulated in biosafety level-4 laboratories. In the first part of this thesis, we study the role of glyco-amino-glycans on Henipavirus infection and their potential use as treatment. In the second part, we describe the interaction between the host immune system and the pathogen. To investigate these interactions, we took advantage of different transgenic mouse models deficient for some immune pathways. Indeed, although mice possess the viral entry receptor for Henipaviruses, they do not succumbed to intraperitoneal infection. We analyzed the susceptibility to Nipah virus (NiV) infection of mice deleted for different components of innate and adaptive immune systems. Obtained results showed that some of these mice can be used as new models for NiV immunopathogenesis study. This study also suggests that type I interferon system plays a major role in limitation of viral spreading to the brain and that T cells are necessary for full viral clearance. Macrophages act at the crossroad of immunity, between innate and adaptive system. Finally, we deal with the preliminary phases of a project which aims to identify the differences, at a molecular level, of interaction between non-structural viral proteins and innate immunity proteins in mice and human. Such differences could explain the different clinical patterns that are observed in these species. In conclusion, this thesis allowed to identify new animal models and to better characterize host-pathogen interactions, from molecular to whole organism level. However, the precise mecanisms of these interactions remain to be elucidated and would probably help to understand the great diversity of pathogeny of Henipaviruses.