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Escherichia coli O157:H7 detection in food is conducted mainly by DNA/PCR, immunoassay or conventional methods. However, all the methods require multiple incubation steps. Antibiotic and isolation agars were found as the main factors that lead to false-positive results. An improved rapid detection method was developed by decreasing detection time and enhancing easiness of detection without the need for any analytical instrumentation. A combination of selective ingredients and temperature was utilized to allow the growth of Escherichia coli O157:H7 in the detection. The detection method minimized the effects of the main false positive bacteria, Pseudomonas spp. and Enterobacter spp. The sensitivity, specificity and accuracy of the 24h detection method in foodstuffs were 96.2%, 99.6% and 97.0%, respectively when the original inoculation was 10-100cfu/g in food. This method can be utilized to detect Escherichia coli O157:H7 in foodstuffs more rapidly, economically and conveniently when compared to the methods that are currently used.

Three studies regarding Escherichia coli O157:H7 in apple cider were conducted. The objectives were: to evaluate the effectiveness of wash and sanitizers for removing E. coli O157:H7 from apples; to survey cider producer practices; and to determine the efficacy of ultraviolet light for reducing E. coli O157:H7 in cider. Apples with a five-strain acid resistant mixture of E. coli O157:H7 were treated with 200 ppm hypochlorite, a phosphoric acid-based fruit wash, 5% acetic acid, 5% acetic acid followed by 3% hydrogen peroxide, a peroxyacetic acid-based solution, and distilled water. The water wash caused insignificant reductions. All other treatments caused significant reductions. Acetic acid and peroxyacetic acid were the most effective with reductions of 3.1 and 2.6 logs, respectively. The survey determined that most producers are small, seasonal operations. Most use sound orchard management practices, clean and sanitize daily, sort and wash apples, use refrigeration, and try to prevent contamination. However, some use drop and damaged apples. Few use chemical sanitizers on apples, preservatives, pasteurize cider, or have HACCP programs. Cider inoculated with the same mixture of E. coli O157:H7 was processed using a thin- film ultraviolet disinfection unit operating at 254 nm. Dosages ranged from 9,402 to 61,005 æW- sec/cm2. Treatment significantly reduced E. coli O157:H7 (pó 0.0001) with a mean reduction of 3.81 log CFU/ml. Reduction was also affected by the level of background microflora in cider. Results indicate that ultraviolet light can reduce this pathogen in cider. However, additional reduction measures are necessary to achieve the required 5 log reduction. / Master of Science

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No description available.

The number of food borne illnesses associated with the consumption of fresh fruits and vegetables and their minimally processed products (juices) has increased over the past years. Of particular interest is the ability of microbial pathogens to internalize and survive in fresh produce that are commonly used for juices. This research project addresses the issue of the ability of Escherichia coli O157:H7 to internalize and survive in whole apples before and after harvest. Four cultivars of apples, Redfree, Red Delicious, Golden Delicious, and York, were inoculated under field conditions with a surrogate strain of E. coli, Escherichia coli ATCC 25922. The Redfree cultivar was inoculated at the beginning of its growth stage (day 0), and again 30 days later, and sampled for two weeks, until E. coli was not recoverable through microbiological methods after three successive sampling days. Red Delicious, Golden Delicious, and York cultivars were spray inoculated with the surrogate strain two weeks before their anticipated harvest date and sampled every other day until E. coli was not recoverable for three successive sampling days. For each cultivar, the presence of E. coli ATCC 25922 was not detectable after 7 to 9 days. In the laboratory study the Red Delicious, Golden Delicious, Rome, and York cultivars received one of three treatments; unblemished control, bruising, or puncturing. The apples were inoculated by immersion in cold water containing E. coli O157:H7 GFP, incubated for three days then microbiologically analyzed for presence of the bacteria. In all cases, the punctured apples of each cultivar showed the greatest uptake of E. coli O157:H7 GFP. Escherichia coli O157:H7 GFP was visualized in flesh and core sections of untreated, bruised, and punctured apples of all cultivars. The microbe was found in between cells, but not within cells of the apple. Internalization of Escherichia coli in whole apples on the tree is not likely, and leads to the conclusion that internalization is a post-harvest problem. Internalization may occur before pressing or processing of apples, leading to an increased risk of infection with E. coli for consumers of apple products that are not properly treated to destroy pathogens. Internalization does occur when apples are immersed in solutions containing the pathogen Escherichia coli O157:H7, and better post harvest controls need to be implemented in order to prevent this in whole apples that are used for cider and juice production. / Master of Science

The main objective of this project is to gain an understanding of the internalization of Escherichia coli in the tissues of apples. This broad statement includes the rate of internalization in young versus mature apples as well as injured versus non-injured apples. Five apple varieties, Redfree, Red Delicious, Golden Delicious, Rome Beauty and York Imperial, were used to compare differences and similarities in structure and ability to internalize the pathogen. Both the surrogate species, E. coli ATCC 25922, and the pathogen, E. coli O157:H7, were used for field and lab studies, respectively. Internalization of E. coli in apples under natural environmental conditions was addressed in the first study using a controlled outdoor setting. Escherichia coli species (ATCC 25922) was used as an alternative to the pathogenic species. The bacterial culture was applied to topsoil and spread evenly on a 6x6-foot area. Red Delicious, Golden Delicious, and Rome Beauty apples were placed randomly on the soil much like a drop or windfall apple. The position was noted as to whether the apple fell calyx up, down or on its side. Apples were examined for the presence of E. coli and sampled on days 1, 3, 8, and 10. Skin, flesh, inner, and outer core samples were plated on MacConkey agar supplemented with cycloheximide and MUG to ease in identification. Escherichia coli was found in the inner core and flesh samples of all apple varieties, indicating the potential for infiltration by the organism outside laboratory conditions. The second study determined the rate of internalization in immature apples. Redfree was used in a long-term study in which individual apples were spray inoculated at the beginning of the growing season with E. coli ATCC 25922 at 104 cfu/apple. The apples were picked on days 1, 30 and 60, and sectioned into skin, flesh, inner and outer cores. The remaining four apples species were used in an intensive, two-week study. In the long-term study, apples were inoculated two weeks prior to harvest and picked every other day until harvest. The surrogate E. coli was not found in the apples after day 1. Other coliforms, such as E. vulneris, Klebsiella pneumoniae and Kl. ozaenae were present in each pick. The two-week study showed higher rates of internalization in Red and Golden Delicious than in Rome and York, with the E. coli present in all four sections of the apples. Red Delicious apples showed a trend of increasing counts of bacteria over the two-week period with initial counts ranging from less than one cfu/ml to final counts as high as 2.64±1.90 log cfu/ml. Again Klebsiella species and E. vulneris were found in the apples. Microscopy was used for imaging of the apples tissues. Morphological differences were found in the skin, where lenticel presence or absence may affect internalization. Differences were also shown in the flesh where cell wall thickness was shown to vary depending on variety. Imaging thick sections of skin showed cuticle cracks and thickness, which also vary depending on the apple variety. This study indicates that internalization occurs at a high degree in drop apples and to a limited extent in tree apples. However, with the low infective dose required for illness, it is necessary to instate strict regulations to ensure safety. The most effective treatment involves the inclusion of a five-log reduction of the target organism, E. coli O157:H7. This reduction can be obtained through one step or the combination of two or more steps. / Master of Science

Numerous food-borne outbreaks of Escherichia coli O157:H7 have been linked to leafy greens in recent years. An overwhelming amount of lettuce and spinach on the market is sold in modified atmosphere packaging as ready to eat salad mixes. The objectives of this study were to determine the effects of modified atmosphere, storage temperature, and inoculum size on survival of E. coli O157:H7 on cut and whole leaf lettuce and spinach. E. coli O157:H7 H1730 was inoculated onto cut and whole leaves of leaf lettuce and spinach. Samples were held under normal atmospheric conditions or in a modified atmosphere package at either 4ºC or 10ºC to simulate display and abuse temperatures. Leaves were sampled at Days 0, 1 and every other day until visual spoilage occurred (7 days for lettuce, 9 days for spinach). E. coli O157:H7 was able to survive at 4° and 10°C regardless of atmosphere and inoculum size for 7 days on cut and whole lettuce and 9 days on cut and whole spinach. Overall, numbers of E. coli O157:H7 increased (1 log) throughout the storage period on spinach, and decreased on lettuce (1-1.5 log). Significantly higher (P < 0.05) numbers of E. coli O157:H7 were found on lettuce and spinach stored at 10ºC than when stored at 4ºC. There were no significant differences (P > 0.05) in Numbers of E. coli O157:H7 with respect to atmosphere, leaf type or inoculum size. If contamination of lettuce or spinach with E. coli O157:H7 occurred, the pathogen may survive well under typical packaging and storage conditions. / Master of Science in Life Sciences

The fate of E. coli O157:H7 and Salmonella spp. during osmotic dehydration of apples was determined at different processing temperatures, times and calcium chloride (CaCl2) concentrations. Apple slices were inoculated to achieve an 8 log CFU/ apple slice concentration of a five strain mixture of E. coli O157:H7 or Salmonella spp. and were soaked in sucrose solutions (60% w/w). In the first study, apple slices were subjected to osmotic dehydration at three different temperatures: 20°C, 45°C and 60°C. In a second study, CaCl₂ was added in the sucrose solution at concentrations of 2%, 4% and 8% to determine its efficacy as an antimicrobial agent. The storage effect of osmotic dehydrated apples on pathogen survival was also tested for seven days at 4°C. Samples were withdrawn at appropriate time intervals, diluted with 0.1% peptone water and surface plated onto recovery media. Recovery of E. coli O157:H7 was compared on Tryptic Soy Agar + 50 ppm nalidixic acid (TSAN) and MacConkey Sorbitol agar (MCS). Recovery of Salmonella was compared on TSAN and XLD agar. There was lower microbial reduction at the lower temperatures tested with approximately 1.0 and 3.0 log CFU/apple slice reduction at 20°C and 45°C, respectively. The population reduction of cells was highest at 60°C, with an approximate five log reduction for both microorganisms (P<0.001). CaCl₂ used as an additive in the osmotic solution, was associated with slightly higher reduction of both E. coli O157:H7 and Salmonella spp. Greater than a 5 log reduction was observed when the combination of CaCl₂ (8%) and 60°C processing temperature was used. During refrigerated storage E. coli O157:H7 and Salmonella decreased by approximately 4.5 log CFU/apple slice, but were still recoverable via direct plating at Day seven. The results of this study show that the survival of E. coli O157:H7 and Salmonella in osmotically dehydrated fruit is influenced by the osmotic processing method used and the level of additive (i.e., CaCl₂) utilized. Parameters associated with decreased survival of pathogens, and therefore, improve product safety, include increasing temperature and time of processing and increasing concentration of CaCl₂. However, E. coli O157:H7 and Salmonella in artificially contaminated apple slices, survived osmotic dehydration processing and subsequent storage under processing and storage parameters of this study. Therefore, processors who produce osmotically dehydrated fruit must consider the potential food safety impact of the osmotic dehydration processes they choose. / Master of Science

Escherichia coli O157:H7 is an enteric pathogen that can cause severe gastrointestinal disease, sometimes leading to hospitalization and death. These bacteria have a variety of virulence factors that can be encoded for on pathogenicity islands (PAIs). The goal of this study was to characterize specific E. coli O157:H7 PAI deletion mutants using three methods: Phentotype Microarrays (PM), growth curves and survival curves were used to elucidate possible roles for the PAIs. Results from the PM study suggest that PAIs have a role in carbon substrate utilization; i.e., four of the O-island (OI) deletion mutants (OI-87, 98, 102 and 172) and an S-Loop (SL-72) deletion mutant exhibited differences in substrate utilization (gains and losses in utilization) compared to parental O157:H7 strains EDL933 (OI) and Sakai (SL), respectively. All of the mutants with the exception of the OI-135 mutant exhibited differences in level of substrate utilization for substrates shown to have important roles in the bacterium. Cell growth results showed that three OI deletion mutants (OI-55, 87 and 102) and the SL (SL-72) mutant exhibited a difference in rate of growth compared to the parental strains. Cell viability results showed that seven of the OI deletion mutants (OI-51, 55, 98, 108, 135, 172 and 176) exhibited different rates of decline in cell number when transferred to sterile water compared to the parental strain. The results show that removal of PAIs from E. coli O157:H7 can affect carbon utilization, growth and survival demonstrating the importance of PAIs in the ecology of these bacteria. / xx, 208 leaves : ill. (some col.) ; 29 cm