The entrainment of microbes from agricultural land into overland flow during rainfall events is recognised as an important source of pathogenic microbes to surface water bodies and yet this transport process is poorly understood. In this study, a method has been developed to separate bacteria into the forms in which they have been postulated to exist in overland flow. Then Escherichia coli was used as a model organism to investigate the transported state of bacteria eroded from cowpats and their subsequent transport in overland flow. Simulated rainfall experiments were used to generate runoff direct from cowpats. Concentrations of E. coli in the runoff direct from cowpats were found to be directly proportional to the concentration in the cowpat, regardless of the age of the cowpat. It was also observed that E. coli were predominantly eroded from cowpats as individual cells. The interactions between E. coli and soil particles in overland flow were then examined in a small laboratory scale model system and showed that E. coli attached to large (>45 [mu]m) soil particles were transported significantly less than unattached cells. However, in the runoff from the model system, E. coli were found to be attached mainly to clay particles that were similar in size to the bacterial cells. Furthermore, the transport of E. coli through the model system appeared to follow the transport of a conservative chemical tracer implying that (a) the cells were being transported as a solute with the bulk of the water flow, and (b) that E. coli attached to small clay particles were as mobile in the overland flow as unattached cells. These observations imply that E. coli predominantly interact with small clay particles that are also being carried along in the overland flow. The transport of E. coli at a larger scale was then investigated using 5-metre long, 1-metre wide buffer strips operated under saturation excess conditions. In buffer strips using intact soils and existing pasture cover, E. coli removal was very poor (26 % removal) at the low flow rate of 2 L min⁻� with no removal observed at the higher flow rates of 6 and 20 L min⁻�. E. coli removal rates were increased to 41 % removal at 2 L min⁻� by cultivating the soils, with the removal rate again decreasing with increasing flow rate. E. coli in the overland flow from the buffer strips did not form into large flocs or attach to large soil particles, but were transported in small neutrally buoyant particles that remain entrained in the overland flow. Under saturation excess runoff conditions, E. coli in overland flow were not effectively removed by buffer strips as the small particles are transported either over the soil surface or, through large pores in the soil. This Thesis has shown that E. coli is transported in overland flow in small particle sizes that are difficult to trap or remove from overland flow thereby explaining the high fluxes of faecal bacteria observed in overland flow from agricultural land.

<p>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.</p><p>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.</p><p>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.</p><p>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.</p>

Escherichia coli double-strand uracil-DNA glycosylase (Dug) was purified to apparent homogeneity from bacteria that were defective in uracil-DNA glycosylase (Ung). After cloning the dug gene, recombinant Dug was overexpressed, purified, and characterized with respect to activity, substrate specificity, product DNA binding, and mechanism of action. Purified Dug excised both uracil and ethenocytosine specifically from double-stranded DNA substrates. One distinctive characteristic of Dug was that the purified enzyme removed a near stoichiometric amount of uracil from DNA containing U/G mispairs. The observed lack of turnover was attributed to tight binding of Dug to the apyrimidinic-site (AP) contained in the DNA reaction product. Catalytic activity was stimulated in the presence of E. coli endonuclease IV that caused AP-site incision and dissociation of Dug. By using enzyme complementation experiments, Dug was shown to initiate uracil-initiated base excision repair (BER) in E. coli (ung) cell-free extracts. The relative rate of repair of uracil- and ethenocytosine-containing DNA in isogenic E. coli cells that were proficient or deficient in Ung and/or Dug was measured using a novel competition assay. Complete ethenocytosine-initiated BER displayed an absolute requirement for Dug and occurred at the same rate as uracil-initiated BER in the presence of both Ung and Dug. However, the rate of Dug-mediated ethenocytosine-DNA repair was 8-fold faster than that of uracil-DNA mediated by Dug. The distribution of BER patch sizes associated with both uracil- and ethenocytosine-containing DNA showed similar results. In both cases, DNA repair synthesis utilized predominantly a long patch BER mechanism involving the incorporation of 2-20 nucleotides. A previously unidentified "very long patch" mechanism of BER involving the incorporation of more than 200 nucleotides was identified and shown to be mediated by DNA polymerase I. The rate-limiting step associated with uracil-initiated BER was found to involve DNA ligase and the distribution of BER patch size was modulated by the ratio of DNA polymerase I and DNA ligase. The fidelity of DNA repair synthesis associated with complete uracil-DNA BER was measured using E. coli cell-free extracts that were proficient or deficient in Ung activity and determined to be 5.5 x 10������ and 19.7 x 10������, respectively. / Graduation date: 2003

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.