Escherichia coli O157:H7 can contaminate dropped apples used for juicing via contact with manure or fecally tainted irrigation water and attach to the flesh of the apple through bruises and wounds where surface sanitizers are not effective. The goal of this project was to determine the efficacy of gamma irradiation at the maximum allowed dose of 1000 Gy to inactivate Escherichia coli O157: H7 in whole apples used for juicing. Whole apples were punctured to simulate wounds which were then inoculated with an outbreak strain of E.coli O157:H7 and subjected to gamma irradiation at doses upto 1000 Gy. The D-value of the E.coli O157:H7 strain was 334 Gy indicating that irradiation at 1000 Gy would result in a 3-log reduction of this pathogen. Contaminated apples were also stored for 3 weeks at refrigerated temperature during which time E.coli O157:H7 survived but did not grow. The inoculated apples were juiced, and the juice was stored up to 72 h. There was no change in counts of E.coli O157:H7 in the juice from the control apples, but irradiation at >600 Gy reduced counts by >3 logs, and survivors were not detected after 72 h storage. Sensory testing of juice treated at 652 Gy indicated consumers could tell the difference from control juice, due mostly to greater sweetness of the juice from irradiated apples. These results show that E.coli O157:H7 can easily survive in bruised apples and the juice made from them. Irradiation at 1000 Gy can provide significant lethality of E.coli O157:H7 in apples and juice conferring a greater level of safety without negative effects on sensory quality.

<p>The presence of human pathogens in water indicates the sanitary risk associated with different types of water utilization. This study surveyed the sources of human pathogens in urban waters. In order to evaluate the microbiological water quality of urban water, the enumeration of various indicator bacteria (total coliform, fecal coliform, E.coli and enterococci) is usually used.</p><p>The abundance of indicator bacteria in urban water indicates the level of fecal contamination and the presence of other human pathogens such as protozoan pathogens (Giardia lamblia & Cryptosporidium parvum).</p><p>Fecal pollution of urban waters can be from human and animal origin. Point sources of fecal contamination in an urbanized area are the effluents of urban wastewater treatment plants. While non-point sources are usually originated from diffuse sources such as (runoff from roads, parking lots, pets, leaks, failing septic systems and illegal sewer connections to storm drains). urban stormwater is considered as a major carrier for delivering human pathogens from diffuse sources to receiving waters. Increases in urban stormwater volumes have resulted from increasing urbanization and growth of impervious surfaces.</p><p>In order to reduce high amounts of human pathogens in urban waters, different methods are used nowadays to develop urban wastewater treatment plants technologies and urban stormwater management practices.</p>

The high incidence of pathogens is one of the main causes for impaired surface water quality designations in the United States. Pathogen presence in fresh water is monitored through the detection of indicator bacteria. Indicator bacteria concentrations, spatial and temporal variability, and microbial risks were evaluated in two rural watersheds, the Bosque and Leon Rivers, and one predominantly urban watershed, the San Jacinto River, all in Texas. Human health risk was predicted from contaminated waters as indicated by ingestion of Escherichia coli found in surface water for contact recreation scenarios. The watersheds were chosen because many segments were previously placed on the 303 (d) list (published by the TCEQ) for failing the indicator bacteria standards. Predominantly urban areas of the San Jacinto River and rural portions of the Bosque and Leon Rivers, where Concentrated Animal Feeding Operations (CAFOs) are numerous, were compared to relatively pristine rural watersheds. Spatial analysis of the watersheds with E.coli concentrations exceeding the single sample (394 MPN/100mL) and the geometric mean standards (126 MPN/100mL) indicated that land use is a significant factor influencing the incidence of bacterial concentrations. Non-agricultural rural areas of the watersheds, such as forests and rangelands, had significantly lower E.coli concentrations compared to the agricultural areas and urban land uses. Human health risk due to ingestion of E.coli as an indicator organism indicated a similar pattern to that of their concentrations in that urban and agricultural areas had a greater risk compared to the other rural areas of the watersheds. The risk estimate for urban and agricultural areas exceeded the acceptable limit of one in ten thousand (10^-4), indicating a potential for adverse health effects to humans. Temporal variability in the watersheds as a function of streamflow, rainfall, and temperature indicated a positive correlation between bacterial concentration and high streamflow, rainfall and temperature. The positive correlation for these effects was greater in the rural areas compared to urban areas, indicating the presence of multiple factors responsible for E.coli concentrations in urban areas. Thus, land use was confirmed to be a major factor contributing to the presence of indicator bacteria in surface waters.

RNA is a highly versatile molecule with functions that span from being a messenger in the transfer from DNA to protein, a catalytic molecule important for key processes in the cell to a regulator of gene expression. The post-genomic era and the use of new techniques to sequence RNAs have dramatically increased the number of regulatory RNAs during the last decade. Many of these are antisense RNAs, as for example the miRNA in eukaryotes and most sRNAs in bacteria. Antisense RNAs bind to specific targets by basepairing and thereby regulate their expression. A major step towards an understanding of the biological role of a miRNA or an sRNA is taken when one identifies which target it regulates. We have used RNA libraries to study the RNA interference pathway during development in the unicellular model organism Dictyostelium discoideum. We have also, by combining computational and experimental methods, discovered the first miRNAs in this organism and shown that they have different expression profiles during development. In parallel, we have developed a novel approach to predict targets for sRNAs in bacteria and used it to discover sRNA/target RNA interactions in the model organism Escherichia coli. We have found evidence for, and further characterized, three of these predicted sRNA/target interactions. For instance, the sRNA MicA is important for regulation of the outer membrane protein OmpA, the sRNAs OmrA and OmrB regulate the transcription factor CsgD, which is important in the sessile lifestyle of E. coli, and MicF regulates its own expression in a feed forward loop via the regulatory protein Lrp. In conclusion, we have discovered novel antisense RNAs, e.g. miRNAs in D. discoideum, developed an approach to identify targets for antisense RNAs, i.e. a target prediction program for sRNAs in bacteria, and verified and characterized some of the predicted antisense RNA interactions.

The presence of human pathogens in water indicates the sanitary risk associated with different types of water utilization. This study surveyed the sources of human pathogens in urban waters. In order to evaluate the microbiological water quality of urban water, the enumeration of various indicator bacteria (total coliform, fecal coliform, E.coli and enterococci) is usually used. The abundance of indicator bacteria in urban water indicates the level of fecal contamination and the presence of other human pathogens such as protozoan pathogens (Giardia lamblia & Cryptosporidium parvum). Fecal pollution of urban waters can be from human and animal origin. Point sources of fecal contamination in an urbanized area are the effluents of urban wastewater treatment plants. While non-point sources are usually originated from diffuse sources such as (runoff from roads, parking lots, pets, leaks, failing septic systems and illegal sewer connections to storm drains). urban stormwater is considered as a major carrier for delivering human pathogens from diffuse sources to receiving waters. Increases in urban stormwater volumes have resulted from increasing urbanization and growth of impervious surfaces. In order to reduce high amounts of human pathogens in urban waters, different methods are used nowadays to develop urban wastewater treatment plants technologies and urban stormwater management practices.

A survey was developed and sent out to each sector of the beef industry (slaughter, non-intact processing and grinding) by using the FSIS Meat, Poultry and Egg Product Inspection Directory. Survey questions were specific to processes and interventions being applied, and the use and familiarity with Industry Best Practices documents for beef processing. Returned completed surveys. A total of 469 beef processing operations responded and of survey respondents, 119 establishments were called and asked additional questions. Critical Control Points (CCPs) and testing for E. coli O157:H7 were common discussion point during phone calls. Plant visits were made to confirm the answers that were provided in the written survey. Plants that further processed beef were found to need to reassess their HACCP plan based on their response to the question, "Is E. coli O157:H7 a reasonably likely to occur food safety hazard?" E. coli O157:H7 is considered an adulterant in the products that they produced if they answered yes to this question. Based on survey responses, slaughter establishments were using available technologies to reduce or eliminate possible microbiological contamination. Further process operations, especially those plants that produced intact steaks and roasts, marinated/enhanced steaks and roasts, and plants that produced needle/blade tenderized steaks and roasts, used documentation such as supplier purchasing specifications instead of using processes to control, reduce, or eliminated microbiological food safety hazards. Industry Best Practices were being utilized most frequently by slaughter and ground beef operations. Plants that further process beef still need to implement the use of the Industry Best Practices specific to them. Plants used testing for E. coli O157:H7 throughout the beef industry regardless of plant size or type.

Increase in consumption of fresh cut produce over the past decade has resulted in a rise in incidents of food borne outbreaks due to pathogens. Conventional techniques of sanitizing washes may not be effective since the organic matter released from the fresh produce use up the free chlorine thus reducing the sanitizing potential of wash water just when it is needed most and a heat treatment step to kill pathogens cannot be applied if the purpose is to consume fresh produce. Electron beam (e-beam) irradiation was used to treat cut cantaloupe, cut roma tomatoes, baby spinach, romaine lettuce which were surface inoculated with a cocktail of Salmonella and E. coli O157:H7. Results showed that irradiation reduced Salmonella and E. coli O157:H7 significantly with increasing doses at 0.2, 0.4, 0.6, 0.8, and 1.0 kGy. The D10-value for Salmonella on irradiated cut cantaloupe, cut roma tomatoes, baby spinach, and romaine lettuce was found to be 0.71 kGy, 0.64 kGy, 0.19 kGy, and 0.23 kGy respectively. The D10-value for E. coli O157:H7 on the produce listed above was found to be 0.73 kGy, 0.54 kGy, 0.18 kGy, and 0.20 kGy respectively. Low dose e-beam irradiation was found to be an excellent tool for ensuring the reduction of spoilage organisms and extending shelf life in cut cantaloupe, cut roma tomatoes, baby spinach, romaine lettuce, strawberries, and green onion. The produce were tested for 12 days of storage for aerobic plate count, yeast and mold, lactic bacteria, color, texture, and respiration rate as a function of irradiation doses 0, 1, 3, and 5 kGy. Aerobic plate counts, yeast counts, and lactic acid bacteria were reduced appreciably at all doses tested on all commodities. Molds did not grow on any samples including control for cut cantaloupe, cut tomatoes, and green onion but for the other commodities, mold was reduced at the same rate as yeasts and vegetative bacteria. Lactic acid bacteria were reduced at all doses while the reduction was highest with 5 kGy in all commodities. When irradiated with 5 kGy, during storage, strawberries, spinach, and green onion displayed wet, soggy and mushy appearance, romaine lettuce leaves were wilted, had a translucent midrib and brown pigmentation. E-beam irradiation increased respiration rate for all samples on day 0 compared to non-irradiated control irrespective of the commodity type and the effect was dose dependent. Firmness reduced appreciably for cut roma tomatoes, baby spinach, strawberries, romaine lettuce, and green onion with increasing doses. Cut cantaloupe was low in firmness but the effect was not dose dependent. Irradiation at low doses is a promising tool to reduce pathogens and enhance keeping quality of cut cantaloupe, cut tomatoes, baby spinach, romaine lettuce, strawberries, and green onion. Irradiation is to be implemented as part of an overall HACCP plan and is not meant to replace existing control measures.

科学研究費補助金 研究種目:一般研究(C) 課題番号:61560140 研究代表者:大澤 俊彦 研究期間:1986-1987年度

科学研究費補助金 研究種目:一般研究(C) 課題番号:05660116 研究代表者:大澤 俊彦 研究期間:1993-1994年度

Bacterial cells reproduce by doubling in size and dividing. The molecular control systems which regulate the cell cycle must do so in a manner which maintains a similar cell size over many generations. A cell can under conditions of fast growth conclude cell cycles in shorter time than the time required to replicate its chromosome. Under such conditions several rounds of replication are maintained in parallel and a cell will inherit replication processes which were initiated by an ancestor. To accomplish this the cell has to initiate and terminate one round of replication during each cell cycle. To investigate the effects of the cell cycle on gene-regulation in the gut bacterium Escherichia coli, an experimental method combining microfluidics, single molecule fluorescence microscopy and automated analysis capable of acquiring an arbitrary number of complete cell cycles per experiment was developed. The method allowed for the rapid exchange of the chemical environment surrounding the cells. Using this method it was possible to measure the dissociation time of the transcription factor molecule, LacI-Venus, from the native lactose operator sequence, lacO1, and an artificially strong operator, lacOsym, in vivo. The results indicated that regulation of gene-expression from the lactose operon does not occur at equilibrium in living cells. Furthermore, by studying the intracellular location of non-specifically interacting transcription factor molecules it was possible to determine that these do not form long-lived gradients inside the cell as was previously proposed. By studying the replication machinery and the origin of replication it was found that replication is initiated according to a cell volume per origin which did not vary over different growth conditions. Further, division timing was found to be determined by the initiation event to occur after a fixed time-delay. A consequence of this mode of regulation is an uncertainty relation between the size at birth and the cell cycle time, in which cells will vary more in in the cycle time during conditions of slow growth as compared to fast growth and vary more in birth length during conditions of fast growth as compared to slow growth.