Thesis(M.S.)--Auburn University, 2006. / Abstract. Vita. Includes bibliographic references.

QUETZ, Josiane da Silva. Estudo sobre Campylobacter jejuni e Campylobacter coli em crianças da área urbana de Fortaleza, Ceará/Brasil : identificação genética, inflamação intestinal e impacto no estado nutricional. 2009. 142 f. Dissertação (Mestrado em Farmacologia) - Universidade Federal do Ceará. Faculdade de Medicina, Fortaleza, 2009. / Submitted by denise santos (denise.santos@ufc.br) on 2012-04-12T12:44:16Z No. of bitstreams: 1 2009_dis_jsquetz.pdf: 1671276 bytes, checksum: 7b86044a2d433a11e1abdc0273d371d4 (MD5) / Approved for entry into archive by Eliene Nascimento(elienegvn@hotmail.com) on 2012-04-13T16:16:02Z (GMT) No. of bitstreams: 1 2009_dis_jsquetz.pdf: 1671276 bytes, checksum: 7b86044a2d433a11e1abdc0273d371d4 (MD5) / Made available in DSpace on 2012-04-13T16:16:02Z (GMT). No. of bitstreams: 1 2009_dis_jsquetz.pdf: 1671276 bytes, checksum: 7b86044a2d433a11e1abdc0273d371d4 (MD5) Previous issue date: 2009 / 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. / Campylobacter jejuni e Campylobacter coli são importantes agentes etiológicos de doença diarréica na população mundial. A infecção por Campylobacter sp. é usualmente identificada por cultivo microbiológico que leva aproximadamente 72 horas para identificação do gênero. Nosso objetivo principal foi pesquisar a prevalência de C. jejuni e C. coli em população infantil, com idade entre 2-36 meses, da área urbana de Fortaleza/CE, Brasil, em estudo do tipo epidemiológico observacional caso-controle, utilizando, como ferramenta de detecção, a reação em cadeia da polimerase (PCR). Outros objetivos consistiram em: investigar o impacto nutricional da infecção (casos) ou da colonização (controles) por Campylobacter sp.; determinar a presença de três genes de virulência para a toxina citoletal distensora (CDT) de C. jejuni e avaliar a ocorrência de inflamação intestinal nas infecções causadas por Campylobacter sp. A população estudada consistiu de 83 casos e 83 controles, sendo os casos, crianças com histórico de diarréia nos 14 dias pregressos à seleção para o estudo. Foram avaliados parâmetros sócio-econômicos através de questionário epidemiológico. Medidas antropométricas foram coletadas para determinação de escores-z no intuito de avaliar o perfil nutricional das crianças. A detecção de Campylobacter nas amostras congeladas foi realizada por ensaio imuno-enzimático (ELISA) e PCR. Pela PCR também investigamos a presença dos genes cdtA, cdtB e cdtC da CDT de C. jejuni. A avaliação da inflamação intestinal foi realizada pela pesquisa de lactoferrina fecal (LFF), através 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 diferença significativa (p<0,05) dos escores WAZ e WHZ entre casos e controles portadores de C. jejuni, sendo que casos portadores apresentaram média inferior de WAZ e WHZ, quando comparados com os controles portadores. No grupo Casos, os portadores de C. jejuni apresentavam valor médio de WHZ inferior ao valor médio apresentado pelos casos não-portadores. Mais de 80,0% das crianças estudadas apresentaram inflamação intestinal caracterizada por elevados níveis de LFF, independente da presença de diarréia e Campylobacter sp. Em conclusão, nossos achados corroboram dados da literatura científica relacionados à prevalência de C. jejuni e C. coli na população infantil, existência de portadores assintomáticos e associação entre a detecção do microorganismo e desnutrição. Além disso, nossos dados apontam para ocorrência de variabilidade genética dentre as cepas de C. jejuni detectadas na população estudada em relação à presença ou ausência dos genes de CDT.

Tesis para optar al Grado de Magíster en Ciencias Animales y Veterinarias . / Campylobacter jejuni (C. jejuni) y Campylobacter coli (C. coli) son microorganismos comensales en animales productivos y constituyen una de las principales causas de enteritis de transmisión alimentaria. C. jejuni es responsable del 90% de las campylobacteriosis humanas y C. coli cerca del 10%. Ambas especies son aisladas en proporciones similares de la carne de pollo, la principal fuente de infección del ser humano, representando cada una de ellas cerca del 50% de los Campylobacter spp. aislados. Para estas bacterias, la adherencia e invasión a células intestinales son mecanismos fundamentales de patogenicidad. Se han identificado diversos factores de virulencia asociados a estos mecanismos, como también diferencias entre C. jejuni y C. coli en las prevalencias y tamaños de algunos de los genes que los codifican. El propósito del presente trabajo fue caracterizar la capacidad de adherencia e invasión a células intestinales humanas de cepas de C. jejuni y C. coli aisladas de personas y animales productores de alimentos y relacionar esta capacidad con la presencia de siete genes de virulencia (cadF, flaA, racR, dnaJ, virB11, ciaB y pldA). La hipótesis fue que las cepas de C. jejuni tendrían mayor capacidad de adherir e invadir células intestinales humanas que las cepas de C. coli, y que esta capacidad se relacionaría positivamente con la presencia de genes de virulencia. Se emplearon 15 cepas de C. jejuni y 17 de C. coli aisladas desde pacientes humanos, cerdos, bovinos y pollos broiler. La presencia de los genes de virulencia de cada una de las cepas fue caracterizada en un estudio previo mediante la técnica de PCR convencional. Para evaluar la capacidad de adherencia e invasión a células intestinales humanas se realizaron estudios in vitro empleando la línea celular T84 de epitelio colónico T84 y midiendo el número de bacterias adheridas luego de una h de infección y las bacterias internalizadas luego de tres. La asociación con los genes de virulencia se valoró mediante análisis de regresión logística, el que se complementó con el test Kruskal-Wallis para evaluar diferencias en adherencia e invasión entre cepas portadoras y no portadoras de los genes. Los resultados demostraron que tanto las cepas humanas como las aisladas desde animales productores de alimento tienen la capacidad de adherir e invadir células intestinales in vitro y que esta capacidad varía entre las diferentes cepas. Estadísticamente, no se encontraron diferencias en la capacidad de adherencia e invasión entre C. jejuni y C. coli. El análisis Kruskal- Wallis (y test post-hoc Dunn) reveló que las cepas de C. coli portadoras del gen dnaJ tenían una mayor capacidad de invasión que las cepas de C. coli no portadoras del gen y que las cepas C. jejuni portadoras del mismo. Asimismo, con el análisis de regresión logística se encontró una asociación significativa entre la presencia del gen dnaJ y una mayor capacidad invasora en la especie C. coli. Los resultados de este trabajo sugieren que C. jejuni y C. coli tendrían la misma capacidad de adherir e invadir células intestinales humanas y que solo existiría una asociación positiva entre el gen dnaJ y la invasión de cepas de C. coli. / Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli) are commensals microorganisms of food-producing animals, and they are considered one of the major causes of food-borne enteritis. 90% of human campylobacteriosis is caused by C. jejuni and most of the rest by C. coli. Both species are isolated in similar proportions from chicken meat, the main source of human infection, representing each of them almost the 50% of Campylobacter spp. isolated. The adherence to and invasion of human intestinal epithelial cells are essential mechanisms in Campylobacter pathogenesis. There have been identified several virulence factors related to these mechanisms, besides differences between C. jejuni and C. coli in the prevalence and size of some of the genes that encode them. The aim of this work was studied the adherence to and invasion of human intestinal epithelial cells by C. jejuni and C. coli isolated from humans and foodproducing animals, and to relate those abilities to the presence of seven virulence genes (cadF, flaA, racR, dnaJ, virB11, ciaB y pldA). The hypothesis was that C. jejuni strains would have more ability to adhere to and invade human intestinal epithelial cells than C. coli strains, and those abilities would be associated with the presence of virulence genes. We used 15 C. jejuni strains and 17 C. coli strains isolated from human patients, broiler chickens, swine, and bovines. The presence of virulence genes of each of the strains was determined using PCR before this work. We employed the human colonic epithelial cell line T84 to test in vitro the adherence and invasion abilities, and we checked them after one and three hours of infection to determine the number adhered and internalized bacteria, respectively. To test the association with the virulence genes we used logistic regression, and to evaluate differences of adherence and invasion between strains carrying and non-carrying virulence genes we used the Kruskal-Wallis test. We observed that both Campylobacter isolates from humans and from food-producing animals are capable of adhering to and of invading intestinal epithelial cells in vitro, and there are variations between strains in those abilities. Statistically, no significant differences were detected between C. jejuni and C. coli in their abilities to adhere to and to invade T84 cells. The Kruskal-Wallis test (and post-hoc Dunn test) showed that C. coli strains carrying dnaJ gene invaded more than C. coli strains non-carrying the gene, and more than C. jejuni strains carrying the same gene. Besides, a significant association was detected between the presence of the dnaJ gene and a higher invasion in C. coli strains by logistic regression. Our results suggest that C. jejuni and C. coli would have the same adherence and invasion abilities, and that, statistically, it would exist only a positive association between the dnaJ gene and the invasion ability of C. coli strains. / Financiamiento: Programa de Apoyo Económico de Actividades de Investigación para estudiantes de Magíster en Ciencias Animales Veterinarias.

Memoria para optar al Título Profesional de Médico Veterinario / La resistencia a los antibióticos es un problema de salud pública mundial, ya que complica y encarece el tratamiento de las enfermedades infecciosas. En el caso de Campylobacter spp., este es un problema emergente, dado que en los últimos años se ha observado un incremento en la resistencia a antibióticos principalmente a las fluoroquinolonas y macrólidos. Esto es de gran importancia dado que estos fármacos son utilizados como primera elección para el tratamiento de campilobacteriosis. El uso indiscriminado, no solo en producción animal, sino también en medicina humana se describe como unas de las principales causas de este fenómeno. En Chile, existen muy pocos estudios sobre la susceptibilidad antimicrobiana en cepas de Campylobacter spp. El objetivo de este trabajo fue evaluar la susceptibilidad a los antibióticos en 120 cepas de Campylobacter spp., provenientes de cerdos y 60 cepas aisladas de bovinos de carne. Los antibióticos analizados fueron ciprofloxacino, tetraciclina, eritromicina y gentamicina. Se utilizaron dos métodos, primero se realizó un screening con la técnica de Kirby Bauer y todas aquellas cepas que resultaron resistentes, fueron sometidas a determinación de concentración mínima inhibitoria en placa mediante el Método Etest. Se analizaron 180 cepas y se observó que el 10,5% de ellas fueron resistentes a gentamicina, el 57,9% a eritromicina, el 82,6% a ciprofloxacino y el 91,4% lo fue a tetraciclina, mientras que el 87,1% de las cepas fueron clasificadas como multiresistentes. Nuestros resultados indican que los niveles de resistencia a los antimicrobianos en las cepas de Campylobacter spp., son elevados especialmente para ciprofloxacino y tetraciclina. Esto hace necesario proponer y establecer sistemas de vigilancia de la resistencia en este patógeno, con un enfoque integral entre Medicina Veterinaria, Medicina Humana y en producción de alimentos, con el fin de resguardar la salud pública. / Antibiotic resistance is a public health problem, because it complicates and increases the cost of treatment of infectious diseases. As for Campylobacter spp., this is a relevant emerging problem, since in recent years it has seen an increase in antibiotic resistance mainly to fluoroquinolones and macrolides. This is of great importance since drugs are used as the first choice for the treatment of campylobacteriosis. Their indiscriminate use not only animal production but also in human medicine, is described as one of the main causes of this phenomenon. In Chile, there are only a few studies on the antimicrobial susceptibility of Campylobacter spp strains. This study was meant to evaluate the susceptibility to antibiotics in 120 strains of Campylobacter spp isolated from pigs and 60 strains isolated from beef cattle. The antibiotics analyzed were: ciprofloxacin, tetracycline, erythromycin and gentamicin. Two methods were used, the first, the Kirby Bauer screening technique and all of those strains that performed resistant on this test, were subjected to determination of minimum inhibitory concentration MIC`s through Etest Method. A total of 180 strains were evaluated, and of these 10.5% strains were resistant to gentamicin, 57.9% to erythromycin, 82.6% to ciprofloxacin and 94.1% to tetracycline. Also, a total of 87.1% from the tested strains were multiresistant. Our results indicate that levels of antimicrobial resistance in strains of Campylobacter spp., they are higher especially for ciprofloxacin and tetracycline. Making it necessary to propose and establish systems for monitoring and reporting of resistance in this pathogen, an integrated approach between Veterinary Medicine, Food production and Human Medicine, in order to protect public health. / Financiamiento: Proyecto Fondecyt no. 11110200.

Campylobacter spp., principalmente as espécies C. coli e C. jejuni, são a causa mais comum de doença bacteriana veiculada por alimentos na Europa, Estados Unidos e alguns outros locais do mundo. No Brasil, há uma escassez de estudos de C. coli, o que dificulta avaliar a dimensão do envolvimento dessa bactéria como causadora de doença nos seres humanos e em animais, bem como, determinar o impacto de sua presença em alimentos e no meio ambiente. O objetivo desse trabalho foi caracterizar molecularmente linhagens de C. coli isoladas de origens diversas no Brasil pela pesquisa da presença de genes relacionados à virulência por PCR, perfil de sensibilidade a antimicrobianos e pela análise da similaridade genotípica por métodos de tipagem molecular. Adicionalmente, o Índice de Discriminação (D) de tais metodologias foi verificado. Foram estudadas 63 linhagens de C. coli, isoladas de humanos (12), animais (21), alimentos (10) e ambiente (20), entre os anos de 1995 e 2011, nos Estados do Rio de Janeiro, São Paulo e Minas Gerais. Todas as linhagens apresentaram os genes flaA, cadF e sodB. O gene cdtB foi detectado em 20 (31,7%) linhagens, o gene flhA foi detectado em 11 (17,5%) linhagens, o gene dnaJ foi encontrado em 10 (15,9%) linhagens, o gene pldA foi detectado em sete (11,1%) linhagens, o gene iamA foi detectado em três (4,8%) linhagens, os genes cdtC e docA foram encontrados em duas (3,2%) linhagens, os genes cdtA e crsA foram encontrados em uma (1,6%) linhagem e os genes ciaB, wlaN, virB11 e racR não foram detectados. Dentre as 63 linhagens estudadas, 42 foram susceptíveis a todos os antimicrobianos testados. Das 21 linhagens resistentes, 10 (15,9%) foram resistentes a tetraciclina e doxaciclina, seis (9,5%) foram resistentes a ciprofloxacina e uma (1,6 %) foi resistente a eritromicina. Somente quatro (6,3%) linhagens foram resistentes a pelo menos duas diferentes classes de antibióticos testados simultaneamente. O dendrograma de similaridade genética de Pulsed field gel electrophoresis (PFGE) agrupou as 63 linhagens estudadas em dois grupos principais denominados PFGE-A e PFGE-B com similaridade genômica de 44,9% entre eles. Entretanto, algumas linhagens isoladas de humanos, animais, ambiente e alimentos apresentaram uma alta similaridade genotípica acima de 80% entre elas e, foram agrupadas em sete subgrupos denominados PFGE-A1 a PFGE-A7. O dendrograma de similaridade genômica das sequências da SVR do gene flaA agrupou as linhagens ii estudadas em dois grupos principais designados SVR-A e SVR-B, com similaridade acima de 83,1 % entre eles. Ademais, o depósito das sequências da SVR do gene flaA no banco de dados online demonstrou que os alelos 30 e o 1647 foram os mais frequentemente encontrados e permitiu a comparação das linhagens estudadas com os alelos descritos no banco de dados. Sete alelos, dentre os 22 encontrados, não haviam sido previamente descritos. A análise do locus CRISPR por HRMA dividiu as linhagens de C. coli em quatro diferentes perfis de melting. O Multilocus sequence typing (MLST) foi utilizado para tipar 20 linhagens de C. coli e foram obtidos 18 STs diferentes dos quais apenas dois já haviam sido previamente descritos. O D das metodologias de PFGE, sequenciamento da SVR do gene flaA, análise do locus CRISPR por HRMA e MLST foi de 0,986, 0,916, 0,550 e 0,989, respectivamente. Pode- se concluir que o potencial patogênico das linhagens de C. coli não foi evidenciado o que pode estar relacionado ao fato da maioria dos estudos envolvendo patogênese terem sido realizados para a espécie C. jejuni. Algumas linhagens apresentaram-se resistentes aos antimicrobianos testados, o que é preocupante uma vez que tais linhagens podem disseminar genes de resistência a outras isoladas de diversas fontes. Os resultados gerados pelos métodos de tipagem molecular por PFGE e sequenciamento da pequena região variável (SVR) do gene flaA demonstraram uma alta similaridade genotípica entre algumas linhagens de C. coli, sugerindo que uma possível contaminação tenha ocorrido entre linhagens isoladas de fontes clínicas e não clínicas ao longo de 16 anos no Brasil. Ademais, a análise dos alelos da SVR do gene flaA nos permitiu concluir que os alelos prevalentes nas linhagens estudadas diferem daqueles encontrados nos países Europeus. Os dados obtidos por MLST sugerem que as linhagens estudadas possuem uma grande diversidade genética entre si e em comparação com as linhagens isoladas em diferentes locais do mundo. Finalmente, as técnicas de MLST e PFGE foram as mais eficientes e adequadas na genotipagem das linhagens de C. coli estudadas. / Campylobacter spp., mainly the C. coli and C. jejuni species, are the most common cause of bacterial disease conveyed by food in Europe, United States, and other places worldwide. In Brazil, there is a paucity of studies on C. coli, 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 molecularly characterize C. coli strains isolated from diverse origins in Brazil by searching for the presence of virulence-related genes by PCR, antimicrobial sensitivity profile, and analysis of the genotypic similarity by molecular typing methods. Addicionaly, the Discriminatory Index (D) of those methodologies was acessed. Sixty-three C. coli strains isolated from humans (12), animals (21), food (10), and the environment (20) between 1995 and 2011, in the States of Rio de Janeiro, São Paulo, and Minas Gerais were studied. All strains presented the flaA, cadF and sodB genes. The cdtB gene was detected in 20 (31.7%) strains; the flhA gene was detected in 11 (17.5%) strains; the dnaJ gene was detected in 10 (15.9%) strains; the pldA gene was detected in 7 (11.1%) strains ; the iamA gene was detected in three (4.8%) strains; the cdtC and docA genes were found in two (3.2%) strains; the cdtA and crsA were found in one (1.6%) strain and the ciaB, wlaN, virB11 and racR genes were not detected. Among the 63 strains studied, 42 were susceptible to all antimicrobials tested. Of the 21 resistant strains, 10 (15.9%) were resistante to tetracycline and doxaciclyne, six (9.5%) showed resistance to ciprofloxacin, and one (1.6%) was resistant to erythromycin. Only four (6.3%) strains were simultaneously resistant to at least two different classes of the antibiotics tested. The dendrogram of genetic similarity of Pulsed field gel electrophoresis (PFGE) grouped the 63 strains studied into two groups namely PFGE-A and PFGE-B with a genomic similarity of 44.9% among them. However, some strains isolated from humans, animals, the environment and food presented a high genotypic similarity above 80% and were subdivided into seven groups designated as PFGE-A1 to PFGE-A7. The dendrogram of genetic similarity of the SRV-flaA gene sequences grouped the strains studied into two groups namely SVR-A and SVR-B, with similarity above 83.1% among them. Besides, the deposit of the SVR sequences of the flaA gene in the online database showed that the alleles 30 and 1647 were the iv most frequently found and allowed the comparison between the strains studied with the alleles described in the database. Seven alleles, among the 22 found have never been described before. The CRISPR locus analysis divided the C. coli strains into four different melting profiles. The Multilocus sequence typing (MLST) was used to type 20 C. coli strains and revealed 18 different STs among which just two had been previously described. The D of PFGE, SVR- flaA sequence, HRMA of CRISPR locus analysis and MLST was 0.986, 0.916, 0.550 and 0.989, respectively. In conclusion, the pathogenic potential of the C. coli strains was not highlighted, which could be related to the fact that the majority of the pathogenicity studies were performed with C. jejuni species. Some strains showed resistance to the antibiotics tested what is a concern once those strains may spread the resistance genes to other strains isolated from different sources. The results obtained by PFGE and SVR-flaA sequence showed a high genomic similarity among some C. coli strains which may suggest that a possible contamination may have occurred among clinical and non-clinical sources during 16 years in Brazil. Furthermore, the analysis of SVR- flaA alleles allowed the conclusion that the prevalent alleles in the strains studied were different from those found in European countries. The data obtained by MLST suggests that the strains studied had a high genomic diversity among them and in comparison with strains isolated from different places worldwide. Finally, the MLST and PFGE technicques were the most efficient and adequate in genotyping the C. coli strains studied.

No description available.

Campylobacter spp., principalmente as espécies C. coli e C. jejuni, são a causa mais comum de doença bacteriana veiculada por alimentos na Europa, Estados Unidos e alguns outros locais do mundo. No Brasil, há uma escassez de estudos de C. coli, o que dificulta avaliar a dimensão do envolvimento dessa bactéria como causadora de doença nos seres humanos e em animais, bem como, determinar o impacto de sua presença em alimentos e no meio ambiente. O objetivo desse trabalho foi caracterizar molecularmente linhagens de C. coli isoladas de origens diversas no Brasil pela pesquisa da presença de genes relacionados à virulência por PCR, perfil de sensibilidade a antimicrobianos e pela análise da similaridade genotípica por métodos de tipagem molecular. Adicionalmente, o Índice de Discriminação (D) de tais metodologias foi verificado. Foram estudadas 63 linhagens de C. coli, isoladas de humanos (12), animais (21), alimentos (10) e ambiente (20), entre os anos de 1995 e 2011, nos Estados do Rio de Janeiro, São Paulo e Minas Gerais. Todas as linhagens apresentaram os genes flaA, cadF e sodB. O gene cdtB foi detectado em 20 (31,7%) linhagens, o gene flhA foi detectado em 11 (17,5%) linhagens, o gene dnaJ foi encontrado em 10 (15,9%) linhagens, o gene pldA foi detectado em sete (11,1%) linhagens, o gene iamA foi detectado em três (4,8%) linhagens, os genes cdtC e docA foram encontrados em duas (3,2%) linhagens, os genes cdtA e crsA foram encontrados em uma (1,6%) linhagem e os genes ciaB, wlaN, virB11 e racR não foram detectados. Dentre as 63 linhagens estudadas, 42 foram susceptíveis a todos os antimicrobianos testados. Das 21 linhagens resistentes, 10 (15,9%) foram resistentes a tetraciclina e doxaciclina, seis (9,5%) foram resistentes a ciprofloxacina e uma (1,6 %) foi resistente a eritromicina. Somente quatro (6,3%) linhagens foram resistentes a pelo menos duas diferentes classes de antibióticos testados simultaneamente. O dendrograma de similaridade genética de Pulsed field gel electrophoresis (PFGE) agrupou as 63 linhagens estudadas em dois grupos principais denominados PFGE-A e PFGE-B com similaridade genômica de 44,9% entre eles. Entretanto, algumas linhagens isoladas de humanos, animais, ambiente e alimentos apresentaram uma alta similaridade genotípica acima de 80% entre elas e, foram agrupadas em sete subgrupos denominados PFGE-A1 a PFGE-A7. O dendrograma de similaridade genômica das sequências da SVR do gene flaA agrupou as linhagens ii estudadas em dois grupos principais designados SVR-A e SVR-B, com similaridade acima de 83,1 % entre eles. Ademais, o depósito das sequências da SVR do gene flaA no banco de dados online demonstrou que os alelos 30 e o 1647 foram os mais frequentemente encontrados e permitiu a comparação das linhagens estudadas com os alelos descritos no banco de dados. Sete alelos, dentre os 22 encontrados, não haviam sido previamente descritos. A análise do locus CRISPR por HRMA dividiu as linhagens de C. coli em quatro diferentes perfis de melting. O Multilocus sequence typing (MLST) foi utilizado para tipar 20 linhagens de C. coli e foram obtidos 18 STs diferentes dos quais apenas dois já haviam sido previamente descritos. O D das metodologias de PFGE, sequenciamento da SVR do gene flaA, análise do locus CRISPR por HRMA e MLST foi de 0,986, 0,916, 0,550 e 0,989, respectivamente. Pode- se concluir que o potencial patogênico das linhagens de C. coli não foi evidenciado o que pode estar relacionado ao fato da maioria dos estudos envolvendo patogênese terem sido realizados para a espécie C. jejuni. Algumas linhagens apresentaram-se resistentes aos antimicrobianos testados, o que é preocupante uma vez que tais linhagens podem disseminar genes de resistência a outras isoladas de diversas fontes. Os resultados gerados pelos métodos de tipagem molecular por PFGE e sequenciamento da pequena região variável (SVR) do gene flaA demonstraram uma alta similaridade genotípica entre algumas linhagens de C. coli, sugerindo que uma possível contaminação tenha ocorrido entre linhagens isoladas de fontes clínicas e não clínicas ao longo de 16 anos no Brasil. Ademais, a análise dos alelos da SVR do gene flaA nos permitiu concluir que os alelos prevalentes nas linhagens estudadas diferem daqueles encontrados nos países Europeus. Os dados obtidos por MLST sugerem que as linhagens estudadas possuem uma grande diversidade genética entre si e em comparação com as linhagens isoladas em diferentes locais do mundo. Finalmente, as técnicas de MLST e PFGE foram as mais eficientes e adequadas na genotipagem das linhagens de C. coli estudadas. / Campylobacter spp., mainly the C. coli and C. jejuni species, are the most common cause of bacterial disease conveyed by food in Europe, United States, and other places worldwide. In Brazil, there is a paucity of studies on C. coli, 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 molecularly characterize C. coli strains isolated from diverse origins in Brazil by searching for the presence of virulence-related genes by PCR, antimicrobial sensitivity profile, and analysis of the genotypic similarity by molecular typing methods. Addicionaly, the Discriminatory Index (D) of those methodologies was acessed. Sixty-three C. coli strains isolated from humans (12), animals (21), food (10), and the environment (20) between 1995 and 2011, in the States of Rio de Janeiro, São Paulo, and Minas Gerais were studied. All strains presented the flaA, cadF and sodB genes. The cdtB gene was detected in 20 (31.7%) strains; the flhA gene was detected in 11 (17.5%) strains; the dnaJ gene was detected in 10 (15.9%) strains; the pldA gene was detected in 7 (11.1%) strains ; the iamA gene was detected in three (4.8%) strains; the cdtC and docA genes were found in two (3.2%) strains; the cdtA and crsA were found in one (1.6%) strain and the ciaB, wlaN, virB11 and racR genes were not detected. Among the 63 strains studied, 42 were susceptible to all antimicrobials tested. Of the 21 resistant strains, 10 (15.9%) were resistante to tetracycline and doxaciclyne, six (9.5%) showed resistance to ciprofloxacin, and one (1.6%) was resistant to erythromycin. Only four (6.3%) strains were simultaneously resistant to at least two different classes of the antibiotics tested. The dendrogram of genetic similarity of Pulsed field gel electrophoresis (PFGE) grouped the 63 strains studied into two groups namely PFGE-A and PFGE-B with a genomic similarity of 44.9% among them. However, some strains isolated from humans, animals, the environment and food presented a high genotypic similarity above 80% and were subdivided into seven groups designated as PFGE-A1 to PFGE-A7. The dendrogram of genetic similarity of the SRV-flaA gene sequences grouped the strains studied into two groups namely SVR-A and SVR-B, with similarity above 83.1% among them. Besides, the deposit of the SVR sequences of the flaA gene in the online database showed that the alleles 30 and 1647 were the iv most frequently found and allowed the comparison between the strains studied with the alleles described in the database. Seven alleles, among the 22 found have never been described before. The CRISPR locus analysis divided the C. coli strains into four different melting profiles. The Multilocus sequence typing (MLST) was used to type 20 C. coli strains and revealed 18 different STs among which just two had been previously described. The D of PFGE, SVR- flaA sequence, HRMA of CRISPR locus analysis and MLST was 0.986, 0.916, 0.550 and 0.989, respectively. In conclusion, the pathogenic potential of the C. coli strains was not highlighted, which could be related to the fact that the majority of the pathogenicity studies were performed with C. jejuni species. Some strains showed resistance to the antibiotics tested what is a concern once those strains may spread the resistance genes to other strains isolated from different sources. The results obtained by PFGE and SVR-flaA sequence showed a high genomic similarity among some C. coli strains which may suggest that a possible contamination may have occurred among clinical and non-clinical sources during 16 years in Brazil. Furthermore, the analysis of SVR- flaA alleles allowed the conclusion that the prevalent alleles in the strains studied were different from those found in European countries. The data obtained by MLST suggests that the strains studied had a high genomic diversity among them and in comparison with strains isolated from different places worldwide. Finally, the MLST and PFGE technicques were the most efficient and adequate in genotyping the C. coli strains studied.

No description available.

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

Submitted by Ubirajara Cruz (ubirajara.cruz@gmail.com) on 2017-06-26T12:47:49Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Camile_Milan.pdf: 974433 bytes, checksum: de53680fe197bd4ba3ac70ba2bac3df5 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-06-26T13:50:13Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Camile_Milan.pdf: 974433 bytes, checksum: de53680fe197bd4ba3ac70ba2bac3df5 (MD5) / Made available in DSpace on 2017-06-26T13:50:13Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Camile_Milan.pdf: 974433 bytes, checksum: de53680fe197bd4ba3ac70ba2bac3df5 (MD5) Previous issue date: 2016-02-18 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / As doenças transmitidas por alimentos são causadas pela ingestão de alimentos contaminados, sendo que em 2013 os principais agentes causadores destas doenças foram Salmonella spp. e Campylobacter spp. No homem, Campylobacter spp. provoca sintomatologia gastrointestinal, que pode variar de uma forma mais suave até formas mais severas. Suínos podem ser portadores destes micro-organismos e produtos de suínos têm sido implicados em casos e surtos de campilobacteriose em humanos. Logo, o objetivo deste trabalho foi rastrear Campylobacter spp. no fluxograma de abate de suínos para compreender o comportamento destes patógenos na linha de produção. Foi acompanhado um total de 40 suínos oriundos de 10 baias de uma granja no Rio Grande do Sul durante o abate. Amostras de fezes foram coletadas no reto do animal logo após a insensibilização. Amostras da superfície da carcaça foram coletadas após a saída dos animais da depiladeira, após a evisceração, momentos antes da carcaça entrar na câmara de resfriamento e da papada. Amostras da água do tanque de escaldagem foram coletadas antes de iniciar o abate de cada lote e após a passagem dos animais também. Os isolados foram obtidos através de culturas microbiológicas e a identificação das espécies foi realizada pela técnica de PCR. Os perfis de bandas das cepas foram determinados por rep-PCR e comparados entre si. Das 200 amostras coletadas, 19 (9,5%) foram positivas para Campylobacter, sendo todas caracterizadas como C. coli. Destes isolados, sete (36,8%) foram obtidos das amostras do reto, sete (36,8%) da pós-evisceração e cinco (26,3%) antes do ingresso na câmara de resfriamento. Através da análise por rep-PCR, observou-se que os animais, uma vez contaminados por C. coli na granja, podem carrear o micro-organismo durante as etapas do fluxograma de abate e que contaminações cruzadas também têm papel importante na contaminação final da carcaça. O manejo higiênico-sanitário adotado nas boas práticas de fabricação é fundamental no controle da contaminação por C. coli. / The foodborne illnesses are caused by contaminated food, and in 2013 the main causative agents of these diseases were Salmonella spp. and Campylobacter spp. In man, Campylobacter spp. causes gastrointestinal symptoms, which can vary more smoothly even more severe forms. Pigs can be carriers of microorganisms and pork products have been implicated in cases of campylobacteriosis outbreaks in humans. Therefore, the aim of this study was to track Campylobacter spp. in pig slaughtering flowchart to understand the behavior of these pathogens in the production line. A total of 40 pigs originating from 10 bays a farm in Rio Grande do Sul was accompanied during slaughter. Stool samples were collected in the animal's rectum immediately after stunning. Surface samples were collected after removal of the animals from scrap machine, after evisceration, before the refrigeration chamber and from jowls. Samples from the scalding tank water before and after the passage of animals were collected too. The isolates were obtained for microbiological analysis and the confirmation of species was performed by PCR. Strains bands profiles were determined by rep-PCR and compared. Of the 200 samples 19 (9.5%) were positive for Campylobacter, which are all characterized as C.coli. In these isolates, 7 (36.8%) were the rectum, 7 (36.8%) after evisceration and 5 (26.3%) before the refrigeration chamber. Through rep-PCR analysis, it was observed that the animals contaminated by C. coli in the farm may carry the micro-organism through the steps of the flowchart slaughter and the cross contamination also has an important role on the final contamination. The hygienic-sanitary management of care adopted in good manufacturing practices are key in controlling the contamination by C. coli.