Progress 08/15/06 to 08/14/09
Outputs
OUTPUTS: Viable cells (10^0 to 10^5 cfu/ml) and dead cells (10^6 to 10^8 cfu/ml) of Salmonella Typhimurium were treated with ethidium monoazide (EMA) and propidium monoazide (PMA). Bacterial DNA was extracted from the treated cells using PrepMan Ultra Sample Preparation reagent and subjected to PCR targeting the invasion protein A gene (invA). In addition, the ability of acid and cold stressed Salmonella Typhimurium to regain culturability under reduced oxidative tension was evaluated. Overnight culture of this pathogen was inoculated at 10^7 cfu/ml into tryptic soy broth adjusted to pH 3.5 with citric, malic, acetic, lactic, or hydrochloric acids. After inoculation, all samples were held statically at 4 degree Celsius. At designated intervals, 1 ml of each sample was diluted in phosphate-buffered saline and enumerated on tryptic soy agar (TSA) and TSA containing catalase (TSA-CL, 2,000 U/plate). PARTICIPANTS: Jin Dong, Junior Researcher, Department of Human Nutrition, Food and Animal Science, University of Hawaii at Manoa; Hongfei He, Graduate Research Assistant, Department of Human Nutrition, Food and Animal Sciences, University of Hawaii at Manoa TARGET AUDIENCES: Farmers, juice processors, and consumers PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
A 288-bp amplicon was amplified from Salmonella Typhimurium DNA. Both EMA and PMA treatments could effectively exclude DNA from the dead cells up to 10^8 cfu/ml. In contrast, PMA performed better than EMA in treating viable cells of Salmonella Typhimurium. It did not affect the detection limit of PCR on the living cells (ca. 10^2 to 10^3 cfu/ml). Quantitative differentiation between viable and dead cells of E. coli O157:H7, Salmonella, and L. monocytogenes in fruit juice by PMA-real time PCR is currently in progress. Moreover, the TSA plate counts of acetic and lactic acids treated cells declined rapidly to undetectable level within 3 and 9 days, respectively. Malic, citric, and hydrochloric acids treated cells maintained their culturability on TSA up to 5 weeks. The addition of catalase resulted in an acidulant-dependent recovery of the stressed cells on TSA. During the cold storage periods, this method was found to promote the TSA plate counts of Salmonella Typhimurium cells treated by acetic and lactic acids, but not by malic, citric, or hydrochloric acids. For instance, TSA-CL increased recovery of lactic acid-treated cells over 1,000 fold after 7 days of storage. These experiments lay the groundwork for further study of recovering stressed cells of the pathogenic bacteria in tropical fruit juices. This grant has been renewed (HAW00207-08G).
Publications
- No publications reported this period
|
Progress 08/15/07 to 08/14/08
Outputs
OUTPUTS: Survival of Escherichia coli O157:H7, Salmonella Typhimurium and Listeria monocytogenes (ca. 10^7 CFU ml-1) in orange juice (pH 3.9) and guava juice (pH 3.2) during refrigerated storage was studied for up to 3 weeks. Culturability of bacterial cells was determined by surface plating on tryptic soy agar every week. During 3 weeks, E. coli O157:H7 populations decreased by 3.2 log10 CFU ml-1 in orange juice and by 4.9 log10 CFU ml-1 in guava juice. The reduction in the count of S. Typhimurium was similar to that of E. coli O157:H7 in orange juice during the storage. However, after 2 weeks, the S. Typhimurium count had decreased by 4.3 log10 CFU ml-1 in guava juice, and was below the plating detection limit (1 CFU ml-1) after 3 weeks. Reverse transcription-polymerase chain reaction (RT-PCR) was employed to monitor the viability of S. Typhimurium in guava juice after 3 weeks. Two sets of primers were used to characterize gene expression in the cells. Expression of the invA gene (coding for invasion protein A) ceased when S. Typhimurium became nonculturable. However, the rpoS gene, encoding the stress sigma factor Rpos, remained expressed up to 3 weeks. Moreover, we exploited the use of ethidium monoazide (EMA) combined with real-time PCR to detect viable cells of pathogenic bacteria. EMA can selectively penetrate dead cells due to their compromised cell wall or membrane and bind to intracellular DNA. This staining step can selectively remove DNA from dead cells without influencing the amplification of DNA from live cells by PCR. In this study, EMA concentration, photo exposure time and other parameters were optimized for DNA binding and simultaneous inactivation of free unbound EMA. DNA was then extracted and amplified by TaqMan real-time PCR. The primers and probe used in this system were designed based on the uidA gene encoding glucuronidase. Results showed a reproducible application of this technique to detect viable cells of E. coli O157:H7. EMA, at a final concentration of 10 ug/ml, could effectively bind the DNA from all dead cells. A 1-min ice treatment resulted in a significant improvement in PCR amplification efficiency. PARTICIPANTS: Dr. Jin Dong, Junior Researcher, Department of Human Nutrition, Food and Animal Sciences, University of Hawaii at Manoa; Hongfei He, Graduate Research Assistant, Department of Human Nutrition, Food and Animal Sciences, University of Hawaii at Manoa TARGET AUDIENCES: Farmers, juice processors, and consumers PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
These results highlight the presence of pathogenic bacterial cells in the VBNC state induced in acidic foods. Since VBNC cells can escape conventional culture-based detection, they may pose serious threats to public health.
Publications
- No publications reported this period
|
Progress 08/15/06 to 08/14/07
Outputs
A two-color fluorescence assay based on integrity of the cell membrane has been established to determine the viability of bacterial cells. The assay involves two nucleic acid dyes, Syto 9 and propidium iodide, which can permeate viable and dead cells in different manners. The dye mixture stains viable cells fluorescent green and dead cells fluorescent red. This assay has been successfully applied to both gram-positive and gram-negative bacteria isolated from juice samples. The method is critical for investigating the survivability of Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes in pineapple, guava, and orange juices. Moreover, we have established two polymerase chain reaction (PCR) assays for rapid detection of E. coli O157:H7 and Salmonella. One PCR assay targets a 252-bp sequence in the uidA gene of E. coli O157:H7. It is highly specific for E. coli O157:H7 and its virulent variant, E. coli O157:NM. The other PCR assay amplifies a 429-bp
genus-specific sequence from Salmonella. It can identify most Salmonella serotypes commonly found in food. The culture-independent assays can be easily completed within 4 h. The detection limits are approximately 103 CFU/ml. The target is to develop corresponding real-time PCR systems. Then ethidium monoazide can be incorporated for direct quantification of viable cells of target pathogenic bacteria in the acidic fruit juices. A graduate research assistant has worked on the project since August 2006. The student holds a B.S. degree in Food Science and Technology and has research experience in postharvest physiology of fruits. He has focused on the evaluation of the growth potential of pathogenic bacteria in the fruit juices. Additionally, a junior researcher was hired for the project. She holds a Ph.D. degree in Agriculture and has solid background in molecular biology and food safety microbiology. The junior researcher is responsible for enhancing recovery and detection of the
acid-stressed cells, and exploiting EMA-real time PCR for quantification of E. coli O157:H7, Salmonella, and L. monocytogenes in pineapple, guava, and orange juices.
Impacts
Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes accounted for 313 cases of food-borne illnesses in Hawaii in 2002. Growth potential and survivability of these pathogenic bacteria in pineapple, guava, and orange juices are critical issues in order to address and manage the related microbial safety risks. As the largest agricultural commodity in Hawaii, pineapple was planted on 13,000 acres in the state in 2004. The total production was approximately 215,000 tons, estimated at $79.9 million in revenue. According to Hawaii Agricultural Statistics, guava output totaled 8.1 million pounds in 2004 and rebounded substantially after three years of continuous decline. The statewide farm value reached $1.2 million, up 26 percent from 2003. Meanwhile, orange is the most consumed fruit in America. The U.S. orange production for the 2004-2005 season is estimated at 9.98 million tons. In addition to being freshly consumed, most of the tropical and subtropical fruits
have been processed into juices. The information obtained from this project would provide a better understanding of the survival of E. coli O157:H7, Salmonella, and L. monocytogenes in the acidic fruit juices. It could assist the fruit juice industry in ensuring the microbial safety of final products and avoiding economic losses due to product recalls and human illnesses. The results will be valuable in quantitative risk assessment and HACCP programs to identify potential sources of contamination and to verify critical limits.
Publications
- No publications reported this period
|
|