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.

The thermophilic Campylobacters, Campylobacter jejuni and Campylobacter coli are found as commensals in the intestinal tract of healthy mammals and birds. Campylobacter jejuni is one of the leading causes of sporadic food-borne bacterial disease in humans which is predominantly contracted from poultry products. Although the vast majority of these infections are mild, life-threatening complications should be treated with antimicrobials. Patients are usually treated with either macrolides of fluoroquinolones. However, globally there is an increased trend in the development of resistance to these antibiotics. This trend has also been observed in infection of poultry and pigs. The aim of this investigation was to determine antimicrobial sensitivity of thermophilic Campylobacters isolated from pigs and poultry by broth microdilution minimum inhibitory concentration testing. A total of 482 samples of the small intestinal content from poultry and pigs from the Western Cape and Gauteng Provinces were collected and analysed. Thirty-eight Campylobacter isolates were obtained. Statistical analyses included percentage resistance, minimum inhibitory concentrations (MIC50 and MIC90) as well as the distribution percentages of the MICs. The non-parametric Mann-Whitney U test was used to establish any significant differences at an interspecies, interhost and interprovincial level. Analyses of the data obtained revealed indications of decreasing susceptibility to several antibiotic groups including the tetracyclines, macrolides, erythromycin and tylosin, as well as the lincoasamides, and fluoroquinolones. It was found that isolates from the Western Cape were more likely to be resistant to the fluoroquinolones (p = 0.0392), macrolides (p = 0.0262), and lincoasmides (p = 0.0001) and, as well as to a certain extent the pleuromutulins (p = 0.0985), whereas isolates from Gauteng were more resistant to the tetracycyclines (p = <.0001). Poultry Campylobacter spp. were more prone to be resistant to enrofloxacin (p = 0.0021). Campylobacter jejuni, mainly isolated from poultry, was more liable to be resistant to the tetracyclines (chlrotetracycline p= 0.0307), whereas C. coli, predominatly isolated from pigs was more likely to be resistant to the macrolides (tylosin p= 0.063). Four of the bacteria isolated from the Western Cape were resistant to three or more antibiotic classes, namely; tetracyclines, macrolides, lincosamides, pleuromutulins and fluoroquinolones. No multi-resistant Campylobacter spp. were isolated from the flocks in Gauteng. With the exception of tiamulin, the bacterial populations could clearly be divided into resistant and susceptible populations. As consequence of the increased resistance to the antimicrobial classes used for human therapy and the geographical differences in antimicrobial susceptibility, it is recommended that an antimicrobial resistance monitoring system for the thermophilic Campylobacter spp. be initiated in the South Africa National Veterinary Surveillance and Monitoring Programme for Resistance to Antimicorbial Drugs (SANVAD) Copyright / Dissertation (MSc)--University of Pretoria, 2009. / Veterinary Tropical Diseases / unrestricted

Campylobacteriosis is a leading foodborne illness worldwide, primarily caused by Campylobacter jejuni (C. jejuni) which is associated with poultry consumption. The emergence of antibiotic resistance has emphasized the need for alternative strategies to control C. jejuni colonization in poultry. To assess the prevalence of C. jejuni in poultry, 270 cloacal swab samples were collected from broilers raised under No-Antibiotics Ever system. Among these samples, 16.3% were identified as C. jejuni positive. Notably, these isolates exhibited a diverse range of virulence factors and antimicrobial resistance genes, with 61.36% of isolates showing hyper-motile and 20.45% demonstrating multidrug resistance. Following isolation, whole genome sequencing was conducted on four selected strains using a hybrid sequencing approach. Subsequently, the complete genomes of these C. jejuni strains were analyzed to identify vaccine candidates using reverse vaccinology. Three conserved potential vaccine candidates were identified as suitable targets for vaccine development, namely phospholipase A (PldA), TonB dependent transporter (ChuA), and cytolethal distending toxin (CdtB). Furthermore, the gene expression of these candidates was examined in four C. jejuni strains during host-pathogen interactions using avian macrophage cell line HD11. Significant upregulation of all three candidate genes were observed in the four tested C. jejuni strains during interaction with host cells, indicating their crucial role in C. jejuni infection. Additionally, the expression of immune genes was evaluated in avian macrophage cells to understand the immune responses during C. jejuni infection. The infection resulted in the upregulation of toll-like receptor genes (TLR-4), pro-inflammatory genes (IL-1β, IFN-γ, IL-6, IL-8L1), anti-inflammatory gene (IL-10), and iNOS2 gene expression. The observed immune response demonstrates the potential of C. jejuni to induce host immunity for protection. In conclusion, our study identifies three conserved potential vaccine candidates and provides insights into the immune responses induced by C. jejuni infection in avian macrophage cells. These findings are crucial for the development of an effective vaccine against C. jejuni, aiming to reduce C. jejuni transmission through poultry consumption and the risk of human infection.

Chez les bactéries à chromosomes circulaires, le crossing-over introduit par la recombinaison homologue peut conduire à des échanges de chromatides soeurs. Des nombres impairs de ces échanges aboutissent à la dimérisation des deux chromatides nouvellement répliquées compromettant ainsi leur ségrégation. Par conséquent, la plupart des bactéries utilisent le système de recombinaison spécifique de site Xer pour convertir les dimères de chromosomes et de plasmides en monomères stables. Ce système comporte deux recombinases de la famille Tyrosine recombinase, XerC et XerD, agissant sur le site dif. Cependant, quelques ε-protéobactéries n’ont besoin que d'une seule recombinase XerH agissant sur un site difH. Il parait intéressant d’étudier le système de recombinaison XerH de Campylobacter jejuni, surtout que l'augmentation spectaculaire de l'incidence de campylobactériose est alarmante. Cette étude vise à mieux comprendre comment la protéine XerH catalyse la réaction de recombinaison au niveau du site difH en mettant en évidence les séquences indispensables pour la liaison et le clivage. Grâce à ces expériences, nous avons pu confirmer que XerH est capable de se lier à la séquence entière difH; XerH est capable de cliver les deux brins supérieurs et inférieurs de difH avec une réaction plus efficace au niveau du brin inférieur; les nucléotides conservés du site de liaison sont indispensables pour la réaction de liaison; la modification de la longueur de l’espaceur améliore la réaction de liaison et de clivage et les modifications apportées au site de clivage prédit ont aboli la réaction de liaison et affecté la réaction de clivage au niveau du brin supérieur et inférieur du site difH. Ces expériences aideront à comprendre comment la recombinase XerH/difH contrôle la résolution des dimères chromosomiques chez Campylobacter jejuni en identifiant les séquences et les facteurs indispensables pour qu’un certain système soit fiable. Notre étude représente un pas vers l’avant pour comprendre un mécanisme important chez un agent pathogène ayant un grand impact sur la santé publique. / In bacteria with circular chromosomes, cross-over induced by homologous recombination can lead to sister chromatid exchanges, odd numbers of these exchanges result in dimerization of the two newly replicated chromatids compromising their segregation. Therefore, most bacteria use the Xer site-specific recombination system to convert chromosomal and plasmid dimers into stable monomers. This system involves two recombinases of the Tyrosine recombinase family, XerC and XerD, acting at the dif site. However, some ε-proteobacteria require only one XerH recombinase acting on a difH site. It seems interesting to study the XerH recombination system of Campylobacter jejuni, especially since the dramatic increase in the incidence of campylobacteriosis is alarming. This study aims to better understand how the XerH protein catalyzes the recombination reaction at the difH site by identifying the sequences required for binding as well as the factors regulating this reaction. As a result of these experiments, we were able to confirm that XerH is able to bind to the entire difH sequence; it is able to cleave both the top and bottom strands of difH with a more efficient reaction at the bottom strand; The conserved nucleotides in the binding site are essential for the binding reaction, modification of the spacer length improves the binding and cleavage reaction, and modifications in the predicted cleavage site abolished the binding reaction and affected the cleavage reaction at both the top and bottom strands of the difH site.. These experiments will help to understand how the XerH/difH recombinase controls the resolution of chromosomal dimers in Campylobacter jejuni by identifying the essential sequences and factors required for a certain system to be reliable. Our study represents a step forward in understanding an important mechanism in a pathogen with great impact on public health.

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Bacterial pathogens assemble complex surface structures in an attempt to circumvent host immune detection. A great example is the glycolipid known as lipopolysaccharide or lipooligosaccharide (LPS), the major surface molecule in nearly all gram-negative organisms. LPS is anchored to the bacterial cell surface by a anionic hydrophobic lipid known as lipid A, the major agonist of the mammalian TLR4-MD2 receptor and likely target for cationic antimicrobial peptides (CAMPs) secreted by host cells (i.e. defensins). In this work we investigate LPS modification machinery in related ε-proteobacteria, Helicobacter pylori and Campylobacter jejuni, two important human pathogens, and demonstrate that enzymes involved in LPS modification not only play a role in evasion of host defenses but also an unexpected role in bacterial locomotion. More specifically, we identify the enzyme responsible for 4'-dephosphorylation of H. pylori lipid A, LpxF. Demonstrating that lipid A depohsphorylation at the 1 and 4'-positions by LpxE and LpxF, respectively, are the primary mechanisms used by H. pylori for CAMP resistance, contribute to attenuated TRL4-MD2 activation and are required for colonization of a the gastric mucosa in murine host. Similarly in C. jejuni, we identify an enzyme, EptC, responsible for modification of lipid A at both the 1 and 4'-positions with phosphoethanolamine (pEtN), also required for CAMP resistance in this organism. Suprisingly, EptC was found to serve a dual role in modifying not only lipid A with pEtN but also the flagellar rod protein FlgG at residue Thr75, required for motility and efficient flagella production. This work links membrane biogenesis with flagella assembly, both shown to be required for colonization of a host and adds to a growing list of post-translational modifications found in prokaryotes. Understanding how pathogens evade immune detection, interphase with the surrounding environment and assemble major surface features is key in the development of novel treatments and vaccines. / text

Campylobacter jejuni is the leading bacterial cause of gastrointestinal diarrheal disease in humans worldwide. This zoonotic pathogen has a complex epidemiology due to its presence in many different host organisms. The overall aim of this thesis was to explore the role of amoebae of the genus Acanthamoeba as an intermediate host and vector for survival and dissemination of C. jejuni. Earlier studies have shown that C. jejuni can enter, survive and replicate within Acanthamoebae spp. In this thesis, I have shown that C. jejuni actively invades Acanthamoeba polyphaga. Once inside, C. jejuni could survive within the amoebae by avoiding localization to degradative lysosomes. We also found that A. polyphaga could protect C. jejuni in acid environments with pH levels far below the range in which the bacterium normally survives. Furthermore, low pH triggered C. jejuni motility and invasion of A. polyphaga. In an applied study I found that A. polyphaga also could increase the survival of C. jejuni in milk and juice both at room temperature and at +4ºC, but not during heating to recommended pasteurization temperatures. In the last study we found that forty environmental C. jejuni isolates with low bacterial concentrations could be successfully enriched using the Acanthamoeba-Campylobacter coculture (ACC) method. Molecular genetic analysis using multilocus sequence typing (MLST) and sequencing of the flaA gene, showed no genetic changes during coculture. The results of this thesis have increased our knowledge on the mechanisms behind C. jejuni invasion and intracellular survival in amoebae of the genus Acanthamoeba. By protecting C. jejuni from acid environments, Acanthamoebae could serve as important reservoirs for C. jejuni e.g. during acid sanitation of chicken stables and possibly as vectors during passage through the stomach of host animals. Furthermore, Acanthamoeba spp. could serve as a vehicle and reservoir introducing and protecting C. jejuni in beverages such as milk and juice. Validation of the ACC method suggests that it is robust and could be used even in outbreak investigations where genetic fingerprints are compared between isolates. In conclusion, Acanthamoeba spp. are good candidates for being natural hosts and vectors of C. jejuni.

This thesis is concerned with the molecular epidemiology of Campylobacter jejuni and Salmonella in New Zealand and the development of source attribution tools for these pathogens. Although campylobacteriosis is the leading enteric zoonosis worldwide, the pathogen's complex epidemiology and di culties with existing typing schemes, have posed challenges for the control of this disease. The rst study of this thesis gives an overview of existing approaches to microbial risk assessment and source attribution, with particular respect to campylobacteriosis, and describes their advantages and shortcomings. Further, the chapter discusses phenoand genotyping techniques for Campylobacter spp. and the value of including microbial typing data in risk assessments. In the second study, data from a sentinel surveillance site in the Manawatu region was used to investigate the molecular epidemiology of human campylobacteriosis cases. This analysis revealed the presence of a dominant C. jejuni clone, namely sequence type (ST) 474, which accounted for 30.7 % of human cases in the study and identi ed risk factors for infection with ruminant and poultry associated STs. The third study investigated the link between C. jejuni in human cases and samples taken from poultry. By applying epidemiological and population genetic techniques this part of the thesis provided further evidence that poultry is a major contributor to human infection. In the fourth study an existing Bayesian source attribution model was modi ed and consecutively applied to New Zealand's major foodborne zoonoses: campylobacteriosis and salmonellosis. The majority (80 %) of human campylobacteriosis cases attributable to C. jejuni were estimated to have been acquired from poultry sources, whereas wildlife source were estimated to contribute only a minor proportion of cases. In the fth study the Salmonella dataset was descriptively analysed and a large proportion of human cases was found to be caused by `exotic' Salmonella types. In the nal study of this thesis four di erent genetic and epidemiological source attribution methodologies were applied to the same dataset in a comparative modelling framework. iv The studies in this thesis show that epidemiological studies combined with molecular tools and modeling can provide valuable risk-based tools to inform the surveillance and control of zoonotic pathogens. Methods from these studies may be readily applied to the control of other (food borne) zoonoses and provide new opportunities for epidemiological investigations and source attribution modelling of major pathogens.