Salmonella é um dos principais agentes de enfermidades transmitidas por alimentos em diversos países, sendo a carne de frango um dos principais veículos envolvidos em surtos. O Brasil vem se destacando como um dos maiores exportadores mundiais deste alimento. O ambiente de criação das aves é apontado como um importante foco de infecção das aves e o ambiente industrial de abate e processamento é importante na disseminação deste ·microrganismo. Na busca pela produção de alimentos seguros do ponto de vista microbiológico, uma das ferramentas utilizadas é a subtipagem de microrganismos isolados ao longo da cadeia de produção, que permite determinar rotas de contaminação do produto final. Os objetivos deste trabalho são: o estudo da disseminação dos subtipos de Salmonella Enteritidis nas várias etapas de uma cadeia de produção industrial de carne de frango, empregando-se diversos métodos de subtipagem e a determinação da resistência a antimicrobianos destas cepas. 108 isolados de Salmonella Enteritidis dos fagotipos PT1, PT4 e PT7a foram obtidos nos anos de 2002 e 2003, a partir de amostras ambientais e de frango relativas a sete sub-regiões de uma cadeia produtiva industrial avícola. Os perfis de resistência destes isolados foram determinados frente a antimicrobianos de uso humano e veterinário e eles foram submetidos a subtipagem por PFGE, RAPO, ribotipagem e PCR-ribotipagem. Foram detectados 21 perfis de resistência diferentes, com 6,5% das cepas sensíveis a todas as drogas, 33,3% resistentes a um ou dois antimicrobianos e 83,3% apresentando resistência intermediária a até quatro deles. Os níveis relativamente elevados de resistência são preocupantes e a diminuição da pressão seletiva deve ser um objetivo para os produtores de aves. De modo geral, a subtipagem permitiu separar as cepas em 13 genótipos, com elevada similaridade entre si. Porém, a maior parte das cepas (69,4%) pertenceu a apenas três deles, que foram encontrados ao longo de toda a cadeia produtiva. A ribotipagem foi o método que apresentou o melhor poder discriminatório (D = 0,701), porém nem todas as cepas foram tipáveis por este método. Não foram encontradas correlações entre os perfis de resistência a antimicrobianos e fagotipos, nem entre genótipos e fagotipos. Porém, dois genótipos proximamente correlacionados e predominantemente encontrados em uma sub-região reuniram apenas cepas com resistência intermediária ou resistentes exclusivamente à furazolidona. A similaridade elevada entre os genótipos evidencia a origem clonal das cepas. / Salmonella is one of the most important foodborne disease agents all over the world, and chicken is recognized as an important vehicle of the infection. Chicken production in Brazil has increased in the last couple of years and the country is now ranked 2nd as producer/exporter of this commodity. For this reason there is an increased concern over the safety of these goods. This study deals with the dissemination, antimicrobial resistance, and genetic characterization of S. Enteritidis strains isolated from an industrial chicken production chain. 108 isolates, phagetypes PT1, PT4 and PT7a, were obtained at different steps of the commercial production from farm to frozen cuts, and the broilers were from different producers supplying the same processing plant. Tests for susceptibility to 12 human and veterinary antimicrobial agents were performed. The strains were also typed by PFGE, RAPO, ribotyping, and PCR-ribotyping. 6.5% of the strains were susceptible to the 12 drugs tested and 33.3% were resistant to 1 or 2 of them. Intermediate resistance to up to 4 agents was observed in 83.3% of the isolates. Combining all the typing methods allowed the division of the strains in 13 genotypes with elevated degree of similarity. However, 69.4% of the strains belonged to 3 main phagetypes spread along the production chain. There was no correlation between phagetypes and genotypes, or phagetypes and resistance profiles. However, most strains from one sub-region were from 2 genotypes and showed intermediate resistance to, or were resistant to furazolidone. The high degree of similarity amongst the genotypes indicates the clonal origin of the strains. The relatively high resistance to antimicrobial agents is a cause of concern and trying to diminish the selective pressure has to be a goal for broiler producers.

Salmonella é um dos mais importantes patógenos causadores de enfermidades transmitidas por alimentos (ETA) no Brasil e em outros países. Devido ao surgimento de fenótipos multi-resistentes (MOR) a agentes antimicrobianos em Salmonella, a caracterização dos genes envolvidos neste processo, sua localização e diversidade são importantes para identificação e compreensão dos fatores envolvidos na resistência. O objetivo deste trabalho foi caracterizar determinantes genéticos de virulência, resistência e integrons classe 1 presentes em cepas de diferentes sorotipos de Salmonella enterica multi-resistentes a antibióticos, isoladas a partir de alimentos de origem suína, avícola e fontes relacionadas. As cepas empregadas pertenceram a nove perfis PFGE distintos com a enzima Xbal, com similaridade genética variando de 38% a 68%. Integrons classe 1 foram detectados em 9 (45%) das 20 cepas de Salmonella enterica, incluindo cinco diferentes sorotipos: Brandenburg, Panama, Agona, Mbandaka e Alachua, e variando de 0,7Kb a 2,7Kb. Os genes de resistência aadA, sul1, sul2, tetA, dhfr, qacEΔl e blatem, que conferem resistência a aminoglicosídeos, sulfonamidas, tetraciclinas, compostos de amônio quaternário e β-lactâmicos, respectivamente, foram identificados no interior dos integrons, no cromossomo bacteriano, ou em ambos. Os genes aadB, floR, tetB e tetG não foram detectados. A resistência às quinolonas foi caracterizada nas 11 cepas que apresentaram resistência ao ácido nalidixico pela análise do gene gyrAM e mutações Ser-83-Fen foram confirmadas após sequenciamento das amostras. Estudos de conjugação demonstraram que apenas uma cepa de Salmonella Mbandaka foi capaz de transferir o gene sul2, para uma cepa de E. coli K12. Com relação ao perfil de virulência, as 20 cepas de Salmonella enterica foram caracterizadas e os genes slyA, invA, sopB e aceK estiveram presentes em 100% delas e o gene h-1i esteve presente em 18 cepas (90%). O gene spvC não foi detectado nas cinco cepas que possuíam plasmídeos. Os dados do presente estudo sugerem que alimentos de origem animal podem ser considerados como reservatórios de cepas de Salmonella enterica virulentas, resistentes a antimicrobianos e apresentando integrons classe 1. Isto caracteriza os produtos de origem suína e avícola como uma importante fonte de patógenos multi-resistentes para humanos. / Salmonella is one of the most important foodborne pathogens in Brazil and worldwide. Due to the emerging of multiresistant phenotypes in Salmonella the characterization of the genes involved in this process, their localization and diversity are important for identifying and understanding the factors involved in the resistance. The purpose of this study was to characterize virulence and antimicrobial determinants in different serovars of antibiotic multiresistant Salmonella enterica strains isolated from pork, poultry and related sources. The isolates belonged to nine different PFGE profiles obtained with Xbal restriction enzyme and showing genetic similarity ranging from 38% to 68%. Class 1 integrons were detected in 9 (45%) of 20 S. enterica strains ranging in size from 0,7Kb to 2,7Kb and comprising five different serotypes: Brandenburg, Panama, Agona, Mbandaka and Alachua. Resistance genes aadA, qacEΔl, sul1, tetA, sul2, dhfr, blatem, that confer resistance to aminoglicosides, sulphonamides, tetracyclines, ammonium quaternary compounds and beta-Iactams, respectively, were identified within class 1 integrons, chromosome, or both. Genes aad8, floR, tetB and tetG were not detected. The resistance to quinolones was characterized in 11 strains that showed resistance to nalidixic acid analyzing gyrA genes and Ser-83-Fen mutations were confirmed after sequencing of the samples. Conjugation studies demonstrated that only one S. Mbandaka strain was able to transfer sul2 gene to the E.coli K12. Regarding virulence profile Salmonella enterica strains were characterized and PCR analysis revealed the presence of the virulence genes invA, aceK, sop8, slyA in all isolates and the presence of virulence gene h-1i in 18 (90%) of them. The spvC gene was not detected in the five strains that harbored plasmids. The data of the present study suggest that foods of animal origin can be considered reservoirs of Salmonella enterica that are virulent, resistant and show class 1 integrons. This characterizes pork and poultry products as important sources of multi-resistant pathogens to human beings.

Salmonella é um dos mais importantes patógenos causadores de enfermidades transmitidas por alimentos (ETA) no Brasil e em outros países. Devido ao surgimento de fenótipos multi-resistentes (MOR) a agentes antimicrobianos em Salmonella, a caracterização dos genes envolvidos neste processo, sua localização e diversidade são importantes para identificação e compreensão dos fatores envolvidos na resistência. O objetivo deste trabalho foi caracterizar determinantes genéticos de virulência, resistência e integrons classe 1 presentes em cepas de diferentes sorotipos de Salmonella enterica multi-resistentes a antibióticos, isoladas a partir de alimentos de origem suína, avícola e fontes relacionadas. As cepas empregadas pertenceram a nove perfis PFGE distintos com a enzima Xbal, com similaridade genética variando de 38% a 68%. Integrons classe 1 foram detectados em 9 (45%) das 20 cepas de Salmonella enterica, incluindo cinco diferentes sorotipos: Brandenburg, Panama, Agona, Mbandaka e Alachua, e variando de 0,7Kb a 2,7Kb. Os genes de resistência aadA, sul1, sul2, tetA, dhfr, qacEΔl e blatem, que conferem resistência a aminoglicosídeos, sulfonamidas, tetraciclinas, compostos de amônio quaternário e β-lactâmicos, respectivamente, foram identificados no interior dos integrons, no cromossomo bacteriano, ou em ambos. Os genes aadB, floR, tetB e tetG não foram detectados. A resistência às quinolonas foi caracterizada nas 11 cepas que apresentaram resistência ao ácido nalidixico pela análise do gene gyrAM e mutações Ser-83-Fen foram confirmadas após sequenciamento das amostras. Estudos de conjugação demonstraram que apenas uma cepa de Salmonella Mbandaka foi capaz de transferir o gene sul2, para uma cepa de E. coli K12. Com relação ao perfil de virulência, as 20 cepas de Salmonella enterica foram caracterizadas e os genes slyA, invA, sopB e aceK estiveram presentes em 100% delas e o gene h-1i esteve presente em 18 cepas (90%). O gene spvC não foi detectado nas cinco cepas que possuíam plasmídeos. Os dados do presente estudo sugerem que alimentos de origem animal podem ser considerados como reservatórios de cepas de Salmonella enterica virulentas, resistentes a antimicrobianos e apresentando integrons classe 1. Isto caracteriza os produtos de origem suína e avícola como uma importante fonte de patógenos multi-resistentes para humanos. / Salmonella is one of the most important foodborne pathogens in Brazil and worldwide. Due to the emerging of multiresistant phenotypes in Salmonella the characterization of the genes involved in this process, their localization and diversity are important for identifying and understanding the factors involved in the resistance. The purpose of this study was to characterize virulence and antimicrobial determinants in different serovars of antibiotic multiresistant Salmonella enterica strains isolated from pork, poultry and related sources. The isolates belonged to nine different PFGE profiles obtained with Xbal restriction enzyme and showing genetic similarity ranging from 38% to 68%. Class 1 integrons were detected in 9 (45%) of 20 S. enterica strains ranging in size from 0,7Kb to 2,7Kb and comprising five different serotypes: Brandenburg, Panama, Agona, Mbandaka and Alachua. Resistance genes aadA, qacEΔl, sul1, tetA, sul2, dhfr, blatem, that confer resistance to aminoglicosides, sulphonamides, tetracyclines, ammonium quaternary compounds and beta-Iactams, respectively, were identified within class 1 integrons, chromosome, or both. Genes aad8, floR, tetB and tetG were not detected. The resistance to quinolones was characterized in 11 strains that showed resistance to nalidixic acid analyzing gyrA genes and Ser-83-Fen mutations were confirmed after sequencing of the samples. Conjugation studies demonstrated that only one S. Mbandaka strain was able to transfer sul2 gene to the E.coli K12. Regarding virulence profile Salmonella enterica strains were characterized and PCR analysis revealed the presence of the virulence genes invA, aceK, sop8, slyA in all isolates and the presence of virulence gene h-1i in 18 (90%) of them. The spvC gene was not detected in the five strains that harbored plasmids. The data of the present study suggest that foods of animal origin can be considered reservoirs of Salmonella enterica that are virulent, resistant and show class 1 integrons. This characterizes pork and poultry products as important sources of multi-resistant pathogens to human beings.

Chapter Introduction Salmonellae are gram negative bacteria that cause gastroenteritis and entericfever. S. enterica is divided into seven phylogenetic groups, subspecies 1, 2,3a, 3b, and 4, 6, 7. Subspecies1 includes 1,367 serovars, some of which are commonly isolated from infected birds and mammals. The other subspecies mainly colonize cold blooded animals. Salmonella typhimurium, Salmonella typhiandSalmonella enteritidis are some of the serovars, which belong to s.enterica species. S. typhimurium is one of the important causes for food poisoning in humans. It causes typhoid like fever in mice. In immuno compromised patients the infection is often fatal if it is not treated with antibiotics. Clinical features of food poisoning include abdominal pain, vomiting, nausea, abdominal cramps, dehydration etc. S. typhi causes typhoid fever in humans. No other host has been identified for this serovar. Main source of infection is contaminated food and water. No age is exempted but it is less common before2 years. Incubation period is 360 days. Clinical features include stepladder type fever, malaise, headache, hepato splenomegaly, coated tongue, Neutrogena etc. It may be fatal if untreated. Among the serovars of Salmonella infecting humans S. typhimurium and S. typhi are the most important. While S. typhimurium infects many host species including birds and mammals, S. typhi is single host adapted and infects only human. The single host adaptation of S. typhi presents it with the need for establishing are servoir of infection in the community which can serve as a source of fresh infection. Also the single host adaptation of S. typhi has made it a highly specialized pathogen which has evolved certain unique genes needed for human colonization at the same time has lost a set of genes which are needed for survival in other hosts and in the highly variable external environment. This has led to the accumulation of a vast number of pseudo genesin S. Typhi. A comparative study of the two serovars is useful in many ways. Due to varied host defense systems encountered by the two serovars owing to different niche of infection the bacterial counter defense mechanisms are also different. By focusing on the differences between genes involved in the bacterial defense of host immune response we can decipher the role played by various genes in combating the antibacterial host response. Chapter 2 The role of TolA and peptidoglycan modification in detergent resistance of pathogenic Salmonella The major Salmonella serovars that infect human are Salmonella enterica serovar Typhi (S.typhi) which cause systemic typhoid and Salmonella enterica serovar Typhimurium (S. typhimurium) which cause gastro enteritis. S. typhi resides in the gall bladder during chronic infection and S .typhimurium infects intestine .Thus both pathogens encounter high concentrations of bile and have developed mechanisms to counter it. The Tol Pal complex spanning the outermembrane and the inner cytoplasmic membrane plays an important role in maintaining the stability of the outer membrane and providing detergent resistance. The tolA gene of S. Typhi Is shorter by 27 aminoacid than S. typhimurium. The tolA gene knockout of S. typhimurium and S. typhi differed in their tritonX resistance behavoiur, morphology and low osmolality tolerance. S. typhi tolA was unable to complement the tolA defect in S. typhimurium which could probably due to the difference in the peptidoglycan layer. An analys is of the peptidoglycan modifying genes of both the serovars revealed that dacD, pbgP, ynhG are different. dacD, pbgP genes are pseudogenes in S. typhi and ynhG has a major deletion in S. typhi. Further studies reveal that a double knockout of dacD and pbpG in S. typhimurium makes it sensitive to low osmolality similar to S. typhi. Based on these results we propose a mechanism, where shortening of TolA increases detergent resistance by bringing the outer membrane into closer contact with the peptidoglycan layer, but this is achieved at the cost of reduced Lpp (Bruan’slipoprotein) peptidoglycan linkage which plays a major role in low osmolality tolerance. The pathogen S. typhi is highly adapted to the human host and cannot infect any other host. The single host adaptation and the need to survive in high concentrations of bile have made S. typhi to acquire higher bile resistance at the cost of lowered osmotic tolerance through shortening TolA and reduced Lpp and peptidoglycan binding. Chapter 3 Development of a DNA vaccine against Salmonella The immune response against Salmonella is multifaceted involving both the innate and the adaptive immune system. The characterization of specific Salmonella antigens inducing immune response could critically contribute to the development of epitope based vaccines for Salmonella. We have tried to identify aprotective Tcellepitope (s) of Salmonella, as cell mediated immunity conferred by CD8+T cells is the most crucial subset conferring protective immunity against Salmonella. It being a proven fact that secreted proteins are better in inducing cell mediated immunity than cell surface and cytosolic antigens, we have analyzed all the GenBank annotated Salmonella pathogenicity island 1 and 2 secreted proteins of S. typhimurium and S. typhi. They were subjected to BIMAS and SYFPEITHI analysis to map MHCI and MHC II binding epitopes. The huge profile of possible T cell epitopes obtained from the two classes of secreted proteins were tabulated and using a scoring system that considers the binding affinity and promiscuity of binding to more than one allele, SopB and SifB were chosen for experimental confirmation in murine immunization model. The entire Sop Band SifB genes were cloned into DNA vaccine vectors and were administered along with live attenuated Salmonella and it was found that SopB vaccination reduced the bacterial burden of organs by about 5fold on day4 and day8 after challenge with virulent Salmonella and proved to be a more efficient vaccination strategy than live attenuated bacteria alone. Chapter 4 PCR based diagnosis and Serovar Determination of Blood Borne Salmonella Typhoid fever is becoming an ever increasing threat in the developing countries. We have improved considerably upon the existing PCR based diagnosis method by designing primers against a region which is unique to S. typhiand S. paratyphiA, corresponding to the gene STY0312 in S. typhi and its homolog SPA2476 in S. paratyphiA. An additional set of primers amplify another region in S. typhi CT18 and S. typhiTy2 corresponding to the region between the genes STY0313 toSTY0316 but which is absent in S.paratyphi A. The threat of false negative result arising due to mutation in hypervariable genes has been reduced by targeting a gene unique to typhoidal Salmonella as a diagnostic marker. The amplified region has been tested for genomic stability by amplifying them from clinical is olates of patients from various geographical locations in India, there by showing that this region is potentially stable. These set of primers can also differentiate between S. typhiCT18, S. typhiTy2 and S. paratyphi A which have stable deletions in this specific locus. The PCR assay designed in this study has a sensitivityof95%ascompared to the Widal test which had only 63%. As observed, in certain cases the PCR assay was more sensitive than the blood culture test as the PCR based detection could also detect dead bacteria.

Salmonella é um dos principais agentes de enfermidades transmitidas por alimentos em diversos países, sendo a carne de frango um dos principais veículos envolvidos em surtos. O Brasil vem se destacando como um dos maiores exportadores mundiais deste alimento. O ambiente de criação das aves é apontado como um importante foco de infecção das aves e o ambiente industrial de abate e processamento é importante na disseminação deste ·microrganismo. Na busca pela produção de alimentos seguros do ponto de vista microbiológico, uma das ferramentas utilizadas é a subtipagem de microrganismos isolados ao longo da cadeia de produção, que permite determinar rotas de contaminação do produto final. Os objetivos deste trabalho são: o estudo da disseminação dos subtipos de Salmonella Enteritidis nas várias etapas de uma cadeia de produção industrial de carne de frango, empregando-se diversos métodos de subtipagem e a determinação da resistência a antimicrobianos destas cepas. 108 isolados de Salmonella Enteritidis dos fagotipos PT1, PT4 e PT7a foram obtidos nos anos de 2002 e 2003, a partir de amostras ambientais e de frango relativas a sete sub-regiões de uma cadeia produtiva industrial avícola. Os perfis de resistência destes isolados foram determinados frente a antimicrobianos de uso humano e veterinário e eles foram submetidos a subtipagem por PFGE, RAPO, ribotipagem e PCR-ribotipagem. Foram detectados 21 perfis de resistência diferentes, com 6,5% das cepas sensíveis a todas as drogas, 33,3% resistentes a um ou dois antimicrobianos e 83,3% apresentando resistência intermediária a até quatro deles. Os níveis relativamente elevados de resistência são preocupantes e a diminuição da pressão seletiva deve ser um objetivo para os produtores de aves. De modo geral, a subtipagem permitiu separar as cepas em 13 genótipos, com elevada similaridade entre si. Porém, a maior parte das cepas (69,4%) pertenceu a apenas três deles, que foram encontrados ao longo de toda a cadeia produtiva. A ribotipagem foi o método que apresentou o melhor poder discriminatório (D = 0,701), porém nem todas as cepas foram tipáveis por este método. Não foram encontradas correlações entre os perfis de resistência a antimicrobianos e fagotipos, nem entre genótipos e fagotipos. Porém, dois genótipos proximamente correlacionados e predominantemente encontrados em uma sub-região reuniram apenas cepas com resistência intermediária ou resistentes exclusivamente à furazolidona. A similaridade elevada entre os genótipos evidencia a origem clonal das cepas. / Salmonella is one of the most important foodborne disease agents all over the world, and chicken is recognized as an important vehicle of the infection. Chicken production in Brazil has increased in the last couple of years and the country is now ranked 2nd as producer/exporter of this commodity. For this reason there is an increased concern over the safety of these goods. This study deals with the dissemination, antimicrobial resistance, and genetic characterization of S. Enteritidis strains isolated from an industrial chicken production chain. 108 isolates, phagetypes PT1, PT4 and PT7a, were obtained at different steps of the commercial production from farm to frozen cuts, and the broilers were from different producers supplying the same processing plant. Tests for susceptibility to 12 human and veterinary antimicrobial agents were performed. The strains were also typed by PFGE, RAPO, ribotyping, and PCR-ribotyping. 6.5% of the strains were susceptible to the 12 drugs tested and 33.3% were resistant to 1 or 2 of them. Intermediate resistance to up to 4 agents was observed in 83.3% of the isolates. Combining all the typing methods allowed the division of the strains in 13 genotypes with elevated degree of similarity. However, 69.4% of the strains belonged to 3 main phagetypes spread along the production chain. There was no correlation between phagetypes and genotypes, or phagetypes and resistance profiles. However, most strains from one sub-region were from 2 genotypes and showed intermediate resistance to, or were resistant to furazolidone. The high degree of similarity amongst the genotypes indicates the clonal origin of the strains. The relatively high resistance to antimicrobial agents is a cause of concern and trying to diminish the selective pressure has to be a goal for broiler producers.