Memoria para optar al Título Profesional de Médico Veterinario / La resistencia antimicrobiana es un fenómeno de gran importancia a nivel mundial, debido a sus consecuencias en la salud tanto humana como animal y también por las consecuencias económicas que genera. Entre las bacterias que presentan multirresistencia se encuentra Salmonella spp., patógeno distribuido mundialmente, generalmente transmitido por los alimentos, pero también por contacto directo o indirecto con su reservorios animales. Dentro de estos, adquieren relevancia los reptiles por ser portadores asintomáticos de la bacteria, constituyendo un reservorio que cada vez adquiere mayor importancia debido a la creciente popularidad que presentan como mascota en las familias actuales. En Chile existen escasos estudios sobre resistencia antimicrobiana en cepas de Salmonella spp. aisladas de reptiles que asocien genes a la resistencia fenotípica, razón por la cual, el objetivo del presente estudio apunta a la detección de genes de resistencia antimicrobiana en cepas de Salmonella spp. aisladas de reptiles en cautiverio, en la Región Metropolitana en Chile. Para ello, se realizó una prueba de susceptibilidad antimicrobiana por el método de Kirby-Bauer y posteriormente, mediante la técnica de PCR, se buscaron 11 genes de resistencia en las cepas en análisis, independiente de su fenotipo. Dentro de los genes a detectar, 3 de ellos están asociados a resistencia a beta-lactámicos (blaTEM, blaOXA y blaPSE-1), 4 a resistencia frente a estreptomicina (aadA1, aadA2, strA y strB), 3 para resistencia a tetraciclinas (tet(A), tet(B), tet(C)) y un gen que confiere resistencia a cloranfenicol (cmlA). Se observó una elevada sensibilidad fenotípica, donde el 23,5% de las cepas fue resistente a estreptomicina. De las 34 cepas analizadas, sólo se detectó el gen blaTEM en un 61,7%. El resto de las cepas resultaron negativas a la detección de los otros genes en análisis. La alta sensibilidad detectada puede deberse a fenómenos de curación plasmidial, mutaciones cromosomales, regulación de la expresión génica, entre otros, como mecanismo de restauración del fitness bacteriano en cepas conservadas en un medio carente de presión selectiva. Se concluye que las cepas analizadas presentan elevada sensibilidad fenotípica frente a beta-lactámicos, tetraciclinas, cloranfenicol y estreptomicina, encontrando sólo cepas portadoras del gen blaTEM. / Antimicrobial resistance is a phenomenon of great importance worldwide because of its impact on both human and animal health and the economic consequences it generates. Among resistant bacteria is Salmonella spp., worldwide distributed pathogen, usually transmitted by food, but also by direct or indirect contact with animal reservoirs. Within these, reptile become relevant because they are asymptomatic carriers of the bacteria, forming a reservoir that increasingly becomes more important due to the growing popularity as pets in today's families. In Chile there are few studies on antimicrobial resistance in strains of Salmonella spp. isolated from reptiles that associated genes to phenotypic resistance, reason why the aim of this study points to the detection of antimicrobial resistance genes in strains of Salmonella spp. isolated from reptiles in captivity, in Región Metropolitana in Chile. For this, an antimicrobial susceptibility test, by Kirby-Bauer method was performed and subsequently, by PCR, 11 resistance genes were searched in strains tested, regardless of their phenotype. Among genes to detect, 3 of them are associated with resistance to beta-lactam (blaTEM, blaOXA and blaPSE-1), 4 to resistance to streptomycin (aadA1, aadA2, strA and strB), 3 for tetracycline resistance (tet(A), tet(B), tet(C)) and a gene conferring chloramphenicol resistance (cmlA). High phenotypic susceptibility was observed, where 23,5% of the strains were streptomycin resistant. Of the 34 strains tested, only blaTEM gene was detected in 61,7%. The remaining strains were negative for detection of the other genes analyzed. High sensitivity detected may be due to a phenomenon of plasmidial healing, chromosomal mutations, gene expression regulation, among others, as a mechanism for restoring the bacterial fitness of strains preserved in a medium lacking of selective pressure. It is concluded that strains tested possess high phenotypic sensitivity to beta-lactams, tetracyclines, chloramphenicol and streptomycin, finding strains carrying only the blaTEM gene.

Orientador: Edir Nepomuceno Silva / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-07-19T02:06:01Z (GMT). No. of bitstreams: 1 Afonso_MagalyAnanias_M.pdf: 2768658 bytes, checksum: 3b0cd61fc672cafefd6f641b93b7bf53 (MD5) Previous issue date: 1994 / Resumo: Determinou-se a sobrevivência de Salmonella enteritidis em ovos artificialmente, contaminados e submetidos à cocção em chama de gás e forno de microondas (FMO), e, em alimentos preparados com ovos contaminados c submetidos a diferentes temperaturas de armazenagem. Foi utilizado Salmonella enteritidis patogênico, fagotipo 28 isolado de aves e ovos brancos de galinhas comerciais. Os alimentos contaminados com aproximadamente 106 a 108 UFC e examinados foram: ovos cozidos em água em ebulição por até oito minutos, ou em forno microondas por 1 minuto, ovos mexidos; ovos tipo pochê; maionese, glacê de clara, gemada e sorvete de creme. A recuperação da Salmonella enteritidis contaminante foi feita quantitativamente através do plaqueamento em duplicata de alíquotas de diluições decimais da amostra sobre Agar a, qualitativamente, com enriquecimento da mesma para verificar presença ou ausência de células viáveis na amostra original. A cocção de ovos em FMO foi mais eficiente na eliminação de Salmonella enteritidis que a em água, num mesmo período de tempo. A cocção de ovos em água em ebulição, até 8 minutos, reduziu drasticamente, mas não foi capaz de eliminar totalmente a contaminação por Salmonella enteritidis. A cocção de ovos em por 1:00 minuto reduziu drasticamente, mas não foi capaz de eliminar totalmente a contaminação por Salmonella enteritidis. O preparo convencional de ovos tipo pochê em chama de gás reduziu drasticamente, mas não foi capaz de eliminar totalmente a contaminação por Salmonella enteritidis. Novamente, a cocção de ovos tipo poché em FMO eliminou totalmente a contaminação por Salmonella enteritidis de ovos inoculados, quando o tempo de cocção era igual ou superior a 1m:30s. A cocção de ovos mexidos em ambos os métodos foi capaz de eliminar totalmente a contaminação de ovos inoculados. Sorvete contaminado, mantido a -18ºC, teve grande redução na sua contami¬nação original por Salmonella enteritidis mas deixou, ainda, uma população residual após 35 dias de armazenagem. A população inicial de Salmonella enteritidis contaminante de glacê de ciara reduziu gradativamente na armazenagem refrigerado a (8ºC) mas, continha, ainda, população residual após 7 dias. A escaldarem com o leite fervente no preparo de gemada artificialmente inoculada exerceu efeito "past eur isante" destruindo quase totalmente a contaminação por Salmonella enteritidis. A detecção de células viáveis sobreviventes (101 UFC/g) só foi possível através de cultivo qualitativo. Tanto a variação do pH (3,80 a 4,04), do tempo (4 a 72 horas), quanto a variação da temperatura de armazenagem (8 e 25ºC) tiveram pequeno ou nenhum efeito na redução ou aumento da contaminação por Salmonella enteritidis de maionese. A população de fungos. e leveduras aumentou gradativamente quando maionese contaminada com Salmonella enteritidis. Foi mantida a 25ºC, mas nao quando estocada a 8ºC, no período de 72 horas, independente da variação do pH da amostra de 3,80 a 4,04. Nossos experimentos determinaram que alimentos que contenham ovos e os ovos propriamente ditos, quando apresentam uma carga alta de contaminante como Salmonella enterit idis podem, em alguns casos, trazer risco à saúde consumidor quando sofrem preparo doméstico convencional. / Abstract: The survival of Salmonella enteritidis was determined in artificially contaminated eggs cooked using both a gas flame and microwave oven (MWO). It was also determined in foods prepared with contaminated eggs and stored at different temperatures. The pathogenic Salmonella enteritidis used was phagotype 28, isolated from chicken meat and white eggs produced commercially. The following foods were examined, having been contaminated with approximately to 106 to 108 UFC: eggs cooked in boiling water for 8 minutes or in the microwave oven for 1 minute, scrambled eggs, poached eggs; mayonnaise, egg white glaze; milk drink containing beaten egg yolk, vanilla ice-cream. The quantitative recuperation of Salmonella enteritidis contamination was carried out by plating decimal dilutions of the sample on agar in duplicate. For the qualitative determination, an enrichment procedure was used to check for the presence or absence of viable cells in the original sample. Using the same time period, MWO cooking was more efficient than boiling water in eliminating Salmonella enteritidis. Boiling in water for 8 minutes reduced the count drastically, but was not capable of eliminating the Salmonella enteritidis completely. Similarly, poaching eggs over a gas flame reduced the count drastically but did not completely eliminate Salmonella enteritidis contamination, whereas MWO totally eliminated it if a cooking time equal or greater to 1 min 30 sees was used. Both methods of cooking scrambled eggs completely eliminated the contamination. When contaminated ice-cream was stored at -18ºC, a considerable reduction in the Salmonella enteritidis count occurred, but after 35 days of storage, there was still a residual population. In egg white glaze- the- initial population of Salmonella enteritidis declined gradually during refrigerated storage (8ºC), but there was still a residual population after 7 days. In the milk drink containing beaten egg yolk, the scalding of the contaminated yolk with boiling milk effectively pasteurized the yolk, almost completely destroying the Salmonella enteritidis contamination. The detection of surviving viable cells- was only possible by way of qualitative cultivation (101 UFC/g). With respect to mayonnaise, variations in pH (3.80 - 4.04), time (4-7S hours) and storage temperature (8 e 25ºC) had little or no effect on the reduction or increase of the Salmonella enteritidis contamination. The yeast and mould count increased gradually when maionese contaminated with Salmonella enteritidis was held at 25ºC, but not when held at 8ºC for a period of 72 hours, independent of the variation in sample pH (from 3.80 to 4.04). These experiments showed that foods containing eggs can, in some cases, present health risks to the consumer when prepared by conventional domestic methods, if the original eggs show a high level of contamination by Salmonella enteritidis. / Mestrado / Mestre em Tecnologia de Alimentos

Orientador: Marcelo Brocchi / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T16:38:13Z (GMT). No. of bitstreams: 1 Neves_MeirieledaSilvadas_M.pdf: 2620199 bytes, checksum: 70b2df72cfdb93196c91a87c813e12e9 (MD5) Previous issue date: 2015 / Resumo: O gênero Salmonella pertence à família Enterobacteriaceae que agrupa bacilos Gram-negativos, anaeróbios facultativos, fermentadores e geralmente flagelados. S. enterica é um dos patógenos de origem alimentar mais prevalente, sendo que infecções causadas por essa bactéria podem estar relacionadas a praticamente todos os tipos de alimentos. O trabalho foi proposto com o intuito de realizar a caracterização fenotípica e molecular de linhagens atenuadas de Salmonella enterica Typhimurium para genes codificadores de proteínas associadas ao nucleóide (NAPs Nucleoid associated Proteins). As características fenótipicas dos mutantes nulos de Salmonella enterica para os genes ihfA ou ihfB, codificadores das subunidades A e B de IHF, foram avaliadas através de crescimento in vitro, motilidade, sobrevivência frente ao estresse nutricional (sobrevivência em fase estacionária), sob condições ácidas, na presença de sais biliares e quanto à capacidade de invasão e sobrevivência em macrófagos (linhagem J774A.1). Testes de confirmação da atenuação e avaliação da capacidade de induzir proteção em caso de infecção por S. enterica foram realizados utilizando o modelo murino. Os mutantes não apresentaram diferença no crescimento in vitro e na capacidade de sobreviver na presença de sais biliares em comparação com a linhagem selvagem. As linhagens mutantes para os genes ihfA ou ihf ihf ihfB) apresentaram uma menor capacidade de sobrevivência sob condições ácidas quando comparadas com a linhagem selvagem. A motilidade dos mutantes simples também foi reduzida. Os mutantes simples e duplo apresentaram maior capacidade de sobreviver sob estresse nutricional quando comparados com a linhagem selvagem. O mutante para o gene ihfA e o duplo mutante apresentaram um aumento na capacidade de invadir macrófagos. ihf ihfB mostraram uma capacidade aumentada em sobreviver no interior de macrófagos quando comparadas com a linhagem selvagem. Os mutantes nulos viii de Salmonella enterica para os genes ihfA ou ihfB apresentam atenuação, em diferentes graus, quanto à virulência e apresentaram capacidade de induzir proteção no modelo murino de infecção por S. enterica. Esses resultados demonstram que essa proteína apresenta função relacionada com a virulência bacteriana, sendo um importante alvo de estudo na busca de linhagens atenuadas / Abstract: The genus Salmonella belongs to the Enterobacteriaceae family that comprises Gram-negative bacillus, facultative anaerobe, fermenting and generally flagellate. S. enterica is one of the most prevalent food-borne pathogen, and infections caused by this bacterium can be associated to almost all types of food. The work was proposed with the purpose of performing phenotypic and molecular characterization of attenuated strains of Salmonella enterica Typhimurium for genes encoding proteins associated with the nucleoid (NAPs - Nucleoid associated Proteins). The phenotypic characteristics of the null mutants of Salmonella enterica for genes ihfA or ihfB, encoding the A and B subunits of IHF, were evaluated by in vitro growth, motility, survival under nutritional stress (survival in the stationary phase), under acidic conditions, in the presence of bile salts and for the ability of invasion and survival in macrophages (J774A.1 strain). Attenuation tests and evaluation of the capacity to induce protection in case of infection by S. enterica were performed using the murine model. The mutants showed no difference in the in vitro growth and the ability to survive in the presence of bile salts in comparison with the wild type strain. The single mutant for ihfA or ihf ihf ihfB) showed decreased survival under acidic conditions when compared to the wild type strain. Motility of single mutants was also reduced. Single and double mutants showed higher ability to survive under nutritional stress when compared with the wild type strain. The mutant gene for ihfA and the double mutant showed an increased ability to invade ihf ihfB mutants showed an increased ability to survive within macrophages when compared with the wild type strain. Null mutants of Salmonella enterica for ihfA or ihfB genes exhibited attenuation, to varying degrees, for virulence and showed ability to induce protection in a murine model of infection by S. enterica. x These results demonstrate that this protein has function associated to bacterial virulence and is an important subject of study in search for attenuated strains / Mestrado / Genetica de Microorganismos / Mestra em Genética e Biologia Molecular

Memoria para optar al Título Profesional de Médico Veterinario / La alta prevalencia de enfermedades infecciosas bacterianas ha generado la continua búsqueda de nuevos elementos terapéuticos que ayuden a su control y prevención. Entre estas herramientas terapéuticas se encuentran los bacteriófagos o fagos, virus que, a través de su ciclo reproductivo lítico, destruyen bacterias. Es por esto que la investigación sobre el uso de fagos en Medicina Veterinaria se ha vuelto cada vez más importante. Fagos que infectan Salmonella han sido documentados, siendo abundantes y ampliamente distribuidos en ambientes relacionados a producciones bovinas. En este estudio investigamos la abundancia y diversidad de fagos de Salmonella en muestras de bovinos provenientes de 6 sectores distintos de Isla de Pascua. Como resultado, se aislaron en total 90 fagos, provenientes de los 6 sectores muestreados. La caracterización del rango de hospederos (RH) determinó que los fagos aislados lisan entre 1 y 19 cepas de Salmonella hospederas, siendo S. Javiana, S. Dublin, S. Heidelberg, S. Enteritidis y S. Agona las más susceptibles a la lisis. Además, según el número de cepas lisadas, 26 de los 90 fagos aislados (28,9%) fueron clasificados en estrecho RH, mientras los otros 63 (70%) en amplio RH. Solo 1 fago fue clasificado en muy amplio RH, por lisar 19 cepas hospederas. Nuestro estudio no solo mostró la considerable diversidad fenotípica que presentan los fagos de Salmonella que se encuentran común y ampliamente distribuidos a lo largo del territorio de Isla de Pascua, sino también muestra que fagos que lisan serotipos que comúnmente causan salmonelosis en bovinos, son obtenidos con frecuencia, sugiriendo que los fagos podrían jugar un rol importante en la ecología de Salmonella en estos animales. / The high prevalence of bacterial infectious diseases has generated the continuous search for new therapeutic elements that help its control and prevention. Among these therapeutic tools are bacteriophages or phages, viruses that cross their lytic reproductive cycle, destroy bacteria. Therefore research on the use of phages in Veterinary Medicine has become increasingly important. Phages that infect Salmonella have been documented, being abundant and widely distributed in environments related to bovine production. In this study, we investigated the abundance and diversity of Salmonella phage in samples of cattle from 6 different sectors of Easter Island. As a result, a total of 90 phage were isolated from the 6 sampled sectors. The characterization of the host ranges (HR) determined that phages isolated lysed between 1 and 19 Salmonella host strains, being S. Javiana, S. Dublin, S. Heidelberg, S. Enteritidis and S. Agona, the most susceptible to lysis. In addition, according to the number of lysed strains, 26 of the 90 isolated phages (28.9%) were classified in narrow RH, while the other 63 (70%) in broad RH. Only 1 phage was classified in very broad RH, by lysing 19 host strains. Our study not only showed the considerable phenotypic diversity of the Salmonella phages that are common and widely distributed throughout the Easter Island territory, but also shows that phage lysing serotypes that commonly cause salmonellosis in cattle are obtained with frequency, suggesting that phages could play an important role in the ecology of Salmonella in these animals.

Salmonellosis is one of the most important foodborne zoonotic diseases throughout the world and poultry represents an important source of infection in humans. Chickens may become infected during incubation, in the brooding houses, through various vectors, such as feed and rodents or during slaughtering and processing. The use of antibiotics have been reduced and even banned in some countries, due to the risk of bacterial populations developing resistance against the antibiotics. This lead to the exploration of alternative products for antibiotics as growth promoters, which include prebiotics, probiotics, organic acids, essential oils, plant extracts and many more. These products may improve animal health, productivity and microbial food safety in a natural way. A feeding trial was conducted to investigate the effects of the dietary inclusion of probiotics and a prebiotic to improve the health and performance of broilers, which were challenged with Salmonella typhimurium. 1800 chicks (900 chicks exposed to Salmonella and 900 chicks not exposed) were randomly assigned to 6 dietary treatments for 5 weeks. The dietary treatments were: 1) No feed additives added, 2) A prebiotic (fructooligosaccharide) added to the feed, 3) Probiotic type 1 (Spore-forming bacteria) added to the feed, 4) Probiotic type 1 combined with the prebiotic added to the feed, 5) Probiotic type 2 (Lactobacillus spp.) added to the feed, 6) Probiotic type 2 combined with the prebiotic added to the feed. The feed intake, average daily gain and body weight of the control (nonchallenged) birds were significantly higher (P<0.05) than the Salmonella (challenged) birds. The Probiotic type 2 combined with the prebiotic improved the feed intake of the non-challenged birds compared to the non-challenged birds that received no supplementation or only a prebiotic. The challenged and non-challenged birds that did not receive any supplementation had lower body weights and average daily gains compared to the birds that received supplementation. The feed conversion ratio showed significant differences among the treatments (P<0.003) and between the control and Salmonella birds (P<0.05). The non-challenged birds fed the Probiotic type 2 combined with a prebiotic and the challenged birds fed only the prebiotic, displayed a decrease in liver weight, compared to the other treatments. However, the duodenum, jejunum and caeca weights of the broilers were significantly (P<0.05) enlarged shortly after Salmonella exposure through the inclusion of Probiotic type 1 and the two combination treatments in the diet. No significant differences were observed in the ileal weights after Salmonella exposure or after dietary supplementation. The total serum protein and the aspartate amino transferase (AST) levels showed no significant differences between the groups and treatments. However, the albumin levels of the challenged birds were significantly lower (P<0.05) than the non-challenged birds. The globulin levels were higher for the challenged birds that did not receive any supplementation than those that received a prebiotic and probiotics. The albumin: globulin ratios were higher for the non-challenged birds than the challenged birds. In general, the challenged birds tended to have more lesions than the non-challenged birds on the gastrointestinal tract (GIT). There were no significant differences in the villous height, mucosal thickness and crypt depth of the duodenum, jejunum and ileum. However, the control birds supplemented with Probiotic type 2 combined with a prebiotic showed a thicker mucosa layer than the control that received no supplementation. These findings indicate that the supplementation of a basal diet with probiotics and combination treatments of probiotics with a prebiotic can be used as growth promoters for broilers. These products, especially the Probiotic type 2 combined with the prebiotic, show promising effects as alternatives for antibiotics as pressure increases to eliminate the growth promotant antibiotics from being used in the livestock industry. / Dissertation (MSc(Agric))--University of Pretoria, 2011. / Animal and Wildlife Sciences / unrestricted

Salmonella Typhimurium is a Gram-negative, facultative intracellular and anaerobic bacterium with high importance as an intestinal pathogen. For successfully colonization and persistence within the host S. Typhimurium utilizes a set of virulence-associated secretion systems. One of these systems is the type I secretion system (T1SS) encoded by Salmonella pathogenicity island 4 (SPI-4). SPI-4 encodes for an adhesin which is necessary to establish initial contact between the bacteria and host cells. Subsequently, the type 3 secretion system encoded by SPI-1 (T3SS-1) injects its effector proteins into the host cell leading to the internalization of the bacteria. SPI-4 harbors six genes, siiAF. SiiCDF form the canonical subunits of the T1SS, SiiE is the only known substrate and the two accessory proteins SiiAB probably form a proton channel. SiiE is an approximately 600 kDa large secreted adhesion. Interestingly, there is only a temporal stable retention of SiiE at the bacterial surface where it can exert its adhesin function. SiiAB is thought to play an important role in the switch between secretion and stable retention of SiiE as their deletion leads to reduced adhesion and invasion of polarized epithelial cells while secretion is only mildly affected.The aim of this thesis was to further characterize the role of SiiAB for SiiE-mediated adhesion and to identify possible environmental signals for the switch between retention and secretion of SiiE. Invasion of polarized epithelial cells was used as a main readout to quantify SPI-4 function. For that the “Virtual Colony Count” method was established as an alternative analytical method and compared to the classical counting of colony-forming units (CFUs). With this automated evaluation method it was possible to increase the throughput of infection experiments and to minimize errors by manual CFU counting. SiiAB has sequence and structural homologies to MotAB and other ion- conducting channels such as PomAB, ExbBD or TolQR, which is why a similar function is assumed. By ratiometric pH measurements in living S. Typhimurium cells it could be shown that overexpression of the supposed proton channel SiiAB actually leads to a drop in intracellular pH. The mutation of a conserved aspartate residue at position 13 of SiiA resulted in loss of proton-conducting function of the SiiAB complex. The same holds true for aspartate 32 of MotB, which could confirm the hypothesis of the functionality of SiiAB. In the present study, the methyl-accepting chemotaxis protein CheM was identified and characterized as an interaction partner of SiiAB. It could be shown that the perception of aspartate by CheM shifted the ratio of secreted and retained SiiE towards the retention of the adhesin. While this effect was independent of other motility or chemotaxis components, the transduction of this signal is probably mediated through the interaction with SiiAB. The stimulation of CheM thus has a direct influence on the SPI-4 mediated adhesion of the bacteria and thus represents an example of a noncanonical signal transduction of a MCP to a virulence factor. It remains to be clarified whether this interaction is also important in vivo for a successful infection of Salmonella. Here, components of the mucus layer could provide the signals for the CheM sensor molecule resulting in precise triggering of SiiE-mediated adhesion in the enterocyte vicinity.

Salmonella is one of the leading foodborne pathogens causing illness today. Because of this, Salmonella rapid detection methods are under immense study for use in food. The traditional method, using the Food and Drug Administration- approved Bacterial Analytical Manual procedure, takes 4-6 days for Salmonella detection in food. Other rapid methods still take at least 16 h for detection due to their enrichment steps. The hypothesis of this study was that the use of immobilized antibodies coupled with polymerase chain reaction (PCR) can be used for the rapid capture and detection of Salmonella spp. in food without the need for pre-enrichment. The rapid detection system was developed using immobilized anti-Salmonella antibody beads to capture and separate Salmonella from food without using an enrichment step. Detection of the immunocaptured Salmonella was done through PCR or an enzyme-linked immunosorbent assay (ELISA)-based system entitled Rapid Immuno-Capture (RIC). The detection limit for buffer, chicken rinse, and shell eggs with static antibody capture PCR and RIC, was determined by inoculation of the Salmonella-free samples. The RIC assay detected Salmonella spp. in buffer at concentrations as low as 4 x 101 CFU/ml, and in chicken rinse and shell eggs, the assay detected Salmonella at 4 x 103 CFU/ml. The antibody capture with PCR detected Salmonella in buffer at concentrations as low as 4 x 102 CFU/ml, in chicken rinse at concentrations as low as 4 x 105 CFU/ml, and in shell egg at bacteria concentrations of 4 x 106 CFU/ml.

Ziel dieser Arbeit war es, die porzine Salmonelleninfektion unter Berücksichtigung der drei beim Schwein am häufigsten vorkommenden Salmonella-Serovaren anhand des klinischen Bildes, der quantitativen und qualitativen Erregerausscheidung, des Kolonisationsverhaltens, der Serokonversion bezüglich der unterschiedlichen Immunglobulin-Isotypen IgA, IgG und IgM sowie durch die Labordiagnostik näher zu charakterisieren. Bisherige Studien an Schweinen wurden überwiegend mit S. Typhimurium durchgeführt, weitere Serovare sind bisher nur unzureichend erforscht worden, obwohl sie ebenfalls zoonotisches Potential besitzen, aber vergleichsweise nur als gering virulent und kaum invasiv gelten. Die Durchführung dieser Studie erfolgte mit 6 Wochen alten Absatzferkeln, welche nach einem bereits etablierten Modell mit den jeweiligen Salmonella-Serovaren infiziert wurden. Nach dem Challenge wurden die Tiere täglich klinisch untersucht und es wurden Kotproben zur qualitativen und quantitativen Erregerausscheidung entnommen. Parallel dazu wurden im Abstand von zwei bzw. drei Tagen Blutproben für ein Differentialblutbild, Blutchemie sowie für die serologische Untersuchung gewonnen. Nach einer Woche wurden die Tiere getötet und es wurden insgesamt 13 sterile Organproben gewonnen, um eine Aussage über das Kolonisationsverhalten treffen zu können. Die Infektionsversuche zeigten, dass nach oraler Verabreichung von 1x1010 KbE die Tiere der mit S. Infantis infizierten Gruppe, entgegen den Erwartungen, die stärksten klinischen Symptome einer Salmonellose mit Diarrhoe, Fieber, Anorexie sowie reduziertem Allgemeinbefinden zeigten. Nach dem Challenge schieden alle Tiere der drei Infektionsgruppen den jeweiligen Challengestamm zu allen Zeitpunkten des Experimentes aus. Dabei war die quantitative Erregerausscheidung bei der S. Infantis-Infektionsgruppe stets am höchsten, bei der S. Derby-Gruppe stets am geringsten. Die Kolonisationsrate in den untersuchten Organproben war bei der S. Derby-Gruppe mit insgesamt 80,7 % am höchsten, gefolgt von der S. Typhimurium DT104-Gruppe mit 80,3 %. S. Infantis wurde mit einer Kolonisationsrate von 73,6 % am seltensten isoliert. Bei der quantitativen bakteriologischen Untersuchung der Organproben wurden bei der S. Typhimurium DT104-Gruppe die meisten Salmonellen vorwiegend aus den lymphatischen Organen isoliert, während bei der S. Infantis-Gruppe die Salmonellenbelastung in den essbaren Organen am höchsten war. Die serologische Untersuchung mittels isotypspezifischen ELISA offenbarte für IgG einzig bei der S. Typhimurium DT104-Gruppe einen deutlichen Anstieg der spezifischen Antikörperaktivität, während für IgM die höchste Aktivität bei der S. Derby-Gruppe gemessen wurde. Für IgA fand bei allen Tieren der drei Infektionsgruppen keine Serokonversion statt. Im Differentialblutbild stellten sich bei allen Tieren der drei Infektionsgruppen nach dem Challenge mit einer Leukozytose, einer Linksverschiebung und einer Monozytose typische Anzeichen einer bakteriellen Infektion ein. Die blutchemische Untersuchung offenbarte besonders bei der S. Typhimurium DT104-Gruppe und der S. Infantis-Gruppe größere Verluste von Natrium und Chlorid nach dem Challenge, während es bei der S. Derby-Gruppe zu keinen Elektrolytverschiebungen im Plasma kam. Im Rahmen dieser Arbeit wurde deutlich, dass S. Infantis auch beim Schwein schwere klinische Verläufe verursachen kann und auch in essbaren Organen kolonisiert. Diese invasiven Isolate stellen neben den wirtschaftlichen Verlusten ein großes Verbraucher- schutzproblem dar. Obwohl die Prävalenz von S. Infantis beim Schwein nicht so hoch wie die von S. Typhimurium ist, so sollte diese Serovar in Zukunft besonders beobachtet werden. Für S. Derby wurde trotz schwacher Virulenz die höchste Kolonisationsrate nachgewiesen, was deren Potential, klinisch inapparente Salmonelleninfektionen auszulösen, deutlich macht. Somit belegt diese Arbeit, dass S. Infantis und S. Derby für den Menschen sehr bedeutsam sein können und daher ein entsprechendes Monitoring erfordern.

The presence of Salmonella enterica throughout the production and processing continuum is a concern in broiler industry. While federal regulations have lowered the acceptable level of Salmonella contamination on broiler carcasses, the mechanisms that contribute to pathogen attachment are not fully understood. Salmonella Kentucky has become the predominant Salmonella serovar isolated from broilers carcasses at the end of the immersion chill tank. In Europe and Africa this serovar has been shown to acquire antibiotic resistance genes that may lead this non-typhoidal serotype to become a potential public health concern. To investigate the genes that are involved in colonization of the bacteria to broiler skin, a mutant library of the bioluminescent strain of S. Kentucky was constructed. According to the chicken attachment assay, it was concluded that attachment is a multifactorial process with the following elements contributing: i), flagella, ii), LPS structure, iii), amino acid metabolism, iv), TCA cycle pathway; v), conjugative transfer system, vi), multidrug resistant protein, vii), signaling and transportation system, viii), metabolism, ix), different enzymes, x), phage tail fiber protein H, xi), fimbrial export usher proteins, xii), membrane proteins xiii), and several unnamed proteins. The role of flagella between all of these contributing elements appeared to be the most significant. The flagella motor gene, filament sub-units and hook associated protein were deleted by using the ë red recombination method. The mutants’ ability to colonize broiler skin was compared to their parental strain, and the motility and flagellin main sub-unit (FliC) were recognized as the key factors contributing to bacterial attachment. Using Caco-2 cell lines as a cell model to assess adhesion and invasion capacity of flagella mutants, similar results were observed. Based on the result of the experiments conducted in this study, it appears that the active flagella FiC sub-unit plays an important role in colonization of epithelial cells outside and inside of the broilers.

Center for Disease Control and Food and Drug Administration reports indicate that cantaloupe is one of the five most likely fruits and vegetables to cause a foodborne disease outbreak. Cantaloupe is a potential hazardous food based on the FDA food code since it is capable of supporting pathogen growth due to its low acidity and high moisture content. The objectives of this study were to determine the effect of strain and temperature on growth and biofilm formation of L. monocytogenes and Salmonella spp. in cantaloupe flesh and peel extracts on different food-contact processing surfaces. Growth of L. monocytogenes and Salmonella strains was greater in high cantaloupe flesh and peel extract concentration at 22°C and 10°C. In 50 mg/ml of cantaloupe flesh or peel extract, the cell numbers of L. monocytogenes and Salmonella increased by 5.0-5.5 log CFU/ml in 40 h at 22°C and 1-3.5 log CFU/ml in 72 h at 10°C. In 2 mg/ml of cantaloupe flesh or peel extract, the cell numbers of L. monocytogenes and Salmonella increased by 4.0-4.5 log CFU/ml in 72 h at 22°C but no change in log CFU/ml in 72 h at 10°C. There were no differences (P ˃ 0.05) among L. monocytogenes orSalmonella strains for biofilm formation in cantaloupe extracts, but biofilm formation was greater (P < 0.05) at high temperature and high cantaloupe flesh or peel extract concentration. In 50 mg/ml cantaloupe flesh or peel extract, L. monocytogenes and Salmonella produced biofilms of 7 log CFU/coupon in 4 days at 22°C and 4-5 log CFU/coupon in 7 days at 10°C. In 2 mg/ml cantaloupe flesh or peel extract, L. monocytogenes and Salmonella produced biofilms of 5-6 log CFU/coupon in 4 days at 22°C and 3-4 log CFU/coupon in 7 days at 10°C. L. monocytogenes and Salmonella spp. formed less biofilms (P < 0.05) on buna-n rubber when compared to stainless steel, polyethylene and polyurethane surfaces. These findings indicate that a very low concentration of nutrients that are leaked from cantaloupe flesh or peel can induce growth and biofilm formation in L. monocytogenes and Salmonella spp. on different food-contact surfaces.