The Epsilonproteobacteria Helicobacter pylori and Campylobacter jejuni are major human pathogens. Both bacteria encode only a small number of transcription factors, but employ small non-coding RNAs (sRNAs) to regulate gene expression at the post-transcriptional level. While these regulatory RNAs often need the help of a protein chaperone to fulfill their function in well-studied enterobacterial model organisms, H. pylori and C. jejuni, like half of all bacteria, lack homologs of this protein named Hfq. In all domains of life, RNA-binding proteins (RBPs) play diverse roles in post-transcriptional regulation beyond their function in supporting base-pairing RNA regulators, and can themselves also directly influence mRNA stability or translation. The global identification of RBPs in eukaryotes by oligo(dT)-capture methods has previously identified a number of interesting novel candidates, including metabolic enzymes, that play a role in post-transcriptional regulation. However, these approaches are not applicable to bacteria, which lack polyA-tails on their mRNAs, and the RBPome of prokaryotes remains incompletely studied. This thesis aimed to identify auxiliary protein factors that are involved in post-transcriptional regulation, especially by sRNAs, in Epsilonproteobacteria. First, the H. pylori sRNA NikS, which we characterize as a nickel-regulated and possibly phase-variable regulator of at least five virulence and colonization factors, served as a model sRNA for a number of biochemical and genetic approaches to capture a potential RBP. NikS was chosen as it regulates its targets at the post-transcriptional level by binding to the site of translation initiation, a mechanism common to Hfq-dependent sRNAs in other bacteria. For biochemical assays, a number of RNA fusions combining the MS2, S1, and D8 aptamers with the sRNAs NikS and RepG were constructed. Fusions that were still able to regulate their targets in vivo were used for affinity purification to identify proteins associated with the sRNAs. In addition to these approaches, H. pylori sRNAs were also fused to a 14nt-long bait sequence that was then used for purification using an antisense oligonucleotide. In addition to these biochemical approaches, a functional genetic screen based on transposon mutagenesis was also used. Fluorescent reporter fusions of NikS targets were combined with transposon mutant libraries to identify genes that are necessary for sRNA function. While these genetic and biochemical screens revealed proteins such as known nickel-associated factors that likely affect NikS transcription, and known RBPs such as RNA degradosome components, thereby validating the approaches, a promising sRNA chaperone candidate was not identified. I therefore aimed to develop two novel methods to identify RBPs. One approach is based on the CRISPR/Cas-system of C. jejuni. Here, the sRNA of interest is fused to either a single-guide RNA (sgRNA) or tracrRNA, two RNAs that are specifically bound by the Cas9 protein. Cas9 is immobilized on affinity resin, in principle allowing purification of the sRNA with associated protein factors from a lysate. In proof-of-principle experiments in E. coli with the Hfq-dependent sRNA MicA, sRNA fusions could be co-purified with Cas9, but Hfq was not detected. Therefore, a second novel method, termed CoCap (Co-purification of RNA-binding proteins with biotin-capped primary transcripts), was developed to investigate the protein interactome of primary transcripts. Here, primary transcripts (including many sRNAs) were selectively biotinylated and purified via Streptavidin, along with associated proteins including potential RBPs, which were identified by mass spectrometry. The method was first established in Salmonella as a model organism for a bacterium encoding the global RNA chaperones Hfq and ProQ, and then applied to C. jejuni. CoCap identified known and novel RBPs in both organisms. In Salmonella, a number of proteins that were previously not associated with RNA binding were validated and investigated independently via RIP-seq (RNA immunoprecipitation followed by deep sequencing), including the plasmid-encoded ProQ-like protein YafB, which turned out to be a global binder of mRNAs and sRNAs. The CoCap dataset for C. jejuni revealed a KH domain-containing protein as a novel RBP candidate. We show that this protein forms a complex with another KH domain-containing protein, and that both proteins can bind RNA and potentially interact with a number of cell envelope proteins. RNA binding of the H. pylori homologs of these proteins could also be verified, although these proteins bind different and smaller subsets of RNAs. While this thesis revealed these KH domain proteins as RNA binders and gives first insights into their potential functions, the roles of these proteins in vivo remain elusive. In summary, I applied diverse genetic and biochemical screens to search for auxiliary protein factors in RNA-based regulation in Epsilonproteobacteria. I further developed a novel approach to globally identify the primary RNA-binding proteome named CoCap and applied it to Salmonella and C. jejuni. This led to the identification of novel RBPs in both organisms and established CoCap as a method applicable to a large number of bacteria. / Die Epsilonproteobakterien Helicobacter pylori und Campylobacter jejuni sind wichtige Humanpathogene. Beide Bakterien kodieren nur eine begrenzte Anzahl an Transkriptionsfaktoren, nutzen jedoch kleine nicht-kodierende RNAs (sRNAs), um die Genexpression auf der posttranskriptionellen Ebene zu regulieren. In gut erforschten enterobakteriellen Modellorganismen benötigen diese regulatorischen RNAs häufig die Hilfe eines Proteins namens Hfq. In H. pylori und C. jejuni, wie in der Hälfte aller Bakterien, fehlt jedoch ein Hfq-Homolog. In allen Domänen des Lebens können RNA-Bindeproteine (RBP) verschiedene Funktionen in der posttranskriptionellen Regulation übernehmen. Sie können zum Beispiel die Basenpaarung von RNAs ermöglichen oder auch direkt die Stabilität oder Translation von mRNAs beeinflussen. Die globale Identifizierung von RBP in Eukaryoten mittels sogenannter „oligo(dT)-capture“-Methoden hat einige interessante Kandidaten zutage gebracht, einschließlich metabolischer Enzyme, die eine Rolle in der posttranskriptionellen Regulierung übernehmen. Diese Methoden können jedoch nicht in Bakterien angewandt werden, da deren mRNAs nicht polyadenyliert sind, weshalb es an einer vollständigen Liste der bakteriellen RBP mangelt. Ziel dieser Arbeit war es, Proteinfaktoren der posttranskriptionellen Regulierung, vor allem durch sRNAs, in Epsilonproteobakterien zu identifizieren. Zuerst wurde die sRNA NikS aus H. pylori, welche wir als einen nickelabhängigen, potenziell phasenvaribalen Regulator von mindestens fünf Virulenz- und Kolonisierungsfaktoren charakterisieren, als Modell-RNA in verschiedenen biochemischen und genetischen Ansätzen verwendet, um potenzielle RBP zu identifizieren. NikS wurde ausgewählt, da es seine Zielgene durch die Interaktion mit der Region der Translationsinitiations reguliert, was ein Mechanismus ist, den auch Hfq-abhängige sRNAs in anderen Bakterien verwenden. Für die biochemischen Ansätze wurden einige RNA-Fusionen aus MS2-, S1- und D8-Aptameren mit den sRNAs RepG und NikS konstruiert. Funktionale Fusionen wurden dann zur Affinitätsaufreinigung verwendet, um Proteine zu identifizieren, die mit den sRNAs interagieren. Zudem wurde eine Ködersequenz an die sRNAs fusioniert, welche dann in einem „Antisense-Oligo“-Ansatz zur Aufreinigung genutzt wurde. Neben diesen biochemischen Ansätzen wurde eine funktionelle Suche basierend auf einer Transposonmutantenbibliothek durchgeführt. Hier wurden Fusionen aus NikS-Zielen und fluoreszenten Proteinen mit der Bibliothek kombiniert, um Gene zu identifizieren, die für die sRNA-Funktion benötigt werden. Die genetischen und biochemischen Experimente konnten Proteine identifizierten, die durch ihre Verbindung zu Nickel vermutlich die NikS-Expression beeinflussen, oder die als Teil des Degradosoms bekannte RBP sind, was die Methoden validiert. Es konnte jedoch kein guter Kandidat für ein sRNA-Hilfsprotein ausgemacht werden. Daher wurden zwei neue Methoden entwickelt. Eine davon basiert auf dem CRISPR/Cas-System von C. jejuni. Hier wird die sRNA an eine “sg RNA” oder “tracrRNA” fusioniert. Diese beiden RNAs werden von Cas9 gebunden, wodurch eine Immobilisierung von Cas9 auch eine Aufreinigung der sRNA und gebundener Proteine erlauben sollte. Um die Machbarkeit zu überprüfen, wurden Fusionen der Hfq-abhängigen sRNA MicA mittels Cas9 aus E. coli aufgereinigt. Eine Anreicherung von Hfq konnte jedoch nicht nachgewiesen werden. Daher wurde eine zweite Methode namens CoCap (Co-purification of RNA-binding proteins with biotin-capped primary transcripts) entwickelt, um Proteine, die mit primären Transkripten, inklusive zahlreicher sRNAs, interagieren, zu identifizieren. Diese Transkripte werden biotinyliert und zusammen mit gebundenen Proteinen aufgereinigt, welche dann mittels Massenspektrometrie identifiziert werden können. Die Methode wurde in Salmonella, als Modell für ein Bakterium, das die globalen RBP Hfq und ProQ exprimiert, entwickelt und dann in C. jejuni angewandt. In beiden Organismen wurden bekannte und neue RBP identifiziert. Einige Proteine aus Salmonella konnten mittels einer unabhängigen Methode verifiziert wurden. So z.B. YafB, ein plasmidkodiertes ProQ-ähnliches Protein, das sich als globales mRNA- und sRNA-Bindeprotein herausstellte. In C. jejuni wurde mittels CoCap ein KH-Domänen-Protein als neues RBP identifiziert. Wir zeigen, dass dieses mit einem weiteren KH-Protein interagiert und dass beide Proteine RNA und wahrscheinlich auch Zellhüllenproteine binden können. Die Interaktion mit RNAs wurde auch für die homologen Proteine aus H. pylori verifiziert, wobei diese eine unterschiedliche und kleinere Gruppe von RNAs binden. Obwohl die KH-Proteine in dieser Arbeit als RBP validiert werden konnten, ist deren Funktion in der Zelle noch unklar. Zusammenfassend habe ich diverse genetische und biochemische Ansätze angewandt, um Hilfsproteine der RNA-basierten Regulierung in Epsilonproteobakterien zu identifizieren. Zudem habe ich eine neue Methode namens CoCap entwickelt, die das primärtranskriptbindende Proteom global ermittelt, und diese in Salmonella und C. jejuni angewandt. Dies führte zur Identifizierung neuer RBP in beiden Organismen und etablierte CoCap als Methode, die in zahlreichen Bakterien angewandt werden kann.

Campylobacter jejuni é a espécie bacteriana mais comumente relacionada como causa de gastroenterite em humanos em vários países. Porém, o isolamento e o estudo de C. jejuni não são muito frequentes no Brasil, o que dificulta avaliar a dimensão dessa bactéria como causadora de doença em humanos e animais, bem como, determinar o impacto de sua presença em alimentos e no meio-ambiente. O objetivo desse trabalho foi avaliar a diversidade genética por cinco diferentes técnicas de tipagem molecular, o potencial patogênico pela pesquisa de 16 genes de virulência por PCR e o perfil de resistência pela concentração inibitória mínima por Etest® frente a quatro antimicrobianos e pela análise in silico de genes de resistência e pontos de mutação de linhagens de C. jejuni isoladas no Brasil. Foram estudadas 121 linhagens de C. jejuni isoladas de humanos (51), animais (35), alimentos (33) e ambiente (02) nos estados de Minas Gerais, São Paulo, Rio de Janeiro e Rio Grande do Sul, no período de 1996 a 2016. Todas as linhagens apresentaram os genes flaA, flhA, iamA, docA, ciaB, cdtA, cdtB, cdtC, racR, dnaJ, pldA, cadF, sodB e csrA. O gene wlaN foi detectado em 15 linhagens, e uma linhagem apresentou o gene virB11. Dentre as 121 linhagens estudadas, 68 linhagens foram resistentes a pelo menos um dos antimicrobianos testados. A resistência à ciprofloxacina, doxiciclina, tetraciclina e eritromicina foi observada em 43,8%, 34,7%, 34,7% e 4,9% das linhagens, respectivamente. O dendrograma de similaridade genética de Pulsed field gel electrophoresis (PFGE) agrupou as 121 linhagens estudadas em três grupos com similaridade genômica de 46,9% entre eles. Apesar da alta diversidade genômica entre as linhagens estudadas, algumas linhagens isoladas de diferentes fontes, locais e anos, apresentaram uma similaridade genotípica acima de 80% entre elas e, foram agrupadas em 21 subgrupos. Pelas sequências da SVR do gene flaA as linhagens estudadas foram agrupadas em dois grupos com linhagens isoladas de fontes clínicas e não clínicas e de humanos e animais com similaridade acima de 80,9 % entre elas e tipadas em 40 SVR-flaA alelos, sendo os alelos 57, 49 e 45 os mais frequentemente detectados. A análise do locus CRISPR por HRMA tipou as linhagens de C. jejuni em 23 diferentes variantes sendo que algumas variantes continham linhagens de origem clínica e não clínica e de humanos e animais. A árvore de SNPs gerada a partir dos dados do sequenciamento do genoma completo alocou as 116 linhagens sequenciadas em dois principais grupos. O grupo SNP-A agrupou 97 linhagens e o grupo SNP-B agrupou 19 linhagens, com linhagens de fontes clínicas e não clínicas e de humanos e animais, respectivamente. A técnica de Multilocus sequence typing (MLST) tipou as 116 linhagens de C. jejuni em 46 STs, e não foi observada a predominância de um ST. O índice de discriminação das metodologias de análise de SNPs no genoma completo, PFGE, MLST, sequenciamento das SVR do gene flaA e análise do locus CRISPR por HRMA foi 1,0, 0,982, 0,941, 0,939 e 0,874, respectivamente. Na análise in silico de genes de resistência e pontos de mutação, 95 linhagens apresentaram ao menos um gene de resistência ou ponto de mutação conhecido, sendo que a porcentagem de correlação entre os resultados de resistência fenotípicos e genotípicos foi maior que 66,7%; 94,6% e 96,8% para eritromicina, tetraciclina e ciprofloxacina, respectivamente. Conclui-se que a alta frequência da maioria dos genes de virulência pesquisados evidenciou o potencial patogênico das linhagens de C. jejuni estudadas. A resistência a antimicrobianos de primeira escolha utilizados para o tratamento da campylobacteriose encontrada nas linhagens estudadas é preocupante, podendo levar à falha terapêutica quando o tratamento é necessário. Os resultados obtidos pelas metodologias de tipagem molecular realizadas sugerem que uma possível contaminação possa ter ocorrido entre fontes clínicas e não clínicas e entre humanos e animais, ao longo de 20 anos no Brasil. Pelo índice de discriminação, foi observado que as metodologias de análise de SNPs no genoma completo e PFGE, em comparação com as outras técnicas de tipagem, foram as mais eficientes em discriminar as linhagens de C. jejuni do presente estudo. / Campylobacter jejuni is the most commonly bacterial species related as a cause of gastroenteritis in humans in several countries. However, the isolation and the study of C. jejuni have not been very frequently in Brazil, which makes it difficult to evaluate the involvement of this bacterium as a cause of diseases in humans and animals, as well as to determine the impact of its presence in food and the environment. The aim of this study was to evaluate the genetic diversity by five different molecular typing techniques, the pathogenic potential by searching for the presence of 16 virulence genes by PCR and the resistance profile by the minimum inhibitory concentration by Etest® against four antibiotics and by the in silico analyses of resistance genes and mutation points of C. jejuni strains isolated in Brazil. A total of 121 C. jejuni strains isolated from humans (51), animals (35), food (33) and the environment (02) in the States of Minas Gerais, Sao Paulo, Rio de Janeiro and Rio Grande do Sul, between 1996 to 2016 were studied. All strains presented the genes flaA, flhA, iamA, docA, ciaB, cdtA, cdtB, cdtC, racR, dnaJ, pldA, cadF, sodB and csrA. The wlaN gene was detected in 15 strains, and one strain presented the virB11 gene. Among the 121 strains studied, 68 strains were resistant to at least one of the antibiotics tested. Resistance to ciprofloxacin, doxycycline, tetracycline and erythromycin was observed in 43.8%, 34.7%, 34.7% and 4.9% of the strains, respectively. The Pulsed field gel electrophoresis (PFGE) dendrogram of genetic similarity clustered the 121 strains studied in three groups with a genomic similarity of 46.9% among them. Despite the high genomic diversity among the strains studied, some strains isolated from different sources, places and years, presented a genotypic similarity above 80% among them and were grouped into 21 subgroups. By flaA-SVR sequencing the strains studied were clustered into two groups with strains isolated from clinical and non-clinical sources and from humans and animals with a similarity above 80.9% among them and typed in 40 flaA-SVR alleles, being the alleles 57, 49 and 45 the most frequently detected. The analysis of the CRISPR locus by HRMA typed the C. jejuni strains in 23 different variants, with some variants containing strains from clinical and non-clinical origin and from humans and animals. The SNP tree generated from the whole genome sequencing data grouped the 116 strains sequenced into two major groups. SNP-A grouped 97 strains and SNP-B grouped 19 strains, with strains from clinical and non-clinical sources and from humans and animals, respectively. Multilocus sequence typing (MLST) technique typed the 116 C. jejuni strains in 46 STs, and it was not observed a predominant ST. The discrimination index of the analysis of SNPs in the whole genome, PFGE, MLST, flaA-SVR sequencing and analysis of the CRISPR locus by HRMA was 1.0, 0.982, 0.941, 0.939 and 0.874, respectively. In the in silico analyses of resistance genes and mutation points, 95 strains showed at least one resistance gene or known mutation point, and the percentage of correlation between phenotypic and genotypic resistance results was greater than 66.7%; 94.6% and 96.8% for erythromycin, tetracycline and ciprofloxacin, respectively. In conclusion, the high frequency of the majority of the virulence genes studied highlighted the pathogenic potential of the C. jejuni strains studied. Resistance to antimicrobials of first choice used for the treatment of campylobacteriosis found in the strains studied is worrying and may lead to therapeutic failure when treatment is required. The results obtained by the molecular typing methodologies performed suggest that a possible contamination may have occurred between clinical and non-clinical sources and between humans and animals over 20 years in Brazil. By the discrimination index, it was observed that the methodologies of analysis of SNPs in the whole genome and PFGE, in comparison to the other typing techniques, were the most efficients in discriminating the C. jejuni strains of the present study.

D. Tech. (Department of Biotechnology, Faculty of Applied and Computer Sciences), Vaal University of Technology. / Salmonella enterica serovar Typhimurium and Campylobacter jejuni are the leading causes of Salmonellosis and Campylobacteriosis that is characterised by gastroenteritis. These waterborne diseases can be easily prevented by home water treatment methods such as solar disinfection (SODIS). The SODIS process involves placing microbiologically unsafe water in clear plastic or glass bottles and exposing them to direct sunlight for approximately six to eight hours. SODIS kills microbes through a combination of DNA-damaging effects of ultraviolet (UV) radiation and thermal inactivation from solar heating. The result is microbiologically safe water. Continuous drinking of SODIS treated water may confer some immunological effects on the consumer. These immunological effects have not been thoroughly explored. Therefore, the objectives of this study were to firstly, characterise the effects of solar irradiation on the viability of S. Typhimurium and C. jejuni; secondly, to determine the cytotoxicity and modulation of cell death of solar irradiated S. Typhimurium and C. jejuni on macrophages. Thirdly, to analyse the chemokine and cytokine profiles of macrophages infected with solar irradiated S. Typhimurium and C. jejuni. Lastly, to analyse the host-cell interactions of macrophages infected with solar-irradiated and non-solar irradiated S. Typhimurium and C. jejuni using a proteomic approach. In all the experiments, S. Typhimurium and C. jejuni were (i) heat/chemically treated, (ii) solar and non-solar irradiated for 4 and 8 hours. A murine macrophage cell line RAW264.7 was co-cultured with the differentially treated bacteria species for 3 and 24 hours. Appropriate controls were included. The impact of solar irradiated S. Typhimurium and C. jejuni on intracellular growth, proliferation, cytotoxicity, and apoptosis on macrophages was assessed. Intracellular growth of the both bacterial species was assessed with the gentamicin protection assay, and cytotoxicity was determined by Lactate Dehydrogenase Assay (LDH). The macrophages treated with solar irradiated S. Typhimurium and C. jejuni showed no intracellular growth after 48 hours post-infection. However, the non-irradiated S. Typhimurium survived within the macrophages and were highly toxic to the macrophages (average cytotoxicity of 91%±32). The non-solar irradiated C. jejuni were metabolically active but non-culturable, whereas the solar-irradiated C. jejuni was metabolically inactive. Thus, solar irradiated C. jejuni showed a lower percentage cytotoxicity (2.57% ± 0.32%) in comparison to non-solar irradiated C. jejuni at 24 hours post-infection (p.i.) (30.28% ± 0.05%). Flow cytometric analysis showed that the non-irradiated S. Typhimurium brought about a statistically significant increase in the percentage of necrotic cells (48% ± 2.99%), whereas bacteria irradiated for 8 hours produced a lower percentage of necrotic cells (25% ± 5.87%). The heat/chemical attenuated samples had the lowest percentage of necrotic cells (21.15% ± 5.36%) at 24 h p.i. Macrophages treated with solar irradiated and non-solar irradiated C. jejuni did not induce necrosis, but apoptotic cell death. At 24 h p.i., the highest proportion of apoptotic cell death was observed in macrophages treated with non-solar irradiated C. jejuni whereas the solar irradiated C. jejuni showed a lower percentage of apoptotic cell death. Therefore, there is great possibility that S. Typhimurium and C. jejuni could become avirulent after SODIS treatment and this could prevent gastroenteritis in consumers of SODIS-treated water. The activation of macrophages infected with solar irradiated S. Typhimurium and C. jejuni was also assessed in this study. The production of nitric oxide (NO) was determined using the Greiss Reagent Assay, whereas the production of chemokines, cytokines, and growth stimulating factors by the RAW264.7 cells in vitro was measured using the Luminex 200. The results showed that both solar and non-solar irradiated S. Typhimurium inhibited the production of nitric oxide in the RAW264.7 cells. The heat/chemically attenuated S. Typhimurium induced a significant increase (p<0.0.5) in the production of NO2− in the macrophages when compared to the unstimulated RAW264.7. The chemokine and cytokine levels produced by the macrophages were similar in the solar inactivated S. Typhimurium and the live untreated S. Typhimurium. However, macrophages treated with heat/chemically attenuated S. Typhimurium showed an anti-inflammatory response by inhibiting the production of pro-inflammatory cytokines such as IL-1, IL-1, IL-2, IL-6, and IL-17 in macrophages. The macrophages treated with solar and non-solar irradiated C. jejuni possibly produced an anti-inflammatory effect since the amount of pro-inflammatory cytokines in the samples was significantly reduced during the late infection period (24 h p.i.). This study also analysed the proteomic profiles of macrophages treated with LPS, non-solar irradiated, solar irradiated, heat/ chemical inactivated S. Typhimurium, and C. jejuni. This was carried out using SWATH-mass spectrophotometry-based proteomics. Proteins were extracted from infected macrophages after 24 hours p.i. HILIC-based sample clean-up and digestion, DDA LCMS-MS (spectral library), SWATH LCMS-MS, and data processing were carried out. A total of 15,077 peptides matching to 2,778 proteins were identified at 1% FDR with numerous differentially expressed proteins (DEPs) detected in macrophages treated with lipopolysaccharide (LPS), non-solar irradiated C. jejuni (NS), heat-attenuated C. jejuni (HA) and 4h-solar irradiated (SI4) and 8h-solar irradiated (SI8) C. jejuni, respectively. Pathway analysis revealed that most of the upregulated proteins in macrophages treated with solar irradiated C. jejuni were involved in oxidation-reduction processes, endoplasmic reticulum stress, transport, antigen processing and presentation of exogenous peptide antigens via MHC class I (TAP-dependant) and ATP-biosynthetic processes. The KEGG-pathways also revealed the roles of some upregulated proteins in lysosomal and phagosome pathways. In conclusion, our results revealed that there is coordinated up-regulation of MHC-I processing pathways occurred at 24 h p.i. It is likely that proteins from solar irradiated C. jejuni may undergo proteasomal degradation, and the peptides are transported to the endoplasmic reticulum (ER) and loaded onto MHC-I molecules. Peptide loading results in class I complexes consolidation and transit to the cell surface where antigens can be presented to circulating CD8 + T cells. Additionally, solar irradiated C. jejuni also undergoes degradation in the phagosome. The phagosome has the potential to create antigens that can be expressed on the cell surface of macrophages to stimulate different lymphocytes and induce appropriate immune responses, thus, connecting the innate to adaptive immunity, and this could also have health benefits via the consumption of SODIS treated water. However, proteomic analysis of S. Typhimurium showed no significant differentially expressed proteins in macrophages treated with LPS, non-solar irradiated, and solar irradiated S. Typhimurium. This may be due to an overestimation of the extracted protein. However, DEPs in macrophages treated with heat-attenuated S. Typhimurium showed that macrophages may have adapted an anti-inflammatory M2 phenotype because the IFN-γ signalling pathway was downregulated. This may have contributed to non-expression of the chemokine IFN-γ in RAW264.7 cells. Moreover, proteins such as Hmox1 and Sqstm1 were upregulated, and this is also characteristic of M2 macrophages. This study provided new insights on the effect of solar irradiated Salmonella Typhimurium and Campylobacter jejuni on the proliferation and activation of macrophages in vitro.

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.

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.

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.