Clostridium difficile est la principale cause de diarrhées nosocomiales liées à la prise d'antibiotiques. La souche hypervirulente NAP1/027 est apparue récemment et a causé de nombreuses épidémies en Amérique du Nord et en Europe. On considère généralement que cette souche produit plus de toxines, sporule davantage, provoque des infections plus sévères menant à des complications et est souvent associée aux cas de récurrence. Toutefois, des études récentes ont montré des données contradictoires à ce sujet. L'objectif de mes travaux de recherche est donc de déterminer si l'issue clinique des infections à C. difficile (ICD) peut être prédite en fonction du génotype et de certains phénotypes bactériens associés à la virulence, comme la production de toxines et la sporulation. Pour ce faire, 21 isolats cliniques associés à des ICD de sévérité différente (légère à modérée, sévère, compliquée) ont été caractérisés par des méthodes de typage courantes, incluant le ribotypage par PCR, le typage des répétitions en tandem, l'analyse de loci multiples de répétitions en tandem polymorphe, la détection des toxines A, B et CDT, ainsi que le séquençage du gène tcdC. Les taux de sporulation et la production des toxines A et B ont aussi été évalués in vitro, de même que la résistance des isolats à certains antibiotiques. La mobilité, la sensibilité des isolats à certains bactériophages et leur contenu en prophages ont aussi été étudiés. Les résultats de mes travaux démontrent que les méthodes de typage utilisées ne permettent pas de prévoir avec certitude le phénotype bactérien ni de prédire la sévérité des ICD. En effet, les souches NAP1/027 peuvent autant provoquer des ICD non-sévères que mener à des complications. Le phénotype n'est pas non plus nécessairement un indice de la sévérité. Les souches NAP1/027 produisent généralement plus de toxines, mais ne possèdent pas forcément la capacité de sporulation qu'on leur attribue généralement. Par conséquent, les généralisations à propos des souches NAP1/027 devraient être évitées.

BACKGROUND:Clostridium difficile is an important cause of intestinal infections in some animal species and animals might be a reservoir for community associated human infections. Here we describe a collection of animal associated C. difficile strains from 12 countries based on inclusion criteria of one strain (PCR ribotype) per animal species per laboratory.RESULTS:Altogether 112 isolates were collected and distributed into 38 PCR ribotypes with agarose based approach and 50 PCR ribotypes with sequencer based approach. Four PCR ribotypes were most prevalent in terms of number of isolates as well as in terms of number of different host species: 078 (14.3% of isolates / 4 hosts), 014/020 (11.6% / 8 hosts) / 002 (5.4% / 4 hosts) and 012 (5.4% / 5 hosts). Two animal hosts were best represented / cattle with 31 isolates (20 PCR ribotypes / 7 countries) and pigs with 31 isolates (16 PCR ribotypes / 10 countries).CONCLUSIONS:This results show that although PCR ribotype 078 is often reported as the major animal C. difficile type, especially in pigs, the variability of strains in pigs and other animal hosts is substantial. Most common human PCR ribotypes (014/020 and 002) are also among most prevalent animal associated C. difficile strains worldwide. The widespread dissemination of toxigenic C. difficile and the considerable overlap in strain distribution between species furthers concerns about interspecies, including zoonotic, transmission of this critically important pathogen.

Chez une bactérie, la régulation de l’expression génétique est essentielle afin de maintenir l’équilibre, s’assurer du bon fonctionnement des processus cellulaires et mieux s’adapter aux changements environnementaux. Elle peut s’effectuer à plusieurs niveaux (la transcription, la traduction et la synthèse ou la dégradation des protéines), et par le bais de différents mécanismes, dont les protéines, font le plus grand part de cette régulation. Cependant, au début des années 2000, une découverte fascinante a mis en évidence un nouveau mécanisme de régulation dont l’ARN est l’acteur principal. Ce sont les riborégulateurs (riboswitches). Ces derniers sont localisés dans la partie non traduite de certains ARNmessagers (ARNm) et capable de lier un ligand spécifique sans l’intervention des protéines, afin de réguler l’expression génique du gène d’intérêt. Aujourd’hui, plusieurs familles de riborégulateurs sont caractérisées, entre autres les riborégulateurs c-di-GMP. Ces derniers sont présents chez plusieurs espèces bactériennes notamment les bactéries pathogènes telles que Clostridium difficile, une bactérie nosocomiale opportuniste qui a causé des problèmes majeurs durant les dernières années, vu sa multirésistance aux antibiotiques. Le séquençage de son génome a révélé la présence de 66 riborégulateurs dont 16 sont des riborégulateurs c-di-GMP. Il a été proposé que parmi ces derniers, certains régulent l’expression des gènes impliqués dans deux phénotypes essentiels chez C. difficile : la motilité et la formation du biofilm. La présente étude porte sur la caractérisation structurale et fonctionnelle de deux riborégulateurs c-di-GMP chez le C. difficile, le Cdi1-1 et Cdi1-12, qui se trouvent en amont du gène CD1990 et le gène CD2830 (ZmpI) respectivement. Au début, nous avons prédit les deux structures liées (en présence du ligand) et non liées (en absence du ligand). Nous avons ainsi démontré qu’un des deux riborégulateurs (Cdi1-12) est fonctionnel et capable de lier le c-di-GMP in vitro. Ensuite, nous avons caractérisé les changements structuraux potentiels lors de l’interaction riborégulateur Cdi1-12/ligand. Nous avons également caractérisé le mécanisme de régulation en cis du riborégulateur Cdi1-12 in vitro et nous avons constaté que c’est un riborégulateur transcriptionnel Rho-indépendant. À la fin de notre étude, nous avons confirmé le mode de régulation de ce riborégulateur in vivo dans la bactérie modèle Bacillus subtilis.

Clostridium difficile is an increasingly common cause of nosocomial infection. C. difficile infection (CDI) presents as a spectrum ranging from asymptomatic carriage to mild diarrhoea, pseudomembranous colitis, toxic megacolon and intestinal perforation. It is not yet fully understood why this spectrum is seen, however, it is believed that the immune response mounted by an individual plays an important role in determining the outcome of infection. This thesis comprises three studies. Firstly, a comparative study of immune cell populations within the lamina propria of colonic tissue not exhibiting pathological changes and taken from individuals with symptomatic CDI (cases); asymptomatic carriers; and non-colonised controls. Effector T cells, B cells, plasma cells and macrophages were enumerated by means of immunohistochemical staining of tissue sections. Secondly, a study to establish the prevalence within these three study groups of specific host single nucleotide polymorphisms (SNPs) in the TLR2, TLR5 and IL-8 genes by PCR genotyping and to determine whether an association existed between these genotypes and susceptibility to CDI. Thirdly, an examination of the mechanisms of moxifloxacin resistance in a collection of clinical isolates. This study also sought to determine whether the competitive advantage conferred by resistance to moxifloxacin influenced the fitness of C. difficile isolates, in particular growth and the expression of the virulence factors toxins A and B. Carriers were found to have fewer of all four immune cell types quantified than both cases and controls. However, in only one instance, that of plasma cells, was this difference statistically significant. Cases had fewer of all cell types than controls but these differences were not significant. These findings suggest that individuals who become infected, both symptomatically and asymptomatically, with C. difficile display altered mucosal immune cell populations when compared with those of uninfected individuals. The data regarding host polymorphisms are suggestive of an association between the presence of SNPs and increased susceptibility to CDI. The variant IL-8 and TLR2 genotypes were carried by cases and carriers while the variant TLR5 genotype was carried by cases only. No variant genotypes were present in control subjects. All moxifloxacin resistant isolates characterised in this study, with the exception of an isolate with intermediate resistance and a third-generation mutant with reduced susceptibility, carried the common gyrA mutation ACT→ATT (Thr82→Ile). Efflux pumps are known to play a role in multi-drug resistance in many bacterial species. Semiquantitative PCR analysis of expression of the putative efflux pumps cme and cdeA found no correlation between overexpression and moxifloxacin resistance, suggesting that these genes do not play a role. Three novel mutations in the putative promoter region of CD3197, a MerR family transcriptional regulator found immediately upstream of cme, were identified. No association between the presence of these mutations and overexpression of cme or resistance or sensitivity to moxifloxacin was found. The competitive advantage conferred by resistance to moxifloxacin does not influence the fitness of C. difficile isolates, as measured in terms of growth and toxin production.

The number of cases of Clostridium difficile infection (CDI) has been increasing globally. CDI is the main cause of nosocomial diarrhoea, which may be life-threatening in complicated cases, and also costs the health care societies millions of pounds annually. The predominant types and their resistance to antibiotics have been changing and one of the major selective pressures which causes this is antimicrobial use. Although much is known about the role of the toxins in pathogenesis of CDI, the role of immunogenic cell wall components is unclear. They may play a role in colonisation and pathology and a study of these could clarify the infection process. It is therefore important to study the immune responses against these bacterial wall components from different strains and their effects on stimulation of leukocytes to produce cytokines and chemokines. This study was divided into four parts: 1. An epidemiological study to determine frequencies of the predominant types of C. difficile, thus 140 C. difficile isolates from surgical patients and their environment during 2009 were investigated to define their PCR ribotype. This utilised capillary sequencing gel electrophoresis for their analysis. 2. The determination of antimicrobial susceptibility to six antibiotics (ampicillin, erythromycin, tetracycline, metronidazole, moxifloxacin and vancomycin) was assessed and MIC determination by agar dilutions. 3. Investigation of host immunity to molecules with conserved molecular patterns. Surface-layer proteins (SLPs), lipocarbohydrate (LC) and flagellar proteins were separated and purified from five ribotypes of C. difficile (001, 002, 027, 078 and106) predominant in Scotland. a) The immune responses to these molecules were assessed by ELISA by exposing serum of patients and healthy donors and measuring specific IgG levels. b) Innate immunity was investigated by distinguishing responses of a macrophage cell line (THP1) to the above molecules. Induction of interleukins (IL)-1β, IL-6, IL- 8, IL-10 and IL-12 interleukins and TNF-α was detected by ELISA. In this study 15 different ribotypes were identified. The most frequent were 001, 020, 106 ribotypes (52.8%, 7.4% and 5.7%), respectively, while 13 isolates could not be assigned a ribotype. However, all isolates were sensitive to vancomycin, metronidazole and moxifloxacin, but 74.28% of isolates were resistant to erythromycin. The IgG level against bacterial antigens (SLPs, LC and flagella proteins) in donors’ serum showed almost normal distribution to all antigens from the different ribotypes and the sensitivity of the assays was increased by raising the concentration of antigens. Levels to SLPs were generally the highest, but the flagellar protein exceeded the SLPs of the 027 ribotype. The donors, controls, patients and carrier sera gave similar results. The greatest induction of interleukins was obtained using 50μg of antigen with the THP-1 cells activated with 50ng of PMA. The highest induction of all antigens was for IL-10. The highest values for the control LPS was with IL-12. But the best effect for SLPs of 027 was for IL-10 (109.1ng/ml), while the weakest for TNF for SLPs of 027 (4.7ng/ml). In general the IL-1β, IL-6, IL-8 and TNF concentrations ranged from 4.7-60ng/ml for all antigens and in contrast IL-12 and IL-10 average ranged 11- 109.1ng/ml. To conclude, the prevalence of C. difficile and their antibiotic susceptibility are constantly changing. IgG antibodies to SLPs and flagellar proteins from the hypervirulent ribotype 027 were highest in the community and hospitalized individuals. The molecules of conserved molecular patterns are immunogenic with various levels of response in the monocytic THP1 cells. SLPs were best in inducing interleukins. Flagellar proteins from 027 ribotypes accompanied SLPs in IL-10 induction levels. Consequently SLPs and flagellar proteins from 027 ribotypes appeared the best immunogenic bacterial molecules.

Clostridium difficile infection (CDI) has proven to be a constantly evolving disease periodically posing new diagnostic and clinical dilemmas. Different regions of the world have reported specific local genomic characteristics of the infecting strains, which may be related to variation in disease presentation and outcome. This study was performed to determine the clinical and molecular features of severe, recurrent and community-associated disease in the Lothian region of Scotland, UK among patients diagnosed from August 2010-July 2011. Three hundred and thirty-five patients with laboratory confirmed CDI were studied for epidemiological features, clinical presentation, and laboratory markers. They were followed up for one year to determine recurrence and mortality. Four hundred and thirty-two episodes were recorded. Ribotypes, presence of toxin genes and MLVA subtypes of isolates from these episodes were determined. During the course of the study, PCR ribotype 078 was identified as an important emerging type and concerns of “hypervirulence” were raised when an outbreak was recorded in 2012. This ribotype was studied to compare its clinical and molecular characteristics with other endemic ribotypes and between its own outbreak-related and endemic subtypes. Asymptomatic children were also sampled to determine their role as pools of potential pathogens. Severe episodes accounted for 40.4% of total and 29.3% patients had multiple episodes on record. One-year mortality was 32.8% of which CDI was listed on 25.5% death certificates. Ribotype 078 was confirmed in 6.8% episodes. Community-associated disease was identified in 25.3% patients, which differed significantly from hospital-associated disease in the number of antibiotics and gastrointestinal manipulation prior to CDI. Endemic PCR ribotype 078 caused significantly less recurrent disease and more community- associated disease when compared to the most prevalent ribotype 001. Patients who died from ribotype 078 within 30d had a lower Charlson comorbidity index than ribotype 001 counterparts suggesting that the former may infect healthier patients. MLVA subtyping of ribotype 078 proved useful in identifying epidemiological relationships during the outbreak. CDI had contributed to the death of 50% of all patients infected with the outbreak related ribotype 078 strain compared to 14.3% of those infected with the endemic strains. This study documents the changing epidemiology of CDI in the region and demonstrates differences in epidemic and endemic disease.

The recent increase in availability of next generation sequencing methodologies has led to extensive analysis of the genome of Clostridium difficile. In contrast, protein expression analysis, crucial to the elucidation of mechanisms of disease, has severely lagged behind. In this study, in-depth proteomic analysis of three strains of varying virulence, demonstrated previously in an animal model, has been undertaken against a background of the sequenced genomes. Strain B-1 is a historic, virulent, ribotype 005 clone, strain A represents the emerging hypervirulent 027 ribotype, while strain Tra5/5, ribotype 001, is of low virulence. To undertake a comprehensive overview of the expressed proteome, both 1D and 2D gel electrophoresis were used to separate and display the protein content of each isolate. This was coupled to MALDI-TOF and LC-MS/MS mass spectrometry for protein identification. A total of 888 different proteins were characterised by comparative analysis of isolates grown in parallel for 64 hours on blood agar. Of these, only 38% were shared between all isolates. An additional 350, 243 and 398 proteins were detected from broth cultures, and the use of a hexapeptide bead library, designed to capture low abundance proteins, led to the detection of a further 148, 127, and 171 proteins in strains A, B-1 and Tra5/5 respectively. Relative differential expression was investigated using Differential In Gel Electrophoresis (DIGE), and five proteins were shown to have a statistically higher concentration in strain A, twelve in strain B-1 and eight in strain Tra5/5. A number of these were surface proteins, with selected S-layer proteins found to be up-regulated in each strain, and the flagellar protein, FliC, up-regulated in both A and B-1. Furthermore, differential post-translation modification events were seen in flagellar and S-layer proteins. In-vivo expression of these proteins was mapped using Western blotting. Immunodetection of the majority of these, including FliC and the high molecular weight S-layer protein, were conserved between the three strains, but a notable series of immunoreactive protein spots were present in strains A and Tra5/5 but not B-1, most likely corresponding to an additional S-layer protein present in the genomes these strains, but not that of B-1. Protein expression differences for a number of previously proposed virulence proteins were evident between strains, including toxin B, sporulation, flagella and the S-layer proteins, metabolic enzymes, stress response proteins and ABC transporters. This study strongly supports the view that the virulence of Clostridium difficile is multifactorial, and that a number of related factors, although not directly required for pathogenicity, may serve to modulate the virulence of individual strains.

Antibiotic-associated diarrhoea (AAD) and colitis, with the causative agent being the Gram-positive anaerobe, Clostridium difficile, are some of the most important hospital-acquired infections and significant burdens to healthcare services worldwide. Treatment of the infection is often ineffective and currently no vaccine is available against C. difficile infection (CDI). Research to identify novel virulence factors potentially leads to the development of new therapeutic and prophylactic drugs. As lipoproteins have been shown to play key roles in the virulence of several pathogens, the aim of this project was to investigate whether lipoproteins are involved in the virulence of C. difficile. Lipoproteins are anchored to the extracellular side of the cytoplasmic membrane in Gram-positive bacteria. Two enzymes are involved in the biosynthesis of lipoproteins: lipoprotein diacylglycerol transferase (Lgt) attaches lipoproteins to the membrane, and lipoprotein signal peptidase (Lsp) cleaves the signal peptide from the amino-terminus of lipoproteins. In order to study lipoprotein processing in C. difficile, lgt and lsp mutants of the C. difficie 630Δerm strain were generated using the ClosTron system. Antibody reactivity of 14 C. difficile lipoproteins was also investigated. It was shown in this study that lgt mutation caused changes in the lipoproteome of C. difficile. Therefore, inactivation of the lgt gene allowed investigation of the global contribution of lipoproteins to bacterial processes. The physiology and virulence of the lgt mutant was studied in vitro and in vivo. Surprisingly, many of the assayed phenotypes were not significantly affected by disruption of the lgt gene. Nevertheless, the ability of the lgt mutant to adhere to Caco-2 cells was markedly reduced. In addition, the phenotype of the lgt mutant observed in mice suggests that the faecal shedding of C. difficile is affected by Lgt inactivation. In further studies, the CD0873 lipoprotein as a potential adhesin of C. difficile was identified by in silico approach. Contribution of the CD0873 lipoprotein to the adherence of C. difficle was investigated by several different assays and the results strongly suggest that the CD0873 lipoprotein is directly involved in adhesion

Résumé: De nos jours, les bactériophages (c.-à-d. des virus bactériens, ou phages) sont reconnus comme un des principaux facteurs qui influencent l’évolution et la biologie bactérienne. De plus, la nature dynamique des relations phage-hôte engendre des adaptations mutuelles au niveau des stratégies d’infection et de défense, phénomène communément appelé « course à l’armement ». Malgré une importance démontrée chez de nombreuses espèces bactériennes, l’étude du rôle des phages dans la biologie du pathogène Clostridium difficile est demeurée très limitée. Or, les infections à C. difficile sont considérées comme étant la principale cause des diarrhées associées à la prise d’antibiotiques. Alors, l’objectif de la présente étude avait pour but de mieux caractériser l’implication des phages dans la biologie de C. difficile. Des travaux préalables ont montré que la lysogénisation par le phage tempéré φCD38-2 pouvait mener à une augmentation de la production de toxines chez certaines souches de C. difficile suggérant une implication des phages dans la virulence bactérienne. En utilisant cette étude comme point de départ, nous avons évalué l’influence de la lysogénisation du phage φCD38-2 sur le transcriptome global d’une souche de C. difficile d’importance clinique. Ainsi, nous avons montré que la lysogénisation par le phage φCD38-2 a un impact significatif sur la transcription de 39 gènes bactériens dont près de la moitié encodent des protéines reliées au métabolisme des sucres, suggérant une implication du phage dans les processus métaboliques de l’hôte. Cependant, le gène présentant la plus grande altération transcriptionnelle encode une protéine de surface nommée CwpV. À partir de sa localisation sur la surface bactérienne, nous avons démontré que son expression a un effet protecteur sur les cellules face aux infections par les phages. Les expériences subséquentes ont permis de lier l’activité antiphage au domaine carboxy-terminale variable de la protéine. Étant donné que l’adsorption virale n’est pas affectée par la présence de CwpV, nous avons établi que le mode d’action du système consiste à bloquer l’injection d’ADN virale dans la cellule bactérienne. De plus, l’effet antiphage est plus prononcé envers les siphophages comparé aux myophages suggérant un mode d’action dépendant de la morphologie virale. Finalement, les expériences préliminaires suggèrent que les cellules qui expriment la CwpV ont un avantage sélectif par rapport aux cellules qui ne l’expriment pas dans un essai de co-culture soumise à une infection virale. / Abstract: Bacteriophages (or simply phages) are viruses that specifically infect and kill bacteria. They are omnipresent in every niche where bacteria thrive and as such are considered as the most abundant biological entities in the biosphere. Their massive impact on bacterial biology has incited scientific community to consider the phages as the major driving force in bacterial evolution. Nowadays, it is also assumed that phages act as the principal vectors for horizontal transfer of genetic information among prokaryotes. Moreover, highly dynamic nature of phage host relationships usually results in mutual adaptations that effectively stimulates acquisition of new offensive and defensive strategies. This phenomenon is generally described as the “phage-host arms race”. Despite their obvious importance, the contribution of phages to the biology of Clostridium difficile, the main cause of nosocomial infectious diarrhea, has not been extensively explored. Thus, the main objective of this study was to assess the overall impact of phages to C. difficile lifestyle. Our previous work has revealed the potential of a specific C. difficile phage termed φCD38-2 to stimulate the production of bacterial toxins. Based on those results, we have performed a global study of the impact of the φCD38-2 lysogeny on the bacterial transcriptome. Thus, we have found a total of 39 genes whose expression was altered during the lysogeny of φCD38- 2 with near half of them encoding proteins implicated in bacterial sugar metabolism. This suggests phage implication in the regulation of bacterial utilization of carbon sources. However, the largest transcriptional alteration has been observed for cwpV which encodes a phase-variable surface-anchored protein. Owing to its variable nature, we have hypothesized that CwpV might play a role in phage infection and indeed, we have shown that CwpV expression protects bacterial cells from phage infection. Moreover, variable C-terminal domain of CwpV was found to be essential for antiphage phenotype since its deletion restored bacterial susceptibility to infection. Additionally, CwpV did not significantly affect phage adsorption, but phage DNA replication was prevented suggesting that CwpV act as a superinfection exclusion system. Interestingly, the antiphage effect was more pronounced against phages from Siphoviridae family compared to phages from Myoviridae family suggesting that structural differences are important for the antiphage phenotype. Finally, our preliminary data suggest that CwpV expression confers selective advantage when mixed cocultures are challenged by phage infection.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.