Expt. 1 evaluated effects of 0 or 10 ppm CAP in the starter phase (d 1-16) on chicks challenged with SE on d of age. Therapeutic inclusion of 10ppm CAP increased (P < 0.05) L/S and ceca positives. In Expt. 2, capsaicin oleoresin (CO) was included in finisher diets (d 30-37) at 0, 5, or 20 ppm with SE challenge on day 31. Inclusion of 5 ppm CO increased (P < 0.05) ceca SE positives and demonstrated 1.05 and 1.39-log fewer SE cfu at CO concentration of 5 or 20 ppm, respectively. A linear decrease (P < 0.05) in lamina propria thickness of SE challenged birds was observed with increased CO. Expt. 3 evaluated prophylactic CO treatment at 0, 5, or 20 ppm in starter, grower, and finisher diets for resistance to SE or ST challenge on d 14 or 29. With challenge on d 14, 5 ppm CO reduced ceca (P<0.005) SE positives and 1.1-log fewer SE cfu. Likewise, 20 ppm CO reduced (P < 0.05) SE ceca positives. Salmonella typhimurium isolation rate was reduced (P<0.05) with 5 ppm CO, and ST cfu were reduced 1.4-log with 5 ppm CO compared to 20 ppm. Lamina propria thickness increased (P < 0.05) linearly as CO concentration increased. With d 29 challenge birds fed 5 ppm CO exhibited 1.08-log fewer SE cfu, and 20 ppm CO reduced L/S positives (P < 0.025) for SE and resulted in 1.39-log fewer SE cfu. Lamina propria thickness decreased with 5 ppm CO and SE or ST challenge compared to non-challenged birds fed 5 ppm (P < 0.0005). An increase was observed in ST or SE, birds fed 20 ppm CO compared to non-challenged, birds fed 20 ppm CO (P < 0.01). No differences were observed in mast cell number in either Expt. 2 or 3. These data provide evidence that prophylactic or therapeutic dietary CAP differentially affect broiler susceptibility to Salmonella and prophylactic administration may provide non-antibiotic means to reduce Salmonella in broilers. / Master of Science

Fresh and fresh-cut tomatoes have been associated with numerous outbreaks of salmonellosis in recent years. While the exact routes of contamination are unknown, high pressure processing (HPP) is being evaluated as a post harvest treatment to eliminate Salmonella enterica from tomatoes. The objectives of the study were to determine the potential for of HPP to reduce S. enterica serovars Newport, Javiana, Braenderup and Anatum (clinical isolates from tomato outbreaks) in tryptic soy broth (TSB) and to determine the effect of HPP to reduce the most pressure resistant S. enterica serovar from fresh diced and whole tomatoes. Five ml portions of broth containing 8 log CFU/ml of one of the four serovars (nalidixic acid resistant) were packaged in sterile stomacher bags and subjected to one of three different pressures (350, 450, or 550 MPa) for 120s. Samples were enumerated by surface plating onto tryptic soy agar supplemented with 50 ppm nalidixic acid (TSAN) and incubated at 35°C for 48 hours. The most pressure resistant S. enterica serovar evaluated was Braenderup. Subjecting the broth culture to 350, 450 and 550 MPa resulted in a 4.53, 5.74 and 7.09 log reduction in S. Braenderup, respectively. Diced tomatoes (150g) and whole red round tomatoes (150g; packaged in 350ml of 1% CaCl2) were inoculated with S. Braenderup, to obtain 6 log CFU/g throughout the sample and subjected to the same pressure treatments as described above. After HPP, diced tomatoes were homogenized for 1 minute and then plated on TSAN. Whole tomatoes were surface sampled, and then homogenized for 1 minute. Surface and homogenate samples were plated on TSAN supplemented with 1% pyruvic acid (TSANP). Significant reductions of S. Braenderup concentrations in diced tomatoes (P < 0.05) were seen after processing at 350 (0.46 CFU/g), 450 (1.44 log CFU/g), and 550 MPa (3.67 log CFU/g). In whole tomatoes, significant reductions (P < 0.05) were also seen at 350 (1.41 log CFU/g), 450 (2.25 log CFU/g) and 550 MPa (3.35 log CFU/g). There were no differences in visual appearance between fresh and HPP diced and whole tomatoes. HPP may be an effective post harvest strategy to reduce low levels of S. enterica contamination in diced tomatoes. / Master of Science in Life Sciences

Memoria para optar al Título Profesional de Médico Veterinario / Los serotipos de Salmonella tradicionalmente se han clasificado mediante métodos serológicos que determinan antígenos somáticos y flagelares específicos. Sin embargo, este método diagnóstico es caro y tarda mucho tiempo en dar un resultado ya que requiere implementar una batería de anticuerpos para detectar los más de 2.500 serotipos de Salmonella enterica que se han identificado. Por otra parte, la identificación molecular de los genes responsables de la expresión de antígenos flagelares son más rápidos y más sensibles que la identificación serológica. Es por esto que, en la presente memoria se implementaron pruebas de PCR para identificar S. enterica serotipos Typhimurium y Hadar, ambas incluidas en un plan nacional de control de Salmonella en establecimientos comerciales de aves. Se analizaron 135 cepas, 50 correspondientes a S. Typhimurium, 50 a S. Hadar y 35 enterobacterias como controles negativos. Estas cepas, previamente serotipificadas en el Instituto de Salud Pública (ISP), fueron sometidas a la prueba de PCR para determinar si existen diferencias de diagnóstico entre ambas técnicas. Del total de cepas analizadas, sólo 46 cepas de S. Typhimurium dieron positivas a la prueba de PCR y cuatro dieron negativas, mientras que las 50 cepas de S. Hadar dieron positivas a las dos pruebas de PCR. Las 35 enterobacterias dieron negativas a las 3 pruebas de PCR. De acuerdo a los resultados obtenidos en el estudio, la detección de antígenos mediante serotipificación y PCR para las cepas de S. Typhimurium fue menor al esperado (92%), a diferencia de lo que ocurrió con las cepas de S. Hadar en que hubo 100% de acuerdo entre ambas técnicas, sugiriendo que esta prueba de detección de Salmonella es posible reemplazarla por los métodos tradicionales de identificación y así poder acelerar el diagnóstico de esta bacteria de gran importancia para la salud pública

Manifiesta que el cuy es una especie de producción que se ve afectada principalmente por Salmonella Typhimurium, la cual expresa factores de virulencia codificados por diferentes genes, que, en conjunto, cumplen funciones coordinadas para llevar a cabo la patogenia y desarrollar la enfermedad. Por ello, el presente estudio evaluó la presencia de 10 genes codificantes de diversos factores de virulencia de importancia biológica de un total de 100 aislados de Salmonella Typhimurium, compuestos por 90 cepas procedentes de cuyes enfermos y 10 cepas procedentes de cuyes aparentemente sanos, confirmados en estudios previos. El ADN de los aislados fue extraído y analizado mediante la técnica de PCR múltiple, para evaluar la presencia de los genes spvB, spiA, cdtB, sipB, tolC, sitC, lpfC, sifA, sopB y pefA, obteniéndose un patrón genético similar, con frecuencias de detección variables mayores al 60%, tanto en aislados de cuyes sanos como enfermos, excepto el gen cdtB, el cual no fue detectado; concluyéndose que no existe diferencia entre los factores de virulencia presentes en cepas de Salmonella Typhimurium aisladas de cuyes enfermos y cuyes aparentemente sanos; sin embargo, debe tenerse en cuenta el potencial peligro que representan los animales portadores dentro de la producción. / Universidad Nacional Mayor de San Marcos (Lima). Vicerrectorado de Investigación y Posgrado Perú. Ministerio de la Producción. Programa Nacional de Innovación para la Competitividad y Productividad (Innóvate Perú) / Tesis

Wong Yin Wai. / Thesis submitted in: June 2002. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 82-93). / Abstracts in English and Chinese. / Acknowledgement --- p.ii / Abstract --- p.iii / 摘要 --- p.v / Table of Content --- p.vi / List of Tables --- p.ix / List of Figures --- p.x / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Taxonomy of salmonellae --- p.1 / Chapter 1.2 --- Clinical significance --- p.2 / Chapter 1.3 --- Treatment of Salmonella infections --- p.4 / Chapter 1.4 --- Global and local prevalence of Salmonella --- p.5 / Chapter 1.5 --- Antimicrobial Susceptibilities --- p.8 / Chapter 1.5.1 --- Salmonella Typhimurium --- p.8 / Chapter 1.5.2 --- Other salmonellae --- p.9 / Chapter 1.5.3 --- Emergence of quinolone-resistant salmonellae --- p.9 / Chapter 1.6 --- Mechanisms of β-lactam resistance --- p.10 / Chapter 1.6.1 --- Enzymatic deactivation of β-lactam antibiotics --- p.10 / Chapter 1.6.2 --- Modifications of normal PBPs --- p.11 / Chapter 1.6.3 --- Alternative routes of peptidoglycan synthesis --- p.12 / Chapter 1.6.4 --- Impermeability and active efflux system --- p.12 / Chapter 1.7 --- Classification and nomenclature of β-lactamases --- p.13 / Chapter 1.7.1 --- Functional classification --- p.13 / Chapter 1.7.2 --- Molecular classification --- p.15 / Chapter 1.7.3 --- Nomenclature of β-lactamases --- p.16 / Chapter 1.8 --- β-Lactamases in salmonellae --- p.17 / Chapter 1.8.1 --- Salmonella Typhimurium --- p.17 / Chapter 1.8.2 --- Other salmonellae --- p.18 / Chapter 1.9 --- Methods for the characterization of β-lactamases --- p.18 / Chapter 1.9.1 --- Isoelectric focusing (IEF) --- p.19 / Chapter 1.9.2 --- β-Lactamase activity assays --- p.20 / Chapter 1.9.3 --- Hybridization with DNA probes --- p.20 / Chapter 1.9.4 --- Amplification of β-lactamase genes by polymerase chain reaction (PCR) --- p.21 / Chapter 1.9.5 --- Polymerase chain reaction - Single strand conformational polymorphism (PCR-SSCP) analysis --- p.23 / Chapter 1.9.6 --- Gene sequencing --- p.23 / Chapter 1.10 --- Objectives --- p.25 / Chapter 2 --- Materials and Methods --- p.26 / Chapter 2.1 --- Bacterial Strains --- p.26 / Chapter 2.1.1 --- Identification of salmonellae --- p.26 / Chapter 2.1.2 --- Antibiotics and chemicals used --- p.26 / Chapter 2.1.3 --- Antimicrobial susceptibility testing --- p.28 / Chapter 2.2 --- Localization of β-lactamase genes --- p.30 / Chapter 2.2.1 --- Transferability study --- p.30 / Chapter 2.3 --- Characterization of β-lactamases --- p.31 / Chapter 2.3.1 --- Extraction of crude β-lactamases --- p.31 / Chapter 2.3.2 --- Isoelectric focusing (IEF) --- p.31 / Chapter 2.4 --- Molecular characterization of β-lactamase genes --- p.33 / Chapter 2.4.1 --- Detection of TEM-type β-lactamase genes using polymerase chain reaction (PCR) --- p.33 / Chapter 2.4.2 --- Detection of OXA-type β-lactamase gene using PCR --- p.36 / Chapter 2.4.3 --- Detection of TEM mutations by polymerase chain reaction 一 single strand conformational polymorphism (PCR-SSCP) analysis --- p.37 / Chapter 2.4.4 --- Detection of OXA mutations by PCR-SSCP analysis --- p.38 / Chapter 2.4.5 --- Sequencing of β-lactamase genes --- p.39 / Chapter 2.4.5.1 --- Preparation of sequencing template --- p.39 / Chapter 2.4.5.2 --- Sequencing reaction --- p.39 / Chapter 2.4.5.3 --- Preparation of sequencing gel --- p.40 / Chapter 2.4.5.4 --- Silver staining of the sequencing gel --- p.41 / Chapter 2.5 --- Relatedness of ampicillin-resistant S. Typhimurium --- p.42 / Chapter 2.5.1 --- Pulsed field gel electrophoresis (PFGE) --- p.42 / Chapter 2.5.2 --- Cluster analysis --- p.44 / Chapter 3 --- Results --- p.46 / Chapter 3.1 --- Bacterial Strains --- p.46 / Chapter 3.1.1 --- Antimicrobial susceptibilities --- p.46 / Chapter 3.2 --- Characterization of β-lactamases by isoelectric focusing --- p.52 / Chapter 3.3 --- Characterization of β-lactamase genes --- p.53 / Chapter 3.3.1 --- Transferability of β-lactamase genes --- p.53 / Chapter 3.3.2 --- Detection of OXA-type β-lactamase gene by polymerase chain reaction (PCR) --- p.53 / Chapter 3.3.3 --- Detection of OXA-type mutations by polymerase chain reaction- single strand conformational polymorphism (PCR-SSCP) analysis --- p.56 / Chapter 3.3.4 --- Detection of TEM-type β-lactamase gene by PCR --- p.56 / Chapter 3.3.5 --- Detection of TEM-type mutations by PCR-SSCP analysis --- p.56 / Chapter 3.3.6 --- Sequencing of β-lactamase genes --- p.61 / Chapter 3.3.7 --- Pulsed-field gel electrophoresis --- p.64 / Chapter 4 --- Discussion --- p.67 / Chapter 4.1 --- Antimicrobial susceptibilities of S. Typhimurium in Hong Kong --- p.67 / Chapter 4.2 --- Transferability of resistance --- p.69 / Chapter 4.3 --- β-Lactamases of S. Typhimurium --- p.70 / Chapter 4.4 --- DNA sequence of β-lactamase genes --- p.72 / Chapter 4.5 --- Relatedness of ampicillin-resistant S. Typhimurium --- p.73 / Chapter 4.6 --- Methods for the characterization of β-lactamases --- p.75 / Chapter 4.7 --- Significance of this study --- p.78 / Chapter 4.8 --- Conclusions --- p.79 / Chapter 4.9 --- Further studies --- p.80 / References --- p.83

by Koo Ching Irene. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 105-118). / Abstract also in Chinese. / Acknowledgments --- p.i / Abstract --- p.ii / Content --- p.iv / List of tables --- p.viii / List of figures --- p.x / Chapter Chapter 1: --- Introduction --- p.1 / Chapter A. --- Classification of salmonellae --- p.1 / Chapter B. --- Salmonellae enterica ser Enteritidis --- p.4 / Chapter C. --- Global increase in the prevalence of S. Enteritidis --- p.6 / Chapter D. --- Susceptibility of S. Enteritidis to antimicrobial agents --- p.13 / Chapter E. --- Methods for the epidemiological typing of S. Enteritidis --- p.17 / Chapter 1. --- Phenotypic methods --- p.17 / Chapter (1) --- Biotyping --- p.17 / Chapter (2) --- Antibiotic resistance pattern --- p.18 / Chapter (3) --- Phage typing --- p.18 / Chapter (4) --- Characterization of plasmids --- p.19 / Chapter a. --- Resistance plasmids --- p.20 / Chapter b. --- Transferability of plasmids --- p.20 / Chapter c. --- Incompatibility --- p.21 / Chapter 2. --- Molecular methods --- p.21 / Chapter (1) --- Plasmid analysis --- p.21 / Chapter a. --- Plasmid profile --- p.22 / Chapter b. --- Plasmid fingerprinting --- p.22 / Chapter (2) --- Chromosomal DNA fingerprinting --- p.24 / Chapter a. --- Restriction fragment length polymorphism (RFLP) of chromosomal DNA --- p.25 / Chapter b. --- Pulsed-field gel electrophoresis (PFGE) --- p.25 / Chapter c. --- Hybridization with specific gene probes --- p.26 / Chapter d. --- Ribotyping --- p.27 / Chapter e. --- Insertion sequence IS200 fingerprinting --- p.29 / Chapter (3) --- Polymerase chain reaction (PCR) --- p.30 / Chapter 3. --- Other --- p.32 / Chapter (1) --- Lipopoly saccharide (LPS) analysis --- p.32 / Chapter (2) --- "Whole cell protein profile analysis, fatty acid profile analysis, multilocus enzyme electrophoresis and Fourier-transform injfrared spectroscopy" --- p.33 / Chapter F. --- Epidemiology of S. Enteritidis in different parts of the world --- p.34 / Chapter G. --- Objectives --- p.35 / Chapter Chapter 2: --- Materials and Methods --- p.36 / Materials --- p.36 / Methods --- p.38 / Chapter A. --- Identification --- p.38 / Chapter 1. --- Biochemical tests --- p.38 / Chapter 2. --- Serotyping --- p.38 / Chapter B. --- Antimicrobial susceptibility testing --- p.39 / Chapter C. --- Characterization of β-lactamases --- p.39 / Chapter 1. --- Extraction of β-lactamases --- p.39 / Chapter 2. --- Determination of isoelectric points (pIs) --- p.41 / Chapter D. --- Characterization ofplasmids --- p.42 / Chapter 1. --- Genetic studies --- p.42 / Chapter (1) --- Transferability of resistance plasmids --- p.42 / Chapter (2) --- Mobilization of resistances --- p.43 / Chapter 2. --- Molecular studies --- p.44 / Chapter (1) --- Plasmid profile analysis --- p.44 / Chapter a. --- Plasmid extraction --- p.44 / Chapter b. --- Agarose gel electrophoresis --- p.45 / Chapter (2) --- Plasmid DNA fingerprinting --- p.45 / Chapter a. --- Preparation of pure plasmid DNA --- p.46 / Chapter b. --- Preparation of individual plasmid from strains harbouring more than one plasmid --- p.46 / Chapter c. --- Restriction endonuclease digestion of plasmid DNA --- p.47 / Chapter E. --- Total DNA fingerprinting --- p.47 / Chapter 1. --- Total DNA preparation --- p.48 / Chapter 2. --- Restriction endonuclease digestion of total DNA --- p.48 / Chapter 3. --- Ribotyping --- p.49 / Chapter (1) --- Restriction enzyme digestion of total DNA --- p.49 / Chapter (2) --- Transfer of DNA fragments to solid support --- p.49 / Chapter (3) --- Reverse transcription of rRNA into cDNA and labelling of cDNA --- p.50 / Chapter (4) --- Hybridization --- p.50 / Chapter (5) --- Detection of hybridized fragments --- p.51 / Chapter 4. --- AP-PCR (Arbitrary primed-PCR) --- p.51 / Chapter F. --- Experimental design --- p.52 / Chapter Chapter 3: --- Results --- p.54 / Chapter A. --- Prevalence of S. Enteritidis in the Prince of Wales Hospital --- p.54 / Chapter B. --- Antimicrobial susceptibilities --- p.58 / Chapter C. --- β-lactamases produced by β-lactam-resistant S. Enteritidis --- p.66 / Chapter D. --- Plasmid profile analysis --- p.66 / Chapter E. --- Characterization of resistance plasmids --- p.69 / Chapter F. --- Plasmid fingerprinting --- p.72 / Chapter G. --- Total DNA fingerprinting --- p.76 / Chapter H. --- Ribotyping --- p.82 / Chapter I. --- AP-PCR --- p.85 / Chapter J. --- "Correlation of plasmid analysis, total DNA fingerprinting, ribotyping and AP-PCR results" --- p.88 / Chapter Chapter 4: --- Discussion --- p.91 / Chapter A. --- Prevalence of S. Enteritidis --- p.91 / Chapter B. --- Susceptibilities of S. Enteritidis to antimicrobial agents --- p.92 / Chapter C. --- Evaluation of methods for epidemiological typing of S. Enteritidis --- p.94 / Chapter D. --- Molecular epidemiology of S. Enteritidis in Hong Kong --- p.99 / Chapter E. --- Areas for future research --- p.102 / References --- p.105 / Appendix --- p.119

Foodborne diseases are one among the diseases with high morbidity and mortality rate. The concern is raised with the emergence of pathogenic strains that are resistant to the available set of antibiotics. Conventional regimens fail to treat the infections caused by these pathogens prolonging the sickness leading to increased morbidity and mortality. The situation can get further complicated with the dietary intake of the host. Of late it has been understood that the dietary flavonoids play an important role in regulating the immune system. Curcumin, a pigment from turmeric, is one among such bioflavonoid with an immunomodulatory potential. Curcumin has been a front-line topic of mainstream scientific research for a variety of diseases from cancer to Alzheimer’s to infectious diseases. Curcumin being considered as a spicy panacea is not a remedy for all diseases. Its ability to act differentially as an antioxidant or pro-oxidant can be either beneficial or harmful for the host. It exhibits antioxidant properties at concentrations achievable in the body; this can make the host vulnerable to infections due to the suppression of innate immune responses. Curcumin also suppresses the type 1 immune response, which might lead to alleviation of type 1 immune response disorders. However, the inhibition of type 1 immune response might invite infections with opportunistic pathogens. We have chosen curcumin to assess the effect of diet on the regulation of pathogenesis of Salmonella along with few medically important pathogens like Yersinia enterocolitica, Staphylococcus aureus, Shigella flexneri and Listeria monocytogenes. The thesis is divided into five chapters. As the main focus of the thesis is on Salmonella, in Chapter 1 we introduce diverse aspects of curcumin and the basic biology of Salmonella. Initially the properties of curcumin, the molecule of interest are introduced followed by brief overview to Salmonella biology and pathogenesis. Various activities of curcumin dealing with the variety of diseases are discussed. Further, the introduction to the intricate underlying mechanisms and the functional determinants of curcumin is given. The subsequent sections give an overview of different phases of Salmonella pathogenesis and the molecular mechanisms of Salmonella virulence and host defense. Towards the end of the chapter we discuss the strength, limitations and the distinctive characteristics of the murine model of typhoid fever. Curcumin has gained immense importance for its vast therapeutic and prophylactic applications. Its anti-bacterial effect has been demonstrated in bacteria, like B. subtilis, H. pylori and E. coli. Contrary to this, the results of the Chapter 2 reveals that curcumin at a nontoxic concentration to both host and pathogen, regulates the defense pathways of Salmonella enterica serovar Typhimurium (S. Typhimurium) to enhance its pathogenicity. In a murine model of typhoid fever, we observed higher bacterial load in reticuloendothelial organs when infected with curcumin-treated Salmonella. Curcumin increased the resistance of S. Typhimurium against antimicrobial agents like antimicrobial peptides, reactive oxygen and nitrogen species. It up-regulated the genes involved in resistance against antimicrobial peptides - pmrD and pmrHFIJKLM and genes with antioxidant function - mntH, sodA and sitA. We implicate that the iron chelation property of curcumin has a role in regulating mntH and sitA. Interestingly, we see that the curcumin-mediated modulation of pmr genes is through the PhoPQ two-component regulatory system (TCS). Curcumin downregulates SPI-1 genes required for entry into epithelial cells and upregulates SPI-2 genes required for intracellular survival, through PhoPQ TCS. Thus, this common regulator (PhoPQ) could explain curcumin's mode of action. Another important factor for the pathogen’s success is its ability to counteract the action of antibiotics. Almost all the bactericidal antibiotics act via production of reactive oxygen species in the bacteria. Curcumin has anti-oxidant property that might interfere with the action of antibiotics. Ciprofloxacin is a commonly used anti-typhoidal drug. It kills the bacteria by inhibiting DNA replication and increasing reactive oxygen species in bacterial cell. In Chapter 3 we present the results obtained after the investigation of the interference of curcumin with the anti-bacterial action of ciprofloxacin against Salmonella. We found that curcumin indeed increased the proliferation of Salmonella Typhi and Salmonella Typhimurium in ciprofloxacin treated macrophages by reducing the ciprofloxacin-induced reactive oxygen species. It also inhibited ciprofloxacin mediated DNA damage and the resultant SOS response and filamentation. However, curcumin was unable to rescue the ciprofloxacin induced gyrase inhibition. The reduced antibiotic (ciprofloxacin) efficacy against Salmonella by curcumin might aggravate the disease. Thus, the results of chapter 1 and 2 urge us to rethink the indiscriminate use of curcumin especially during Salmonella outbreaks. Bacteria modulate its virulence determinants in response to the environmental cues. Salmonella being a foodborne pathogen has a very likely chance of getting exposed to turmeric and hence curcumin. In Chapter 4 we have assessed the modulation of motility of S. Typhimurium, an important virulence determinant, by curcumin. We show that curcumin reduced the motility of the S. Typhimurium by decreasing the flagellar density around it. Surprisingly, this was achieved without affecting the expression of the flagellin gene and protein. Curcumin physically adhered to the flagella making it fragile and breaking it into fragments. This can hinder bacterial motility, chemotaxis, adherence and invasion into the host cells. However, aflagellate bacteria are hypervirulent as is the case with our experimental results with curcumin treated bacteria. Curcumin regulates myriad of bacterial (Salmonella) activities increasing its pathogenicity. Curcumin is known to regulate the host defenses in response to the disease. In Chapter 5 we have sought to address the effect of curcumin treatment of host cells on the outcome of infection by different pathogens. Pathogens have evolved different strategies to evade the host innate immune system, one of them being avoiding lysosome mediated degradation. Pathogens like Salmonella, Yersinia, Mycobacterium and Staphylococcus have acquired molecular machinery to inhibit the fusion of the pathogen containing vacuole with lysosomes and multiply within the vacuole whereas other pathogens like Shigella, Listeria and Rickettsia escape into and multiply in the cytosol. In our study we show that pretreatment of macrophage with curcumin increased the fold proliferation of S. Typhimurium, S. aureus and Y. enterocolitica whereas decreased that of S. flexneri and L. monocytogenes. From the results obtained, we can state that curcumin differentially regulates the pathogenesis of vacuolar and cytosolic pathogen. We hypothesized that curcumin pretreatment stabilizes the membrane of pathogen containing vacuole retarding the lysis of the phagolysosome harboring the cytosolic pathogen and hence facilitating its clearance. We indeed observed that the membrane stabilizing effect of curcumin led to increased fusion of cytosolic pathogen with the lysosome, decreasing its proliferation in the cells. As the vacuolar pathogens have an inherent ability to inhibit this fusion, they proliferate better in curcumin treated cells. In a nutshell curcumin can have multiple and sometimes unexpected effects not only on a pathogen’s potential to successfully cause infection but also on the host’s ability to counter it. A brief summary of the study that does not directly deal with the modulation of bacterial pathogenesis by curcumin is included in the Appendix. In this study a novel, simple, sensitive and efficient PCR based assay was devised to detect Salmonella contamination in milk, fruit juice and ice-cream without any pre-enrichment.

A aplicação de métodos de tipificação baseados na caracterização fenotípica e genotípica em vários pontos da cadeia de produção de suínos pode ser uma importante ferramenta para identificar a principal fonte de contaminação por Salmonella sp. A partir disso, o trabalho propôs tipificar uma coleção de 97 amostras de Salmonella enterica subsp. enterica ser. Typhimurium (ST) isolada de suínos levados ao abate em três diferentes frigoríficos no Rio Grande do Sul, por meio de fagotipificação, hibridização com IS200, resistência a antimicrobianos, amplificação da região spvR, amplificação de sequências repetitivas (rep-PCR) e PFGE. Paralelamente, os isolados de ST foram avaliados frente a dois desinfetantes (amônia quaternária e iodofor) pela técnica da diluição em tubo. Os isolados foram classificados em 12 fagotipos distintos, sendo o DT177 o mais freqüentemente identificado. Houve o predomínio de um padrão único de hibridização com o IS200 e em apenas três isolados, a região spvR foi detectada. Um alto número de amostras resistentes à tetraciclina, sulfonamida e estreptomicina foi encontrado, sendo o perfil de resistência relacionado ao frigorífico de origem dos isolados. No rep-PCR, usando seqüências iniciadoras para REP e ERIC, um único padrão de bandas foi gerado entre os isolados de ST. A análise por PFGE mostrou doze diferentes padrões de bandas. Sessenta e quatro isolados apresentaram um padrão idêntico no PFGE. Todas amostras foram inibidas pelo composto quaternário de amônio, na concentração recomendada pelo fabricante e numa concentração inferior à indicada. Frente ao iodofor, quatro amostras mostraram-se resistentes na concentração indicada e 59 na sub-concentração. A combinação da fagotipificação e do perfil de PFGE permitiu alcançar uma melhor discriminação das amostras, sendo essas técnicas consideradas as mais adequadas. Por outro lado, a presença de linhagens clonais parecem estar presentes na região, indicando possíveis pontos comuns de infecção.

No Rio Grande do Sul, um grupo clonal de Salmonella Enteritidis vem sendo identificada como o principal microrganismo causador de Doenças Transmitidas por Alimentos (DTA) nos últimos anos. Em vista disso, o objetivo deste trabalho foi avaliar a sobrevivência em fluido gástrico simulado (FGS) e a capacidade de invasão intestinal de Salmonella Enteritidis (SE86) em comparação à Salmonella Typhimurium (ST99) em dois modelos animais. Os microrganismos foram cultivados em meio de cultura e meio de cultura suplementado com glicose, objetivando a adaptação ácida. Aproximadamente 9logUFC dos microrganismos SE86 e ST99, ácido-adaptados ou não adaptados, foram expostos ao FGS, com pH 1,5, e também inoculados por via oral em ratos Wistar adultos machos. Um inóculo de 2logUFC foi utilizado nos experimentos com camundongos “germ-free”, de 21 dias, mantidos em condições assépticas. As fezes e porções do trato gastrintestinal foram analisadas microbiologicamente, sendo que os ratos foram também investigados quanto a lesões morfológicas intestinais. Os camundongos foram analisados por histopatologia e submetidos à curva de mortalidade. Os resultados indicaram que SE86 ácido-adaptadas apresentaram uma sobrevivência significativamente maior (p <0,05) que às SE86 e ST99 não adaptadas ao ácido e também às ST99 ácidoadaptadas no FGS. Os experimentos “in vivo” demonstraram que as bactérias inoculadas foram capazes de provocar lesões morfológicas intestinais e foram recuperadas das fezes, do jejuno e da junção íleo-cecal dos ratos, sendo que contagens mais altas de SE86 e ST99 ácido-adaptadas foram encontradas nas fezes. SE86 ácido-adaptada demonstrou maior contagem no ileo-ceco do que as outras linhagens, sugerindo que a adaptação ácida influenciou na virulência deste microrganismo. Nos animais “germ-free” houve rápida multiplicação de todos os microrganismos inoculados, contudo a mortalidade ocorreu de forma mais rápida devido à infecção por SE86 ácido-adaptada. A análise histopatológica revelou maior severidade da infecção provocada pela SE86, o que foi confirmado pela mortalidade dos animais a partir do quarto dia de infecção, ao contrário da ST99 que não provocou a morte dos animais por até 12 dias. A maior sobrevivência e virulência apresentadas pela SE86 podem estar relacionadas com o freqüente envolvimento desta cepa com salmoneloses alimentares na última década no Rio Grande do Sul. / In Rio Grande do Sul (RS) State, Southern Brazil, a clonal group of Salmonella Enteritidis has been identified as the main cause of foodborne diseases, in the last years. Given that, the objective of this study was to evaluate the survival in simulated gastric fluid (FGS) and the intestinal invasion ability of Salmonella Enteritidis (SE86) and Salmonella Typhimurium (ST99) submitted or not to acid adaptation. The microorganisms were grown in culture media and culture media supplemented with glucose, aiming to promote acid adaptation. After that, approximately 9log of SE86 (Isolated from a foodborne outbreak) and ST99 (not involved with foodborne outbreaks), acid-adapted or not adapted, were exposed to FGS with pH 1.5, and inoculated in adult male Wistar rats. Germ-free mice were also inoculated but with approximately 2 log, and the animals were observed during 21 days, at aseptic conditions. Animal feces and portions of the gastrointestinal tract were examined by microbiological analysis, and the rats were also investigated for intestinal morphological damage. The mice were analyzed by histopathology and submitted to the mortality curve. The results indicated that acid-adapted SE86 had a significant higher survival rate (p<0.05) than not adapted SE86, not adapted ST99, and also than acid-adapted ST99, in the FGS. The “in vivo" experiments demonstrated that the strains were capable of causing intestinal morphological damage and were recovered from feces, jejunum and ileum-cecal junction of rats, with higher counts being demonstrated for acid-adapted SE86 and acid-adapted ST99. Acid-adapted SE86 showed higher counts in the ileum-cecal junction than the other strains, suggesting that acid adaptation influenced the virulence of this microorganism. After inoculation, all strains were able to rapidly multiply in germ-free animals, but mortality caused by acid-adapted SE86 was more intense. Histopathological analyses revealed greater infection’s severity caused by SE86, and it was confirmed by the death of animals starting at the fourth day of infection, unlike the ST99 which did not cause the death of animals until twelve days of inoculation. Based on these results, the higher survival rates in FGS and the higher virulence presented by the SE86 can be related to the frequent involvement of this strain with foodborne salmonellosis occurred in the last decade in RS.

Destaca-se a Salmonella como um dos mais importantes patógenos veiculados por alimentos, devido ao fato de estar amplamente distribuído na natureza, possuir um grande número de reservatórios, apresentar sorotipos inespecíficos quanto aos hospedeiros e apresentar cepas multiresistentes aos antimicrobianos. Salmonella Enteritidis tornou-se um grande problema de saúde pública no Brasil, a partir da década de 80, quando foi associada a produtos avícolas. Desde 1962, a Salmonella Heidelberg tem sido isolada de aves e produtos derivados e aves no Brasil e tem reconhecida importância para saúde humana e em poedeiras comerciais na América do Norte. O presente trabalho teve por objetivo o estudo de diferentes cepas de Salmonella Heidelberg (SH) em comparação com Salmonella Enteritidis (SE) em pintinhos e frangos de corte no que diz respeito à capacidade de invasibilidade de órgãos, persistência de excreção fecal e alterações na morfometria intestinal. Para o estudo in vivo, 65 isolados de SH foram avaliados quanto à presença dos genes de virulência invA, lpfA e agfA e diferenciados por eletroforese em campo pulsado (PFGE). Outro objetivo foi testar uma mistura de ácidos orgânicos e óleos essenciais no controle de excreção fecal de SE. Duas amostras de SH não relacionadas pelo método de PFGE (SH23 e SH35) e uma cepa de SE padrão foram selecionadas para a inoculação das aves. As três cepas promoveram alteração na medida de vilosidades e criptas dos pintinhos, frente a grupo controle. A cepa SH35 promoveu maior reação inflamatória nos cecos. Ambas as cepas testadas foram capaz de atingir o fígado de pintinhos às 6 horas pós-inoculação. Tanto a SE como a SH demonstram persistência e intermitência de excreção fecal em frangos de corte até os 21dias pós-inoculação. A mistura de ácidos e óleos essenciais foi eficaz na redução da excreção fecal de SE, porém somente com o uso contínuo da mistura na ração. As cepas apresentaram comportamentos diferentes quando inoculadas em frangos de corte e devido à persistência destas nos intestinos dos frangos, prejuízo para as aves podem ocorrer, enquanto a subseqüente contaminação de carcaças, representa risco para a saúde humana. / Salmonella, one of the most important food borne pathogen due to the widely distribution in nature, large number of reservoirs, to show unspecific host serotypes and provide multiresistant strains to antibiotics. Salmonella Enteritidis became a big public health problem in Brasil, since 80’s, when it was correlated with poultry products. Since 1962, Salmonella Heidelberg has been isolated from birds and poultry products in Brazil, with importance in human health and layers in North America. The present work aimed to the study of different strains of Salmonella Heidelberg (SH) and compared with Salmonella Enteritidis (SE) in chicks and broiler chickens with regard to the ability of invasibilidade of organs, persistence of fecal excretion and changes in intestinal morphometry. For the in vivo study, 65 isolates of SH were evaluated for the presence of virulence genes invA, lpfA and agfA and differentiated by pulsed field gel electrophoresis (PFGE). Another objective was to test a organic acids and essential oils mixture for controlling SE fecal excretion. Two not related SH samples by the method of PFGE (SH23 and SH35) and a standard strain of SE were selected to the poultry inoculation. The three strains promoted changes in the measures of villi and crypts of the chicks intestine, when compared to the control group. The SH35strain promoted greater inflammatory reaction in the caeca. Both strains tested were able to achieve the chicks liver at 6 hours post inoculation. Both strains, SE and SH, showed persistence and intermittence of fecal excretion in broiler chickens until the 21days post-infection. The organic acids and essential oils mix was effective in reducing the SE fecal excretion, but only with the continuous use of the mix added to the feed. The strains showed different behavior when inoculated in broiler chickens, and because of the persistence of these strains in the chickens intestines, injury to birds may occur, while the subsequent carcasses contamination represents risk to human health.