Campylobacter jejuni is a major cause of bacterial gastroenteritis in the developed world; however, many aspects of its biology remain poorly understood, including its colonization of the mucus layer lining the gastrointestinal tract. In this study, we utilized microarray transposon tracking to compile a list of 195 genes essential for the growth of C. jejuni in vitro under microaerophilic conditions. Then we characterized C. jejuni growing in an extracted intestinal mucus medium. We found that C. jejuni will grow efficiently in a medium comprised of either chick and piglet intestinal mucus, and that these media have a dramatic impact on its transcriptome. Within the genes identified as differentially expressed during growth in a mucus medium, we identified a single operon, (cj0481-cj0490), which we have subsequently characterized as being responsible for both the uptake and metabolism of L-fucose. This represents the first observation of carbohydrate metabolism by the otherwise asaccharolytic C. jejuni. We further found that the inability to utilize L-fucose puts C. jejuni at a competitive disadvantage when colonizing the piglet intestine, but not the chick cecum. Finally, we examined C. jejuni’s ability to utilize mucins as a carbon source while growing within the mucus layer. We found that despite mucins being a major source of L-fucose and amino acids within the intestine, C. jejuni has a minimal ability to degrade and utilize mucins on its own. However, close proximity to mucolytic bacteria within the microbiota of the intestine, allows for increased C. jejuni growth. Together, this paints the picture of an organism that is well adapted to survival within the mucus lining of the intestine and establishing itself as part of the intestinal microbiota.

Regensburg, Univ., Diss., 2008.

Campylobacter spp., especially C. jejuni and C. coli, are the leading cause of bacterial gastroenteritis in Europe. There is a recognised need to develop detection tools which can be performed on farms to facilitate reducing the presence of Campylobacter in poultry. A similar application could be beneficial for detection of C. fetus, a veterinary pathogen which causes significant economic loss in the cattle industry. Campylobacter species perform protein N-linked glycosylation and in C. jejuni at least 150 proteins, many of which are surface-exposed, may be modified. Therefore, the first portion of this thesis investigated the feasibility of using N-linked glycans as targets for antibody-based detection of Campylobacter species. To do this, a His-tagged N-glycoprotein was expressed and purified from C. fetus and used as immunogen to raise an antiserum termed CfNgp. The Campylobacter N-glycan reactivity of this antiserum was characterised and it was shown to react with N-glycoproteins and cells of C. fetus and other emerging Campylobacter species such as C. concisus. Immunoblotting techniques and flow cytometry were used to characterise an antiserum (CjNgp) raised against a C. jejuni N-linked glycoprotein and demonstrated that it can specifically detect cells of C. jejuni, C. coli and other emerging Campylobacter species found in poulty. This thesis also describes the investigation of the relatively uncharacterised C. fetus N-linked glycosylation system. Functional analysis of C. fetus predicted glycosyltransferases was acheived by developing glycocompetent E. coli containing a hybrid C. jejuni/C. fetus pgl system. The N-glycan structures biosynthesised were analysed using mass spectrometry and this novel approach discovered the activity of two C. fetus glycosyltransferase enzymes. Finally, this work used a bioinformatics pipeline to produce a C. fetus predicted N-linked glycoproteome and experimentally verified a newly identified N-linked glycoprotein. This pipeline was also applied to investigate the putative conservation of N-linked glycoproteins throughout the Campylobacter genus and highlighted ‘core’ N-linked glycoproteins which are key targets for experimental investigation. Overall, this work demonstrates that Campylobacter N-linked glycans are attractive targets for antibody-based detection, expands our knowledge of C. fetus N-linked glycosylation and contributes to the broader understanding of this intriguing aspect of Campylobacter biology.

Membros termofílicos do gênero Campylobacter são reconhecidos como importantes enteropatógenos para o ser humano e animais. A grande diversidade ecológica destes microorganismos em diferentes habitats tais como: água, animais e alimentos predispõem ao aparecimento de novos fatores de virulência. Este trabalho teve por objetivo detectar os genes codificantes da Toxina Distensiva Citoletal (CDT) por meio da técnica de PCR, pesquisar a atividade de hemolisinas e a influência de soluções quelantes e de íons nesta atividade. Foram utilizadas 45 amostras de C. jejuni de origem avícolas para pesquisa de atividade hemolítica, cultivadas em Caldo Triptona de Soja (TSB). Após o crescimento bacteriano, as amostras foram semeadas em Ágar tríptico de soja (TSA) contendo 5% de sangue de ovino, equino e bovino, sendo cada sangue testado isoladamente. Para verificar a influência de agentes quelantes e solução de íons na atividade hemolítica, as amostras de C. jejuni foram cultivadas em TSB contendo separadamente os quelantes EDTA, ácido acético, soluções de íons CaCl2 , MgCl2 e FeCl3, em atmosfera de microaerofilia. Quanto à atividade de hemolisina de Campylobacter jejuni em placas de TSA – sangue, foi possível observar que houve hemólises em 48,89% das amostras quando utilizado sangue eqüino, em 40% em sangue de bovino e em 31,11% quando de ovino. Quanto à influência de agentes quelantes e íons em caldo TSB na atividade de hemolisinas em amostras de Campylobacter jejuni semeadas em placas de TSA – sangue ovino, foi observada atividade hemolítica em 26,67% quando utilizado CaCl2, 15,55% (FeCl3), 22,22% (EDTA), 11,11% (MgCl2) e apenas 2,22% (ácido acético). No tocante à atividade hemolítica, o TSA - sangue bovino apresentou 15,55% (CaCl2), 24,44% (FeCl3), 26,26% (EDTA), 20% (MgCl2) e 11,11% (ácido acético). A atividade hemolítica para o sangue equino foi de 24,44% (CaCl2), 22,22% (FeCl3), 28,89% (EDTA), 28,89% (MgCl2) e 8,89% (ácido acético). Para detecção dos genes cdtA, cdtB e cdtC através da técnica da Reação da Polimerase em Cadeia (PCR), foram utilizadas 119 amostras de C. jejuni de origem avícolas. Foi possível observar que 38% possuíam os três genes, e foram identificados somente os genes cdtA e cdtC em 19% do total de amostras, sendo que o gene cdtB foi encontrado em 14%, o gene cdtC foi observado em 12%, os genes cdtA e cdtB em somente 1%, os genes cdtB e cdtC em 1% e para cdtA em 1%. Observou-se que os resultados são dignos de atenção, pois demonstraram em amostras avícolas a presença de estirpes de C. jejuni com potencial virulento. A atividade hemolítica apresentou significativo aumento quando utilizado sangue de origem equina. A mesma foi diminuída quando utilizados agentes quelantes ou íons, nos três tipos de sangue. / Thermophilic members of the Campylobacter genus are recognized as important enteropathogenics for humans and also for other animals. The great diversity of ecological habitats in different organisms such as water, food, and animals may promote new virulence factors. This study aimed at detecting the distending cytolethal toxin (CDT) encoding genes by PCR, studying the activity of hemolysin and also the influence of chelation solutions and ions. A total of 45 samples of C. jejuni from poultry origin, grown in Tryptone Soy Broth (TSB) were used for investigating hemolytic activity. After bacterial growth, samples were plated on Tryptic Soy Agar (TSA) containing 5% sheep, equine or bovine blood, being each blood tested individually. In order to check the influence of chelation agents and ions solution on the hemolytic activity, samples of C. jejuni strains were grown in TSB containing chelation agents individually: EDTA, acetic acid, CaCl2 ion, MgCl2 and FeCl3 solutions, all in microaerophilic atmosphere. Regarding the detection of Campylobacter jejuni hemolysin activity on TSA plates, blood hemolysis were observed in 48.89 % of samples when equine blood was used; in 40% of samples when bovine blood was used and in 31.11 % when the blood used was of sheep origin. The influence of ions and chelation agents in hemolysin activity in TSB when Campylobacter jejuni was plated on TSA with sheep blood can be described as: hemolytic activity was observed at 26.67% of samples when CaCl2 was used, at 15.55 % for FeCl3, 22 22 % for EDTA, 11.11 % for MgCl2 and only 2.22% when acetic acid was used. The hemolytic activity detected when bovine blood - TSA was used indicated 15.55% for CaCl2, 24.44% for FeCl3, 26.26 % for EDTA, 20 % for MgCl2 and 11.11% for acetic acid. In terms of the hemolytic activity when equine blood was used, the results indicated 24.44% for CaCl2, 22.22 % for FeCl3, 28.89 % for EDTA, 28.89 % for MgCl2 and 8.89% for acetic acid. Finally, regarding the detection of cdtA, cdtB and cdtC through PCR, 119 samples of C. jejuni from poultry origin were used. The results indicated that all three genes were present in 38 % of the samples, whereas only two genes were identified in 19 % of samples, while the cdtB gene was singly found in 14%, the cdtC gene was independently observed in 12%, cdtA and cdtB genes together were found in 1% of the samples; the cdtB and cdtC genes associated were detected in 1%, while cdtA alone answered for 1% of detections. The results also showed the presence of C. jejuni strains with virulence potential. The hemolytic activity increased significantly when blood of equine origin was used, and that this activity was reduced when ions or chelating agents were used in combination with the three types of blood cells.

Campylobacter jejuni is the main cause of bacterial diarrhoeal illness in developed countries, with ~7000 cases being reported each year in Sweden. C. jejuni has received growing attention since it’s recognition as a human pathogen in the 1970s, but its epidemiology is complex and much still remains unknown. There are several potential reservoirs for C. jejuni, including environmental sources as water and soil, wild and domesticated animals, particularly poultry, but also other livestock and pets. In this study 348 Swedish C. jejuni isolates from the year 2000 from humans (n = 164) and broilers (n = 184) were characterized with multilocus sequence typing (MLST) with the aim of comparing the population structures and diversity of C. jejuni between isolates from the two hosts. MLST is a method for characterization of bacterial isolates that indexes the variation in DNA sequence of multiple protein encoding housekeeping genes. A secondary aim in this study was to compare populations of C. jejuni from 11 subgroups of isolates based on location of the sampling. The overlap between the populations was analyzed numerically based on genotypes detected and with analysis of phylogeny, gene flow and molecular variation. It was shown that the population structure of C. jejuni isolates from broilers and humans show a high degree of similarity, supporting broilers as an important source of human infection. However, even though the population structure of human and broiler C. jejuni were almost genetically indistinguishable other sources of C. jejuni infections in humans cannot be ruled out since the same genotypes can be found in other sources as well. Analysis of the 11 subgroups suggested that there may be a difference in populations infecting humans in different Swedish regions, and between populations of C. jejuni in broilers from different slaughterhouses. But this could be a result of chance since most of the subgroups were small. Future studies to improve the understanding of C. jejuni epidemiology, for which MLST has proven itself as a valid method, is important to develop control strategies to prevent infection with this common cause of diarrhoeal illness.

Memoria para optar al Titulo Profesional de Médico Veterinario / El objetivo del presente estudio fue aislar, cuantificar y caracterizar molecularmente a Campylobacter jejuni (C. jejuni) proveniente de muestras de canales obtenidas en la etapa de eviscerado y enfriado en dos plantas faenadoras de pollos broiler de la Región Metropolitana. Además se obtuvo los datos del enfriador de agua de las canales (“chiller”) en ambas plantas para establecer las diferencias. Las cepas de C. jejuni fueron aisladas en medios de cultivo selectivos, posteriormente identificadas por pruebas bioquímicas y caracterizadas a través de “Electroforesis en Gel de Campo Pulsado” (PFGE) usando dos enzimas de restricción, SmaI y KpnI. Los resultados mostraron que, C. jejuni se aisló en 166 de las 259 muestras analizadas (64%). En la planta A se obtuvo un total de 82% (107/130) muestras positivas y en la planta B 46% (59/129). Al analizar la contaminación por etapa de proceso se observó mayor porcentaje de ocurrencia en la etapa de eviscerado 71% (97/136) que enfriado 56% (69/123), mientras que al analizar los datos por planta y etapa se obtuvo que en la planta A la etapa de eviscerado tuvo un 89% y la etapa de enfriado un 74% de ocurrencia para C. jejuni. Comparativamente, la contaminación con Campylobacter en la planta B fue menor en ambas etapas con un 53% de ocurrencia en el eviscerado y un 37% en el enfriado. Los resultados de la caracterización molecular de C. jejuni mostraron 13 patrones distintos de macrorrestricción al usar la enzima SmaI y 12 al aplicar KpnI. En ambos casos se obtuvo 6 patrones comunes, siendo los restantes patrones únicos (6 y 7 respectivamente). Los mismos patrones se observaron tanto en la etapa de eviscerado como de enfriado. Los controles de temperatura en el “chiller” de las plantas A y B fueron en promedio de 1,56ºC y 0,59ºC respectivamente. La planta B presenta temperaturas significativamente menores (p=0,0024). La concentración de cloro del agua del “chiller” medida en la planta A fue de 0,58 ppm y en la planta B de 0,53 ppm. Las diferencias observadas no fueron significativas (p=0,4315). Solo ocurrió una disminución estadísticamente significativa de los porcentajes de ocurrencia desde la etapa de eviscerado a enfriado por parte de la Planta A. Sin embargo, la ocurrencia en la planta A siempre fue mayor que la planta B. Los patrones de macrorrestricción observados fueron específicos para cada una de las plantas y según el día de muestreo, no así para las etapas. Estos resultados, inducen a pensar, que la mayor contaminación de las canales con C. jejuni corresponde a la proveniente de la crianza y no se genera al interior de la planta faenadora de aves

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.

The major outer membrane protein, MOMP, is the main protein in the outer membrane of pathogenic Campylobacter bacteria. Infection with Campylobacter is the principle cause of severe enteritis and untreated may result in non-trauma related paralysis. Studies have shown, that MOMP can act as antigen and thus has the potential to provide protection by induced humoral immunity. In our study, we expressed recombinant MOMP in Escherichia1coli, developed an alternative method to extract the outer membrane protein from its lipid environment and solved and characterised its crystal structure. The information acquired through these structural studies sheds new light on the structural characteristics of this important membrane protein. The West-African Lassa virus can cause deadly haemorrhagic fever. Lassa virus only possesses five proteins, which are synergistically responsible for the virus' life cycle, and virulence. The way in which the individual proteins act with one another and with host cell proteins is not fully understood. The polymerase L is the largest of the five proteins and has multiple functions. In this study, we first divided the L protein into different domains and tested their recombinant expression in Escherichia1coli. For first time, we solved the crystal structure of the putative endonuclease domain of Lassa virus and validated its endonucleolytic function by means of RNA digestion assays and alanine point mutations.

Campylobacter jejuni is a major cause of bacterial gastroenteritis in the developed world; however, many aspects of its biology remain poorly understood, including its colonization of the mucus layer lining the gastrointestinal tract. In this study, we utilized microarray transposon tracking to compile a list of 195 genes essential for the growth of C. jejuni in vitro under microaerophilic conditions. Then we characterized C. jejuni growing in an extracted intestinal mucus medium. We found that C. jejuni will grow efficiently in a medium comprised of either chick and piglet intestinal mucus, and that these media have a dramatic impact on its transcriptome. Within the genes identified as differentially expressed during growth in a mucus medium, we identified a single operon, (cj0481-cj0490), which we have subsequently characterized as being responsible for both the uptake and metabolism of L-fucose. This represents the first observation of carbohydrate metabolism by the otherwise asaccharolytic C. jejuni. We further found that the inability to utilize L-fucose puts C. jejuni at a competitive disadvantage when colonizing the piglet intestine, but not the chick cecum. Finally, we examined C. jejuni’s ability to utilize mucins as a carbon source while growing within the mucus layer. We found that despite mucins being a major source of L-fucose and amino acids within the intestine, C. jejuni has a minimal ability to degrade and utilize mucins on its own. However, close proximity to mucolytic bacteria within the microbiota of the intestine, allows for increased C. jejuni growth. Together, this paints the picture of an organism that is well adapted to survival within the mucus lining of the intestine and establishing itself as part of the intestinal microbiota.

Membros termofílicos do gênero Campylobacter são reconhecidos como importantes enteropatógenos para o ser humano e animais. A grande diversidade ecológica destes microorganismos em diferentes habitats tais como: água, animais e alimentos predispõem ao aparecimento de novos fatores de virulência. Este trabalho teve por objetivo detectar os genes codificantes da Toxina Distensiva Citoletal (CDT) por meio da técnica de PCR, pesquisar a atividade de hemolisinas e a influência de soluções quelantes e de íons nesta atividade. Foram utilizadas 45 amostras de C. jejuni de origem avícolas para pesquisa de atividade hemolítica, cultivadas em Caldo Triptona de Soja (TSB). Após o crescimento bacteriano, as amostras foram semeadas em Ágar tríptico de soja (TSA) contendo 5% de sangue de ovino, equino e bovino, sendo cada sangue testado isoladamente. Para verificar a influência de agentes quelantes e solução de íons na atividade hemolítica, as amostras de C. jejuni foram cultivadas em TSB contendo separadamente os quelantes EDTA, ácido acético, soluções de íons CaCl2 , MgCl2 e FeCl3, em atmosfera de microaerofilia. Quanto à atividade de hemolisina de Campylobacter jejuni em placas de TSA – sangue, foi possível observar que houve hemólises em 48,89% das amostras quando utilizado sangue eqüino, em 40% em sangue de bovino e em 31,11% quando de ovino. Quanto à influência de agentes quelantes e íons em caldo TSB na atividade de hemolisinas em amostras de Campylobacter jejuni semeadas em placas de TSA – sangue ovino, foi observada atividade hemolítica em 26,67% quando utilizado CaCl2, 15,55% (FeCl3), 22,22% (EDTA), 11,11% (MgCl2) e apenas 2,22% (ácido acético). No tocante à atividade hemolítica, o TSA - sangue bovino apresentou 15,55% (CaCl2), 24,44% (FeCl3), 26,26% (EDTA), 20% (MgCl2) e 11,11% (ácido acético). A atividade hemolítica para o sangue equino foi de 24,44% (CaCl2), 22,22% (FeCl3), 28,89% (EDTA), 28,89% (MgCl2) e 8,89% (ácido acético). Para detecção dos genes cdtA, cdtB e cdtC através da técnica da Reação da Polimerase em Cadeia (PCR), foram utilizadas 119 amostras de C. jejuni de origem avícolas. Foi possível observar que 38% possuíam os três genes, e foram identificados somente os genes cdtA e cdtC em 19% do total de amostras, sendo que o gene cdtB foi encontrado em 14%, o gene cdtC foi observado em 12%, os genes cdtA e cdtB em somente 1%, os genes cdtB e cdtC em 1% e para cdtA em 1%. Observou-se que os resultados são dignos de atenção, pois demonstraram em amostras avícolas a presença de estirpes de C. jejuni com potencial virulento. A atividade hemolítica apresentou significativo aumento quando utilizado sangue de origem equina. A mesma foi diminuída quando utilizados agentes quelantes ou íons, nos três tipos de sangue. / Thermophilic members of the Campylobacter genus are recognized as important enteropathogenics for humans and also for other animals. The great diversity of ecological habitats in different organisms such as water, food, and animals may promote new virulence factors. This study aimed at detecting the distending cytolethal toxin (CDT) encoding genes by PCR, studying the activity of hemolysin and also the influence of chelation solutions and ions. A total of 45 samples of C. jejuni from poultry origin, grown in Tryptone Soy Broth (TSB) were used for investigating hemolytic activity. After bacterial growth, samples were plated on Tryptic Soy Agar (TSA) containing 5% sheep, equine or bovine blood, being each blood tested individually. In order to check the influence of chelation agents and ions solution on the hemolytic activity, samples of C. jejuni strains were grown in TSB containing chelation agents individually: EDTA, acetic acid, CaCl2 ion, MgCl2 and FeCl3 solutions, all in microaerophilic atmosphere. Regarding the detection of Campylobacter jejuni hemolysin activity on TSA plates, blood hemolysis were observed in 48.89 % of samples when equine blood was used; in 40% of samples when bovine blood was used and in 31.11 % when the blood used was of sheep origin. The influence of ions and chelation agents in hemolysin activity in TSB when Campylobacter jejuni was plated on TSA with sheep blood can be described as: hemolytic activity was observed at 26.67% of samples when CaCl2 was used, at 15.55 % for FeCl3, 22 22 % for EDTA, 11.11 % for MgCl2 and only 2.22% when acetic acid was used. The hemolytic activity detected when bovine blood - TSA was used indicated 15.55% for CaCl2, 24.44% for FeCl3, 26.26 % for EDTA, 20 % for MgCl2 and 11.11% for acetic acid. In terms of the hemolytic activity when equine blood was used, the results indicated 24.44% for CaCl2, 22.22 % for FeCl3, 28.89 % for EDTA, 28.89 % for MgCl2 and 8.89% for acetic acid. Finally, regarding the detection of cdtA, cdtB and cdtC through PCR, 119 samples of C. jejuni from poultry origin were used. The results indicated that all three genes were present in 38 % of the samples, whereas only two genes were identified in 19 % of samples, while the cdtB gene was singly found in 14%, the cdtC gene was independently observed in 12%, cdtA and cdtB genes together were found in 1% of the samples; the cdtB and cdtC genes associated were detected in 1%, while cdtA alone answered for 1% of detections. The results also showed the presence of C. jejuni strains with virulence potential. The hemolytic activity increased significantly when blood of equine origin was used, and that this activity was reduced when ions or chelating agents were used in combination with the three types of blood cells.