Over the past decade, the Eastern Shore of Virginia (ESV) has been implicated in at least four outbreaks of Salmonellosis associated with tomato all originating from the same strain, Salmonella enterica serovar Newport.  In addition to S. Newport contamination, the devastating plant disease, bacterial wilt, caused by the phytopathogen Ralstonia solanacearum threatens the sustainability of ESV tomato production.  Bacterial wilt is present in most ESV tomato fields and causes devastating yield losses each year.  Due to the ESV\'s endemic population of R. solanacearum and S. Newport, the relationship between the two pathogens is of interest and has never been investigated.  Two separate studies were conducted to assess the relationship between these two bacteria.  One study consisted of a series of greenhouse trials that involved root-dip inoculations of tomato plants with one of four treatments: 1) S. Newport, 2) R. solanacearum, 3) a co-inoculation of S. Newport + R. solanacearum, and 4) a control group with no inoculation. Leaf, stem, and fruit samples were collected from the plants and S. enterica presence from the internal tissues was observed.  S. enterica was recovered from a low percentage of fruit and leaf samples.  There were significantly more stem samples from plants co-inoculated with S. Newport + R. solanacearum positive for S. enterica (17.46%) than from other treatments.  Another study examined the relationship between the two bacteria via vacuum infiltration inoculations of tomato fruit collected from commercial production fields on the ESV with S. Newport.  Tomato fruit were collected from plants expressing symptoms of bacterial wilt (symptomatic) and plants not expressing bacterial wilt symptoms (asymptomatic).  After fruit infiltration with S. Newport, recovery concentration of S. enterica from internal tissues was measured.  S. enterica populations were greater in fruit originating from asymptomatic (5.15 log CFU/g) versus symptomatic (4.91 log CFU/g) plants across five studies.  Fruit collected from asymptomatic plants had a significantly higher internal pH (4.60) than fruit collected from symptomatic plants (4.37).  These results suggest that R. solanacearum can influence S. enterica survival and transportation throughout the internal tissues of tomato plants as well as the influence internal tomato fruit pH, which could potentially impact S. Newport survival in the fruit. / Master of Science in Life Sciences

Produce is responsible for 46% of all foodborne illnesses in the USA. Salmonella enterica causes 19,000 hospitalizations each year, and has been associated with produce. Presently, chlorine based sanitizers are most often used, however organic matter reduces its antimicrobial activity. Bacteriophage treatments are an all-natural, alternative method for pathogen inactivation. The objective of this study was to determine the efficacy of a five-strain bacteriophage treatment against a S. enterica cocktail in simulated wash waters at different temperatures. Bacteriophage and S. enterica were enumerated in simulated wash water solutions. One set of experiments studied bacteriophage and S. enterica growth in TSB+vegetable solutions. Bacteriophage behavior was not statistically different (p < 0.05) in spinach, romaine, or iceberg lettuce across different concentrations of organic matter. S. enterica reduction was approximately 2 log over 135 minutes for vegetable solutions and for the TSB control. S. enterica reduction was only 0.5 log in water solutions. The next set of experiments studied bacteriophage and S. enterica growth in vegetable solutions. Spinach wash water and tryptone soy broth solutions (TSB) at 20 °C and 37 °C. S. enterica was not reduced in spinach solution studies at 20 °C and 37 °C or at broth solutions at 20 °C. However, S. enterica was effectively reduced 4 log in broth solutions at 37 °C up to 7.5 hours, but grew to high levels after 24 hours. These results indicate that bacteriophage could not effectively control bacteria levels in produce wash water, and may need to be optimized.

No description available.

Fresh market tomatoes have been associated with 15 multistate Salmonella outbreaks between 1973 and 2010. While, S. enterica survival has been studied in tomato plants, field studies have been limited. To understand pathogen growth and survival, in crop fields, surrogate or attenuated organisms must be developed and validated. The purpose of this study was to compare the growth and survival of seven attenuated S. enterica Typhimurium and Newport strains against virulent strains S. Typhimurium ATCC14028 and S. Newport J1892 in optimum (TSB and TSB+kan) and minimal M9 growth media, and in commercial, red ripe tomatoes. Bacterial growth in media was assessed via BioScreen. Tomatoes were separately inoculated with 7 Log CFU/g of each isolate via vacuum infiltration, surface spot inoculation, or diced inoculation. Populations of each strain were determined on Days 0, 1, 3, and 5. In media, there were few differences in overall growth and growth rates between mutant isolates and wild-type (P<0.05). Growth in M9 was less (P<0.01), while growth rates were higher (P<0.01) than in TSB. In tomatoes (per treatment), there were no significant differences between growth rates of each isolate compared to WT (P>0.05); however, Salmonella strains in diced tomatoes had a higher growth rate than that in spot treated tomatoes (P>0.05). The growths of all the isolates in tomatoes indicated that under the tested conditions, isolates acted similarly to their WT counterparts. Thus, these strains may be able to be used as surrogate organisms in field studies. / Master of Science in Life Sciences

Since 2006 there have been four Salmonella enterica and one Listeria monocytogenes foodborne outbreaks linked to whole cantaloupe fruit. No post-harvest intervention to reduce potential contamination on cantaloupe currently exists. The complex surface topography of netted cantaloupes aids bacterial attachment. This research evaluates the use of allyl isothiocyanate (AITC; a natural antimicrobial) to reduce populations of S. enterica Michigan and L. monocytogenes on the surface of cantaloupe. Fifty μl of S. Michigan or L. monocytogenes was inoculated onto whole ‗Athena‘ or ‗Hales Best Jumbo‘ (‗HBJ‘) cantaloupe fruit in 22 mm diameter circles and allowed to dry for 90 min. resulting in 6.60 log CFU/g. Cantaloupe received either AITC liquid or vapor, sterile deionized water, 200 ppm sodium hypochlorite per circle, or no treatment. All cantaloupes were stored in separate sealed glass desiccators for 1 or 24 h at 25°C or 35°C. To enumerate the bacteria following treatment, 22 mm sections of the rind were removed, homogenized and plated onto appropriate agar. Headspace analysis using Gas Chromatography-Mass Spectrometry (GC-MS) quantified the concentration of each AITC vapor treatment. The texture quality of the pericarp tissue of whole cantaloupes was evaluated after 24 h treatments, followed by two weeks of storage at 4°C. The concentration of vapor ranged from 3.4 to 19.6 μl AITC/L inside the desiccators. The liquid treatment reduced (P < 0.05) S. Michigan populations on ‗Athena‘ (3 log CFU/g) and L. monocytogenes on ‗HBJ‘ (2.6 log CFU/g). The longer exposure time to the AITC vapor (24 h versus 1 h) resulted in a greater reduction of both S. Michigan and L. monocytogenes on ‗Athena‘ and treatments at 35°C reduced microbial populations up to 4.5 times greater (P < 0.05). The highest vapor concentration reduced (P < 0.05) both pathogens at least 3.0 log CFU/g on ‗Athena‘ at 25°C. Generally, bacterial pathogens from the surface of ‗Athena‘ cantaloupe were reduced more than pathogens inoculated on the surface of ‗HBJ.‘ The application of AITC liquid or vapor is a natural alternative post-harvest treatment to 200 ppm free chlorine to reduce the level of bacterial contamination on cantaloupe surfaces for certified organic production. / Ph. D.

Environmental sampling is a pathogen monitoring technique that has become important in the food industry. Many food processing companies have adopted environmental sampling as a way to verify good manufacturing practices and sanitation plans in their facilities. Environmental sampling is helpful because it gives better information on the source of product contamination than end product sampling. Two specific pathogens of concern to the food industry are Listeria monocytogenes and Salmonella enterica. Environmental samples are rarely analyzed immediately, but instead may be batched for later analysis or shipped to an off site testing facility. Multiple media on the market today is used for storage and transport of environmental samples. These various media types, differences in holding temperatures and time create variability in test sample conditions. Select time, temperature and media combinations were tested to determine their effect on Listeria monocytogenes and Salmonella enterica populations during transport and storage of samples. Cocktails of Listeria monocytogenes and Salmonella enterica were added separately to sample tubes containing D/E Neutralizing Broth, Neutralizing Buffer or Copan SRK Solution. Bacterial counts at 0, 12, 24 and 48 hours post inoculation were compared. Neutralizing Buffer and Copan SRK Solution maintained consistent bacterial populations at all temperatures. At 10° and 15°C, D/E Broth supported bacterial growth. This study helps validate the use of D/E Neutralizing Broth, Neutralizing Buffer and Copan SRK Solution for environmental sample transport and storage at proper holding temperatures. At temperatures >10°C Neutralizing Buffer or Copan SRK solution should be used if quantifying microbial recovery. / Master of Science

No description available.

La bactérie Salmonella enterica sérovar Typhi (S. Typhi) provoque la fièvre typhoïde chez les humains et constitue un problème de santé publique important. La majorité de nos connaissances sur la pathogenèse de cette bactérie provient du modèle de fièvre entérique chez la souris causée par le sérovar Typhimurium. Peu d’études se sont penchées sur les facteurs de virulence uniques au sérovar Typhi, ni sur la possibilité que les pseudogènes retrouvés dans son génome puissent être fonctionnels. Le fimbria stg, unique au sérovar Typhi, renferme un codon d’arrêt TAA prématuré dans le gène stgC qui code pour le placier responsable de l’assemblage des sous-unités fimbriaires à la surface de la bactérie. Ainsi, le fimbria stg a été classifié dans la liste des pseudogènes non-fonctionnels. Les objectifs de cette étude étaient d’évaluer l’implication du fimbria stg lors de l’interaction avec les cellules humaines, puis de vérifier l’importance du pseudogène stgC lors de la biogenèse fimbriaire. Dans une première partie, la transcription de stg a été évaluée à l’aide d’une fusion lacZ. Malgré des niveaux d’expression observés généralement faibles en milieu riche, la croissance en milieu minimal a favorisé la transcription de l’opéron. La délétion complète de l’opéron fimbriaire stgABCD du génome de S. Typhi a été réalisée par échange allélique, puis a été complémentée sur un plasmide. Il a été démontré que la présence de stg chez S. Typhi, S. Typhimurium et E. coli contribue à une adhérence accrue sur les cellules épithéliales humaines. De plus, ce fimbria semble agir comme une structure anti-phagocytaire lors de l’interaction avec des macrophages humains. Ainsi, l’opéron stg semble fonctionnel, malgré son codon d’arrêt prématuré, puisque des phénotypes ont été observés. La seconde partie de cette étude consistait à vérifier le rôle joué par le pseudogène stgC dans la biogenèse du fimbria. Différentes variantes de l’opéron ont été générées, clonées dans un vecteur inductible à l’arabinose, puis transformées dans la souche afimbriaire d’E. coli ORN172. La translocation de la sous-unité fimbriaire StgD à la surface de la bactérie a été évaluée chez ces différents mutants par immunobuvardage de type Western. Cette expérience a permis de démontrer que le pseudogène stgC est essentiel pour l’exportation de la sous-unité StgD à la surface. L’ajout d’une étiquette de 6-histidines en C-terminal de StgC a permis de confirmer la traduction complète du gène, malgré le codon d’arrêt TAA prématuré. Le séquençage peptidique a révélé l’insertion d’une tyrosine à ce codon. Une fusion traductionnelle avec la protéine verte fluorescente a révélé qu’environ 0.8% de l’ARNm peut être traduit et permet la production complète du placier. Ce projet a permis la caractérisation d’un facteur de virulence unique à S. Typhi et constitue une étape de plus vers la compréhension de ses mécanismes de pathogenèse. Il s’agit de la première démonstration chez les bactéries de la fonctionnalité d’un gène interrompu prématurément par un codon d’arrêt TAA. / Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever in humans and is considered as an important health problem. Most of our knowledge on the pathogenesis of this bacterium comes from an enteric fever model in mice caused by serovar Typhimurium. Few studies have examined the virulence factors unique to serovar Typhi or the possibility that pseudogenes harbored in its genome may be functional. stg fimbriae are found only within the serovar Typhi genome and contain a premature TAA stop codon in the stgC gene encoding the usher responsible for the assembly of fimbrial subunits at the bacterial surface. Thus, the stg fimbria has been classified among the list of non-functional pseudogenes. The objectives of this study were to assess the involvement of stg fimbriae during interaction with human cells, and then to evaluate the importance of the stgC pseudogene in fimbrial biogenesis. First, stg transcription was evaluated using a lacZ fusion. Despite low expression levels generally observed in rich medium, growth in minimal medium promoted transcription of the operon. Complete deletion of the stgABCD fimbrial operon from S. Typhi was performed by allelic exchange and was complemented on a plasmid. It has been shown that the presence of stg in S. Typhi, S. Typhimurium and E. coli contributes to increased adherence to human epithelial cells. In addition, the fimbriae seem to act as an anti-phagocytic structure during the interaction with macrophages. Thus, the stg operon appears to be functional despite its premature codon, as phenotypes were observed. The second part of this study involved testing the role of the stgC pseudogene in fimbrial biogenesis. Different variants of the operon were generated, cloned into an arabinose inducible vector, and then transformed into afimbriated E. coli strain ORN172. Translocation of the StgD subunit to the cell surface of the different mutants was evaluated using Western blot. This experiment demonstrated that stgC is essential for export of the StgD subunit to the cell surface. The addition of a 6-histidine tag at the C-terminal end of StgC confirmed the complete translation of the gene, despite the premature TAA stop codon. Peptide sequencing revealed the insertion of a tyrosine at this codon. A translational fusion with the green fluorescent protein demonstrated that approximately 0.8% of the mRNA can be translated to allow full production of the usher. This project allowed characterization of a virulence factor unique to S. Typhi and is a step closer towards better understanding of its pathogenesis mechanisms. This is the first demonstration in bacteria of the functionality of a gene which is interrupted by a premature TAA stop codon.

Les fimbriae sont des structures protéiques extracellulaires retrouvées chez une vaste diversité de bactéries. Ces structures ont fait l’objet de nombreuses études et sont maintenant reconnus pour leur implication dans l’adhésion et l’invasion aux cellules eucaryotes, mais aussi dans la production de biofilms. Ils sont groupés selon leur voie de sécrétion. Certains utilisent une machinerie spécifique et individuelle, c’est le cas des pili de type IV, tandis que d’autres utilisent la voie de sécrétion générale suivit d’une voie spécifique telle que la voie du chaperon-placier (« Chaperon Usher Pathway ») (fimbriae CUP) ou la voie de nucléation précipitation (« nucleation precipitation pathway ») (Curli). Malgré toutes les connaissances actuelles concernant les fimbriae, très peu d’informations sont disponibles quant aux fimbriae de Salmonella enterica sérovar Typhi (S. Typhi). Ce pathogène unique à l’homme est l’agent étiologique de la fièvre typhoïde. Puisque les fimbriae sont reconnus pour être impliqués dans l’adaptation à l’hôte, nous avons décidé d’étudier davantage l’arsenal fimbriaire de S. Typhi, dans l’espoir d’identifier des facteurs de virulence uniques à S. Typhi et impliqués dans la ségrégation de l’hôte. La souche S. Typhi ISP1820 possède 14 opérons codant pour des systèmes d’adhésion, mais plusieurs contiennent des pseudogènes et leur expression n’a jamais été observée in vitro. Afin d’étudier les systèmes d’adhésion de S. Typhi, nous avons supprimé chaque opéron du génome individuellement et cumulativement à l’aide une technique de mutagénèse par échange allélique. Ainsi, nous avons testé chaque mutant individuel et la souche mutante pour tous les systèmes d’adhésion dans plusieurs essais tels que des infections de cellules épithéliales et de macrophages, de mobilité et de formation de biofilm. Nous avons aussi évalué l’expression des fimbriae lors de différentes conditions de croissance en laboratoire par RT-PCR. Tous les tests réalisés nous ont permis de découvrir que plusieurs opérons fimbriaires de S. Typhi sont opérationnels et utilisés pour différentes fonctions par la bactérie. / Fimbriae are extracellular proteinaceous appendages found in many bacteria. They are widely studied and believe to be implicated in several cellular functions such as adhesion, invasion of eukaryotic cells, and biofilm production. They are classified depending on their pathway of secretion: some, like type IV pili, use self-specific machinery, while others use the general secretory pathway followed by their own assembly pathway such as the Chaperon Usher Pathway (CUP fimbriae) and the nucleation precipitation pathway (curli). Despite everything that is known about these structures, little has been discovered regarding fimbrial systems of Salmonella enterica serovar Typhi (S. Typhi). This pathogen is a human restricted serovar and the etiological agent of typhoid fever. Since fimbriae have been implicated in host adaptation, we have decided to further study S. Typhi fimbrial arsenal in the hope of uncovering virulence factors unique to S. Typhi and implicated in host specificity. The S. Typhi ISP1820 strain carries 14 operons encoding for fimbrial structures, but many are believed pseudogenes or are not expressed in vitro. In order to study these different adhesion systems in S. Typhi, we have deleted each one individually and cumulatively by allelic exchange mutagenesis. Hence, we have tested every individual mutation and the mutant strain deprived of all 14 operons in many different assays including epithelial cell and macrophage infection, mobility, and biofilm formation. We also evaluate expression during growth under laboratory conditions by RT-PCR. These experiments have allowed us to discover that many of S. Typhi fimbriae are functional, expressed, and used by the bacteria in many different processes.

Lipopolissacarídeos (LPS) de L. interrogans, S. enterica e B. pertussis foram extraídos e as subfrações monofosforil lipídeo A (MPLA) preparadas por hidrólise. O LPS é responsável por forte resposta imunológica e endotóxica em mamíferos, enquanto o MPLA apresenta baixa endotoxicidade e boa capacidade imunomoduladora. Estudamos a atividade adjuvante de LPS e de MPLA em formulações com antígenos LigA ou OmpA contra leptospirose. Testes de imunização e desafio em hamsters confirmaram alta atividade imunoprotetora de LigA e capacidade adjuvante do MPLA de salmonela e de LPS de leptospiras, mas as formulações não foram capazes de bloquear a colonização renal na leptospirose. Os resultados sugerem diferenças na imunidade protetora sistêmica da imunidade protetora renal. / Lipopolysaccharides (LPS) of L. interrogans, S. enterica and B. pertussis were extracted and the subfraction monophosphoril lipid A (MPLA) prepared by hydrolysis. The LPS is responsible for immunological response and endotoxicity in mammallians, while MPLA has reduced endotoxic activity, but maintain the immunomodulatory capacity. We studied the adjuvant capacity of LPS and MPLA in formulations with LigA or OmpA antigens against leptospirosis. Tests of immunization and challenge in hamsters confirmed the high immune protection activity of LigA and adjuvant capacity of MPLA of salmonela and LPS of lepstopiras, however formulations did not block the renal colonization. The results suggest that there are differences in the systemic immune protection from the kidney immune protection.