<p>Originally considered the sole providence of protein coding sequences, evolutionary biology has begun to recognize the importance of non-coding DNA in dictating phenotypic adaptation. Exclusively examined in eukaryotic anatomical development, <em>cis</em>-regulatory modifications have the power to alter the spatial-temporal dynamics of gene expression without the plieotropic consequences of protein modification. Owing to the need to integrate horizontally acquired DNA into existing regulatory networks, <em>cis</em>-regulatory mutations may also significantly contribute to prokaryotic evolution. The horizontal acquisition of <em>Salmonella</em> Pathogenicity Island (SPI)-2 led to the evolutionary divergence of <em>Salmonella enterica</em> from <em>S. bongori</em>. Use of the type 3 secretion system encoded in SPI-2 allowed <em>S. enterica</em> to exploit an intracellular host niche offered by immune cells and allowed for its systemic dissemination. Here we identify ancestrally encoded <em>srfN</em> and <em>dalS</em> and demonstrate that through acquisition of a binding site for the SPI-2 regulator, SsrB, they have contributed to the pathoadaptation of <em>S. enterica</em> to the host environment. We also demonstrate that ancestral regulatory networks contribute to the establishment of an expression hierarchy for SPI-2 <em>in vitro</em> and to transcriptional priming in the host lumen prior to invasion. These findings demonstrate that <em>cis</em>-regulatory modifications have significantly contributed to the evolution of <em>S. enterica</em> as an intracellular pathogen.</p> / Doctor of Philosophy (PhD)

Infectious diseases are the leading cause of illness worldwide, leading to over 20 million hospitalizations each year in the United States alone. Although numerous diseases are treatable with vaccines and pharmacological agents, including antibiotics, a large fraction of infections remain poorly controlled, mainly due to lack of effective therapies and/or vaccines. Two such infectious agents are influenza A virus and the bacterium Salmonella enterica. Influenza A virus is transmitted through the aerosol route and infects lung epithelial cells, while Salmonella is transmitted via the fecal-oral route and infects the cells lining the intestine of the host. In each case, the first lines of defense against these infectious agents are non-phagocytic cells. How these pathogens are controlled in non-phagocytic cells dictates the overall outcome of infection; however there are significant gaps in our knowledge of how non-phagocytic cells respond to influenza A virus and Salmonella. Therefore, studying the fate of these cells during the course of infection is of crucial importance to disease outcome. In each case, the regulated (or programmed) death of the infected cell may represent an important pathogen clearance mechanism. Programmed cell death can be non-inflammatory (e.g., apoptosis) or pro-inflammatory (e.g., necroptosis and pyroptosis). In this dissertation, I outline experiments carried out to identify the pathways of programmed cell death activated by Salmonella and influenza A virus in their respective target non-phagocytic cells, both in vitro and in vivo. My work outlines new pathways of cell death activated by these pathogens and new mechanisms of both viral and bacterial clearance. This will have broad implications in the clearance of pathogens, and new therapeutic avenues to pursue upon treating infections. / Biomedical Sciences

Salmonella enterica is an enteric pathogen with a broad host tropism that can cause disease ranging from self-limited gastroenteritis to enteric fever. The evolution of S. enterica as a pathogen is driven by the horizontal acquisition of genes that promote virulence and survival within host immune cells, as well as the coordinated regulation of these and ancestral genes by two-component systems (TCS). TCS integrate environmental cues with the transcriptional reprogramming of bacteria, and in the case of Salmonella, result in niche-specific gene expression in response to anti-bacterial cues produced by the host. The TCS SsrA-SsrB in S. enterica is considered the master regulator for intracellular virulence, where SsrA is a sensor kinase that triggers the activation of the DNA binding protein SsrB. The full suite of genes regulated by SsrB in S. enterica, as well as the cues that activate this TCS, have not been fully characterized. Here, we demonstrated that horizontally acquired and ancestral genes in the S. enterica genome have evolved to be regulated by SsrB, and the repression of a set of ancestral genes involved in flagellar motility promotes evasion of the host immune system. Additionally, we identified the production of reactive oxygen species (ROS) by host immune cells as a signal that can activate a cluster of genes regulated by the SsrA-SsrB TCS, likely mediated by SsrA sensing of these ROS. Together, these results expand our understanding of the complex interplay between the pathogen S. enterica and the host that results in bacterial infections. / Thesis / Doctor of Philosophy (PhD) / Salmonella enterica (S. enterica) is a species of bacteria that can cause food poisoning in various animals, including humans, through consumption of contaminated food and water. During an infection, host cells activate numerous defense mechanisms to prevent disease. S. enterica has evolved to turn specific genes on or off in response, resulting in modifications to bacterial and host cell behaviour that promote infection. The timing of these genetic changes is controlled by proteins that can sense specific environmental signals and adjust gene expression accordingly. The specific signals sensed by S. enterica that allow for adaptive gene expression within the host, and the types of genes that are regulated to promote survival, have not been fully identified. Here, we show that S. enterica evolved to repress genes involved in flagellar motility to hide from the host immune response. We further demonstrate that S. enterica can sense anti-bacterial molecules produced by the host, called reactive oxygen species, to trigger specific changes in gene expression. Together, this work reveals novel aspects for the molecular basis of Salmonella enterica pathogenesis.

No description available.

There is an increasing incidence of antimicrobial-resistant organisms isolated from food and food products. Coupled with that rising incidence is increased media scrutiny and coverage of outbreaks of foodborne illnesses. Consequently, consumers increasingly demand safer food, and that the antimicrobial measures used be other than antimicrobial drugs. A possible solution is to use bacteriophages, or the purified holin and endolysin proteins that make them lethal and lytic, as antimicrobial food treatments or additives. The bacteriophage Felix O1 is a promising candidate for development as an anti-Salmonella food treatment. This dissertation describes the work done to determine if these proteins could be of value as bioremedial agents. Endolysin treatments of Gram negative bacteria require two agents: the lytic endolysin, and a second agent to permeabilize the outer membrane of the bacterium. The holin protein was proposed as an outer membrane permeabilization agent. Methods used to locate the holin gene included BLAST analysis, analysis of putative Felix O1 proteins for transmembrane domains, and examination of the lysin sequence for an N-terminal signal sequence. Analyses did not reveal a promising candidate. Cloning of rIIA as a potential holin was attempted without success. Results of various analyses are discussed, as are chemical alternatives to the use of purified holin as a permeabilization agent. The endolysin, LysO1, was successfully cloned and characterized. PHYRE analysis predicted that the enzyme structure is composed of α helices arranged into two lobes, with the active site in a cleft between them. The enzyme lysed all tested strains of Salmonella and a tested strain of the foodborne pathogen Escherichia coli. Campylobacter jejuni susceptibility remains ambiguous, and the enzyme had no effect on Listeria monocytogenes or Micrococcus luteus. LysO1 was most active at alkaline pH and low ionic strength; optimal activity was observed in 25 mM buffer at pH 10. If removed from frozen storage, the enzyme was most thermostable at 30 °C. Lytic activity was adversely affected by the presence of the divalent cations calcium, magnesium, and zinc, and by high ionic strength. Considerable time was devoted to development of the activity assay used to further characterize the enzyme, and details of those experiments are provided. Logical extensions of the research project, such as further characterization and testing needed to obtain government approval for widespread use of the treatment, and possible pursuit of treatment based on an enzyme derivative such as an antimicrobial peptide, are discussed. / Ph. D.

The objective of this work was to determine the effect of high pressure processing (HPP) on fresh tomato-associated outbreak isolates of Salmonella enterica in broth and on green mature tomato fruits. Nalidixic acid resistant (to 50 ppm) cultures of Salmonella enterica ser. Newport and Salmonella enterica ser. Braenderup were suspended in tryptic soy broth to a concentration of approximately 8 log CFU/ml and subjected to 350, 450, and 550 MPa for 120 s. Samples were serially diluted in peptone water, and surface plated onto tryptic soy agar supplemented with nalidixic acid (50 ppm; TSAN) and incubated at 35°C for 48 h. Reductions of 5.64, 6.30, and 6.61 log CFU/ml in S. Newport, and reductions of 4.10, 5.22, and 6.35 log CFU/ml in S. Braenderup at 350, 450, and 550 MPa, respectively, were observed. Green tomato fruits inoculated with S. Newport or S. Braenderup to an initial concentration of approximately 6 log CFU/g were sealed in a bag containing 350 ml of 1% CaCl2 and subjected to the same pressure treatments described above. The whole tomato fruits were pummeled in a stomacher and samples were surface plated onto TSAN supplemented with 1% pyruvic acid. Reductions of 1.55, 2.89, and 4.26 log CFU/g for S. Newport and 1.22, 2.26, and 3.77 log CFU/g for S. Braenderup at 350, 450, and 550 MPa, respectively, were observed. Bagged (350 ml 1% CaCl₂) samples of non-inoculated green tomato fruits were subjected to the same conditions described above. HPP treated tomatoes were then subjected to an ethylene gas (125 ppm; 0.7 cc/min) for 5 to 6 days. Pressured tomato fruits did not ripen. Even though HPP effectively reduced populations of S. enterica, it adversely affects the ripening characteristics of green mature tomato fruits. / Master of Science in Life Sciences

no abstract provided by author / Master of Science

Salmonella embedded biofilms were formed on whole black peppercorns and treated with ethanol vapor under atmospheric pressure conditions and under vacuum assisted pressure conditions. The effect of ethanol vapor, heat and vacuum pressure on the survival of multiple Salmonella enterica serovars within a biofilm contained on low moisture food products (Aw 0.30-0.40) was observed in this study. Samples were exposed to zero, one, five and ten minute ethanol vapor treatments at atmospheric boiling temperature (Atmospheric Pressure Boiling Method, AB), and at reduced temperature and pressure conditions, -20 inHg (Vacuum-Assisted Boiling Method, VB). The AB treatments showed 4.0 log CFU/g reductions on nonselective media that included native microbiota, and 6.0 log CFU/g reductions on Salmonella selective media. The VB treatments showed 2.69 log reductions on Tryptic Soy Agar and 4.55 log reductions on Xylose Lysine Tergitol-4 agar. Ethanol vapor treatments should be further investigated as an alternative to ethylene oxide or ionizing radiation processes to treat dry spices to control Salmonella. / Master of Science in Life Sciences

The consumption of spiced food is steadily increasing, subsequently leading to increased incidence of spice-related food illnesses. Many outbreaks can be traced to human pathogens that can survive in low moisture content of spices, prompting development of additional inactivation treatments that reduce bacterial pathogens while maintaining spice quality. Spices are currently treated by fumigation with ethylene oxide, pasteurization with ionizing radiation, or steam treatment. However, these treatments exhibit flaws pertaining to consumer preference, regulatory issues, and quality degradation. In this study, two novel treatments were evaluated for reduction of Salmonella enterica Tennessee: far infrared radiation (FIR), a short time â " high temperature treatment, and pasteurization with ethanol vapor (EV). Both treatments were effective in reducing levels of Salmonella Tennessee between 3-5 logs. FIR treatment showed increased efficacy at longer treatment times with a maximum reduction of 5 log CFU/g in paprika at 24s. EV reduced Salmonella Tennessee by 3 log CFU/g within 120s when applied to inoculated paprika and black pepper without detrimentally affecting spice quality. However, the samples receiving FIR treatments suffered reductions in volatile content and color changes to the spices. High levels (up to 1% w/w) of residual ethanol were also detected on samples treated for 300s. Concluding, both treatment show similar results when comparing efficacy; however, based on the magnitude of change in volatile content associated with FIR being significantly greater than those samples receiving EV, FIR treatment requires additional research before recommending for use with dried, ground paprika, black pepper, or sage. / Master of Science

Two studies investigating the use ultraviolet light at a wavelength of 253.7nm (UVC) into the inhibition of Salmonella spp. and Escherichia coli O157:H7 were conducted. The objectives of these studies were: to determine the rates for the destruction of Salmonella and Escherichia coli O157:H7 on the surface of agar and to investigate its effectiveness on the surface of fresh produce. Multiple replications of different doses and cocktail concentrations were performed and resulted in a 5 log reduction of Escherichia coli O157:H7 at doses exceeding 8.4 mW / cm2, while a 5 log reduction for Salmonella spp. was observed at doses exceeding 14.5 mW / cm². Samples of Red Delicious apples, green leaf lettuce and tomatoes were subjected to different doses ranging from 1.5 __ 24 mW / cm2 of UVC to determine effective log reductions of microbial populations. UVC applied to apples inoculated with E. coli O157:H7 resulted in the highest log reductions of approximately 3.3 logs at 24 mW/cm2. Lower log reductions (2.19 logs) were seen on tomatoes inoculated with Salmonella spp. and leaf lettuce (2.65 and 2.79) inoculated with both Salmonella spp. and E. coli O157:H7 respectfully. Due to the low capital involved in initiating a UVC system, the use of ultraviolet energy may prove to be a beneficial mechanism to decrease pathogens on fresh produce if used in conjunction with strict adherence to a sanitation program, Good Manufacturing Practices and Good Agricultural Practices in ensuring the safety of fresh produce. / Master of Science