VERAS, H. N. Diagnóstico molecular e detecção de genes de virulência de Campylobacter jejuni em crianças com diarreia moderada a severa na cidade de Fortaleza – CE, Brasil. 2016 .129 f. Dissertação (Mestrado em Microbiologia Médica) - Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2016. / Submitted by Carolinda Oliveira (ppgmm@ufc.br) on 2017-06-29T13:12:19Z No. of bitstreams: 1 2016_dis_hnveras.pdf: 3228642 bytes, checksum: f6abd0d718a4279964069ed09e6aeaa8 (MD5) / Approved for entry into archive by Erika Fernandes (erikaleitefernandes@gmail.com) on 2017-06-29T13:30:59Z (GMT) No. of bitstreams: 1 2016_dis_hnveras.pdf: 3228642 bytes, checksum: f6abd0d718a4279964069ed09e6aeaa8 (MD5) / Made available in DSpace on 2017-06-29T13:30:59Z (GMT). No. of bitstreams: 1 2016_dis_hnveras.pdf: 3228642 bytes, checksum: f6abd0d718a4279964069ed09e6aeaa8 (MD5) Previous issue date: 2016-07-22 / Campylobacter spp. infections are considered one of the most common causes of bacterial gastroenteritis caused by contamination of water and food. Campylobacter jejuni is the most characterized specie, and the investigation of the epidemiology and virulence genes can elucidate some aspects of the microorganism pathogenicity. The aim of this study was to diagnose and identify the presence of virulence genes related to Campylobacter jejuni in children with moderate to severe diarrhea in the city of Fortaleza – CE, Brazil. This work is part of a project entitled “Diarrhea Enteric Card (DEC)”, which goal is to develop PCR-based multiplex diagnostic assays for bacterial enteric pathogens. That project was approved by the local and national ethical committees in Brazil (HIAS 80/06 and CONEPE 13523/2007, respectively). DNA was extracted directly from fecal samples isolated from 436 childrenwith moderate to severe diarrhea from May 2008 to April 2009, in Fortaleza, Ceará, Brazil. The diagnosis of C. jejuni was performed by conventional PCR using hipO gene. The detection of genes that encode proteins associated with virulence of C.jejuni was performed by uniplex and multiplex PCR techniques. C. jejuni was diagnosed in 14% (61/436) of the samples, presenting significant association among children aged 0-12 months (P = 0.0001) and children aged 12, 1-24 months. 51 positive samples for C. jejuniwere used for the detection of the virulence genes. The prevalence of theC. jejuni’s virulence-associated genes were detected in the following proportions of C. jejuni-positive DNA samples: flgE, (92.2%, 47/51) and flaA, (76.5%, 39/51), related to motility;cheW, (90.2%, 46/51); (cheA, 82.4%, 42/51) andcheR, (66.6%, 34/51), related to bacterial chemiotaxis; cadF, (100%, 51/51) andjlpA, (43.1%, 22/51), related to bacterial adhesion; ciaB, (96.1%, 49/51); iamA, (90.2%, 46/51); pldA, (45.1%, 23/51) and pVir(0%, 0/51), related to invasion; cdtABC, (94.1%, 48/51), related to cytolethal distending toxin (CDT); fur, (66.6%, 34/51); cfrA, (31.4%, 16/51) and ceuE, (21.7%, 11/51), related to bacterial iron transport and regulation; racR, (100%, 51/51); sodB, (96.1%, 49/51); dnaJ, (88.2%, 45/51) andkatA, (66.6%, 34/51), related to oxidative stress. The distribution profiles of C. jejuni’s virulence did not correspond to the patient’s clinical presentation of abdominal pain. But the presence of cfrA and dnaJ genes was correlated with fever (P=0,0214)and the presence of jlpA e katA gene were correlated with vomiting (P=0,0211) and pldA and ceuE genes was correlated with the presence of blood in stool (P=0,0013), this data suggests that relationships might be related to the severity of infection by this microorganism. New studies about the expression of proteins associated with the virulence genes must be carried out to better understand the pathobiology mechanisms of Campylobacter jejuni infections. / Infecções porCampylobacter spp. são consideradasuma das causas mais comuns de gastroenterite ocasionada porcontaminação de água e alimentos. Campylobacter jejuni é a espécie mais bem caracterizada, e a investigação da epidemiologia, e dos genes de virulência, podem elucidar algum aspecto da patogenicidade deste micro-organismo. O objetivo desse estudo foi diagnosticar e identificar a presença de genes de virulência relacionados à Campylobacter jejuni em crianças com diarreia moderada a severa na cidade de Fortaleza – CE, Brasil. Este estudo faz parte de um projeto intitulado “Diarrhea Enteric Card (DEC)”, que teve como objetivo desenvolver um ensaio de PCR multiplex para o diagnóstico de bactérias patogênicas. O projeto teve aprovação nos comitês de ética local e nacional no Brasil (HIAS 80/06 e CONEPE 13523/2007, respectivamente). A extração de DNA foi realizada diretamente das amostras fecais oriundas de 436 criançascom diarreia moderada a severa, durantes os meses de maio de 2008 a abril de 2009, na cidade de Fortaleza, Ceará, Brasil. O diagnóstico de C. jejuni foi realizado através de PCR convencional, utilizando o gene hipO. A detecção dos genes de virulência de C. jejuni foi realizada através das técnicas de PCR uniplex e multiplex. C. jejuni foi diagnóstico em 14% (61/436) das amostras, apresentando associação significante da presença do patógeno em crianças com idade entre 0-12 meses (P=0,0001) e com idade entre 12,1-24 meses (P=0,0427). A detecção dos genes de virulência foi realizada em 51 amostras positivas para a C. jejuni.A prevalência dos genes associados à virulência de C. jejuni foram detectados seguindo a proporção de amostras positivas:flgE, (92,2%, 47/51) eflaA, (76.5%, 39/51), relacionados à motilidade;cheW (90,2%, 46/51); cheA, (82,4%, 42/51) echeR,(66,6%, 34/51), relacionados à quimiotaxia bacteriana; cadF, (100%, 51/51) ejlpA, (43,1%, 22/51), relacionados à adesão; ciaB, (96,1%, 49/51); iamA, (90,2%, 46/51); pldA, (45,1, 23/51) epVir (0%, 0/51), relacionados a invasão; cdtABC, (94,1%, 48/51), relacionados à toxina citoletal distensora (CDT); fur, (66,6%, 34/51); cfrA, (31,4%, 16/51) eceuE, (21,7%, 11/51), relacionados ao transporte e regulação de ferro; racR, (100%, 51/51),sodB, (96,1%, 49/51),dnaJ, (88,2%, 45/51) ekatA, (66,6%, 34/51), relacionado à sobrevivência da bactéria e ao estresse oxidativo. A distribuição dos perfis de genes de virulência de C. jejuni não correspondeu com o parâmetro clínico de dor abdominal, mas houve a associação dos genescfrA e dnaJ com a presença de febre (P=0,0214), dos genes jlpA e katA com a presença de vômito (P=0,0211), e dos genespldA e ceuE com a presença de sangue nas fezes(P= 0,0013), sugerindo que essas relações poderiam estar associadas a severidade da infecção causada pelo micro-organismo. Novos estudos sobre a expressão de proteínas relacionadas aos genes de virulência devem ser realizados, para que assim haja uma melhor compreensão sobre os mecanismos da patobiologia das infecções causadas porC. jejuni.

Campylobacter jejuni is a prevalent cause of bacterial gastroenteritis in humans worldwide. The mechanism by which C. jejuni survives stomach acidity remains unknown. In this study, we have demonstrated that the ferric uptake regulator Fur plays an important role in Campylobacter acid survival. C. jejuni with a fur deletion was more sensitive to acid than the wild-type. Profiling the acid stimulon of the C. jejuni ∆fur mutant allowed us to uncover Fur-regulated genes under acidic conditions. The up-regulation of heat shock genes and the down-regulation of genes involved in flagellar and cell envelope biogenesis in the fur mutant highlight the importance of Fur in Campylobacter acid survival. Furthermore, prior exposure of C. jejuni to acid increased its capacity to survive other stresses, such as oxidative stress. This enhanced survival in the presence of oxidative stress was shown to be Fur-dependent through the regulation of catalase katA expression. Interestingly, Fur-mediated repression of katA was alleviated under low-pH conditions, allowing for higher catalase expression and defense against oxidative stress. Additionally, the transcriptome of C. jejuni under acidic conditions revealed that many genes involved in Campylobacter pathogenesis were differentially expressed. Prior exposure of C. jejuni to acid significantly increased its adherence to and invasion of human epithelial cells. Furthermore, in vivo experiments using Galleria mellonella larvae showed that acid exposure markedly enhanced Campylobacter virulence potential. In conclusion, this study demonstrates that the ferric uptake regulator Fur is a potential regulator of Campylobacter acid survival and cross-protection against other stresses. Moreover, our results suggest that the obligate passage of C. jejuni through the stomach acid barrier modulates the expression of its virulence factors and predisposes the bacterium for efficient gut colonization.

Campylobacter jejuni is an enteric bacterium that causes human gastroenteritis worldwide. Some C. jejuni strains exhibiting human ganglioside-like lipooligosaccharide (LOS) structures, such as GM1 ganglioside, can induce an autoimmune neuropathy of the peripheral nervous system known as the Guillain-Barré syndrome (GBS). This GBS-inducible determinant is encoded by a gene cluster, which shows a high degree of variation among C. jejuni strains. The experiments presented in this thesis were conducted to give a better insight into the LOS synthesis genes in relation to the pathophysiology of C. jejuni. Firstly, a C. jejuni strain without GM1-like molecules was shown to be able to take up large DNA fragments, including LOS synthesis genes, from a strain expressing GM1-like molecules and consequently be transformed into a number of potential GBS-inducible transformants, which exhibited a high degree of genetic and phenotypic diversity. The ability of C. jejuni to take up and integrate foreign DNA explains the genome plasticity observed in this pathogen. Secondly, while attempting to analyse transcription of the LOS gene cluster, neither published methods nor any commercially available kits for RNA isolation could produce DNA-free RNA from C. jejuni. Combinations of these methods were trialled and only the combination of RNAzolB, TURBO DNase treatment, and acid phenol extraction was able to produce DNA-free RNA. The RNA isolated from most C. jejuni strains showed different RNA patterns to that of other bacteria. In addition the RNA from C. jejuni seemed closely associated with DNA compaired to RNA from other organisms. This might be caused by species-specific DNA conformation or chromatin structure. Thirdly, bidirectional transcription was observed in the LOS gene cluster. Both DNA strands were transcribed but transcription of the non-coding strands was at a lower rate, and both sense and antisense transcripts of each LOS gene tested were responsive to acid stress. This unusual transcription might have a potential effect on the expression of the GBS-inducing determinant. Finally, one of the LOS genes, the htrB gene, was further analysed. It was shown that expression of the htrB gene affects morphology, viability, growth ability, and sensitivity to stress environments. These results showed that the LOS molecule of C. jejuni is involved in many processes and is an important molecule for survival.

Thesis (Ph. D.)--Michigan State University. Dept. of Microbiology and Molecular Genetics, 2007. / Title from PDF t.p. (viewed on Apr. 16, 2009) Includes bibliographical references. Also issued in print.

Thesis (Ph. D.)--University of North Texas, May, 2009. / Title from title page display. Includes bibliographical references.

Campylobacter jejuni is one of the most frequent causes of gastroenteritis in both the developing and the developed world. Understanding C. jejuni biology is paramount to reducing the amount of Campylobacter jejuni in the food chain, however our understanding of the regulatory networks that enable Campylobacter jejuni to successfully colonize various hosts remains incomplete. Campylobacter jejuni has an absolute requirement for iron in order to grow as iron catalyzes a wide range of essential biochemical reactions. In contrast to many Gram negative bacteria, the genome of Campylobacter jejuni contains two iron activated Fur-family transcriptional regulators, Fur and PerR, which are primarily responsible for regulating iron homeostasis and oxidative stress respectively. We have used an integrated approach that combines genome wide technologies (ChIP-chip, RNA-seq) and structural studies to define the role of iron, Fur and PerR in Campylobacter jejuni. These studies have demonstrated that apo-Fur directly regulates gene transcription in Campylobacter jejuni, identified novel ncRNAs that are Fur and/or iron responsive, and revealed that the Fur and PerR regulons are more extensive than previously characterized. These results provide further insight into the surprisingly complex regulatory networks that allow Campylobacter jejuni to be a successful gut pathogen.

Campylobacter jejuni is one of the most common causes of foodborne diarrheal illness in the U.S. and worldwide. (1-2). C. jejuni infection in humans is most often attributed to undercooked poultry (3-6). However, since 2001, the Centers for Disease Control (CDC) has confirmed 9 outbreaks of campylobacteriosis linked to consumption of beef and beef products, resulting in 297 illnesses and 10 hospitalizations, and cattle isolates have been linked to other human infections (7-10). Because Campylobacter infection is generally sporadic, and not all cases are linked to poultry, other animal reservoirs such as beef likely exist. Because beef is not commonly considered a significant source of Campylobacter, interventions regarding beef cattle are generally geared toward other pathogens, such as E. coli O157:H7. Interventions to prevent Campylobacter spread in poultry houses include reducing flock colonization and bacterial loads, (11), as well as interventions directly targeting consumer behavior. Despite these efforts, many countries have not been able to reduce the prevalence of Campylobacter in poultry. The goals of this research were to 1) determine Campylobacter loads in broilers at poultry farms and processing houses through the 3-tube MPN method, and determine baseline data for poultry production systems, 2) describe temporal relationships and prevalence of Campylobacter strains in a potentially underrepresented host/environment (cattle feedlot environment), and 3) test the efficacy of natural, plant derived compounds against C. jejuni on meat. Our results show that there is a significant positive association between pre-harvest and post-harvest Campylobacter loads in poultry, with Campylobacter levels during the final step of processing remaining at infectious levels. Beef cattle represent another potential and not well-described source of campylobacteriosis, as beef cattle and their environment become rapidly contaminated with Campylobacter from weaning through processing, and cross-contamination of carcasses is possible. This research also determined that natural plant extracts of cinnamon and oregano essential oils, when added to edible films, reduced surface contamination of retail poultry meat with C. jejuni, and thus may be a useful post-harvest intervention for future use in packaging of retail meat with a high risk of Campylobacter contamination.

The transcriptome is defined as the set of all RNA molecules transcribed in a cell. These include protein-coding messenger RNAs (mRNAs) as well as non-coding RNAs, such as ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), and small non-coding RNAs (sRNAs). sRNAs are known to play an important role in regulating gene expression and virulence in pathogens. In this thesis, the transcriptome of the food-borne pathogen Campylobacter jejuni was characterized at single nucleotide resolution by use of next-generation sequencing approaches. The first genome of a C. jejuni strain was published in the year 2000. However, its transcriptome remained uncharacterized at large. C. jejuni can survive in a variety of ecological niches and hosts. However, how strain-specific transcriptional changes contribute to such adaptation is not known. In this study, the global transcriptome maps of four closely related C. jejuni strains were defined using a differential RNA-seq (dRNA-seq) approach. This analysis also included a novel automated method to annotate the transcriptional start sites (TSS) at a genome-wide scale. Next, the transcriptomes of four strains were simultaneously mapped and compared by the use of a common coordinate system derived from whole-genome alignment, termed as SuperGenome. This approach helped to refine the promoter maps by comparison of TSS within strains. Most of the TSS were found to be conserved among all four strains, but some single-nucleotide-polymorphisms (SNPs) around promoter regions led to strain-specific transcriptional output. Most of these SNPs altered transcription only slightly, but some others led to a complete abrogation of transcription leading to differential molecular phenotypes. These in turn might help the strains to adapt to their specific host or microniche. The transcriptome also unveiled a plethora of sRNAs, some of which were conserved among the four strains while others were strain specific. Furthermore, a Cas9-dependent minimal type-II CRISPR-Cas system with only three Cas genes and multiple promoters to drive the transcription of the CRISPR locus was also characterized in C. jejuni using the dRNA-seq dataset. Apart from sRNAs, the role of global RNA binding proteins (RBPs) is also unclear in C. jejuni. Aided by the global transcriptome data, the role of RBPs in post-transcriptional regulation of C. jejuni was studied at a global scale. Two of the most widely studied RNA binding proteins in bacteria are Hfq and CsrA. The RNA interactome of the translational regulator CsrA was defined using another global deep-sequencing technique that combines co-immunoprecipitation (coIP) with RNA sequencing (RIP-seq). Using this interactome dataset, the direct targets of this widespread global post-transcriptional regulator were defined, revealing a significant enrichment for mRNAs encoding genes involved in flagella biosynthesis. Unlike Gammaproteobacteria, where sRNAs such as CsrB/C, antagonize CsrA activity, no sRNAs were enriched in the CsrA-coIP in C. jejuni, indicating absence of any sRNA antagonists and novel modes of CsrA activity regulation. Instead, the CsrA regulatory pathway revealed flaA mRNA, encoding the major flagellin, as a dual-function mRNA. flaA mRNA was the main target of CsrA but it also served to antagonize CsrA activity along with the protein antagonist FliW previously identified in the Gram-positive bacterium Bacillus subtilis. Furthermore, this regulatory mRNA was also shown in this thesis to localize to the poles of elongating C. jejuni cells in a translation-dependent manner. It was also shown that this localization is dependent on the CsrA-FliW regulon, which controls the translation of flaA mRNA. The role and mechanism of flaA mRNA localization or mRNA localization in general is not yet clear in bacteria when compared to their eukaryotic counterparts. Overall, this study provides first insights into riboregulation of the bacterial pathogen C. jejuni. The work presented in this thesis unveils several novel modes of riboregulation in C. jejuni, which could be applicable more generally. Moreover, this study also lays out several unsolved intriguing questions, which may pave the way for interesting studies to come. / Das Transkriptom ist definiert als die Summe aller RNA-Moleküle, die in einer Zelle transkribiert werden. Hierzu gehören sowohl protein-kodierende Boten-RNAs (mRNAs für „messenger RNAs“), als auch nicht-kodierende RNAs, wie ribosomale RNAs (rRNAs), transfer RNAs (tRNAs) und kleine nicht-kodierende RNAs (sRNAs für „small RNAs“). Diese sRNAs spielen eine wichtige Rolle in der Regulierung von Genexpression und Virulenz von Pathogenen. In der vorliegenden Arbeit wurde das Transkriptom des Lebensmittelkeims Campylobacter jejuni mit Hilfe von Next-Generation-Sequencing-Methoden charakterisiert, welche eine Auflösung des Transkriptoms auf Einzelnukleotid-Ebene ermöglichen. Obwohl eine erste Genomsequenz für C. jejuni bereits im Jahr 2000 veröffentlicht wurde, war das Transkriptom bisher größtenteils uncharakterisiert. C. jejuni besitzt die Fähigkeit in vielen ökologischen Nischen und Wirten überleben zu können. Es ist jedoch bislang unbekannt, wie stammspezifische Veränderungen des Transkriptoms zu dieser Adaption beitragen. Mittels eines differenziellen RNA-Sequenzierungsansatzes wurden in dieser Arbeit globale Transkriptomkarten von vier nahverwandten C. jejuni Stämmen erstellt. Diese Analyse beinhaltet auch eine neue automatisierte Methode zur genomweiten Identifizierung von Transkriptionsstartstellen (TSS). Anschließend wurde aus den Genomsequenzen der vier Campylobacter Stämme ein SuperGenom erstellt. Dieses wiederum diente als Referenz, anhand dessen die Transkriptome kartiert und miteinander verglichen werden konnten. Dieser Ansatz ermöglichte eine verfeinerte Kartierung der Promotoren mittels des Vergleichs verschiedener Stämme. Die meisten TSS waren innerhalb der vier Stämme konserviert. Allerdings kam es durch SNPs („single-nucleotide polymorphisms“) in den Promoterregionen zu stammspezifischem Transkriptoutput. Die meisten dieser SNPs hatten nur geringe Veränderungen der Transkription zur Folge. Manche jedoch führten zu einem kompletten Verlust der Transkription und damit zu verschiedenen molekularen Phänotypen. Diese wiederum könnten es den verschiedenen Stämmen ermöglichen, sich an ihre spezifische Wirts- oder Mikronische anzupassen. Das Transkriptom wies auch eine Fülle von sRNAs auf, von denen manche in allen vier Stämmen konserviert, andere jedoch stammspezifisch waren. Zudem wurde mittels des C. jejuni-dRNA-seq-Datensatzes ein minimales Cas9-abhängiges CRISPR-Cas-System des Typs II entdeckt. Dieses beinhaltet lediglich drei Cas-Gene, jedoch mehrere Promotoren, die die Expression des CRISPR-Lokus antreiben. Neben der Funktion von sRNAs ist auch die Rolle globaler RNA-Bindeproteine (RBPs) in C. jejuni weitestgehend unklar. Mithilfe der Transkriptomdaten wurde die Rolle von RBPs in der posttranskriptionellen Regulierung in C. jejuni untersucht. Zwei der am besten untersuchten RNA-Bindeproteine in Bakterien sind Hfq und CsrA. Das RNA-Interaktom des Translationsregulators CsrA wurde mittels eines weiteren globalen Deep-Squencing-Ansatzes definiert. Bei dieser Methode werden Coimmunopräzipitation (coIP) und RNA-Sequenzierung zum so genannten RIP-seq kombiniert. Mithilfe dieses Interaktionsdatensatzes wurden die Zielgene dieses weitverbreiteten, globalen posttranskriptionellen Regulators definiert. Hierbei wurde eine signifikante Anreicherung von mRNAs, die in die Biosynthese von Flagellen involviert sind, erkennbar. Anders als in Gammaproteobakterien, in denen sRNAs wie CsrB und CsrC die CsrA-Aktivität antagonisieren, wurden in C. jejuni keine sRNAs in der CsrA-CoIP angereichert. Dies deutet auf das Fehlen jeglicher sRNA-Antagonisten, und damit auf eine neue Art der CsrA-Aktivitätskontrolle hin. Anstelle der sRNAs wurde die flaA mRNA, welche für das Hauptflagellin kodiert, als mRNA mit dualer Funktion identifiziert. Sie ist zum einen das Hauptzielgen von CsrA, fungiert aber gleichzeitig, zusammen mit dem Protein FliW, als Antagonist von CsrA. FliW wurde bereits zuvor in dem Grampositiven Bakterium Bacillus subtilis identifiziert. In dieser Arbeit konnte zudem gezeigt werden, dass die regulatorische flaA mRNA translationsabhängig an den Polen der wachsenden C. jejuni-Zellen lokalisiert ist. Außerdem war zu erkennen, dass diese Lokalisierung abhängig von dem CsrA-FliW-Regulon stattfindet, welches die Translation der flaA-mRNA kontrolliert. Im Gegensatz zu Eukaryoten ist die Rolle, die die Lokalisation der flaA-mRNA, oder bakterieller mRNA im Allgemeinen, spielt, sowie der Mechanismus, der zu dieser Lokalisierung führt, bisher noch unklar. Zusammenfassend ermöglicht diese Arbeit einen ersten Einblick in die Riboregulierung des bakteriellen Pathogens C. jejuni. Es konnten einige neue Mechanismen dieser Art der Regulierung aufgedeckt werden, welche auch allgemeine Gültigkeit finden könnten. Zudem werden in dieser Arbeit neue, faszinierende Fragen aufgeworfen, die den Weg für weitere interessante Studien bereiten.

Thermophilic Campylobacter species such as Campylobacter jejuni and Campylobacter coli are recognised worldwide as major causes of acute gastroenteritis in humans. Campylobacteriosis is frequently a mild to moderate self-limited illness and most cases do not require antimicrobial therapy; antimicrobial therapy is necessary for patients with systemic Campylobacter infections, for patients with severe disease, or for immunosuppressed patients. Antimicrobial susceptibility testing of Campylobacter species using disk diffusion currently is not standardised by the National Committee for Clinical Laboratory Standards (NCCLS), however, in order to monitor the prevalence of antimicrobial resistance in Campylobacter species, there is a need for standardised or calibrated methods of susceptibility testing. Initially, 90 human clinical isolates of thermophilic Campylobacter species from Southeast Queensland, Australia, were screened for resistance to ampicillin, erythromycin and tetracycline using the disk diffusion susceptibility testing method. Levels of resistance were then determined using E test MIC and agar dilution methods to determine the reliability of disk diffusion results. Results of the disk diffusion testing showed 87 (97%) isolates resistant to ampicillin, 14 (16%) isolates were resistant to tetracycline and three (3.4%) isolates were resistant to erythromycin. Results of disk diffusion testing showed 100% correlation (+1 log2 dilution) with agar dilution for erythromycin and tetracycline, and 77% for ampicillin. E test showed 68% correlation with agar dilution for ampicillin, 100% for erythromycin and 64% for tetracycline. These data suggest that disk diffusion susceptibility testing may be used to screen thermophilic Campylobacter spp. for putative resistance to erythromycin and tetracycline and that the incidence of resistance of Campylobacter spp. to erythromycin and tetracycline is low in Southeast Queensland, Australia. Agar dilution remains the most accurate method for determination of ampicillin susceptibility. Numerical analyses of restriction endonuclease (RE) fragment profiles were performed to elucidate relatedness of the antibiotic resistant isolates and the results suggested a high level of isolate variation. The role of the B-lactamase in the resistance of C. jejuni to various B-lactams has been well documented and B-lactamase production in C. jejuni has been reported in 83-93% of strains. The expression and characterisation of the Campylobacter B-lactamase, however, has not been described. In this work, standard cloning techniques utilising a high-copy number E. coli cloning vector and a previously described E. coli-Campylobacter shuttle cloning vector were unsuccessful in isolation and expression of the C. jejuni B-lactamase gene in E. coli, possibly due to a lack of expression of the campylobacter gene in its host or low efficiency of transformation. Therefore, in order to facilitate the isolation, expression and characterisation of the C. jejuni B-lactamase gene, it was necessary to construct a new E.coli-Campylobacter shuttle cloning vector for the purposes of expressing the C. jejuni B-lactamase in Campylobacter. To aid in the construction of the vector, the sequence and genetic organisation of a 4.0-kb cryptic plasmid, termed pCJ419, identified in a human clinical isolate of C. jejuni was determined. Plasmid pCJ419 is a circular molecule of 4013 bp and contains four open reading frames (ORFs), the products of which share significant sequence similarity with putative proteins from known C. jejuni and C. coli plasmids. ORF-1 encodes a putative mobilisation protein (Mob); ORF-2 and ORF-3 encode proteins which have high identity to putative RepA and RepB proteins, respectively, of known C. jejuni and C. coli plasmids. ORF-4 encodes a protein which has high identity to a hypothetical protein of unknown function, Cjp32, previously described in a pVir plasmid of C. jejuni. Tandem repeating sequences typical of a plasmid replication origin (ori) were identified upstream of the DNA sequences encoding putative replication initiation proteins RepA and RepB. An E. coli-Campylobacter shuttle cloning vector, pGU0202, was constructed using plasmid pMW2 which harbours a Campylobacter-derived kanamycin-resistance gene, aphA(3’)-III. The sequences encoding pCJ419 mob, repA and repB were inserted upstream of aphA(3’)-III resulting in a stable construct of 6174 bp that was used successfully to transform both E. coli and Campylobacter. Subsequently, a novel molecular class D ?-lactamase gene, blaOXA-61, from a B-lactamase-positive, ampicillin-resistant (MIC 64 mg l-1), clinical strain of Campylobacter jejuni, strain GC015 was isolated, cloned and characterized using the newly constructed shuttle vector pGU0202. An open reading frame of 774 bp was identified on a ClaI genomic fragment of 2.2 kb and encodes a protein of 257 amino acids. Conserved motifs composed of identical amino acids typical of penicillin-recognising proteins and specific class D motifs were identified. blaOXA-61 was cloned into the shuttle cloning vector pGU0202 and expressed in B-lactamase-negative, ampicillin-susceptible C. jejuni and E. coli. A conserved 122-bp sequence directly upstream of blaOXA-61 was identified and shown to be required in cis for high-level resistance of Campylobacter to the penicillins although blaOXA-61 expressed only at low levels in E.coli. Southern hybridisation analysis demonstrated that the bla gene was chromosomally encoded and present on the same BglII and ClaI-digested genomic DNA fragments from various strains of Campylobacter with ampicillin MICs of between 4 and 64 mg l-1. In addition, DNA fragments encoding two putative zinc-dependent hydrolases from the metallo-B-lactamase superfamily, designated GLX2-1 and GLX2-2, were identified in a clinical isolate of Campylobacter jejuni, strain 012, cloned and sequenced. A strictly conserved motif, -H-X-H-X-D-, characteristic of the metallo- B-lactamase superfamily of proteins, including the class B metallo- B-lactamases, was identified in both proteins although functional B-lactamase could not be expressed in either E. coli or C. coli transformed using the C. jejuni hydrolase-containing shuttle vector pGU0202. Further work is warranted to determine the exact function of these proteins.

Campylobacter jejuni is one of the leading bacterial causes of human gastroenteritis world wide and has been linked to several severe complications including autoimmune syndromes which can result in paralysis. Despite being the subject of much study, C. jejuni remains a major public health burden in both developing and developed nations. There is currently no vaccine available for protection against this pathogen and the mechanisms important for C. jejuni pathogenesis are not fully defined. This study has employed a range of experimental approaches to investigate the molecular mechanisms involved in C. jejuni pathogenesis. Lipooligosaccharides (LOSs) are surface structures and known virulence factors of C. jejuni which are involved in serum resistance, resistance to phagocytic killing, endotoxicity and adhesion. Mutagenesis studies targeting the putative LOS biosynthesis genes wlaRF, wlaTA, wlaTB, wlaTC and waaV were performed in order to characterise the proteins encoded by each of these six genes and assess their potential role in C. jejuni pathogenesis in vitro. The gene product of wlaTA was found to be essential for C. jejuni survival and therefore a knock out mutant could not be generated. Phenotypic characterisation of four knock-out mutants confirmed that each gene contributed to the construction of the LOS molecule as all four mutants produced a truncated LOS moiety and altered their immunoreactivity. Further analysis determined that the production of complete LOSs was important for C. jejuni to invade and adhere to both human and chicken cells in vitro. This study identified a link between the inactivation of two LOS biosynthesis genes and the loss of motility, another important virulence factor. A major source of human C. jejuni infection is contact with contaminated poultry. However, C. jejuni exists as a commensal in chickens. It is currently not known why C. jejuni is pathogenic to humans and not to chickens and the differences between these two hosts represent pathogenic and non-pathogenic environments respectively. These environmental differences were exploited in this study. The four conditions investigated were temperature, blood, bile and host cells in vitro. Five different C. jejuni strains (NCTC11168, 81116, HB93-13, a recent human enteritis isolate and a recent chicken isolate) were subjected to modelled