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

Aus Materialien einer mesophilen Biogasanlage wurden Untersuchungen zur natürlichen Inaktivierung von Salmonellen durchgeführt. In dieser Biogasanlage werden zur alternativen Energiegewinnung im zweistufigen Prozess Rinderflüssigmist, Hühnerkot und Fettabscheiderinhalte fermentiert. Insgesamt konnten in den Jahren 1997-2000 zwölf verschiedene Salmonella-Serovare (z. B. S. Enteritidis, S. Agona, S. Hadar) in den Ausgangsmaterialien, im Fermentationsmaterial und im fertigen Fermentationsprodukt isoliert werden. Salmonella-positiv waren die Proben zu 95,5% (n = 22) aus der Rindergülle, zu 69,2% (n =13) aus dem Fermenter I, zu 50% (n = 20) aus dem Fermenter II, zu 77,3% (n = 22) aus der Lagune und zu 40% (n = 10) aus dem Fettabscheider. Als Quellen der Salmonellen werden die Gülle der Milchviehanlage (besonders für den Impfstamm) sowie Fettabscheiderinhalte diskutiert. Nach einer Infektion von Rindern mit S. Typhimurium in der gülleliefernden Milchviehanlage war nach der Vakzinierung der Kälber der Zoosaloral®-Impfstamm (LT: DT009) in der Gülle häufig nachweisbar. Bei 13 Untersuchun-gen wurde der Impfstamm zwölfmal in der Gülle der Milchviehanlage, einmal im Fermentationsprodukt der Biogasanlage und in fünf Proben aus der Lagune isoliert. Im Laboratorium wurde das Absterben von S. Typhimurium DT104 in fermen-tierender Rindergülle bei Lagerungstemperaturen von 7°C, 22°C und 37°C un-tersucht. Nach durchschnittlich 10 Tagen waren bei 37°C – dies entspricht etwa der Betriebstemperatur einer mesophilen Biogasanlage – keine Salmonellen nachweisbar. Bei einer Temperatur von 22°C überlebten die Salmonellen neun Wochen, bei 7°C überlebten sie mehr als 52 Wochen. Der mikrobiologische Abbau von Biomasse führt zur Aufspaltung der Makro-moleküle und danach zur Bildung von Karbonsäuren. Nach der Analyse orga-nischer Säuren aus Rindergülle und Cosubstraten wurden Konzentrationen dieser Säuren gegen S. Typhimurium DT104 experimentell geprüft. Es wurde der Einfluss von Ameisen-, Essig-, Propion-, Butter-, Isobutter-, Valerian-, Iso-valeriansäure auf die Inaktivierung von S. Typhimurium DT104 untersucht. In Versuchen mit den Einzelsäuren und Dosen der Salmonellen, die über den Gehalten nativer Gülle lagen, konnte eine Inaktivierung erst bei Konzentratio-nen von 10 bis 40 g/l erzielt werden. Da diese Konzentrationen laut der zu Grunde gelegten Gülleanalyse in den jeweiligen Einzelfällen nicht erreicht wurden, erfolgte die Prüfung der Säuren gegenüber den Salmonellen im Kom-plex. Dazu wurde ein Säuregemisch hergestellt, das den ermittelten Konzentra-tionen der Säuren in der Rindergülle plus Cosubstraten entspricht und auf ei-nen pH-Wert von 7,3 eingestellt. In dieser Säurelösung wurden Salmonellen täglich um durchschnittlich 0,5 Zehnerpotenzen reduziert und in drei Ver-suchsansätzen innerhalb von durchschnittlich 17 Tagen inaktiviert. Mit diesen Daten wird der Einfluß von in der Gülle vorkommenden Konzentrationen or-ganischer Säuren auf S. Typhimurium DT104 erstmals quantifiziert. Aus den Untersuchungen wird der Schluß gezogen, dass für das Absterben von S. Typhimurium DT104 während der 24 bis 33 Tage andauernden natürlichen Fermentation der Gülle in der Biogasanlage der Anstieg und der Einfluß der Karbonsäuren sehr wesentlich ist. Die nach der Vakzinierung der Kälber mit dem Lebendimpfstoff Zoosaloral® ausgeschiedenen Salmonellenimpfstämme waren auch nach Passage der Bio-gasanlage durch ihr auxotrophes Verhalten sicher von Wildstämmen zu unter-scheiden. Bei der Untersuchung von Gülle aus mit Salmonella-Lebendvakzinen geimpften Rinderbeständen ist das Mitführen des Bovisal-Diagnostikums® zu empfehlen. Bei den natürlich vorkommenden Salmonellen-Serovaren wurden zahlreiche Resistenzen gegenüber unterschiedlichen Antibiotika festgestellt. Die Zoosalo-ral®-Impfstämme wiesen nach der Passage der Biogasanlage keine veränderten Resistenzen auf. Die Zoosaloral®-Impfstämme sind resistent gegen Spectinomy-cin, Erythromycin und Penicillin. / We investigated the natural inactivation of Salmonella in the stuff of a meso-philic biogas plant where cattle slurry, poultry waste and fat separator contents are fermented in a two-step process for the use of alternative energy recovery. From 1997 to 2000 we isolated 12 different Salmonella serovars (e. g. S. Enteriti-dis, S. Agona, S. Hadar) in the native sludge, in the fermenter material and in the fermentation product. The following parts of the samples were Salmonella-positive: cattle slurry 95,5% (n = 22), fermenter I 69,2% (n =13), fermenter II 50% (n = 20), storage tank 77,3% (n = 22), and fat separator 40% (n = 10). As source of the Salmonella we assume the slurry of the dairy cattle farm (esp. in the case of vaccine strains) and the fat separator contents. After an infection of cattle with S. Typhimurium in the sludge-producing farm and vaccination of calves with Zoosaloral® the vaccine strain (LT: DT009) was frequently found in the slurry. In the course of 13 tests we isolated the vaccine strain in 12 samples of the biogas plant slurry, in one sample of the fermenta-tion product and in 5 samples of the storage tank. In laboratory investigations we studied the inactivation of S. Typhimurium DT104 in fermented cattle slurry at storage temperatures of 7°C, 22°C, and 37°C. After a mean storage time of 10 days at 37°C (i.e. the working tempera-ture of the biogas plant) all Salmonella were inactivated. At 22°C they survived nine weeks, at 7°C more than 52 weeks. The microbiologic degradation causes the splitting of macromolecules and the formation of free volatile acides (VFA). After analysis of the VFA in cattle slurry and cosubstrates we tested different concentrations of formic, acetic, propionic, butyric, isobutyric, valerianic, and isovalerianic acid. In tests with the single acids and Salmonella concentrations higher than in native slurry an inactivation was achieved at acid concentrations between 10-40 mg/l. Because acid concen-trations in native sludge are lower, we examined an acid mixture with acid con-centrations equivalent to cattle slurry/cosubstrate at pH 7,3. In the mixture Salmonalla were daily reduced about 0,5 orders and inactivated in an average of 17 days. These data quantify the influence of VFA concentrations in slurry for the first time. We concluded that the increase and the influence of VFA are very important for the inactivation of S. Typhimurium DT104 during the 24-33 days of slurry fer-mentation in the biogas plant. After vaccination of calves with the live vaccine Zoosaloral® the excreted Salmonella vaccine strains could be distinguished after the passage of the biogas plant by their auxotrophy from wild strains. We rec-ommend the use of Bovisaloral-Diagnostikum® for investigations of slurry from cattle vaccinated with Salmonella live vaccine. The natural Salmonella serovars were resistant against numerous antibiotics. The Zoosaloral® vaccine strains showed no deviating resistances after passag-ing the biogas plant. The Zoosaloral® vaccine strains were spectinomycine-, erythromycine- and penicilline-resistent.

[ES] Actualmente, una de las principales preocupaciones de las autoridades sanitarias y de la industria alimentaria sigue siendo garantizar la inocuidad microbiológica en la cadena alimentaria ya que a pesar de los notables esfuerzos realizados, el número de brotes y casos de enfermedades de transmisión alimentaria causadas por microorganismos sigue elevándose. Por otro lado, otro de los problemas que debe afrontar la industria alimentaria es la implementación de nuevas alternativas de producción más sostenibles, manteniendo altos estándares de seguridad y garantizando la disponibilidad de alimentos. En este contexto se enmarca la presente tesis doctoral, cuyo principal objetivo es la evaluación in vivo e in vitro de la capacidad antimicrobiana de distintas sustancias naturales procedentes de fuentes alternativas, más sostenibles (extracto de coliflor, espirulina y soluciones de quitosano), frente a microorganismos patógenos de importancia en la industria de alimentos. Los estudios demuestran la capacidad antimicrobiana in vitro del quitosano de insecto y crustáceo frente a microorganismos patógenos como E. coli, L. monocytogenes y S. Typhimurium siendo dependiente del pH del medio estudiado. Además, cuando el quitosano se combina con un tratamiento suave de altas presiones hidrostáticas, se pone de manifiesto una relación sinérgica entre ambas tecnologías llegando a inactivar completamente la carga microbiana de S. Typhimurium. Los estudios in vivo llevados a cabo con Caenorhabditis elegans muestran que el nematodo aumenta su supervivencia en presencia de extractos de coliflor y espirulina. En cuanto a la capacidad antimicrobiana, los extractos de espirulina y de coliflor reducen la colonización intestinal por S. Typhimurium en el nema todo mostrando así su capacidad antimicrobiana in vivo. En la presente tesis doctoral, se hace un recorrido completo sobre la capacidad antimicrobiana de ciertos extractos vegetales o componentes procedentes de animales, fundamentalmente basados en estudios in vivo. Este es un paso importante en la materialización de estos compuestos como alternativas más naturales y sostenibles a otros compuestos sintéticos usados en conservación de alimentos. / [CA] Actualment, una de les principals preocupacions de les autoritats sanitàries i de la indústria alimentària continua sent garantir la innocuïtat microbiològica en la cadena alimentària ja que malgrat els notables esforços realitzats, el nombre de brots i casos de malalties de transmissió alimentària causades per microorganismes continua elevant-se. D'altra banda, un altre dels problemes que ha d'afrontar la indústria alimentària és la implementació de noves alternatives de producció més sostenibles, mantenint alts estàndards de seguretat i garantint la disponibilitat d'aliments. En aquest context s'emmarca la present tesi doctoral, el principal objectiu de la qual és l'avaluació in vivo i in vitro de la capacitat antimicrobiana de diferents substàncies naturals procedents de fonts alternatives, més sostenibles (extracte de coliflor, espirulina i solucions de quitosano), enfront de microorganismes patògens d'importància en la indústria d'aliments. Els estudis demostren la capacitat antimicrobiana in vitro del quitosano d'insecte i crustaci enfront de microorganismes patògens com a E. coli, L. monocytogenes i S. Typhimurium sent dependent del pH del mig estudiat. A més, quan el quitosano es combina amb un tractament suau d'altes pressions hidroestàtiques, es posa de manifest una relació sinèrgica entre totes dues tecnologies arribant a inactivar completament la càrrega microbiana de S. Typhimurium. Els estudis in vivo duts a terme amb Caenorhabditis elegans mostren que el nematode augmenta la seua supervivència en presència d'extractes de coliflor i espirulina. Quant a la capacitat antimicrobiana, els extractes d'espirulina i de coliflor redueixen la colonització intestinal per S. Typhimurium en el nematode mostrant així la seua capacitat antimicrobiana in vivo. En la present tesi doctoral, es fa un recorregut complet sobre la capacitat antimicrobiana d'uns certs extractes vegetals o components procedents d'animals, fonamentalment basats en estudis in vivo. Aquest és un pas important en la materialització d'aquests compostos com a alternatives més naturals i sostenibles a altres compostos sintètics usats en conservació d'aliments. / [EN] Currently, one of the main concerns of the health authorities and the food industry continues to be ensuring microbiological safety in the food chain, as despite considerable efforts, the number of outbreaks and cases of foodborne diseases caused by microorganisms continues to rise. On the other hand, another chalenge facing the food industry is the implementation of new, more sustainable production alternatives while maintaining high safety standards and ensuring food availability. It is in this context, the main objective of this PhD thesis is the in vivo and in vitro evaluation of the antimicrobial capacity of different natural substances from alternative, more sustainable sources (cauliflower extract, spirulina and chitosan solutions) against pathogenic microorganisms of importance in the food industry. Studies demonstrate the in vitro antimicrobial capacity of insect and crustacean chitosan against pathogenic microorganisms such as E. coli, L. monocytogenes and S. Typhimurium, being dependent on the pH of the medium studied. Moreover, when chitosan is combined with a mild treatment of high hydrostatic pressures, a synergistic relationship between both technologies is evident, reaching the point of completely inactivating the microbial load of S. Typhimurium. In vivo studies carried out with Caenorhabditis elegans show that the nematode increases its survival in the presence of cauliflower and spirulina extracts. In terms of antimicrobial capacity, spirulina and cauliflower extracts reduce intestinal colonization by S. Typhimurium in the nematode, thus showing their antimicrobial capacity in vivo. In this doctoral thesis, a complete overview of the antimicrobial capacity of certain plant extracts or animal-derived compounds is presented, mainly based on in vivo studies. This is an important step in the materialisation of these compounds as more natural and sustainable alternatives to other synthetic compounds used in food preservation. / La presente tesis doctoral se enmarca dentro del Programa de Doctorado Ciencia, Tecnología y Gestión Alimentaria de la Universidad Politécnica de Valencia. El trabajo experimental se realizó gracias a fondos del Ministerio de Ciencia e Innovación y del Fondo Europeo de Desarrollo Regional (FEDER), a través del proyecto: Validación de nuevas herramientas y procesos para el análisis y la mejora de la seguridad alimentaria microbiológica: aplicación a nuevas matrices alimentarias (AGL2017-86840-C2-2-R) del Programa Estatal de Investigación, Desarrollo e Innovación Orientada a los Retos de la Sociedad. / Ibáñez Peinado, D. (2022). Estudio de la capacidad antimicrobiana de sustancias naturales in vivo e in vitro [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/184317 / TESIS

No description available.

Grape seed extract (GSE) and peanut skin extract (PSE) are waste products in the wine and peanut industries. Both extracts have high concentrations of polyphenols, known to possess antioxidant and antimicrobial properties. A subcategory of polyphenol is procyanidin, which can be divided into two types, type A and type B. Type A (PSE), contains two single bonds connecting the phenolic groups while type B (GSE), contains one single bond connecting the phenolic groups. The minimum inhibitory concentration (MIC) of the two extracts was evaluated for their antimicrobial effect on Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella Typhimurium using the pour plate method. GSE was found to have a significantly lower MIC (p ≤ 0.05) than PSE for L. monocytogenes (GSE=60.60ppm, PSE=not found), S. aureus (GSE=38.63ppm, PSE=51.36ppm), and S. Typhimurium (GSE=45.73ppm, PSE=60.60ppm). There was no significant difference in inhibition of E. coli O157:H7 (GSE=47.44ppm, PSE=51.13ppm). Since GSE, contributed to greater pathogen inhibition, its extract was fractionated into monomer and oligomers components. Growth curves of all four pathogens inoculated in the monomer and oligomer fractions were compared using the BioScreen method. Oligomers inhibited growth of L. monocytogenes, S. aureus, and E. coli O157:H7 while monomers inhibited growth of S. Typhimurium. These results indicate that an extract with type B procyanidins that are high in oligomers may be more effective as antimicrobials. Type B procyanidins have also been shown to prevent bacterial adhesion, as is the case with urinary tract infections, and may aid in the prevention of biofilms. / Master of Science in Life Sciences

Metabolism of D-amino acids is of considerable interest due to their key importance in cellular functions. The enzymes D-serine dehydratase (DSD) and D-cysteine desulfhydrase (DCyD) are involved in the degradation of D-Ser and D-Cys, respectively. We determined the crystal structure of Salmonella typhimurium DSD (StDSD) by multiple anomalous dispersion method of phasing using selenomethione incorporated protein crystals. The structure revealed a fold typical of fold type II PLP-dependent enzymes. Although holoenzyme was used for crystallization of both wild type StDSD (WtDSD) and selenomethionine labeled StDSD (SeMetDSD), significant electron density was not observed for the co-factor, indicating that the enzyme has a low affinity for the cofactor under crystallization conditions. Interestingly, unexpected conformational differences were observed between the two structures. The WtDSD was in an open conformation while SeMetDSD, crystallized in the presence of isoserine, was in a closed conformation suggesting that the enzyme is likely to undergo conformational changes upon binding of substrate as observed in other fold type II PLP-dependent enzymes. Electron density corresponding to a plausible sodium ion was found near the active site of the closed but not in the open state of the enzyme. Examination of the active site and substrate modeling suggested that Thr166 may be involved in abstraction of proton from the Cα atom of the substrate. Apart from the physiological reaction, StDSD catalyses α, β-elimination of D-Thr, D-Allothr and L-Ser to the corresponding α-keto acids and ammonia. The structure of StDSD provides a molecular framework necessary for understanding differences in the rate of reaction with these substrates. Salmonella typhimurium DCyD (StDCyD) is a fold type II PLP-dependent enzyme that catalyzes the degradation of D-Cys to H2S and pyruvate. We determined the crystal structure of StDCyD using molecular replacement method in two different crystal forms. The better diffracting crystal form obtained in presence of benzamidine illustrated the influence a small molecule in altering protein interfaces and crystal packing. The polypeptide fold of StDCyD consists of a small domain (residues 48-161) and a large domain (residues 1-47 and 162-328) which resemble other fold type II PLP-dependent enzymes. X-ray crystal structures of StDCyD were also obtained in the presence of substrates, D-Cys and βCDA, and substrate analogs, ACC, D-Ser, L-Ser, D-cycloserine (DCS) and L-cycloserine (LCS). The structures obtained in the presence of D-Cys and βCDA show the product, pyruvate, bound at a site 4.0-6.0 Å away from the active site. ACC forms an external aldimine complex while D and L-Ser bind non-covalently suggesting that the reaction with these ligands is arrested at Cα proton abstraction and transimination steps, respectively. In the active site of StDCyD cocrystallized with DCS or LCS, electron density for a pyridoxamine phosphate (PMP) was observed. Crystals soaked in cocktail containing these ligands show density for PLP-cycloserine. Spectroscopic observations also suggested formation of PMP by the hydrolysis of cycloserines. Mutational studies suggested that Ser78 and Gln77 are key determinants of enzyme specificity and the phenolate of Tyr287 is responsible for Cα proton abstraction from D-Cys. Based on these studies, we proposed a probable mechanism for the degradation of D-Cys by StDCyD. The acid-induced arginine decarboxylase (ADC) is part of an enzymatic system in Salmonella typhimurium that contributes to making this organism acid resistant. ADC is a PLP-dependent enzyme that is active at acidic pH. It consumes a proton in the decarboxylation of arginine to agmatine, and by working in tandem with an arginine-agmatine antiporter, this enzymatic cycle protects the organism by preventing the accumulation of protons inside the cell. We have determined the structure of the acid-induced StADC to 3.1 Å resolution. StADC structure revealed an 800 kDa decamer composed as a pentamer of five homodimers. Each homodimer has an abundance of acidic surface residues, which at neutral pH prevent inactive homodimers from associating into active decamers. Conversely, acidic conditions favor the assembly of active decamers. Therefore, the structure of arginine decarboxylase presents a mechanism by which its activity is modulated by external pH.

The global increasing problem of antibiotic resistance necessarily drives the pursuit and discovery of new antimicrobial agents. Antimicrobial peptides (AMPs) initially seemed like promising new drug candidates. Already members of the innate immune system, it was assumed that they would be bioactive and non-toxic. Their common trait for fundamental, non-specific mode of action also seemed likely to reduce resistance development. In this thesis, we demonstrate the ease with which two species of pathogenic bacteria, the gram-negative Salmonella typhimurium (S. typhimurium), and the gram-positive Staphylococcus aureus (S. aureus), can gain increased tolerance and stable resistance to various AMPs. By serially passaging each bacterial species separately under increasing AMP selection pressure we observed increasing AMP tolerance. Resulting in independent bacterial lineages exposed to four different AMPs (including a two-AMP combination) that exhibited 2 to 16-fold increases in MIC. Substantial cross-resistance between the AMPs was observed. Additionally, the S. aureus mutants were found to be cross-resistant to human beta-defensins 1, 2, 3, and 4. The LPS molecule, with mutations in the waaY, pmrB and phoP genes, was the principal target for S. typhimurium resistance development. The main target for S. aureus remained elusive. Reduced membrane potential was a common change for two of the mutants, but not for the others. All sequenced mutants had one or more mutations in various stress response pathways. Fitness of the resistant mutants was assayed by growth rate analysis and in vitro virulence factor testing (e.g. survival response to bile, superoxide, acidic pH). Furthermore an in vivo survival/virulence test involving a mouse competition experiment (S. typhimurium) and sepsis model (S. aureus) was performed. In the absence of AMPs there was often little or no fitness reduction in the mutants. Our results suggest that AMP resistance mechanisms do not irrevocably weaken either species with regard to virulence characteristics or survival within the host. In light of these findings, we suggest that the progression of therapeutic use of AMPs should proceed with great caution since otherwise we might select for AMP resistant mutants that are more resistant to our innate host defenses and thereby potentially more virulent.

A contaminação cruzada por Salmonella spp. durante o processo de abate de suínos contribui para o aumento da prevalência de carcaças positivas no pré-resfriamento. Um dos fatores que pode contribuir para a contaminação cruzada é a execução de cortes e palpação de carcaças durante o processo de inspeção. O presente estudo teve como objetivo estimar, por meio de ensaios laboratoriais, a transferência de Salmonella Typhimurium DT 177 entre faca e carne suína, para subsidiar análises futuras aplicadas ao processo de abate. Foram conduzidas observações independentes e aleatórias da transferência de uma cepa de S. Typhimurium resistente a Ampicilina (AmpR), entre faca e carne suína, as quais formaram quatro coleções de dados: Coleção de dados A: transferência de S. Typhimurium AmpR de faca contaminada para porção de carne suína cortada uma vez (n=20); Coleção de dados B: transferência de S. Typhimurium AmpR de faca contaminada para porção de carne suína cortada cinco vezes no mesmo lugar (n=20); Coleção de dados C: Transferência de S. Typhimurium AmpR de porção de carne suína contaminada para faca após execução de um corte (n=20); Coleção de dados D: Transferência de S. Typhimurium AmpR de porção de carne suína contaminada para faca após execução de cinco cortes no mesmo lugar (n=20). As bactérias transferidas foram quantificadas na lâmina da faca e na superfície da carne, a porcentagem de transferência foi calculada em todas as coleções de dados. As porcentagens de transferência entre as coleções de dados foram comparadas por meio de teste t para amostras independentes usando o programa R Core Team. As percentagens médias de transferência na coleção de dados A e B foram de 6,26% (4,7% – 7,7%) e 8,32% (6,4% - 10,2%). Nas coleções de dados C e D, as percentagens médias de transferência foram, respectivamente, 0,42% (0,3% - 0,5%) e 0,3% (0,2% - 0,4%). Não houve diferença significativa entre as percentagens de transferência após um e cinco cortes consecutivos. A partir disso, conclui-se que há transferência de S. Typhimurium da faca para a carne suína, bem como da carne suína para a faca. A porcentagem de transferência da carne suína contaminada para a faca é baixa, ao passo que a faca contaminada transfere alta percentagem do total de células de S. Typhimurium que carreia, durante a realização dos cortes. / Cross-contamination by Salmonella spp. during the pig slaughtering process contributes to increase the prevalence of positive carcasses in pre-chilling. One of the factors that may contribute to cross-contamination is the implementation of cuts and palpation of carcasses during the inspection process. The present study aimed to estimate, through laboratory tests, the transfer of Salmonella Typhimurium between knife and swine meat, to support future analyzes applied to the slaughter process. Independent and random observations of the transfer of a strain of S. Typhimurium Ampicillin-resistant (AmpR) between knife and swine meat were conducted, which formed four collections of data: Data collection A: Transfer of S. Typhimurium AmpR from contaminated knife to one portion of swine meat cut once (n = 20); Data collection B: Transfer of S. Typhimurium AmpR from contaminated knife to swine meat portion cut five times in the same place (n=20); Data collection C: Transfer of S. Typhimurium AmpR from portion of contaminated meat swine to knife after a cut (n=20); Data collection D: Transfer of S. Typhimurium AmpR from swine meat portion contaminated to knife after five cuts in the same place (n=20). The transfer percentages between the data collection were compared by t-test for independent samples using the R Core Team software. The mean transfer percentages in the data collection A and B were 6,26% (4,7% - 7,7%) and 8,32% (6,4% - 10,2%). In the C and D data collections, mean transfer rates were, respectively, 0.42% (0.3% - 0.5%) and 0.3% (0.2% - 0.4%). There was not significant difference between transfer rates after one and five consecutive cuts. From this, it is concluded that there is transfer of S. Typhimurium from the knife to the swine meat as well as from the swine meat to the knife. The percentage of transfer of contaminated pork to the knife is low, while the contaminated knife transfers at high percentage of the total number of S. Typhimurium cells it carries during cuts.

The ƒÑ-toxin of Clostridium perfringens is a toxin involved in numerous diseases of humans and agriculturally important animals. One of these diseases is necrotic enteritis (NE), a sporadic enteric disease which affects avian species world-wide. This study involved the inactivation of alpha-toxin (ƒÑ-toxin) for use as a potential vaccine candidate to combat NE in chickens, and other diseases caused by C. perfringens type A. During the course of this research a number of ƒÑ-toxin recombinant proteins were developed through molecular inactivation of the ƒÑ-toxin gene, plc. Proteins plc316 and plc204 were developed by the deletion of the first three and seven ƒÑ-helices of the N-terminal domain respectively. These deletions resulted in proteins which were unstable in solution, constantly aggregated into insoluble masses and elicited lower overall antibody responses when administered to mice. A third protein, plcInv3 was developed from the deletion of part of the catalytic domain of the ƒÑ-toxin. PlcInv3 was highly soluble and upon immunisation of mice elicited a significant antibody response which was also capable of protecting mice against a live challenge of C. perfringens. The fourth and final protein developed was plc104. The smallest of the recombinant ƒÑ-toxin proteins, it consisted entirely of the C-terminal domain of ƒÑ-toxin. Its small size did not affect its ability to induce a strong antibody response when administered to mice, the antibodies of which were also protective during a challenge with C. perfringens. STM1, an attenuated strain of S. Typhimurium was used in the development of a vectored vaccine for the expression and oral delivery of plcInv3 and plc104 within the mouse host. The proteins were expressed within STM1 from expression plasmids containing the in vivo inducible promoters PhtrA and PpagC. A measurable humoral immune response against ƒÑ-toxin was absent following three oral vaccinations with the vectored vaccines, although, cytokine profiling of splenocytes from vaccinated mice revealed an increase in the number of interleukin-4 (IL-4)secreting cells and the lack of interferon-gamma (IFN-ƒ×) secreting cells. This indicated the stimulation of a T-helper type 2 (TH2) immune response which also lead to partial protection against a live C. perfringens challenge. This study demonstrates the feasibility of using STM1 as a carrier for the in vivo expression of the C. perfringens ƒÑ-toxin recombinant proteins plcInv3 and plc104. It is the first study to express C. perfringens antigens within an attenuated strain of S. Typhimurium, STM1.The partial protection of mice immunised with these vaccines indicates there is potential for this vectored vaccine system to be used in the protection of diseases caused by the ƒÑ-toxin of C. perfringens.

X-ray crystallography is a powerful method for determining the three-dimensional structures of biological macromolecules at atomic resolution. Crystallography can reliably provide the answer to many structure related questions, from global folds to atomic details of bonding. Crystallographic techniques find wide applications in understanding macromolecular assembly, enzyme mechanism, mode of activation of enzymes, substrate-specificity, ligand-binding properties, domain movement etc. The knowledge of accurate molecular structures is also a prerequisite for rational drug design and for structure based functional studies to aid the development of effective therapeutic agents. The current thesis can be broadly divided into two major parts. The first four chapters deal with assembly studies that have been carried out on Sesbania mosaic virus and the next two chapters describe the structure and function of a stationary phase survival protein, SurE from Salmonella typhimurium. In both studies X-ray crystallographic techniques have been used extensively for the structural studies. Viruses are obligate parasites with a proteinaceous capsid enclosing the genetic material. For genetic economy, several copies of capsid proteins self assemble to form complex virus capsids. Due to their intricate symmetric structures, viruses are considered as minute marvels of molecular architecture and study of virus structures serve as a paradigm for solutions to problems concerning macromolecular assembly and function in general. Crystallography provides a means of visualizing intact virus particles as well as their isolated constituent proteins and enzymes at near-atomic resolution, and is thus an extraordinarily powerful tool for understanding the function of these biological systems. Protein-protein interactions, protein-nucleic acid interactions, metal-ion mediated interactions, interactions between capsid proteins and auxillary or scaffolding proteins and particle maturation or post processing of capsid protein subunits are various elements that play a role in capsid assembly. Many structural and sequential motifs have been proposed as important conformational switches of capsid assembly. A functional analysis of these motifs by way of mutations in the capsid protein and structural studies of these mutants can provide further insight into capsid assembly pathways. Interaction between capsid protein subunits can determine the size and robustness of the capsid. Analysis of protein-protein interactions can help in understanding the principles of self-assembly. Arresting capsid assembly by disrupting intersubunit interactions and trapping the assembly intermediates will be helpful to delineate the changes that happen in capsid protein during the course of assembly and understand assembly pathways. Sesbania mosaic virus (SeMV) is a plant virus with a positive sense single-stranded RNA genome and belongs to the Sobemovirus genus. The protein and nucleic acids of SeMV can be separated and reassembled in vitro. Also, expression of the coat protein (CP) gene of SeMV in E. coli leads to the formation of virus like particles (VLPs). Therefore, SeMV is an excellent model system to study the assembly pathways that lead to the formation of complex virus shells. Earlier structural and functional studies on the native virus and the recombinant capsid protein and its various mutants have revealed the following: SeMV is a T=3 virus with chemically identical A-, B- and C-subunits occupying quasi equivalent positions in the icosahedral asymmetric unit of the virus particle. The A-type subunits form pentamers at the five-fold, and the B- and C- type subunits form hexamers at the icosahedral three-fold axes. The amino terminus of the polypeptide is ordered from residue 72 in the A- and B- subunits whereas it is ordered from residue 44 in the C-subunit. The disordered segment in all the subunits has an arginine rich motif (N-ARM). The segment ordered only in C-subunits has a -annulus structure that promotes intersubunit interactions at the quasi six-fold and a -segment (A). The virus is stabilized by protein-protein, protein–RNA and Ca2+ mediated protein-protein interactions. Virus like particles (VLPs) formed by the expression of full length CP encapsidate 23 S E. coli rRNA and CP mRNA. Expression of a deletion mutant lacking the N-terminal 65 residues (rCP∆N65) which results in the removal of the N-ARM, the -annulus and the A leads to the formation of stable T=1 particles. The -annulus, which was earlier believed to be an important molecular switch controlling the assembly of T=3 VLPs was found to be dispensable. The N-ARM, though important for RNA encapsidation, was not essential for capsid assembly . Depletion of Ca2+ ions led to slight swelling of virus particles and significantly reduced stability. Extensive studies on the VLPs suggested that the assembly is most likely initiated by the dimers of the capsid protein. Following a brief account of the historical highlights in the field of structural virology, a review of current literature on the available crystal structures of viruses and various assembly studies on viruses that have been carried out with emphasis on role of nucleic acid mediated interactions, protein-protein interactions and role of specific residues and ion-mediated interactions in assembly are presented in Chapter I of the thesis. A separate section in this chapter deals with the disassembly experiments that have led to the formation of smaller oligomers of spherical viruses. This chapter also gives an account of the earlier work that has been carried out on SeMV, which is the model system of study for the present thesis. Chapter II describes in detail the structural studies on the β-annulus deletion mutant of SeMV. A unique feature of several T = 3 icosahedral viruses is the presence of a structure called the β-annulus formed by extensive hydrogen bonding between protein subunits related by icosahedral three-fold axis of symmetry. This unique structure has been suggested as a molecular switch that determines the T = 3 capsid assembly. In order to examine the importance of the β-annulus, a deletion mutant of Sesbania mosaic virus coat protein in which residues 48–59 involved in the formation of the β-annulus were deleted retaining the rest of the residues in the amino terminal segment (rCP (Δ48–59)) was constructed. When expressed in Escherichia coli, the mutant protein assembled into virus like particles of size close to that of the wild type virus particles. The purified capsids were crystallized and their three dimensional structure was determined at 3.6Å resolution by X-ray crystallography. The mutant capsid structure closely resembled that of the native virus particles. However, surprisingly, the structure revealed that the assembly of the particles has proceeded without the formation of the β-annulus. Therefore, the β-annulus is not essential for T = 3 capsid assembly as speculated earlier and may be formed as a consequence of the particle assembly. This is the first structural demonstration that the virus particle morphology with and without the β-annulus could be closely similar. Chapter III begins with a detailed description of the interfacial residue mutations that have been carried out in SeMV with the aim of disrupting assembly and trapping an assembly intermediate. These mutations were performed in rCP as well as rCP∆N65 gene. Among these, a single point mutation of a Trp 170 to a charged residue (either Glu or Lys) arrested virus assembly and resulted in stable dimers of the capsid protein. The chapter also gives an account of the biophysical characterization of these mutants. rCP∆N65 dimer mutants showed a characteristic 230 nm peak in CD spectral studies which may be due to the interactions of a stretch of aromatic residues in the capsid protein. The isolated dimers were more susceptible to trypsin cleavage compared to the assembled capsids due to the exposed basic amino terminus. Thermal melting studies showed that the isolated dimer mutants were much less stable when compared to the assembled capsids, probably due to the loss of intersubunit interactions and Ca2+ mediated interactions. The structure of one of the isolated dimer mutant- rCP∆N65W170K was solved to a resolution of 2.65Å. Chapter IV describes the crystal structure analysis of the rCP∆N65W170K mutant dimer and compares its structure with the dimers of native virus, T=3 and T=1 VLPs. A number of structural changes occur especially in the loop and interfacial regions during the course of assembly. The dimer in solution was “more relaxed” than the dimer that initiates assembly. Ca2+ ion is not bound and consequently the C-terminal residues are disordered. The FG loop, which interacts with RNA, was found to be flexible and adopts a different conformation in the unassembled dimer. The present thesis also deals with the structural and functional studies of a phosphatase, SurE, the stationary phase survival protein from Salmonella typhimurium. Chapter V provides a general introduction on Salmonella, which is a mesophilic food borne pathogen, its general features, classification and stress responses. This chapter also gives an account of stationary phase in bacteria and stress responses. A brief description about phosphatases and their classification is also presented in this chapter. Following this, a review of the current literature on the structural, biochemical and functional role of stress related proteins and phylogenetic and enzymatic studies of various homologues of SurE are described in detail. Chapter VI deals with the detailed crystal structure analysis of SurE, the first stationary phase survival protein from a mesophilic organism. SurE, of Salmonella typhimurium forms part of a stress survival operon regulated by the stationary phase RNA polymerase alternative sigma factor. SurE is known to improve bacterial viability during stress conditions. It functions as a phosphatase specific to nucleoside monophosphates. Here we report the X-ray crystal structure of SurE from Salmonella typhimurium (St SurE). The protein crystallized in two forms- orthorhombic F222 and monoclinic C2. The two structures were determined to resolutions of 1.7Å and 2.7Å, respectively. The protein exists as a domain swapped dimer. The residue Asp 230 is involved in several interactions that are probably crucial for domain swapping. A divalent metal ion is found at the active site of the enzyme, which is consistent with the divalent metal-ion dependent activity of the enzyme. Interactions of the conserved DD motif present at the N-terminus with the phosphate and the Mg2+ present in the active site suggest that these residues play an important role in enzyme activity. The divalent metal ion specificity and the kinetic constants of SurE were determined using the generic phosphatase substrate- para- Nitro Phenyl Phosphate. The enzyme was inactive in the absence of divalent cations and was most active in the presence of Mg2+. Thermal denaturation studies showed that St SurE is much less stable compared to its homologues and an attempt was made to understand the molecular basis of the lower thermal stability based on solvation free energy. The thesis concludes with a brief summary of the entire work that have been presented and future prospects. The various crystallographic, biochemical and biophysical techniques employed in the investigations are described under the section experimental techniques in Appendix I and the NCS matrices used in the structure solution of the β-annulus deletion mutant are listed in Appendix II.