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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 enterica subtype Typhimurium (S. Typhimurium) is one of many non-typhoidal Salmonella enterica strains responsible for over one million cases of salmonellosis in the United States each year. These Salmonella strains are also a leading cause of diarrheal disease in developing countries. Nontyphoidal salmonellosis induces gastrointestinal distress that is characterized histopathologically by an influx of polymorphonuclear leukocytes (PMNs), the non-specific effects of which lead to tissue damage and contribute to diarrhea. Prior studies from our lab have demonstrated that the type III secreted bacterial effector SipA is a key regulator of PMN influx during S. Typhimurium infection and that its activity requires processing by caspase-3. Although we established caspase-3 activity is required for the activation of inflammatory pathways during S. Typhimurium infection, the mechanisms by which caspase-3 is activated remain incompletely understood. Most challenging is the fact that SipA is responsible for activating caspase-3, which begs the question of how SipA can activate an enzyme it requires for its own activity. In the present study, we describe our findings that the eukaryotic tetraspanning membrane protein PERP is required for the S. Typhimuriuminduced influx of PMNs. We further show that S. Typhimurium infection induces PERP accumulation at the apical surface of polarized colonic epithelial cells, and that this accumulation requires SipA. Strikingly, PERP accumulation occurs in the absence of caspase-3 processing of SipA, which is the first time we have shown SipA mediates a cellular event without first requiring caspase-3 processing. Previous work demonstrates that PERP mediates the activation of caspase-3, and we find that PERP is required for Salmonella-induced caspase-3 activation. Our combined data support a model in which SipA triggers caspase-3 activation via its cellular modulation of PERP. Since SipA can set this pathway in motion without being cleaved by caspase-3, we propose that PERP-mediated caspase-3 activation is required for the activation of SipA, and thus is a key step in the inflammatory response to S. Typhimurium infection. Our findings further our understanding of how SipA induces inflammation during S. Typhimurium infection, and also provide additional insight into how type III secreted effectors manipulate host cells.

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[ES] La seguridad alimentaria es una prioridad para la población y en la actualidad cobra mayor importancia por ciertas tendencias alimentarias como el consumo de alimentos crudos y la distribución generalizada de alimentos orgánicos, que pueden ser la causa de enfermedades transmitidas por alimentos. Para garantizar la seguridad alimentaria, la detección de estos microorganismos debe realizarse de manera rápida y eficiente. Par eso, el método de cultivo microbiológico se considera el oficial para la detección de estos patógenos. Sin embargo, adolece de importantes inconvenientes, ya que no solo requiere mucho tiempo, sino que también es laborioso y consume muchos recursos. Además, puede ser limitado con respecto a la detección de bacterias fisiológicamente alteradas y/o estresadas durante el almacenamiento y la conservación. En este trabajo se ha desarrollado un protocolo sencillo y rápido para la detección simultánea de E. coli, L. monocytogenes, S. aureus y S. enterica en alimentos, mediante la combinación de una etapa de co-cultivo en medio líquido y la detección por PCR múltiple. Se ha evaluado la eficiencia de varios medios de enriquecimiento y se seleccionó el agua de peptona tamponada como el medio óptimo para el co-cultivo de las cuatro bacterias diana. También se optimizaron las condiciones de PCR múltiple y se aplicaron tanto a co-cultivos como a muestras de alimentos inoculados artificialmente, lechuga orgánica y carne picada. Después de la optimización, la PCR múltiple desarrollada fue capaz de detectar las cuatro bacterias simultáneamente, hasta con una inoculación inicial de 10^0 UFC/mL. En presencia de ambas matrices alimentarias inoculadas, tras la etapa de co-cultivo, la PCR múltiple pudo detectar simultáneamente las 3 bacterias E. coli, S. enterica y L. monocytogenes, mientras que S. aureus se ha detectado por PCR simplex, a partir del mismo extracto de ADN del co-cultivo. Los resultados obtenidos permiten concluir que el uso de un paso de co-cultivo en Agua peptona tamponada, antes de la detección por PCR simple y múltiple, puede facilitar la detección simultánea de las cuatro bacterias potencialmente presentes en las matrices alimentarias. La presencia o ausencia de la bacteria diana en los alimentos se confirma en unas 30 horas, lo que reduce el tiempo requerido para la detección en comparación con el tiempo mínimo de 7 días por método cultural. Asimismo, permite reducir el número de medios de cultivo y reactivos, para el aislamiento e identificación de bacterias que no son detectadas por PCR y que no están presentes en las matrices alimentarias, lo que supone un importante ahorro económico. / [CA] La seguretat alimentària sempre és una prioritat per a la població i en l' actualitat cobra major importància per certes tendències alimentàries, com el consum d' aliments crus i la distribució generalitzada d' aliments orgànics, que poden ser la causa de malalties transmeses per aliments. Per garantir la seguretat alimentària, la detecció d' aquests microorganismes s' ha de realitzar de manera ràpida i eficient. Per a això, el mètode de cultiu microbiològic es considera l' oficial per a la detecció d' aquests patògens. Però, hi ha importants inconvenients, ja que no només requereix més temps, sinó que també és laboriós i consumeix molts recursos. A més, pot ser limitat pel que fa a la detecció de bacteris fisiològicament alterats i/o estressats durant l'emmagatzematge i la conservació. En aquest treball s'ha desenvolupat un protocol senzill i ràpid per a la detecció simultània d' E. coli, L. monocytogenes, S. aureus i S. enterica en aliments, mitjançant la combinació d' una etapa de co-cultiu en medi líquid i la detecció per PCR múltiple. S'ha avaluat l'eficiència de diversos mitjans d'enriquiment i s'ha seleccionat l'aigua de peptona tamponada com el medi òptim per al co-cultiu dels quatre bacteris diana. També es van optimitzar les condicions de PCR múltiple i es van aplicar tant a co-cultius com a mostres d'aliments inoculats artificialment, enciam orgànic i carn picada. Després de l'optimització, la PCR múltiple desenvolupada va ser capaç de detectar els quatre bacteris simultàniament, fins a una inoculació inicial de 10^0 UFC/mL. En presència d' ambdues matrius alimentàries inoculades, després l' etapa de co-cultiu, la PCR múltiple va poder detectar simultàniament els 3 bacteris: E. coli, S. enterica i L. monocytogenes, mentre que S. aureus s' ha detectat per PCR simple, a partir del mateix extracte d' ADN del co-cultiu. Els resultats obtinguts permeten concloure que l' ús d' un pas de co-cultiu en Aigua de peptona tamponada, abans de la detecció per PCR simple i múltiple, pot facilitar la detecció simultània dels quatre bacteris potencialment presents en les matrius alimentàries. La presència o absència del bacteri diana en els aliments es confirma en unes 30 hores, la qual cosa redueix el temps requerit per a la detecció en comparació amb el temps mínim de 7 dies per mètode cultural. Així mateix, permet reduir el nombre de mitjans de cultiu i reactius, per a l' aïllament i identificació de bacteris que no són detectats per PCR i que no estan presents en les matrius alimentàries, la qual cosa suposa un important estalvi econòmic. / [EN] Food safety is a priority for the population and is nowadays more important than ever due to certain dietary trends such as the consumption of raw foods and the widespread distribution of organic foods, which may be the cause of foodborne diseases. To ensure food safety, the detection of these microorganisms must be done quickly and efficiently. Although, the microbiological culture method is considered to be the official method for the detection of these food-borne pathogens, it suffers from significant drawbacks, such as time-consuming, laborious and expensive, in addition it may be limited regarding the detection of physiologically altered and/or stressed bacteria, during storage and preservation. In this work has been developed a simple and rapid protocol for the simultaneous detection of E. coli, L. monocytogenes, S. aureus and S. enterica in food, by combining a liquid co-culture step and detection by multiplex PCR. The efficiency of several enrichment media was evaluated and buffered peptone water was chosen as the optimal medium for the co-culture of the four target bacteria. Then, optimized multiplex PCR conditions were applied to both the co-cultures and the samples of artificially inoculated foods, organic lettuce and ground meat. After optimization, the developed multiplex PCR was able to simultaneously detect the four bacteria, up to an initial inoculation of 10^0 CFU/mL. In the presence of the two inoculated food matrices, after a co-culture step, the multiplex PCR could simultaneously detect the 3 bacteria: E. coli, S. enterica and L. monocytogenes, whereas, S. aureus has been detected by simplex PCR, from the same co-culture DNA template. The results obtained allow conclusion that the use of a co-culture step in Buffered Peptone Water, before detection by simplex and multiplex PCR, can facilitate the simultaneous detection of the four bacteria potentially present in the food matrices. The presence or the absence of the target bacteria in food is confirmed in approximately 30 hours, which reduce the time required for the detection compared to the minimum time of 7 days by cultural method. Also, it allows to reduce the number of culture media and reagents, for the isolation and identification of bacteria that are not detected by PCR and which are not initially present in the food matrices, which represents a significant economic savings. / Boukharouba, A. (2022). Isolation and Identification of Foodborne Pathogens of Special Interest in Food Safety [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/182828