Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2007. / Härtill 6 uppsatser.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Campylobacter jejuni e Campylobacter coli sÃo importantes agentes etiolÃgicos de doenÃa diarrÃica na populaÃÃo mundial. A infecÃÃo por Campylobacter sp. à usualmente identificada por cultivo microbiolÃgico que leva aproximadamente 72 horas para identificaÃÃo do gÃnero. Nosso objetivo principal foi pesquisar a prevalÃncia de C. jejuni e C. coli em populaÃÃo infantil, com idade entre 2-36 meses, da Ãrea urbana de Fortaleza/CE, Brasil, em estudo do tipo epidemiolÃgico observacional caso-controle, utilizando, como ferramenta de detecÃÃo, a reaÃÃo em cadeia da polimerase (PCR). Outros objetivos consistiram em: investigar o impacto nutricional da infecÃÃo (casos) ou da colonizaÃÃo (controles) por Campylobacter sp.; determinar a presenÃa de trÃs genes de virulÃncia para a toxina citoletal distensora (CDT) de C. jejuni e avaliar a ocorrÃncia de inflamaÃÃo intestinal nas infecÃÃes causadas por Campylobacter sp. A populaÃÃo estudada consistiu de 83 casos e 83 controles, sendo os casos, crianÃas com histÃrico de diarrÃia nos 14 dias pregressos à seleÃÃo para o estudo. Foram avaliados parÃmetros sÃcio-econÃmicos atravÃs de questionÃrio epidemiolÃgico. Medidas antropomÃtricas foram coletadas para determinaÃÃo de escores-z no intuito de avaliar o perfil nutricional das crianÃas. A detecÃÃo de Campylobacter nas amostras congeladas foi realizada por ensaio imuno-enzimÃtico (ELISA) e PCR. Pela PCR tambÃm investigamos a presenÃa dos genes cdtA, cdtB e cdtC da CDT de C. jejuni. A avaliaÃÃo da inflamaÃÃo intestinal foi realizada pela pesquisa de lactoferrina fecal (LFF), atravÃs de ELISA semiquantitativa. Foi detectado, por PCR, C. jejuni em 9,6% dos casos (8/83) e 7,2% dos controles (6/83). C. coli foi detectado em 6,0% dos casos (5/83) e 1,2% dos controles (1/83). Os genes cdtA, cdtB e cdtC foram encontrados em 50% das amostras hipO+ (7/14). Houve diferenÃa significativa (p<0,05) dos escores WAZ e WHZ entre casos e controles portadores de C. jejuni, sendo que casos portadores apresentaram mÃdia inferior de WAZ e WHZ, quando comparados com os controles portadores. No grupo Casos, os portadores de C. jejuni apresentavam valor mÃdio de WHZ inferior ao valor mÃdio apresentado pelos casos nÃo-portadores. Mais de 80,0% das crianÃas estudadas apresentaram inflamaÃÃo intestinal caracterizada por elevados nÃveis de LFF, independente da presenÃa de diarrÃia e Campylobacter sp. Em conclusÃo, nossos achados corroboram dados da literatura cientÃfica relacionados à prevalÃncia de C. jejuni e C. coli na populaÃÃo infantil, existÃncia de portadores assintomÃticos e associaÃÃo entre a detecÃÃo do microorganismo e desnutriÃÃo. AlÃm disso, nossos dados apontam para ocorrÃncia de variabilidade genÃtica dentre as cepas de C. jejuni detectadas na populaÃÃo estudada em relaÃÃo à presenÃa ou ausÃncia dos genes de CDT. / Campylobacter jejuni and Campylobacter coli are important etiologic agents of worldwide diarrheal disease. Campylobacter sp. infection is usually identified by a 72 hour microbiological culture that identifies the genus of the responsible organism. Our main goal was to investigate the prevalence of C. jejuni and C. coli in children, aged 2-36 months, from urban Fortaleza, CE, Brazil, in an observational epidemiological case-control study using, as a tool of detection, the polymerase chain reaction (PCR). Our other goals were to investigate the nutritional impact of infection (cases) or colonization (controls) for Campylobacter sp., to determine the presence of three virulence genes of C. jejuni cytolethal distending toxin (CDT), and to evaluate the occurrence of inflammation in intestinal infections caused by Campylobacter sp. The study population consisted of 83 cases and 83 controls, where the cases consisted of children with a history of diarrhea in the 14 days prior to selection for the study. We assessed socioeconomic parameters through an epidemiological questionnaire. Anthropometric measurements were collected to determine z-score parameters for assessing the nutritional status of the children. Detection of Campylobacter from frozen samples was performed by enzyme-linked immunosorbent assay (ELISA) and PCR. Also, using PCR technology, we investigated the presence of C. jejuni genes cdtA, cdtB and cdtC. Intestinal inflammation was assessed by semi-quantitative ELISA detection of fecal lactoferrin (LFF). PCR technology detected C. jejuni in 9.6% of the cases (8/83) and 7.2% of the controls (6/83), while C. coli was detected in 6.0% of the cases (5/83) and 1.2% of the controls (1/83). CDT genes were found in 50% of hipO+ samples (7/14). There was a significant difference (p <0.05) in the weight for age z-scores (WAZ) and the weight for height z-scores (WHZ) between case and control carriers of C. jejuni, where case carriers showed lower average WAZ and WHZ than control carriers. Moreover, in the case group, carriers of C. jejuni showed a lower WHZ average than that of non-carrier cases of C. jejuni. More than 80.0% of the children studied had intestinal inflammation characterized by high levels of LFF regardless of the presence of diarrhea and Campylobacter sp. In conclusion, our findings corroborate data in the scientific literature related to the prevalence of C. jejuni and C. coli in pediatric populations, the existence of asymptomatic carriers and an association between the detection of the microorganism and malnutrition. In addition, our data suggest a genetic variability among the strains of C. jejuni detected in the study population, related to presence o absence of CDT genes.

No description available.

This thesis focuses on the structural and functional characterization of two integral membrane proteins; CfrA, an outer membrane TonB-dependent transporter (TBDT) from Campylobacter jejuni, and ELIC, a pentameric ligand-gated ion channel (pLGIC) from Erwinia Chrysanthemi. The spectroscopic characterization of CfrA revealed a fold consistent with the structural and biophysical properties observed for other TBDT. Both a homology model of CfrA and sequence alignments of CfrA with other ferric-enterobactin transporters suggested a unique mode of ligand binding, thus raising the possibility that C. jejuni can be specifically inhibited. To investigate the molecular determinates of binding to CfrA, I set out to crystallize CfrA. Hundreds of crystal trials led to crystals diffracting to 3.6 Å resolution, with a complete data set acquired at 5 Å resolution that led to a structural model of the CfrA β-barrel. In the second part of this thesis, I reconstituted ELIC into model membranes in order to test the role of intramembrane aromatic interactions in ELIC gating and lipid sensing. ELIC was reconstituted into both asolectin (aso-ELIC) and 1-palmitoyl-2-oleoyl phosphatidylcholine (PC-ELIC), membranes that stabilize the homologous nicotinic acetylcholine receptor (nAChR) in functional coupled versus non-functional uncoupled conformations, respectively. In both membrane environments, ELIC exhibits a mixed α-helical and β-sheet secondary structure, with a thermal denaturation intermediate between those of the nAChR and the close prokaryotic homolog, GLIC, in similar membranes. The data suggest that although ELIC has a decreased propensity to adopt an uncoupled conformation relative to the nAChR, its ability to undergo cysteamine-induced channel gating is sensitive to its lipid environment. The decreased propensity to uncouple may reflect an increased level of aromatics at the interface between the transmembrane α-helices, M1, M3, and M4. To test this hypothesis further, the level or aromatic residues at the M1, M3, and M4 interface in both GLIC and ELIC were varied, and in both cases the levels of intramembrane aromatic interactions correlated with the efficiency of coupling binding to gating. The data provide further evidence for a role of intramembrane aromatics in channel gating and in dictating the propensity of pentameric ligand-gated ion channels to adopt an uncoupled conformation.

No description available.

No description available.

Campylobacter jejuni est l’agent causal de la campylobactériose, infection bactérienne importante en santé publique. Un des vecteurs de transmission de C. jejuni pour l’humain est le poulet via la chaîne alimentaire. Les mécanismes impliqués dans colonisation caecale commensale des oiseaux par C. jejuni sont toujours peu caractérisés, bien qu’une meilleure compréhension de ces mécanismes puisse apporter des solutions pour le contrôle du pathogène à la ferme. Cette étude avait pour buts de caractériser les propriétés phénotypiques et les facteurs génétiques impliqués dans la colonisation du poulet par C. jejuni et d’identifier de nouveaux mécanismes impliqués dans cette association. Des souches, issues d’élevages conventionnels échantillonnés en 2003 et en 2008 ainsi que d’élevages biologiques, ont été caractérisées afin d’obtenir leur profil de résistance aux antibiotiques, leur autoagglutination et leur chimiotactisme. Les souches des élevages conventionnels ont de plus été caractérisées pour leur capacité à adhérer et envahir une culture primaire de cellules caecales de poulet. Une puce à ADN a été développée pour détecter la présence de 254 gènes et variants associés à la colonisation des poulets ainsi qu’à la résistance aux antibiotiques chez les souches issues d’élevages conventionnels. Les propriétés phénotypiques et la présence de certains gènes chez les souches ont par la suite été comparées. Finalement, des souches ayant des caractéristiques différentes ont été utilisées dans un modèle de colonisation du poulet pour évaluer l’efficacité d’un nouvel additif alimentaire à base d’acides organiques et d’huiles essentielles sur le contrôle de C. jejuni. Les propriétés phénotypiques des souches étaient très variées et n’étaient pas corrélées entre elles, à l’exception de l’adhésion et de l’invasion. L’analyse génétique a révélé que le contenu en gènes des souches était variable, notamment au niveau des gènes de l’enveloppe bactérienne, au flagelle, aux récepteurs du chimiotactisme et à la résistance à l’arsenic. Les souches de 2003 et de 2008 étaient semblables lorsque leur contenu en gènes ainsi que leurs propriétés phénotypiques étaient comparés. Des gènes possiblement associés à un fort ou un faible potentiel de colonisation ont été identifiés. L’additif alimentaire a diminué la contamination des carcasses bien qu’une augmentation de la colonisation intestinale ait été observée pour certaines souches. La moitié des lots de poulets d’origine biologique étaient positifs pour C. jejuni. Les souches issues de ce type d’élevage étaient peu résistantes aux antibiotiques et possédaient des phénotypes variés. Cette étude a permis de mieux définir les caractéristiques importantes de C. jejuni qui sont associées à la colonisation intestinale du poulet. Elle a établi pour la première fois au Canada la présence du pathogène dans les élevages de poulets biologiques. Cette étude fait partie des quelques études qui décrivent la présence des gènes de colonisation et de résistance aux antibiotiques dans une collection de souches issues uniquement du poulet. Elle a également remis en doute l’importance de certains gènes dans la colonisation. La caractérisation exhaustive des souches a également permis d’identifier de nouveaux gènes possiblement associés à la colonisation de poulet par C. jejuni. Finalement, elle a indiqué que l’utilisation d’un mélange d’huiles essentielles et d’acide organique encapsulés pouvait être efficace pour réduire la contamination des carcasses de poulet par C. jejuni et que son effet était souche-dépendant. / Campylobacter jejuni is the bacterium responsible for campylobacteriosis. It is a human pathogen of concern for public health. One of the transmission routes of this bacteria to humans is by the food chain through poultry meat products. The mechanisms involved in the commensal caecum chicken colonization by C. jejuni are poorly characterized, despite that increasing the knowledge on these mechanisms would allow a better control of the pathogen at the farm. The objectives of this study were to characterize the phenotypic and genetic factors affecting chicken colonization by C. jejuni and to identify news mechanisms involved in this process. Isolates, recovered from chicken caecal content sampled from conventional farms in 2003 and in 2008 as well as originating from organic farms sampled in 2009, were used. All strains were characterized for their antibiotic resistance profiles (AMR), autoagglutination and chemotaxis properties. Strains originating from conventional farms were further characterized for adhesion/invasion of primary chicken caecal cells. A new microarray was created to detect the presence of 254 genes and variants, all associated with chicken colonization or AMR. An association was made between the strains phenotypic properties and their gene content. Finally, strains possessing different phenotypic and genetic properties were used in a chicken colonization model to assess the efficacy of a novel feed additive to control chicken colonization by C. jejuni. Strains had variable phenotypic properties and these properties were not correlated, with the exception of adhesion and invasion. The microarray analysis revealed that gene presence was highly variable among the strains, especially for genes involved in the bacterial envelope, the flagella, the chemotaxis receptors and arsenic resistance. The 2003 and 2008 strains had similar phenotypic and genetic properties. Some genes, present in strains possessing higher or lower chicken colonization potentials, were identified. The feed additive was successful in reducing chicken carcass contamination but it increased bacterial caecal counts for some strains. C. jejuni was isolated from half of the sampled organic farms and strains originating from this type of production had low AMR and variable phenotypic properties.

Submitted by Cleide Dantas (cleidedantas@ufpa.br) on 2014-07-29T11:13:47Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_PesquisaCampylobactersppGranjas.pdf: 649227 bytes, checksum: c39eeff01123d4d81e68a9da4c4b01ec (MD5) / Approved for entry into archive by Ana Rosa Silva (arosa@ufpa.br) on 2014-08-28T14:41:49Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_PesquisaCampylobactersppGranjas.pdf: 649227 bytes, checksum: c39eeff01123d4d81e68a9da4c4b01ec (MD5) / Made available in DSpace on 2014-08-28T14:41:49Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_PesquisaCampylobactersppGranjas.pdf: 649227 bytes, checksum: c39eeff01123d4d81e68a9da4c4b01ec (MD5) Previous issue date: 2007 / As infecções de origem alimentar no homem, causadas por Campylobacter spp., resultam em grandes perdas econômicas e estão relacionadas à produção e o abate de frangos, etapas importantes na disseminação dessas bactérias. Baseando-se na importância do Campylobacter spp. na saúde pública e tendo em vista os dados constantes na literatura de que as aves comercializadas estão constantemente contaminadas com esse agente, sentiu-se a necessidade de realizar um estudo envolvendo a criação e o abate de frangos na região amazônica para que medidas profiláticas e de controle possam ser adotadas. O trabalho teve como objetivo estudar a ocorrência de Campylobacter spp. em granjas e abatedouro avícolas na mesorregião metropolitana de Belém – PA; isolar e identificar as espécies de Campylobacter spp. e identificar as fontes de contaminação nas granjas e os pontos críticos no abate. Foi coletado um total de 120 amostras em três granjas avícolas: 30 amostras de “swab” cloacal, 30 amostras de cama de frango, 30 amostras de ração e 30 amostras de água dos bebedouros. No abatedouro, foram colhidas 126 amostras: 36 amostras de água em 12 pontos diferentes da linha de abate e mais 30 amostras de pele do conjunto peito/ pescoço, 30 amostras de fígado e 30 amostras de moela. As amostras foram colhidas entre os meses de janeiro e maio de 2007 para o isolamento e identificação das espécies de Campylobacter spp. As amostras foram processadas na Seção de Bacteriologia e Micologia do Instituto Evandro Chagas – IEC da Secretaria de Vigilância em Saúde (SVS), Ministério da Saúde, Ananindeua – PA. Campylobacter spp. foi isolado em 33,3% (40/120) das amostras das granjas. Não houve diferença significativa (p>0,05) entre os percentuais de isolamentos positivos entre as três granjas pesquisadas. Ao analisar as freqüências dos isolados de Campylobacter spp. para cada tipo de amostra das granjas, observou-se que 96,6% (29/30) das amostras de “swab” cloacal, 33,3% (10/30) das amostras de cama e 3,3% (1/30) das amostras de água foram positivas para Campylobacter spp. Não foi isolada a bactéria nas amostras de ração. Campylobacter jejuni foi identificado bioquimicamente em 82,5% (33/40) das cepas isoladas nas granjas. No abatedouro, todas as cepas isoladas foram identificadas como C. jejuni., sendo isolado a bactéria em 8,73% (11/126) das amostras provenientes da linha de abate. Ao analisar as freqüências dos isolados de C. jejuni para cada tipo de amostra do abatedouro, observou-se que 27,8% (10/36) das amostras de água foram positivas para C. jejuni, seguido pela moela com 3,3% (1/30) das amostras positivas. Não foi isolado Campylobacter spp. nas amostras de fígado e de pele do conjunto peito/ pescoço. Houve diferença significativa (p<0,0001) entre os isolamentos positivos, negativos e os tipos de amostras processadas nas granjas e no abatedouro. As principais fontes de contaminação nas granjas foram o “swab” cloacal, a cama e, em menor escala, a água. Os principais pontos críticos observados no abatedouro foram a água, seguido pela moela. C. jejuni foi identificado em elevado percentual entre as cepas isoladas nas granjas e em todas as cepas do abatedouro. / The human infections of food origin caused by Campylobacter spp. result in high economic losses and these infections are correlated to poultry flock and slaughter, important steps in Campylobacter spp. dissemination. Based on the importance of this microorganism in public health and in the literature data that show the high poultry contamination by Campylobacter spp., we realize a study involving the flock and the poultry slaughter in Amazon region for adoption of prophylactic and control measures. The objective of this work was to investigate the occurrence of Campylobacter spp. in poultry flock and slaughterhouse in Amazon region and to isolate and to identify the Campylobacter species and to identify the sources of infection in the flock and the critical points in the slaughterhouse. We collected 120 samples in three flocks: 30 cloacal swab samples, 30 poultry litter samples, 30 feed samples and 30 water samples. In the slaughterhouse, 126 samples were collected: 36 water samples from 12 different points in the abattoir, 30 neck/ chest skin samples, 30 liver samples and 30 gizzard samples. The samples were collected from January to May 2007. The samples were processed in the Bacteriology and Micology Laboratory in Evandro Chagas Institute – Health Surveilance Office – Brazil’s Health Ministery. Campylobacter spp. was isolated in 33,3% (40/120) flock samples. There was no significant difference (p>0,05) between positive isolates in the three flocks. Campylobacter spp. was isolated in 96,6% (29/30) cloacal swab samples, 33,33% (10/30) poultry litter samples and 3,3% (1/30) water samples. There were no positive samples in the feed. Campylobacter jejuni was identified by biochemistry reactions in 82,5% (33/40) of isolates from flocks. In the slaughterhouse, all isolates were identified as C. jejuni. This microorganism was isolated in 8,73% (11/126) of slaughterhouse samples. C. jejuni was isolated in 27,8% (10/36) water samples and in 3,3% (1/30) gizzard samples. Campylobacter spp. was not isolated in liver samples neather in neck/ chest skin samples. There was significant difference (p<0,0001) between positive, negative isolates and among all kinds of samples collected in the flocks and at the slaughterhouse. The infections sources identified in the flocks were the cloacal swab, the poultry litter and the water. In the slaughterhouse, the critical points identified were the water and the gizzard. C. jejuni was identified in high levels in the flocks and in all isolates from the slaughterhouse.

Campylobacter jejuni est l’agent causal de la campylobactériose, infection bactérienne importante en santé publique. Un des vecteurs de transmission de C. jejuni pour l’humain est le poulet via la chaîne alimentaire. Les mécanismes impliqués dans colonisation caecale commensale des oiseaux par C. jejuni sont toujours peu caractérisés, bien qu’une meilleure compréhension de ces mécanismes puisse apporter des solutions pour le contrôle du pathogène à la ferme. Cette étude avait pour buts de caractériser les propriétés phénotypiques et les facteurs génétiques impliqués dans la colonisation du poulet par C. jejuni et d’identifier de nouveaux mécanismes impliqués dans cette association. Des souches, issues d’élevages conventionnels échantillonnés en 2003 et en 2008 ainsi que d’élevages biologiques, ont été caractérisées afin d’obtenir leur profil de résistance aux antibiotiques, leur autoagglutination et leur chimiotactisme. Les souches des élevages conventionnels ont de plus été caractérisées pour leur capacité à adhérer et envahir une culture primaire de cellules caecales de poulet. Une puce à ADN a été développée pour détecter la présence de 254 gènes et variants associés à la colonisation des poulets ainsi qu’à la résistance aux antibiotiques chez les souches issues d’élevages conventionnels. Les propriétés phénotypiques et la présence de certains gènes chez les souches ont par la suite été comparées. Finalement, des souches ayant des caractéristiques différentes ont été utilisées dans un modèle de colonisation du poulet pour évaluer l’efficacité d’un nouvel additif alimentaire à base d’acides organiques et d’huiles essentielles sur le contrôle de C. jejuni. Les propriétés phénotypiques des souches étaient très variées et n’étaient pas corrélées entre elles, à l’exception de l’adhésion et de l’invasion. L’analyse génétique a révélé que le contenu en gènes des souches était variable, notamment au niveau des gènes de l’enveloppe bactérienne, au flagelle, aux récepteurs du chimiotactisme et à la résistance à l’arsenic. Les souches de 2003 et de 2008 étaient semblables lorsque leur contenu en gènes ainsi que leurs propriétés phénotypiques étaient comparés. Des gènes possiblement associés à un fort ou un faible potentiel de colonisation ont été identifiés. L’additif alimentaire a diminué la contamination des carcasses bien qu’une augmentation de la colonisation intestinale ait été observée pour certaines souches. La moitié des lots de poulets d’origine biologique étaient positifs pour C. jejuni. Les souches issues de ce type d’élevage étaient peu résistantes aux antibiotiques et possédaient des phénotypes variés. Cette étude a permis de mieux définir les caractéristiques importantes de C. jejuni qui sont associées à la colonisation intestinale du poulet. Elle a établi pour la première fois au Canada la présence du pathogène dans les élevages de poulets biologiques. Cette étude fait partie des quelques études qui décrivent la présence des gènes de colonisation et de résistance aux antibiotiques dans une collection de souches issues uniquement du poulet. Elle a également remis en doute l’importance de certains gènes dans la colonisation. La caractérisation exhaustive des souches a également permis d’identifier de nouveaux gènes possiblement associés à la colonisation de poulet par C. jejuni. Finalement, elle a indiqué que l’utilisation d’un mélange d’huiles essentielles et d’acide organique encapsulés pouvait être efficace pour réduire la contamination des carcasses de poulet par C. jejuni et que son effet était souche-dépendant. / Campylobacter jejuni is the bacterium responsible for campylobacteriosis. It is a human pathogen of concern for public health. One of the transmission routes of this bacteria to humans is by the food chain through poultry meat products. The mechanisms involved in the commensal caecum chicken colonization by C. jejuni are poorly characterized, despite that increasing the knowledge on these mechanisms would allow a better control of the pathogen at the farm. The objectives of this study were to characterize the phenotypic and genetic factors affecting chicken colonization by C. jejuni and to identify news mechanisms involved in this process. Isolates, recovered from chicken caecal content sampled from conventional farms in 2003 and in 2008 as well as originating from organic farms sampled in 2009, were used. All strains were characterized for their antibiotic resistance profiles (AMR), autoagglutination and chemotaxis properties. Strains originating from conventional farms were further characterized for adhesion/invasion of primary chicken caecal cells. A new microarray was created to detect the presence of 254 genes and variants, all associated with chicken colonization or AMR. An association was made between the strains phenotypic properties and their gene content. Finally, strains possessing different phenotypic and genetic properties were used in a chicken colonization model to assess the efficacy of a novel feed additive to control chicken colonization by C. jejuni. Strains had variable phenotypic properties and these properties were not correlated, with the exception of adhesion and invasion. The microarray analysis revealed that gene presence was highly variable among the strains, especially for genes involved in the bacterial envelope, the flagella, the chemotaxis receptors and arsenic resistance. The 2003 and 2008 strains had similar phenotypic and genetic properties. Some genes, present in strains possessing higher or lower chicken colonization potentials, were identified. The feed additive was successful in reducing chicken carcass contamination but it increased bacterial caecal counts for some strains. C. jejuni was isolated from half of the sampled organic farms and strains originating from this type of production had low AMR and variable phenotypic properties.

Campylobacter jejuni is the commonest cause of bacterial foodborne gastroenteritis in industrialised countries. Despite its significance, it remains unclear how C. jejuni is disseminated in the environment, whether particular strains are more pathogenic than others, and by what routes this bacterium is transmitted to humans. One major factor hampering this knowledge is the lack of a standardised method for fingerprinting C. jejuni. Therefore, the overall aim of this project was to develop systematic and novel genotyping methods for C. jejuni. Chapter Three describes the use of single nucleotide polymorphisms (SNPs) derived from the multilocus sequence typing (MLST) database of C. jejuni and the closely related Campylobacter coli for genotyping these pathogens. The MLST database contains DNA sequence data for over 4000 strains, making it the largest comparative database available for these organisms. Using the in-house software package "Minimum SNPs", seven SNPs were identified from the C. jejuni/C. coli MLST database that gave a Simpson's Index of Diversity (D), or resolving power, of 0.98. An allele-specific real-time PCR method was developed and tested on 154 Australian C. jejuni and C. coli isolates. The major advantage of the seven SNPs over MLST is that they are cheaper, faster and simpler to interrogate than the sequence-based MLST method. When the SNP profiles were combined with sequencing of the rapidly evolving flaA short variable region (flaA SVR) locus, the genotype distributions were comparable to those obtained by MLST-flaA SVR. Recent technological advances have facilitated the characterisation of entire bacterial genomes using comparative genome hybridisation (CGH) microarrays. Chapter Four of this thesis explores the large volume of CGH data generated for C. jejuni and eight binary genes (genes present in some strains but absent in others) were identified that provided complete discrimination of 20 epidemiologically unrelated strains of C. jejuni. Real-time PCR assays were developed for the eight binary genes and tested on the Australian isolates. The results from this study showed that the SNP-binary assay provided a sufficient replacement for the more laborious MLST-flaA SVR sequencing method. The clustered regularly interspaced short palindromic repeat (CRISPR) region is comprised of tandem repeats, with one half of the repeat region highly conserved and the other half highly diverse in sequence. Recent advances in real-time PCR enabled the interrogation of these repeat regions in C. jejuni using high-resolution melt differentiation of PCR products. It was found that the CRISPR loci discriminated epidemiologically distinct isolates that were indistinguishable by the other typing methods (Chapter Five). Importantly, the combinatorial SNP-binary-CRISPR assay provided resolution comparable to the current 'gold standard' genotyping methodology, pulsed-field gel electrophoresis. Chapter Six describes a novel third module of "Minimum SNPs", 'Not-N', to identify genetic targets diagnostic for strain populations of interest from the remaining population. The applicability of Not-N was tested using bacterial and viral sequence databases. Due to the weakly clonal population structure of C. jejuni and C. coli, Not-N was inefficient at identifying small numbers of SNPs for the major MLST clonal complexes. In contrast, Not-N completely discriminated the 13 major subtypes of hepatitis C virus using 15 SNPs, and identified binary gene targets superior to those previously found for phylogenetic clades of C. jejuni, Yersinia enterocolitica and Clostridium difficile, demonstrating the utility of this additional module of "Minimum SNPs". Taken together, the presented work demonstrates the potentially far-reaching applications of novel and systematic genotyping assays to characterise bacterial pathogens with high accuracy and discriminatory power. This project has exploited known genetic diversity of C. jejuni to develop highly targeted assays that are akin to the resolution of the current 'gold standard' typing methods. By targeting differentially evolving genetic markers, an epidemiologically relevant, high-resolution fingerprint of the isolate in question can be determined at a fraction of the time, effort and cost of current genotyping procedures. The outcomes from this study will pave the way for improved diagnostics for many clinically significant pathogens as the concept of hierarchal combinatorial genotyping gains momentum amongst infectious disease specialists and public health-related agencies.