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Salmonella enterica is a foodborne pathogen of major significance, and as such it has been extensively studied by researchers around the world. However, despite the numerous scientific publications on Salmonella, there are still many gaps in our understanding of its biology. One such gap is in the bacteria's interactions with invertebrate hosts, and in particular, oysters. Nearly 70 million pounds of oysters are consumed in the United States each year, and previous work in the Joens' laboratory found Salmonella in roughly 7% of the market oysters they sampled, with the majority of the isolates being the Newport serovar. The majority of oysters are consumed raw, which makes the presence of Salmonella within oysters a potentially significant food safety problem.To more closely examine the interactions between Salmonella and oysters, the Present Study developed a method to consistently and reproducibly raise oysters in a controlled laboratory environment in order to systematically expose them to enteric bacteria and quantify the amount of surviving bacteria at various time points after the initial exposure. Use of this model system throughout the Present Study led to four main conclusions.The first is that Salmonella enterica serovar Newport is capable of surviving in oysters for at least 60 days, from an average concentration of 3.7x103 CFU/g of oyster meat after 10 days, to over 102 CFU/g of oyster meat after 60 days. The second main conclusion is that the Newport serovar of Salmonella, which was found in such predominance in the earlier Joens' laboratory study, does not appear to have any special adaptations for survival within oysters, as other strains of Newport and other serovars of Salmonella survived equally well within our model. The third main conclusion, based on the results of immunohistochemistry, is that the relationship between Salmonella and oysters is not a transient interaction that is limited to the outside of the oyster's gut epithelium, but involves a long-term colonization inside the oysters' connective tissues. Because the survival of Salmonella in oysters could be of a pathogenic nature, the Present Study knocked out two key type III secretion systems (T3SS) found in two distinct Salmonella pathogenicity islands (SPI-1 and SPI-2) known to be critical for pathogenesis in mammalian hosts and examined their role in the bacteria's ability to survive within oysters. The results revealed that neither the SPI-1 nor the SPI-2 T3SS were necessary for Salmonella's survival in oysters, which led to the final conclusion of the Present Study that the nature of Salmonella's infection of oysters is fundamentally different than the pathogenesis that occurs in mammalian hosts and that further study of the mechanisms of the survival of Salmonella in oysters is needed to better understand the important and interesting relationship between a significant source of food and this common, and occasionally deadly, foodborne pathogen.

The usage of antimicrobials either as performance enhancers or for prophylactic and therapeutic purposes in food animals, such as chickens, increases the prevalence of antimicrobial drug resistance among enteric bacteria of these animals. This may be transferred to people working with such animals, e.g. abattoir workers, or the products arising from these animals. In this study antimicrobial drug resistance was investigated for selected enteric bacteria from broilers raised on feed supplemented with antimicrobial growth enhancers, and the people who carry out evisceration, washing and packing of intestines in a high throughput poultry abattoir in Gauteng, South Africa. Poultry farms (n=6) were purposively selected on the basis of allowing for sampling of farms from more than one grow out cycle. Broiler carcases (n=100) were randomly selected per farm five minutes after slaughter and sampled by incising caecae from the rest of the gastro-intestinal tract (GIT). The ends of each caecae were tied off to prevent contamination and to enhance the culturing of anaerobic bacteria. In the laboratory, caecal contents were selectively cultured for Clostridium perfringens, Escherichia coli, Enterococcus faecium, E. faecalis, and vancomycin-resistant enterococci (VRE). Salmonella enterica was isolated using pre-enrichment followed by selective culture. The minimum inhibitory concentration (MIC) micro broth dilution test as prescribed by the Clinical and Laboratory Standards Institute USA (CLSI), previously known as National Committee of Clinical Laboratories (NCCL), was used to determine the susceptibility of the isolates to the following antimicrobials: vancomycin, virginiamycin, doxycycline, trimethoprim, sulphamethoxazole, ampicillin, bacitracin, enrofloxacin, erythromycin, fosfomycin, ceftriaxone and nalidixic acid. The same was done on the faeces of 29 abattoir workers exposed to potentially resistant micro-organisms from broilers and 28 persons used as controls, who had not been equally exposed to potentially resistant micro-organisms from broilers. Both of the human populations had not been treated with antimicrobials within three months prior to sampling. Statistical analysis was done by Fisher’s exact test. No salmonellae and VRE on VRE selective agar (Oxoid UK) were cultured. Two Clostridium perfringens, 168 E. coli, 20 E. faecalis and 96 E. faecium isolates from the broiler caecae were cultured. Fifty four (28 and 26) E. coli, 24 (21 and 3) E. faecalis and 12 (2 and 10) E. faeciumfrom humans were cultured. The figures in brackets represent the abattoir workers and human controls respectively. The majority of E. coli isolates from broilers had MIC’s above the cut off point for the antimicrobials tested. Low resistance was observed among broiler enterococci isolates to vancomycin, virginiamycin, trimethoprim and ampicillin. A comparison of the median MIC’s of isolates from abattoir workers (packers) and the control group revealed significant differences in the median MIC’s for the following antimicrobials; E. faecalis: enrofloxacin (p=0.019). E. faecium, trimethoprim (p=0.01), enrofloxacin (p=0.029) and erythromycin (p=0.03). E. coli: trimethoprim (p=0.012) and ampicillin (p=0.036). Use of antimicrobials as feed additives causes resistance among enteric bacteria from broilers. Significant differences between median MIC’s of abattoir workers (packers) and the control group were observed for therapeutics and not growth enhancers. There was a tendency for isolates from abattoir workers to have a higher median MIC and a higher number of resistant isolates as compared to the control group. In spite of the fact that there was a high level of resistance in the enteric commensal bacteria of broiler caecae, an association could not be shown with that of the human enteric bacteria. It could not be concluded that a significant AMR transfer to poultry abattoir workers existed. This notwithstanding, both the control and experimental group, carried levels of resistance among their enteric bacteria that could be described as being high. / Dissertation (MSc (Veterinary Science))--University of Pretoria, 2007. / Paraclinical Sciences / unrestricted

Nonsteroidal anti-inflammatory drugs (NSAIDs) are a widely used class of drugs, due in part to the effective anti-inflammatory and analgesic properties they exhibit. Unfortunately, NSAIDs also exhibit substantial gastrointestinal (GI) toxicity. The mechanisms underlying the ability of NSAIDs to cause ulceration in the stomach and proximal duodenum are well understood, and this injury can largely be prevented through the suppression of gastric acid secretion by proton pump inhibitors (PPIs) or histamine H2 receptor antagonists (H2RAs). In contrast, the pathogenesis of small intestinal injury induced by NSAIDs (i.e., NSAID-enteropathy) is poorly understood, and there are no proven-effective therapies. This is a major clinical concern as NSAID-induced enteropathy and bleeding occur more frequently than NSAID-induced gastropathy, and is associated with significantly higher rates of morbidity and mortality. There is clear evidence that indicates important contributions to NSAID-enteropathy by bile, enteric bacteria, and the enterohepatic circulation of NSAIDs. However, it is not clear which of these mechanisms is/are the primary driver(s) of intestinal damage and injury. There is also evidence that hydrogen sulfide (H2S) can protect the GI mucosa from ulceration and reduce the severity of NSAID-induced GI damage, although the mechanisms of H2S-induced intestinal protection remain to be determined. Therefore, the central aim of this thesis was to evaluate the roles of bile, enteric bacteria, and the enterohepatic circulation of NSAIDs in the pathogenesis of NSAID-enteropathy, and to investigate the ability of H2S to protect the small intestine from NSAID-induced damage. Chapter 1 is an introduction to the relevant literature and Chapter 2 is an outline of the thesis scope and objectives. In Chapter 3, I demonstrated that the co-administration of an H2S-releasing agent protected rats from NSAID-induced enteropathy, in part by preventing NSAID-induced dysbiosis and bile cytotoxicity. In Chapter 4 and 5, I established that the co-administration of PPIs and H2RAs exacerbated NSAID-enteropathy in part by causing intestinal dysbiosis and enhanced bile cytotoxicity. Lastly, I demonstrated that the small intestine-sparing effects of an H2S-releasing NSAID, ATB-346, are partly attributable to the reduced enterohepatic circulation of ATB-346 or the naproxen liberated from this drug (Chapter 5). In summary, the work presented in this thesis provided novel understanding of the complicated pathogenesis of NSAID-enteropathy by confirming that the nature of the bile, the enterohepatic circulation of NSAIDs, and the nature of the intestinal microbiota are of paramount importance. In addition, the results also demonstrated that hydrogen sulfide represents an effective preventative therapy for NSAID-enteropathy and that H2S-releasing NSAIDs, such as ATB-346, have remarkable preclinical safety. / Thesis / Doctor of Philosophy (PhD)

Orientador: Oswaldo Durival Rossi Junior / Banca: Luiz Augusto do Amaral / Banca: Ana Maria Centola Vidal Martins / Resumo: A deterioração "blown pack" é caracterizada por abundante produção de gás, induzindo a completa distensão da embalagem durante o processo de estocagem sob refrigeração. Quando a embalagem é aberta, há um odor desagradável, levemente fecal. O gás presente na embalagem é composto por dióxido de carbono e hidrogênio e por vários tipos butíricos do metabolismo fermentativo. O objetivo deste experimento foi determinar possíveis causadores deste tipo de deterioração, quantificando as populações de bactérias da família Enterobacteriaceae, e caracterizando-as nos principais gêneros e espécies encontradas, o número de bactérias ácido-lácticas, a freqüência de Clostridium estertheticum e do Clostridium gasigenes, em carnes próprias para o consumo e em carnes que apresentaram a deterioração "blown pack". Para contagem e identificação dos membros da família Enterobacteriaceae e contagem de bactérias ácido-lácticas utilizou-se de técnicas microbiológicas clássicas. Já para pesquisa do C. estertheticum e C. gasigenes fez-se uso de técnicas de biologia molecular. Os microrganismos da família Enterobacteriaceae e bactérias ácido-láticas estavam presentes em populações elevadas e em maior número nas carnes com deterioração "blown pack". A espécie mais freqüentemente encontrada foi a Hafnia alvei. As amostras com deterioração "blown pack' apresentaram maior positividade para o C. estertethicum que amostras não deterioradas. Não houve diferença estatística de positividade para a presença do C. gasigenes entre amostras com deterioração "blown pack" e carnes não deterioradas. A principal forma de controle desta deterioração é a prevenção da contaminação da carne por material fecal. / Abstract: The "blown pack" spoilage is characterised by abundant gas production, leading to complete gross distention pack during refrigerated storage. When the packaging is opened, there is an unpleasant smell, lightly fecal. The gas present in the package is composed of carbon dioxide and hydrogen and also of several butyric types of metabolism fermentation. The purpose of this experiment was to determine possible causes of this spoilage type, quantifying the populations of bacteria of the family Enterobacteriaceae, and characterizing them in the major genera and species found, the number of lactic acid bacteria, the frequency of Clostridium estertheticum and Clostridium gasigenes in meat proper for consumption and meat which showed the "blown pack" spoilage. In order to enumerate and identify the members of the Enterobacteriaceae family, and to enumerate the lactic acid bacteria the procedure was classical microbiological techniques. However to search the C. estertheticum and C. gasigenes the procedure was molecular biology techniques. The microorganisms of the family Enterobacteriaceae and lactic acid bacteria were present in large populations and in greater numbers in meat with "blown pack" spoilage. The species which were found more often was the Hafnia alvei. Samples of "blown pack" spoilage had greater positive features for C. estertethicum than samples not damaged. There was no statistical difference of positive features for the presence of C. gasigenes between samples of "blown pack" spoilage and not damaged meat. The main way to control this spoilage is the prevention of contamination of meat by fecal material. / Mestre

The first recorded observation of bacterial biofilms dates back to the 17th century by Antoine Van Leeuwenhoek. Today, biofilms are known as bacteria encapsulated within a self-produced extracellular matrix adherent to biotic or abiotic surfaces. Since the initial discovery of biofilms, research has explored the structure and function of biofilms. Only until recently has the role of biofilms within the medical setting become apparent. Here, we investigate the role of curli-extracellular DNA (eDNA) complexes in disease pathogenicity and explore the ability to target bacterial amyloid curli as a novel anti-biofilm therapeutic target. Biofilms of enteric bacteria, such as Escherichia coli and Salmonella enterica serovar Typhimurium, are composed of various components that act in consortium to fortify the extracellular matrix. One of the main components of enteric biofilms is amyloid curli. Curli, one of the best characterized bacterial amyloids, is a protein with a conserved cross beta sheet structure that forms basket like structures encapsulating the bacteria. Within the biofilm, curli serves to fortify the extracellular matrix, aids in bacterial attachment and protects bacteria from harsh environmental conditions. Extracellular DNA (eDNA) is another integral component of enteric biofilms. Recent reports from our lab has suggested that curli forms irreversible complexes with eDNA. Even with exposure to DNases, co-localized curli and eDNA can be observed. Other components of enteric biofilms include cellulose and Biofilm Associated Protein A. Biofilms of S. Typhimurium have been associated with significant disease pathologies. In addition to identifying the existence of curli-eDNA complexes within S. Typhimurium biofilms, our lab has also reported that curli-eDNA complexes of S. Typhimurium potentiate the autoimmune disease Systemic Lupus Erythematosus (SLE). SLE is an autoimmune disease characterized by the production of type I interferons and autoantibodies, although the etiology remains unknown. Systemically, curli binds to and activates the Toll like Receptor (TLR)1/2 complex leading to a pro-inflammatory response. In these studies we aimed to identify the innate immune mechanisms leading to the autoimmune phenotype following stimulation with curli-eDNA complexes. As TLR9 is activated by unmethylated bacterial DNA CpG DNA sequences leading to the production of type I interferons we hypothesized a potential role for TLR9 in recognizing eDNA of the curli-eDNA complex leading to the generation of the hallmarks of SLE. To investigate this hypothesis, we stimulated wild-type, TLR2 knockout, TLR9 knockout and TLR2-9 double knockout immortalized macrophages with curli-eDNA complexes purified from S. Typhimurium biofilms. We observed a significant reduction in the transcript level of type I interferons (IFN), Ifnβ, Isg15 and Cxcl10, upon stimulation of TLR2 knockout, TLR9 knockout and TLR2-9 double knockout immortalized macrophages implicating a role in TLR9 recognition of the curli-eDNA complex. As there was a significant reduction of type I interferon levels upon stimulation of TLR2 knockout macrophages, we hypothesized that TLR2 may serve as a carrier to bring the curli-eDNA complex into the endosome containing TLR9. To inhibit phagocytosis, we pretreated cells with endocytosis inhibitors and stimulated wild-type macrophages with curli-eDNA complexes. We observed a reduction in the transcript level of Ifnβ suggesting that curli-eDNA complexes gain access to endosomal TLR9 via TLR2 engagement. Finally, to explore the role of TLR2 and TLR9 in the production of autoantibodies, curli-eDNA complexes were intraperitoneal injected twice weekly for six weeks into C57BL/6 wild-type, TLR2 knockout, TLR9 mutant and TLR2 knockout-TLR9 mutant mice. We observed a robust generation of anti-double stranded autoantibodies within the first three weeks, however the production of autoantibodies was significantly decreased and delayed in the TLR2 knockout, TLR9 mutant and TLR2 knockout-TLR9 mutant mice. Overall, these data suggest that curli acts as a carrier for DNA to elicit an autoimmune response via TLR2 and TLR9. Within biofilms of S. Typhimurium, curli is the main proteinaceous component. Biofilms lacking curli destabilize and fail to form mature biofilms. Recent research has shown that in response to the production of host amyloids, the body will generate anti-amyloid antibodies in the serum. Monoclonal antibodies (mAb) generated from serum antibodies have been shown to have pan anti-amyloid properties in vitro and in vivo due to the β-sheet conformational epitope. As amyloids from both human and bacterial origin share a β-sheet conformational structure, we hypothesized as to if the anti-amyloid mAbs can eradicate S. Typhimurium biofilms by targeting curli. We incubated S. Typhimurium biofilms in the presence of various mAbs (ALZ.4A6, ALZ.4GI, ALZ.2C10 and ALZ.3H3) and observed a significant reduction of biofilm thickness and curli content within the biofilm. We deduced that ALZ.3H3 conferred the greatest anti-biofilm response. When we visualized the three-dimensional architecture of biofilms incubated with ALZ.3H3, we observed that ALZ.3H3 induced the formation of a loose architecture compared to untreated biofilms that were dense and compact. The resulting loose biofilm architecture induced by incubation with ALZ.3H3 enhanced the susceptibility of the biofilms to antibiotic exposure and macrophage clearance. We also observed enhanced biofilm eradication in vivo when catheters precoated with S. Typhimurium biofilms were inserted into the back flanks of mice that were percutaneously injected with ALZ.3H3. Both in vitro and in vivo, combination therapy of ALZ.3H3 and antibiotic enhanced biofilm clearance. In summary, we propose a novel anti-biofilm strategy by targeting the amyloid component of the biofilm, thus satisfying an unmet need in the art of biofilm prevention. Overall, these data in summation significantly broadens our understanding of disease pathogenicity and the role of curli-eDNA complexes in S. Typhimurium biofilms. As amyloid-eDNA complexes may be found in other biofilms, these results may extend beyond enteric bacteria proving novel insight into host-microbe interactions and the generation of novel anti-biofilm therapeutics. / Microbiology and Immunology

Made available in DSpace on 2014-06-11T19:27:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-06-06Bitstream added on 2014-06-13T19:35:10Z : No. of bitstreams: 1 felipe_lm_me_jabo.pdf: 356871 bytes, checksum: 16d6d5f606db8c2a71fd32fc4c6570dc (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A deterioração “blown pack” é caracterizada por abundante produção de gás, induzindo a completa distensão da embalagem durante o processo de estocagem sob refrigeração. Quando a embalagem é aberta, há um odor desagradável, levemente fecal. O gás presente na embalagem é composto por dióxido de carbono e hidrogênio e por vários tipos butíricos do metabolismo fermentativo. O objetivo deste experimento foi determinar possíveis causadores deste tipo de deterioração, quantificando as populações de bactérias da família Enterobacteriaceae, e caracterizando-as nos principais gêneros e espécies encontradas, o número de bactérias ácido-lácticas, a freqüência de Clostridium estertheticum e do Clostridium gasigenes, em carnes próprias para o consumo e em carnes que apresentaram a deterioração “blown pack”. Para contagem e identificação dos membros da família Enterobacteriaceae e contagem de bactérias ácido-lácticas utilizou-se de técnicas microbiológicas clássicas. Já para pesquisa do C. estertheticum e C. gasigenes fez-se uso de técnicas de biologia molecular. Os microrganismos da família Enterobacteriaceae e bactérias ácido-láticas estavam presentes em populações elevadas e em maior número nas carnes com deterioração “blown pack”. A espécie mais freqüentemente encontrada foi a Hafnia alvei. As amostras com deterioração “blown pack’ apresentaram maior positividade para o C. estertethicum que amostras não deterioradas. Não houve diferença estatística de positividade para a presença do C. gasigenes entre amostras com deterioração “blown pack” e carnes não deterioradas. A principal forma de controle desta deterioração é a prevenção da contaminação da carne por material fecal. / The blown pack spoilage is characterised by abundant gas production, leading to complete gross distention pack during refrigerated storage. When the packaging is opened, there is an unpleasant smell, lightly fecal. The gas present in the package is composed of carbon dioxide and hydrogen and also of several butyric types of metabolism fermentation. The purpose of this experiment was to determine possible causes of this spoilage type, quantifying the populations of bacteria of the family Enterobacteriaceae, and characterizing them in the major genera and species found, the number of lactic acid bacteria, the frequency of Clostridium estertheticum and Clostridium gasigenes in meat proper for consumption and meat which showed the blown pack spoilage. In order to enumerate and identify the members of the Enterobacteriaceae family, and to enumerate the lactic acid bacteria the procedure was classical microbiological techniques. However to search the C. estertheticum and C. gasigenes the procedure was molecular biology techniques. The microorganisms of the family Enterobacteriaceae and lactic acid bacteria were present in large populations and in greater numbers in meat with blown pack spoilage. The species which were found more often was the Hafnia alvei. Samples of blown pack“ spoilage had greater positive features for C. estertethicum than samples not damaged. There was no statistical difference of positive features for the presence of C. gasigenes between samples of blown pack spoilage and not damaged meat. The main way to control this spoilage is the prevention of contamination of meat by fecal material.

Depuis quelques années, des Toxi-Infections Alimentaires Collectives causées par la contamination de produits frais, comme les laitues, par des bactéries pathogènes entériques (Salmonella, Escherichia coli productrice de shigatoxines -ou STEC-) apparaissent de plus en plus nombreuses. La présence de ces bactéries dans cet environnement inhabituel est un risque sanitaire émergent majeur, d'autant plus que les bactéries entériques, pathogènes ou non, présentent fréquemment des résistances aux antibiotiques. Afin d’étudier la persistance des bactéries antibiorésistantes ou pathogènes sur des laitues, la caractérisation de plasmides de résistance portés par des souches de E. coli issues d’environnements aquatiques contaminés a été réalisé pour, par la suite, étudier leur implication potentielle dans l’adhésion des souches-hôtes sur différentes variétés de laitues. L’étude de la survie et de l’adhésion de souches de E. coli environnementales et de laboratoire, transformées avec les plasmides d’intérêt, sur de jeunes plants de laitues a permis de mettre en évidence trois points : 1) plus le temps de contact entre bactéries et feuilles augmente et moins la survie bactérienne est importante ; 2) il existe une différence de survie et d’adhésion selon les variétés de laitues étudiées ; 3) il existe une différence de survie et d’adhésion entre les souches de laboratoire et les souches environnementales, ces dernières étant en meilleur état métabolique et montrant une adhésion plus importante durant les 11-12 jours d’expérimentation. Après ces constatations de persistance des E. coli antibiorésistantes en conditions contrôlées, des études en champs sur 4 exploitations maraîchères normandes, possédant des itinéraires techniques différents, ont été réalisées. La recherche de pathogènes entériques, Salmonella et STEC, a été effectuée sur les laitues et une recherche de E. coli, témoin de contamination fécale, a été réalisée sur les laitues ainsi que dans l’eau d’irrigation d’un des sites. Les résultats révèlent une qualité microbiologique satisfaisante des parcelles étudiées (selon l’arrêté européen N°2073/2005) bien que des E. coli aient été régulièrement retrouvées au niveau des laitues, dont certaines antibiorésistantes. L’analyse de l’eau d’irrigation a montré la présence continue de E. coli, dont des souches présentant des profils d’antibiorésistance communs à ceux retrouvés sur les laitues, montrant que l’eau d’irrigation est l’une des sources critiques de contamination des végétaux en champs. / In recent years, foodborne diseases caused by fresh products contaminated, such as lettuce, with enteric pathogenic bacteria (Salmonella, Shigatoxin-producing Escherichia coli-or STEC-) increasingly. The presence of these bacteria in this unusual environment is a major emerging health risk, especially since enteric bacteria, whether pathogenic or not, are frequently resistant to antibiotics. To study the persistence of antibiotic-resistant or pathogenic bacteria on lettuce, the characterization of resistance plasmids carried by E. coli strains from contaminated aquatic environments was carried out in order to study their potential involvement in adhesion of host strains on different varieties of lettuce. The study of the survival and adhesion of environmental and laboratory E. coli strains, transformed with the plasmids of interest, on young lettuce plants allowed to highlight three points: 1) more time contact between bacteria and leaves increases and less bacterial survival is important; 2) there is a difference in survival and adhesion depending on the varieties of lettuce studied; 3) there is a difference in survival and adhesion between laboratory strains and environmental strains, the latter being in better metabolic state and showing greater adhesion during the 11-12 days of experimentation. After the persistence of antibiotic-resistant E. coli strains under controlled conditions, field studies on 4 Normandy vegetable farms, with different technical itineraries, were carried out. The search for enteric pathogens, Salmonella and STEC, was carried out on lettuce and a search for E. coli, a control of fecal contamination, was realized on the lettuce as well as in the irrigation water of one of the sites. The results reveal a satisfactory microbiological quality of the agricultural plots studied (according to the European decree N ° 2073/2005) although E. coli strains were regularly found at the lettuce level, including some antibiotic resistant. Analysis of the irrigation water showed the continued presence of E. coli strains, including strains with common antimicrobial resistance profiles to those found on lettuce, showing that irrigation water is one of the critical sources of plant contamination in the field.