Graduation date: 2000

Graduation date: 2000

Graduation date: 1996

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 152-164).

This study measured and compared different temperatures and dwell times of hot water treatment on the reduction of Escherichia coli O157:H7 and Salmonella Typhimurium on beef carcass surfaces. Two different types of beef surfaces, lean and fat, were inoculated with a fecal slurry containing E. coli O157:H7 and S. Typhimurium at ca. 7-log CFU/g, washed to remove gross fecal matter, and rinsed with hot water between 66 and 82 degrees C (150 to 180 degrees F water) for either 5, 10, or 15 s. There were no differences (P > 0.05) in the log reductions of S. Typhimurium and E. coli O157:H7 on the lean surfaces for all three temperature treatments (66, 74, and 82 degrees C). Although the 15 s treatment resulted in a numerically higher log reduction than the other treatments, each of the times resulted in at least a 1 log reduction of both S. Typhimurium and E. coli O157:H7 for lean surfaces. For the fat surfaces, all time treatments for the 82 degrees C and the 10 and 15 s treatments for the 74 degrees C resulted in the highest log reduction for S. Typhimurium. The 5 and 10 s dwell times for treatments at 66 degrees C and the 5 s dwell time at 74 degrees C resulted in the lowest log reduction of S. Typhimurium and E. coli O157:H7. For E. coli O157:H7 all temperature and time treatments resulted in at least a 1 log reduction for the fat surfaces of the outside round. Therefore, hot water treatment is a proven method for reducing both coliforms and pathogenic bacteria.

Transmission of bacterial strains between patients is a serious problem in hospitals and with the increasing rate of antibiotic resistance the problem has farther escalated. Enterobacteriaceae produced extended-spectrum beta-lactamses (ESBLs), especially Escherichia coli (E-coli), are increasingly important nosocomial pathogens (7, 8). These bacteria are often multiple resistant and are responsible for many intestinal infections and urinary tract infections (2, 5). With the more frequent use of invasive devices in hospital care, these types of nosocomial infections have increased, particularly in seriously ill patients. In order to diminish transmission of bacterial strains between patients and to study the epidemiology of these bacteria, it is of great importance to develop rapid and specific methods to be able to subtype on strain-level, i.e. to create a fingerprint of the isolates. The method may be based on phenotypic or genotypic characteristics of the microorganism. Any typing method must have high reproducibility and discrimination power to differentiate unrelated strains and also to demonstrate relationship of organisms deriving from the same source. In the present project, a Pulsed-Field Gel Electrophoresis (PFGE) assay for genotyping clinical E. coli isolates was used. PFGE can be used as a genotyping tool and is widely used to type bacteria and trace nosocomial infection. However, the method is time-consuming and relatively expensive in compare with other methods like PCR. In this study, a total of 93 strains were collected. The study was aimed to investigate the genotypes of the collected isolates and to identify and potential the outbreak strains. The isolates investigated were genotypically diverse shown by a variety of PFGE banding patterns. However, clusters of closely related isolates involved in outbreaks were also identified. In conclusion, when analyzing a large number of strains, a combination of a rapid phenotyping or genotyping method and a powerful genotyping method like PFGE would be an appropriate strategy for studying clonal relationship among isolates e.g. for detecting cross-transmission of nosocomial pathogens.

The translocation of Escherichia coli O157:H7 as well as the impact of water washing and partial or complete surface trimming as possible pathogen reduction strategies were evaluated for vacuum-packaged beef subprimals destined for non-intact use. Cap-on and cap-off beef top sirloin butts were inoculated with two levels of E. coli O157:H7! a high-inoculum at approximately 10^4 CFU/cm^2 and a low-inoculum at approximately 10^2 CFU/cm^2. Following inoculation, the subprimals were vacuum packaged and stored for either 0, 14, or 28 days. Upon opening, the following sites were evaluated: exterior of the bag, purge, the inoculation site on the subprimal, the area adjacent to the inoculation site, and the surface opposite from the inoculation site. The following treatments then were applied: water wash, water wash followed by full-surface trimming, water wash followed by partial-surface trimming, full-surface trimming, full-surface trimming followed by water wash, partial-surface trimming, and partial-surface trimming followed by water wash. For both high and low inoculated top sirloin butts, contamination of adjacent and opposite surfaces was found after vacuum packaging. Of the treatments applied, water washing alone and partial-surface trimming were the least effective for both high and low inoculated subprimals. Full trimming, with or without a water wash, proved to be the most effective treatment used to reduce E. coli O157:H7 to non-detectable levels.

In order to determine and study a protein structure, large amounts of it is needed. The easiest way to obtain a protein is to recombinantly overexpress it in the well-studied bacterium Escherichia coli. However, this expression host has one major disadvantage, overexpressed proteins might not be folded or be insoluble. Within the field of structural genomics, protein production has become one of the most challenging problems and the recombinant overexpression of viral proteins has in particular proven to be difficult. The first part of the thesis concerns the recombinant overexpression of troublesome proteins in E. coli. A method has been developed to screen for soluble overexpression in E. coli at the colony level, making it suitable for screening large gene collections. This method was used to successfully screen deletion libraries of difficult mammalian proteins as well as ORFeomes from five herpesviruses. As a result soluble expression of previously insoluble mammalian proteins was obtained as well as crystals of three proteins from two oncogenic human herpesviruses, all linked to DNA synthesis of the viral genome. The second part of the work presented concerns the structural studies of three herpesviral proteins. SOX from Kaposi’s sarcoma associated herpesvirus is involved in processing and maturation of the viral genome. Recently SOX has also been implicated in host shutoff at the mRNA level. With this structure, we propose a substrate binding site and a likely exonucleolytic mechanism. The holoenzyme ribonucleotide reductase is solely responsible for the production of deoxyribonucleotides and regulates the nucleotide pool of the cell. The small subunit, R2, has been solved from both Epstein Barr virus and KSHV. Both structures show disordered secondary structure elements in their apo-and mono metal forms, located close to the iron binding sites in similarity to the p53 induced R2 indicating that these two R2 proteins might play a similar and important role.

No description available.

Membrane proteins are key players in many biological processes. Since most membrane proteins are assembled into oligomeric complexes it is important to understand how they interact with each other. Unfortunately however, the assembly process (i.e. their social life) remains poorly understood. In the work presented in this thesis I have investigated when and how membrane proteins assemble with each other and their cofactors to form functional units. We have shown that that cofactor insertion in the hetero-tetrameric cytochrome bo3 occurs at an early state in the assembly process. We also found that the pentameric CorA magnesium ion channel is stabilised by different interactions depending on the magnesium ion concentration in the cell. These studies indicate that the assembly of a functional unit is a dynamic process, which is a result of many different forces. By studying the assembly of membrane proteins we have obtained a deeper insight into their function, which cannot be explained by static crystal structures. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.</p>