Aspartate transcarbamoylase (ATCase; encoded by pyrBI genes) is one of the most studied regulatory enzymes in bacteria. It is feedback inhibited by cytidine triphosphate (CTP) and activated by adenosine triphosphate (ATP). Much is known about the catalytic site of the enzyme, not nearly as much about the regulatory site, to which CTP binds. Until now a positive selection for feedback-modified mutants was not available. The selection we have developed involves the use of a pyrA deletion in S. typhimurium. This strain lacks carbamoylphosphate and requires both a pyrimidine and arginine for growth. In this strain citrulline is used to satisfy the pyrimidine and arginine requirements. The minimal flow through the pyrimidine pathway from the citrulline-produced carbamoylphosphate is exquisitely sensitive to feedback control of ATCase by CTP. By elevating the CTP pool, via exogenous cytidine, in a strain that also contains a cytidine deaminase mutant (cdd) growth can be stopped completely, indicating 100% inhibition. It was therefore possible to measure in vivo feedback inhibition of ATCase among the citrulline users and to isolate a family of ATCase regulatory mutants with either modified or no response to effectors.

This study was undertaken to compare the MIC (minimum inhibitory concentration) and MPC (mutant prevention concentration) values for oxytetracycline and florfenicol against strains of Pasteurella multocida isolated from cattle and pigs, and for enrofloxacin against strains of Salmonella Typhimurium isolated from horses. Isolates of P. multocida from cattle and pigs, and S. Typhimurium from horses were obtained from specimens or isolates from contributing laboratories. All the equine isolates and 50% of the cattle and pig isolates were from clinically sick animals. All isolates were tested in duplicate with both the MIC and the MPC methods. The MIC method used was the standardized microdilution method performed in microtitre plates. The MPC method used was according to the method described by Blondeau. This method was modified, to make use of smaller plates and lower volumes of antimicrobials, but retaining a final bacterial concentration of 109 colony-forming units per ml. The antimicrobials were dissolved as described in the certificates of analyses. Enrofloxacin and oxytetracycline were dissolved in water, and florfenicol was dissolved in alcohol. For the MPC method, an additional control was added to one quadrant of a four-quadrant 90mm plate/petri dish. The antimicrobials were tested as individual antimicrobials and not as combinations. Both the MIC and MPC methods included ATCC (American Type Culture Collection) strains as control organisms and were evaluated according to the guidelines of the CLSI (Clinical and Laboratory Standards Institute). The MIC50 values for enrofloxacin against Salmonella Typhimurium isolates from horses was 0.25 ìg/ml and the MPC50 values 0.5 ìg/ml. A comparative reference range was not available as enrofloxacin is not registered in South Africa for use in horses, and is used extra-labelly. The results for florfenicol against P. multocida yielded an MIC50 value of 0.5 ìg/ml and an MPC50 value of <2 ìg/ml. The close relationship of these two concentrations is an indication of the effectiveness of florfenicol when used against P. multocida. The PD/PK data with a value of 141.78 for AUC/MIC provided additional support for the efficacy of florfenicol against P. multocida. The PD/PK value of >125, is an effective parameter for treatment of Gram-negative bacteria. The corresponding results for oxytetracycline were above the MIC value but fell within the mutant selection window. The results point to the fact that the use of oxytetracycline against P. multocida may not be effective in preventing the appearance of first step mutant strains when used at current recommended dosages. The PK/PD data, using AUC/MIC, yielded a value of 56. Some of the isolates (55.17%) had an MPC value of 16 ìg/ml. Whereas the MIC method is used routinely in diagnostic laboratories, the MPC method can be employed to generate data that can be applied where antimicrobial treatment of certain bacteria is problematic and standard treatment may lead to the development of resistance. Data obtained from such studies will enable manufacturers of antimicrobial drugs to adapt antimicrobial therapy where practical and feasible to prevent the development of first step mutants. / Dissertation (MSc)--University of Pretoria, 2012. / Veterinary Tropical Diseases / unrestricted

The Salmonella typhimurium/mammalian microsome test has provided a simple and sensitive short‐term assay for the detection of environmental mutagens. Metabolic activation of precarcinogens is usually achieved by incubating the compound to be tested, the bacterial strain and mammalian liver homogenates with NADPH. The results presented here utilize Salmonella typhimurium strain TA100, the precarcinogen 2‐aminofluorene and microsomal enzyme preparations prepared from liver homogenate of carp (Cyprinus carpio) taken from aquatic environments of northeastern Tennessee. Those environments range from virtually unpolluted to extremely polluted. The results show that the precarcinogen 2‐aminofluorene is activated either partially or totally in the presence of liver homogenates of carp taken from polluted aquatic environments (e.g., microsomal enzyme preparations made from rat liver with 2‐aminofluorene produced 808 revertants; whereas the liver preparations made from carp, taken from the Pigeon River, with 2‐aminofluorene produced 2,786 revertants). Revertant colony results correlated well with the degree of pollution within those waters. An increase (data were statistically different at the 0.05 level of significance) of TA100 revertant colonies was observed as aquatic contamination worsened. All data pairs of collecting sites in their order of increasing contamination, as well as those between collecting sites, were statistically different at the 0.05 level of significance.

Determina el efecto de la Salmonella Typhimurium sobre los parámetros productivos, composición química y calidad microbiológica de la carne de cuy (Cavia porcellus). El trabajo se realizó en la unidad de experimentación de cuyes del laboratorio de Bioquímica, Nutrición y Alimentación Animal de la Facultad de Medicina Veterinaria, UNMSM. Se utilizaron 40 cuyes machos de engorde que fueron distribuidos en 4 tratamientos con diez (10) repeticiones cada uno; T1: cuyes alimentados con dieta base + solución salina (control), T2: cuyes alimentados con dieta base + APC + solución salina, T3: cuyes alimentados con dieta base y desafiados experimentalmente con Salmonella Typhimurium, T4: cuyes alimentados con dieta base + APC y desafiados experimentalmente con Salmonella Typhimurium. En el día 11, los animales del T1 y del T2 fueron dosificados vía oral con solución salina, mientras que los T3 y T4 fueron desafiados con una dosis infectiva (2 x 106 UFC) de Salmonella Typhimurium, por única vez. Se evaluaron los parámetros productivos (ganancia de peso, consumo de alimento, conversión alimenticia), la composición química y la calidad microbiológica de la carne de cuy. Los cuyes de los tratamientos T3 yT4 presentaron, significativamente (p<0.05), menor ganancia de peso vivo total (T3: 534g; T4: 577 g) y mayor índice de conversión alimenticia (T3: 6.29; T4: 5.92) comparados con el grupo de animales no desafiados (T1: 761g, 4.04; T2: 828 g, 3.66). No se observó diferencia estadística significativa en el rendimiento de la canal de los cuyes en los cuatro tratamientos. El número de casos con mayor presencia de Salmonella sp. se observó en ganglios linfáticos, hígado, bazo, vesícula biliar y pulmón de las muestras de órganos de los grupos T3 y T4. Se concluye que el desafío oral a Salmonella Typhimurium causa, significativamente (p<0.05), una menor ganancia de peso vivo, menor porcentaje de proteína en la canal, mayor índice de conversión alimenticia y menor retribución económica en animales desafiados comparados con el grupo de animales no desafiados. / Tesis

Understanding the composition of camel milk coupled with studying the effects of thermal and non-thermal treatments on its components and bacterial inactivation were the general objectives of this dissertation. In the first study (Chapter 2), the gross composition of camel milk including milk protein, fat, casein, total solids, lactose, ash, and mineral content were analyzed. In addition, fatty acid profile, amino acid profile, protein fractions, and volatile compounds were evaluated as well. Our results revealed that camel milk has its unique nutrients profile. These findings make it easier for the researchers and consumers to understand some of the nutritional attributes of camel milk.The impact of non-thermal ultrasound treatment (900 W, 20 kHz, 100% power level) on some milk-borne microorganisms and the components of camel milk was studied in Chapter3. We reported that continuous ultrasound processing was efficient in inactivating Escherichia coli (E.coli) O157: H7 and Salmonella Typhimurium (S. Typhimurium) in camel milk without detrimental effects on milk fatty acids profile, lipid peroxides, and protein fractions except for some changes in milk volatile compounds (VC). In Chapter 4, another non-thermal technique, ultraviolet-C (UV-C) light, was applied to camel milk to study the effects of different UV-C light doses on the viability of E. coli O157:H7 and S. Typhimurium and the chemical changes to milk components. The main findings of this study were: (i) UV-C treatment at a dose of 12.45 mJ/cm2 resulted in only 3.9-log10 for both bacterial strains which did not meet the Food and Drug Administration (FDA) requirements for the 5-log pathogen reduction; (ii) the UV-C treatment at the above dose, had limited effects on camel milk components. Thermal pasteurization of milk was first introduced to prevent milk-borne infectious diseases, however, its effects on camel milk components and quality are still unknown. Therefore, in Chapter 5, we investigated the efficacy of three previously reported thermal methods: PAST-1 (65ºC/30 min), PAST-2 (72ºC/5 min), and PAST-3 (80ºC/5 min) on bacterial inactivation and some camel milk components such as the fatty acid profile, lipid peroxidation, VC, and milk protein fractions. Complete elimination (6 log10 CFU/ml reduction) of E. coli O157: H7 was achieved using all pasteurization methods, however, only 3.4 log10 CFU/ml reduction of the total viable counts was reported using PAST-1 and PAST-3 methods. We also reported that the PAST-1 and PAST-3 methods did not affect the chemical composition of camel milk. In conclusion, we assessed the main components of camel milk along with the amino fatty acid profile, acid profile, volatile compounds, and protein fractions. Thermal methods were more effective than the non-thermal methods in terms of microbial inactivation and most camel milk components were not significantly influenced by thermal and non-thermal methods.

Regulation of the bacterial second messenger cyclic-di-GMP in Salmonella Typhimurium allows it to delicately alter phenotypes to optimize invasion and survive intracellularly in epithelial cells and macrophages to become virulent and cause infection. The concentration of cyclic-di-GMP is regulated by the presence of external stimuli, sensor CMEs (diguanylate cyclases, DGCs, and phosphodiesterases, PDEs), and cyclic-di-GMP binding effectors. Previous studies established that maintenance of low cyclic-di-GMP concentrations is required for survival in macrophages and that the deletion of 3 active PDEs reduces this survival. This study aimed to further investigate the regulation of cyclic-di-GMP for survival in macrophages and epithelial cells. Salmonella Typhimurium mutants were generated and used for an infection assay with RAW 264.7 macrophage and HeLa epithelial cell lines to determine active CMEs via intracellular survival. Intracellular survival was quantified by plate counting of cell lysates at 1-, 4-, and 24-hours post-infection. Our result showed that the previously identified 3 PDEs also influenced the infection of epithelial cells. We re-established the decreased survival in the RAW 264.7 macrophage cell line and determined that the cyclic-di-GMP-binding cellulose synthase BcsA was responsible for decreased survival in macrophages. Finally, we identified an active DGC whose deletion within the 3xKO PDEs restores survival levels, suggesting that this enzyme is responsible for the synthesis of cyclic-di-GMP during macrophage infection. Further studies on how cyclic-di-GMP regulates Salmonella Typhimurium intracellular survival could lead to identifying a potential alternative drug target for treating its infections.

Regulation of the bacterial second messenger cyclic-di-GMP in Salmonella Typhimurium allows it to delicately alter phenotypes to optimize invasion and survive intracellularly in epithelial cells and macrophages to become virulent and cause infection. Cyclic-di-GMP concentration is regulated by the presence of external stimuli, sensory diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), and cyclic-di-GMP binding effectors. Previous studies established that maintenance of low cyclic-di-GMP concentrations is required for survival in macrophages, and that deletion of 3 active PDEs reduces this survival. Here I showed that these 3 PDEs also influenced the infection of epithelial cells. Further studies re-established the decreased survival in an immortalized macrophage cell line and determined that cyclic-di-GMP-binding cellulose synthase BcsA was responsible for the decreased survival in macrophages. Finally, I also identified an active DGC whose deletion within the 3xKO restores survival levels, suggesting that this enzyme is responsible for the synthesis of cyclic-di-GMP during macrophage infection.

Bacteria-based cancer therapy (BBCT) holds immense promise in addressing the limitations in treatment of solid tumors. Bacterial strains used for BBCT are engineered to express therapeutics, facilitate precise navigation within the tumor microenvironment by enhancing bacteria's motility, chemotaxis (movement toward or away from specific chemicals), or other mechanisms that aid in reaching and infiltrating the tumor tissue effectively, and complementing traditional chemotherapy and immunotherapies while minimizing side effects. Bacterial motility not only influences the ability of bacteria to navigate within the tumor but also plays a pivotal role in optimizing drug delivery, treatment efficacy, and minimizing potential obstacles associated with the complex microenvironment of human tissues. However, the current understanding of bacterial motility remains limited. In this thesis, we use a reductionist approach and study bacteria motile behavior within human tissue phantoms (collagen and agar) and the bacteria-hydrogel interactions. Apart from motility, it is important to analyze the mechanical properties of the hydrogels the bacteria interact with as they play a vital role in overall behavior and physics of bacteria movement. To that extent, there exists a gap in our understanding of the viscoelastic properties of hydrogels. Lastly, systematic and comprehensive investigation of bacteria behavior in hydrogels requires tracking of thousands of individual cells. Thus, there is an unmet need to develop new automated techniques to reduce the labor-intensive manual tracking of bacteria in low-contrast hydrogel environments, with feature sizes comparable to that of bacteria. To address these gaps, this thesis proposes a trident approach towards mechanistic understanding of bacteria motility in time-invariant agar and temporally evolving collagen hydrogels to bridge critical gaps in understanding bacterial motile behavior in these media, non-destructive microelastography-based mechanical characterization of hydrogels with less than 4.7% error compared with rheology, and the development of deep learning-enabled automated bacteria tracking tools with 77% precision. / Master of Science / There exists a huge scope for improvement in cancer therapies. The gold standard chemotherapy and immunotherapies are responsible for a lot of side effects. Over a century ago, bacteria-based cancer therapy started to develop and over the period, it was discovered that they can be effective when used with traditional therapies improving precision and reducing side effects. The motility of bacteria is shown to improve bacterial distribution in solid tumors. However, the physical underpinnings of bacteria motility in the tumor environment remains understudied. This thesis proposes a trident approach, investigating bacteria motility in tissue-like environments (hydrogels), characterizing the mechanical properties of hydrogels using acoustic waves to capture bacteria-induced alterations, and developing deep-learning-enabled automated bacterial tracking approach for high throughput analysis of experimental data. We report bacteria behavior and motility patterns in hydrogels, the mechanics of these hydrogels with less than 4.7% error compared with standard characterization methods, and automated bacteria tracking with 77% precision to inform the development and advancement of bacteria-based drug delivery systems. In summary, these tools can help improve our understanding of bacteria-hydrogel interactions, allowing us to develop innovative bacteria-based cancer therapies in the long term.

The first recorded observation of bacterial biofilms dates back to the 17th century by Antoine Van Leeuwenhoek. Today, biofilms are known as bacteria encapsulated within a self-produced extracellular matrix adherent to biotic or abiotic surfaces. Since the initial discovery of biofilms, research has explored the structure and function of biofilms. Only until recently has the role of biofilms within the medical setting become apparent. Here, we investigate the role of curli-extracellular DNA (eDNA) complexes in disease pathogenicity and explore the ability to target bacterial amyloid curli as a novel anti-biofilm therapeutic target. Biofilms of enteric bacteria, such as Escherichia coli and Salmonella enterica serovar Typhimurium, are composed of various components that act in consortium to fortify the extracellular matrix. One of the main components of enteric biofilms is amyloid curli. Curli, one of the best characterized bacterial amyloids, is a protein with a conserved cross beta sheet structure that forms basket like structures encapsulating the bacteria. Within the biofilm, curli serves to fortify the extracellular matrix, aids in bacterial attachment and protects bacteria from harsh environmental conditions. Extracellular DNA (eDNA) is another integral component of enteric biofilms. Recent reports from our lab has suggested that curli forms irreversible complexes with eDNA. Even with exposure to DNases, co-localized curli and eDNA can be observed. Other components of enteric biofilms include cellulose and Biofilm Associated Protein A. Biofilms of S. Typhimurium have been associated with significant disease pathologies. In addition to identifying the existence of curli-eDNA complexes within S. Typhimurium biofilms, our lab has also reported that curli-eDNA complexes of S. Typhimurium potentiate the autoimmune disease Systemic Lupus Erythematosus (SLE). SLE is an autoimmune disease characterized by the production of type I interferons and autoantibodies, although the etiology remains unknown. Systemically, curli binds to and activates the Toll like Receptor (TLR)1/2 complex leading to a pro-inflammatory response. In these studies we aimed to identify the innate immune mechanisms leading to the autoimmune phenotype following stimulation with curli-eDNA complexes. As TLR9 is activated by unmethylated bacterial DNA CpG DNA sequences leading to the production of type I interferons we hypothesized a potential role for TLR9 in recognizing eDNA of the curli-eDNA complex leading to the generation of the hallmarks of SLE. To investigate this hypothesis, we stimulated wild-type, TLR2 knockout, TLR9 knockout and TLR2-9 double knockout immortalized macrophages with curli-eDNA complexes purified from S. Typhimurium biofilms. We observed a significant reduction in the transcript level of type I interferons (IFN), Ifnβ, Isg15 and Cxcl10, upon stimulation of TLR2 knockout, TLR9 knockout and TLR2-9 double knockout immortalized macrophages implicating a role in TLR9 recognition of the curli-eDNA complex. As there was a significant reduction of type I interferon levels upon stimulation of TLR2 knockout macrophages, we hypothesized that TLR2 may serve as a carrier to bring the curli-eDNA complex into the endosome containing TLR9. To inhibit phagocytosis, we pretreated cells with endocytosis inhibitors and stimulated wild-type macrophages with curli-eDNA complexes. We observed a reduction in the transcript level of Ifnβ suggesting that curli-eDNA complexes gain access to endosomal TLR9 via TLR2 engagement. Finally, to explore the role of TLR2 and TLR9 in the production of autoantibodies, curli-eDNA complexes were intraperitoneal injected twice weekly for six weeks into C57BL/6 wild-type, TLR2 knockout, TLR9 mutant and TLR2 knockout-TLR9 mutant mice. We observed a robust generation of anti-double stranded autoantibodies within the first three weeks, however the production of autoantibodies was significantly decreased and delayed in the TLR2 knockout, TLR9 mutant and TLR2 knockout-TLR9 mutant mice. Overall, these data suggest that curli acts as a carrier for DNA to elicit an autoimmune response via TLR2 and TLR9. Within biofilms of S. Typhimurium, curli is the main proteinaceous component. Biofilms lacking curli destabilize and fail to form mature biofilms. Recent research has shown that in response to the production of host amyloids, the body will generate anti-amyloid antibodies in the serum. Monoclonal antibodies (mAb) generated from serum antibodies have been shown to have pan anti-amyloid properties in vitro and in vivo due to the β-sheet conformational epitope. As amyloids from both human and bacterial origin share a β-sheet conformational structure, we hypothesized as to if the anti-amyloid mAbs can eradicate S. Typhimurium biofilms by targeting curli. We incubated S. Typhimurium biofilms in the presence of various mAbs (ALZ.4A6, ALZ.4GI, ALZ.2C10 and ALZ.3H3) and observed a significant reduction of biofilm thickness and curli content within the biofilm. We deduced that ALZ.3H3 conferred the greatest anti-biofilm response. When we visualized the three-dimensional architecture of biofilms incubated with ALZ.3H3, we observed that ALZ.3H3 induced the formation of a loose architecture compared to untreated biofilms that were dense and compact. The resulting loose biofilm architecture induced by incubation with ALZ.3H3 enhanced the susceptibility of the biofilms to antibiotic exposure and macrophage clearance. We also observed enhanced biofilm eradication in vivo when catheters precoated with S. Typhimurium biofilms were inserted into the back flanks of mice that were percutaneously injected with ALZ.3H3. Both in vitro and in vivo, combination therapy of ALZ.3H3 and antibiotic enhanced biofilm clearance. In summary, we propose a novel anti-biofilm strategy by targeting the amyloid component of the biofilm, thus satisfying an unmet need in the art of biofilm prevention. Overall, these data in summation significantly broadens our understanding of disease pathogenicity and the role of curli-eDNA complexes in S. Typhimurium biofilms. As amyloid-eDNA complexes may be found in other biofilms, these results may extend beyond enteric bacteria proving novel insight into host-microbe interactions and the generation of novel anti-biofilm therapeutics. / Microbiology and Immunology

Attenuated bacterial strains have been investigated on the premise of selective tumor colonization and drug delivery potential for decades. Salmonella Typhimurium VNP20009 was derived from the parental strain 14028 through genetic modification and tumor targeting ability, being well studied for anticancer effects in mice. In 2001 Phase 1 Clinical Trials, patients diagnosed with melanoma were introduced with VNP20009, resulting in safe delivery of the strain and targeting to the tumor, however no anticancer effects were observed. Recently, it was discovered that VNP20009 contains a SNP in cheY, which encodes the chemotaxis response regulator of flagellar motor function, rendering the strain deficient in chemotaxis. Replacement of cheY with the 14028 wild-type copy resulted in a 70% restoration of phenotype in traditional chemotaxis capillary assays compared to the parental strain. We attempted to optimize the chemotactic potential of VNP20009 but were unable without reversing the attenuated state of VNP20009. Due to the role of chemotaxis in bacterial tumor colonization and eradication remaining unclear, we aimed to compare VNP20009 and VNP20009 cheY+ primary tumor colonization and impact on metastasis in an aggressive 4T1 mouse mammary carcinoma model. Bacterial tumor colonization and metastatic potential of the cancerous cells to the lungs appear bacterial chemotaxis independent. Moreover, mice bearing tumors exposed to Salmonella exhibited increased morbidity that was associated with significant liver disease. Our results suggest that in our timeline VNP20009 may not be safe or efficacious when used in the context of immunocompetent animals with aggressive, metastatic breast cancer. In a novel approach, we aimed to understand the bacterial-cancer cell relationship within the tumor microenvironment, with an emphasis on gene expression changes occurring within the eukaryotic transcriptome. We employed the B16-F10 mouse melanoma model because VNP20009 is known to colonize and eradicate these tumors in mice. First, we optimized a timeline for Salmonella treatment of mouse melanoma, finding a dramatic delay in tumor growth between 2 and 7 days due to the presence of Salmonella. Additionally, we observed upregulation of the IFN-gamma signaling pathway within tumor tissue upon exposure to Salmonella after 7 days. In future studies, we aim to analyze the bacterial transcriptome in the tumor microenvironment to gain unique understanding and contribute to knowledge supporting bacterial-mediated cancer therapies. / Ph. D. / Bacteria have become our allies in the fight against cancer. Strains of Salmonella, normally thought of as a cause of gastrointestinal discomfort, are able to target cancer in the body and effectively shrink tumors in several animal models. Specifically, a strain of Salmonella Typhimurium called VNP20009, has shown great promise as an anticancer agent. Research on VNP20009 culminated in a Phase 1 Clinical Trial in which safe delivery of the strain and targeting to the tumor were achieved, however no anticancer effects were observed. We hypothesized further targeting of Salmonella could be achieved using chemotaxis, the coordination of flagellar driven movement with sensing environmental chemical gradients, akin to the nose of the bacterium. We discovered strain VNP20009 to be defective in chemotaxis, due to a genetic mutation that occurred during the strain’s construction. We were able to restore chemotaxis of the strain, at least partially, and discovered we could not further optimize chemotaxis without compromising the safety profile of VNP20009. We tested the effect of chemotaxis on tumor colonization in a mouse breast cancer model and found that the bacteria had an additive effect in causing liver disease and morbidity of the mice. We finally examined genome-wide gene expression changes occurring in the tumor microenvironment, as a response to anticancer agent VNP20009 colonization in a mouse melanoma model of cancer. Overall, this work contributes significantly to the understanding of VNP20009 chemotaxis and its tumor targeting abilities.