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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.

QUETZ, Josiane da Silva. Diagnóstico microbiológico, imunoenzimático e molecular e perfil de genes associados à virulência de Campylobacter. 2013. 166 f. Tese (Doutorado em Farmacologia) – Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2013. / Submitted by denise santos (denise.santos@ufc.br) on 2016-03-23T13:04:24Z No. of bitstreams: 1 2013_tese_jsquetz.pdf: 3530958 bytes, checksum: e95cebb86634d9fd6d6bd11bdbfcb145 (MD5) / Approved for entry into archive by denise santos(denise.santos@ufc.br) on 2016-03-23T13:17:51Z (GMT) No. of bitstreams: 1 2013_tese_jsquetz.pdf: 3530958 bytes, checksum: e95cebb86634d9fd6d6bd11bdbfcb145 (MD5) / Made available in DSpace on 2016-03-23T13:17:51Z (GMT). No. of bitstreams: 1 2013_tese_jsquetz.pdf: 3530958 bytes, checksum: e95cebb86634d9fd6d6bd11bdbfcb145 (MD5) Previous issue date: 2013 / Campylobacter sp. is an important cause of food-borne gastroenteritis with high incidence in children living in developing countries. However, the specific diagnosis of its etiology remains as a challenge, since conventional diagnosis by culture is now challenged by molecular and immunoenzymatic methods, which have greater sensitivity. We postulate that the knowledge of its virulence and specific diagnosis may assist in identifying and potentially controling campylobacteriosis in childhood. We determined the etiology of Campylobacter sp. associated diarrhea, in a cross-sectional study of diarrhea in children aged 0-36 months from the urban area of Fortaleza, Ceará, Brazil, who required emergency medical care because of diarrheal disease. After ethical approval of the study, a questionnaire was applied to describe the clinical conditions presented by each child at the time of admission. DNA was extracted directly from fecal samples collected from 226 children. For the determination of the etiologic agent we used molecular diagnostics (PCR and RT-PCR) and diagnostic immunoassay (ELISA), besides the detection of virulence associated genes of C. jejuni (PCR). Campylobacter sp. was found in 8.9% (20/225) of the samples by conventional microbiological diagnosis. C. jejuni and C. coli were detected in 19.5% (44/226) and 1.3% (3/226) of the diarrheic samples, respectively. The diagnostic RT-PCR and ELISA reached 26.7% (60/225) and 37.9% (58/153) of positivity, respectively. When considering the combination of diagnostic (positive in microbiological diagnosis or immunoassay and at least one of the molecular tests) the prevalence was 16.4% (37/226). The agreement between the tests used for diagnosis was moderate to regular, according to Kappa index. The presence of C. jejuni´s virulence-associated genes that encode proteins related to the pathogenesis of micro-organism were detected in the following proportions of C. jejuni-positive DNA samples: flaA, 79.5% (35/44); racR, 97.7% (43/44) and dnaJ, 88.6% (39/44) – related to bacterial adhesion and colonization; ciaB, 97.7 % (43/44); pldA, 45.4% (20/44) and pVir 0% (0/44) – related to invasion, and cdtABC in 95.4% (42/44) of samples related to citoletal distending toxin (CDT). Specific signs and symptoms such as blood in the stool, vomiting, fever and/or abdominal pain, although quite frequent, were not associated with the detection of C. jejuni. The distribution profile of C. jejuni´s virulence genes was not correlated with the clinical presentation of the disease, even when this profile was categorized according to the function of the proteins encoded by the genes, which leads us to believe that other factors, perhaps related to host susceptibility, may be more important than genetic variability of the microorganism. We conclude that Campylobacter sp. was detected in a significant percentage of the children 0-36 months with diarrhea, especially when the diagnostic methods were used in combination. In general, the virulence genes were detected in a high proportion of C. jejuni-positive samples, although the invasion-related genes have been found less frequently. Our data corroborates findings from other groups on the need to revise the diagnostic for Campylobacter sp. towards the inclusion of more sensitive and species-specific methods, as well as search for extra markers for intestinal inflammation and predictors of negative culture. / Campylobacter sp. é uma importante causa de enterite de origem alimentar com alta incidência na população infantil de países em desenvolvimento. No entanto, o diagnóstico específico de sua etiologia segue como um desafio, visto que métodos moleculares e imunoenzimáticos têm se mostrado mais sensíveis. Postulamos que o conhecimento de sua virulência e o diagnóstico específico possam ajudar na identificação e potencial controle da campilobacteriose na infância. Foi determinada a etiologia de diarreia por Campylobacter sp., em um estudo transversal sobre diarreia em crianças de 0-36 meses residentes da área urbana de Fortaleza, Ceará, Brasil, que necessitaram de atendimento médico de urgência por causa de doença diarreica. Após a aprovação ética do estudo, um questionário foi aplicado para qualificar as condições clínicas apresentadas por cada criança no momento da admissão. O DNA foi extraído diretamente de amostras fecais coletadas de 226 crianças. Para a detecção do agente etiológico, utilizamos diagnóstico molecular (PCR e RT-PCR) e diagnóstico imunoenzimático (ELISA), além da detecção de genes associados à virulência de C. jejuni (PCR). Campylobacter sp. foi encontrado em 8,9% (20/225) das amostras, por diagnóstico microbiológico convencional. C. jejuni e C. coli foram detectados em 19,5% (44/226) e 1,3% (3/226) das amostras diarreicas, respectivamente, por PCR. Os diagnósticos por RT-PCR e ELISA alcançaram 26,7% (60/225) e 37,9% (58/153), respectivamente. Quando considerada a combinação de diagnósticos (positividade no diagnóstico microbiológico ou no imunoenzimático e ao menos em um dos testes moleculares) a prevalência encontrada foi de 16,4% (37/226). A concordância entre os testes para diagnóstico utilizados foi de moderada a regular, de acordo com o índice de Kappa. Genes associados à virulência foram detectados nas seguintes proporções de amostras positivas para C. jejuni: flaA, 79,5% (35/44); racR, 97,7% (43/44) e dnaJ, 88,6% (39/44) – relacionados à adesão bacteriana e colonização; ciaB, 97,7% (43/44); pldA, 45,4% (20/44) e pVir 0% (0/44) – relacionados à invasão; e cdtABC em 95,4% (42/44) das amostras, operon relacionado à produção da toxina citoletal distensora (CDT). Sinais e sintomas específicos, tais como sangue nas fezes, vômito, febre e/ou dor abdominal, apesar de bastante frequentes, não foram associados com a detecção de C. jejuni. O perfil de distribuição dos genes de virulência de C. jejuni não apresentou correlação com a apresentação clínica da doença, mesmo quando tal perfil foi categorizado de acordo com a função das proteínas codificadas pelos genes, o que nos leva a crer que outros fatores, talvez relacionados à susceptibilidade do hospedeiro, possam ser mais importantes do que a variabilidade genética do micro-organismo. Concluímos que Campylobacter sp. foi detectado em percentual relevante da população estudada, principalmente quando os métodos diagnósticos foram utilizados de forma combinada. Em geral, os genes de virulência foram detectados em uma alta proporção das amostras positivas para C. jejuni, embora os genes relacionados à invasão tenham sido menos frequentemente encontrados. Corroboramos dados de outros grupos sobre a necessidade de revisão do diagnóstico para Campylobacter sp. em prol da inclusão de metodologias mais sensíveis e espécie-específicas, além da busca por marcadores para inflamação intestinal e fatores preditivos de cultura negativa.

No description available.

Campylobacter is the leading cause of bacterial food-borne diarrhoeal disease in the developed world and a significant cause of infant morbidity and mortality in developing countries. Epidemiological studies implicate poultry as a key source of infection, with up to 80% of human cases being attributable to the avian reservoir. An effective vaccine for broilers is predicted to limit the incidence of human campylobacteriosis. Vaccination of chickens with CjaA, either in recombinant form or vectored in live-attenuated Salmonella, has been reported to significantly reduce caecal colonisation by C. jejuni, with more invasive carriers eliciting greater protection. However, protection remains modest and is slow to develop. I therefore sought to improve such vaccines, first by vectoring codon-optimised CjaA in a licensed avian pathogenic E. coli ΔaroA vaccine. In two independent trials, White Leghorn birds were vaccinated subcutaneously on the day of hatch and 14 days later then challenged with C. jejuni M1 at 28 days post-hatch. No protection was observed despite significant induction of CjaA-specific serum IgY, however, a previously described S. Typhimurium ΔaroA vaccine vectoring CjaA also failed to protect. Owing to the variability observed with live CjaA-based vaccines in these and previous studies, other candidate antigens were sought and evaluated as subunits. Twenty-one candidate C. jejuni antigens were cloned and expressed as glutathione-S-transferase (GST) fusions. Nine of these could be purified in adequate soluble quantities to be tested in vivo. The intervals of vaccination and challenge were as above, with GST alone or GSTCjaA acting as negative and positive controls, respectively. Each antigen was administered subcutaneously in TiterMax Gold® adjuvant at the molar equivalent of the doses of GSTCjaA. Repeated testing of initially promising candidates revealed that, when averaged across three independent trials, GST-SodB and GST-FliD induced statistically significant reductions in caecal colonisation of 1-2 log10 colony-forming units of C. jejuni at 48 and 56 days post-hatch compared to negative controls. Induction of antigen-specific serum IgY was measured by enzyme linked-immunosorbent assays using maltose-binding protein fusions to each antigen. This revealed significant induction of antigen specific serum IgY for the majority of the antigens tested, even when no protection was observed. In the SodB- and FliD-vaccinated groups, the peak of antigen-specific serum IgY was not coincident with the onset of protection and the fold-change in specific IgY levels in individual birds did not correlate with caecal Campylobacter numbers. Furthermore, sera from SodB-vaccinated birds failed to detect SodB in the outer membrane or surface of Campylobacter cells, indicating that SodB-specific antibodies are unlikely to be neutralising. Taken together, these studies identified two novel protective antigens that, with further optimisation, could form part of an anti-Campylobacter vaccine for broilers. However further studies are required to define the nature and consequences of immune responses required for protection.

No description available.

Campylobacter jejuni is a human pathogen that causes severe diarrhea! disease. However, our understanding of C. jejuni virulence mechanisms and survival during disease and transmission remains limited. Amino acid ATP Binding Cassette (AA-ABC) transporters in C. jejuni have been proposed as being important for bacterial physiology and pathogenesis. We have investigated a novel AA-ABC transporter system, encoded by cj0467-9, by generating targeted deletions of cj0467 (membrane transport component) and cj0469 (ATPase component) in C. jejuni 81-176. Analyses described herein have led us to designate these genes paqP and paqQ, respectively [pathogenesis-ssociated glutamine (q) ABC transporter permease () and ATPase (Q)]. We found that loss of either component resulted in amino acid uptake defects, most notably diminished glutamine uptake. Both ΔpaqP and ΔpaqQ mutants also exhibited a surprising but significant increase in short-term intracellular survival in macrophages and epithelial cells. Levels of resistance to a series of environmental and in vivo stresses were examined. Both mutants were hyper-resistant to aerobic and oxidative stress, and while ΔpaqP was also hyper-resistant to heat and osmotic shock, ΔpaqQ was more susceptible than wild-type to the latter two stresses. Annexin-V staining coupled with fluorescence microscopy revealed that macrophages infected with the ΔpaqP and ΔpaqQ mutants underwent a lower level of apoptosis than cells infected with wild-type bacteria. Macrophages infected with the mutant strains exhibited a transient decrease in ERK activation compared to wild type-infected macrophages, potentially explaining the reduced apoptosis phenotype. The ΔpaqP mutant did not exhibit a defect for short or longer term mouse colonization, consistent with its increased stress survival and diminished host cell damage phenotypes. Collectively, these results demonstrate a unique correlation between an AA-ABC transporter with bacterial stress tolerance, intracellular survival, host cell damage, and host signal transduction in response to pathogen infection. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

>Magister Scientiae - MSc / Campylobacter and related Epsilonproteobacteria (Arcobacter and Helicobacter) are currently viewed as emerging pathogens and are able to cause gastroenteritis, bacteraemia, the Guillain-Barré syndrome, reactive arthritis and other diseases in humans. While poultry, cattle and sheep are known reservoirs for campylobacter, very little is known about ostriches as a vector for these organisms. What is known, however, is that these birds can and sometimes do get infected. Studies by various researchers have provided evidence that various species of animals shed Epsilonproteobacteria in their faeces. In this study, qualitative microbiological assays were performed on liver, caecum and colon samples predominantly from healthy ostriches presented for slaughter, to detect any Epsilonproteobacteria present. Samples were collected at an abattoir in the Western Cape between February and December 2010. Qualitative microbiological assays were also performed on 50 faecal samples collected on a farm. Epsilonproteobacteria were isolated from the tissue samples and characterized following the phenotypic and biochemical scheme presented in the Cape Town protocol. This protocol uses membrane filtration onto antibiotic-free Tryptose blood agar plates, and incubation at 37 ºC in a hydrogen-enhanced microaerobic atmosphere (Lastovica, 2006). The isolates were identified as C. jejuni subsp. jejuni. The multiplex PCR of Neubauer and Hess (2006) was applied to some of these isolates. A panel of isolates consisting of C. jejuni subsp. jejuni, C. fetus and E. coli was used to verify the DNA extraction procedure. The C. fetus and E. coli isolates were used as negative controls, and although DNA was successfully extracted from them, no bands were observed in the respective lanes of electrophoresed gels after application of the PCR.

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.

Las bacterias del género Campylobacter han cobrado importancia como patógenas para el hombre, debido principalmente a su asociación con procesos gastroentéricos, a través del contacto con animales infectados y/o alimentos contaminados como fuente principal de contaminación para el hombre que provoca cuadros gastrointestinales ocasionados por este tipo de bacterias, las personas mas susceptibles son los niños menores de cinco años, las personas de edad avanzada y los inmunocomprometidos. Los objetivos del presente trabajo fueron determinar la presencia e incidencia de Campylobacter (Campylobacter jejuni, C. coli, C. lari) como agente etiológico de diarrea aguda, en niños menores de cinco años atendidos en hospitales de Lima (Perú). / -- The bacteria of the Campylobacter genus have become important as pathogens for humans mainly because of their association to gastroenteric processes. These are caused through contact with infected animals and/or contaminated food as the main source of contamination for humans, which cause gastrointestinal manifestations resulting from this type of bacteria. The most sensitive are children under five, the elderly and the immune deficient people. The objectives of the present work were to determine the presence and incidence of Campylobacter (Campylobacter jejuni, C. coli, C. lari) as etiological agent of acute diarrhea in children under five treated in hospitals in Lima (Perú). / Tesis