Progress 08/15/12 to 08/14/15
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has provided PD Sanglay valuable experience with project management, experimental design, and data analysis. These skills will aid PD Sanglay in his position at the Nestlé Quality Assurance Center (NQAC) in Dublin, Ohio. PD Sanglay is a microbiologist for the Technical Services group, which provides technical expertise to the laboratory, conducts specialized/non-routine analyses, performs matrix and method validation/verification, evaluates new/rapid pathogen detection methodologies for potential incorporation into lab operations, assisting Nestlé factories and operations with method training and technical issues, and ensuring that products meet Nestlé's high standards for quality and safety. How have the results been disseminated to communities of interest?PD Sanglay presented a research poster during the International Association for Food Protection (IAFP) annual meeting in Indianapolis, IN (August 3-6, 2014). Results from this project were shared with fellow food safety researchers as well as members of Columbus Public Health and Ohio Association for Food Protection. The members from Columbus Public Health and OAFP were interested in the research, and recommended publication for IAFP's Food Protection Trends, as information gained from this study would be of great value to the food industry and for the Conference for Food Protection. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We evaluated manual and mechanical warewashing procedures on ceramic plates inoculated with contaminated cream cheese (Salmonella Typhimurium, Tulane virus [TV], Listeria innocua, and murine norovirus [MNV-1]). Manual warewashing was performed in a 3-compartment sink, with each compartment designated for washing, rinsing, and sanitizing. Each compartment held 28 L of 100 ppm Monsoon detergent at 44 + 0.5°C (wash), 23 + 1°C tap water (rinsing), and sanitizing solution at 22 + 2°C. Sanitizers tested included tap water, 200 ppm chlorine, acidic electrolyzed water (AEW;pH 3.30 + 0.26, 248 + 12 ppm chlorine, and an oxidation reduction potential ranging from +1120.1 to +1173.2 mV ), neutral electrolyzed water (NEW; pH 7.06 + 0.06, 199 + 1 ppm chlorine, and an ORP ranging from +873.2 to +882.3 mV), basic electrolyzed water (BEW; pH 11.75 + 0.03, ORP -841.7 to -884.7 mV), and ozonated water (approximately 7 ppm). 3 g of inoculated cream cheese were pasted on the surface of sterile ceramic plates and allowed to air-dry for 1 hr. After drying, plates were washed in the washing compartment with a sponge attached to a spring-loaded device to deliver an average force of 0.8 + 0.22 kg. Plates were washed for 30 sec, using 15 clockwise and 15 counterclockwise strokes. After washing, plates were immersed in the rinsing compartment for 10 sec. After rinsing, plates were immersed in the sanitizing compartment for 30 sec. Plates were air-dried for 1 hr. Mechanical warewashing was performed in a Hobart LxiC Dishwasher. The dishwasher tank held approximately 11.4 L of water. 3,000 ppm of Ecotemp Ultra Klene detergent was used to wash the plates. During the wash cycle, water (49°C) and detergent were sprayed onto the plates for 76.5 seconds at 138 kPa. After wash, the rinse/sanitize cycle sprayed sanitizing solution for 10 seconds. Sanitizers tested included tap water (control), 208 + 8 ppm of chlorine, and 7 ppm ozonated water. Due to time constraints, we were unable to successfully incorporate the electrolyzed waters into the mechanical warewashing procedures. Microbial enumeration was performed by swabbing the surface of the plates with 2 sterile swabs moistened with phosphate buffered saline (PBS). Swabs were vortexed in 2 ml of PBS, serially diluted, and applied to tryptic soy agar (TSA, nonselective medium) and selective agars (XLD, a selective medium for Salmonella; MOX, a selective medium for Listeria) pour plates, or to viral plaque assays (6-well plates seeded with LLC-MK2 cells for TV; RAW 264.7 cells for MNV). Bacterial pour plates were incubated at 37°C for 48 hours, while viral plaque assays were incubated at 37°C + 5% CO2 for 72 hours. Experiments were replicated three times. For manual warewashing, the effect of sanitizers used were significant (P < 0.05). Salmonella appeared to be the most susceptible to washing and sanitization out of all the test organisms. Using tap water, 200 ppm of chlorine, or AEW provided a < 5 log reduction of Salmonella populations on the contaminated plates. NEW, BEW, and ozonated water were the most effective, providing a > 5 log reduction of the bacteria on both TSA and XLD. Listeria was more resistant to the manual warewashing procedures than Salmonella (3.4 to 4.7 log reduction for all sanitizer types). All 6 of the sanitizers failed to provide a 5 log reduction of the bacteria. Both foodborne virus surrogates were also resistant to washing and sanitization. All 6 sanitizers only provided a < 5 log reduction of TV. For MNV, only BEW and ozonated water produced a > 5 log reduction of the virus, however virus was still present on the plates. For mechanical washing, Salmonella was again the most susceptible organism. Washing and sanitizing with tap water provided a 6.7 log reduction of the bacteria, while 200 ppm chlorine produced up to a 7 log reduction. Ozonated water produced a > 6 log reduction of Salmonella. Significant differences were observed between sanitizer types (P < 0.05). Mechanical warewashing and all three sanitizer types only produced a < 5 log reduction of Listeria. For the viruses, TV was more resistant to washing and sanitization than MNV, producing a < 5 log reduction. MNV experienced a > 5 log reduction using tap water or chlorine, but only a < 5 log reduction using ozonated water. For mechanical warewashing of viruses, no significant differences were observed between the different sanitizers (P > 0.05). When comparing bacterial populations recovered from warewashing/sanitization plated on nonselective and selective agars, there was a difference of 0.5 to 1 log reduction of Salmonella and a < 0.5 log reduction of Listeria on the plates. This indicates that warewashing and sanitization may cause sublethal injury to the bacteria, with injured populations being unable to recover on XLD or MOX agars. In general, the results generated from this study indicated that warewashing and sanitization procedures were effective against Salmonella, but were ineffective against Listeria, TV,and MNV. The use of BEW (which was one of the more effective sanitizers) may not be sufficient for use as an alternative sanitizer because it acts as more of a surfactant (rather than microbicidal) and left a residue on the plates after drying. Ozonated water was also effective against certain microorganisms, but also may not be suitable for use due to the high cost of ozone-generating equipment, the water must be generated on-site, ozone degrades rapidly especially with higher temperatures, and off-gassing of ozone from water may pose a health hazard air the concentrations are > 0.2 ppm. Mechanical warewashing and sanitization procedures produced higher log reductions of the organisms, but again, Listeria, TV, and MNV proved to be resistant to the process. The higher log reductions observed with mechanical warewashing may be due to the use of higher water temperature (43°C for manual vs. 49°C for mechanical) as well as the application of a pressurized water spray. Even though washing and sanitization produced a > 5 log reduction of MNV (using tap water or chlorine), the presence of both MNV and TV on plates after washing is still a safety concern. The human norovirus infectious dose is as low as 10 virus particles required to cause illness. Although the human norovirus is currently noncultivable in the laboratory, the use of surrogates such as MNV or TV may give insight to how the human strain would survive on contaminated tableware after washing or sanitization. Based on the results of this study, changes to the Food Code may be required in order to enhance proper cleaning and sanitization of tableware in food establishments, as well as address the issue of foodborne viruses.
Publications
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Progress 08/15/13 to 08/14/14
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Although not previously specified, PD Sanglay mentored a Metro High School student (Elisabeth Spector) with their Capstone project. PD Sanglay trained Ms. Spector in viral cultivation and enumeration techniques so that she would be able to complete her project, which examined the effect of chlorine concentrations (0 to 1000 ppm) on inactivation of human norovirus surrogates. Ms. Spector also assisted PD Sanglay with data collection for his warewashing studies, learning microbiological techniques (bacterial cultivation, serial dilutions, microbial sampling), in addition to the viral techniques she learned for her project. Through her experiences, Ms. Spector has gained an appreciation for the scientific method and gained valuable laboratory experiences that will help prepare her for a future career in a scientific discipline. Although she is undecided on a major, she recently started as an undergraduate at the University of Dayton in their science discovery program. How have the results been disseminated to communities of interest? PD Sanglay presented a research poster during the International Association for Food Protection (IAFP) annual meeting in Indianapolis, IN (August 3-6, 2014). Results from this project were shared with fellow food safety researchers as well as members of Columbus Public Health and Ohio Association for Food Protection.The members from Columbus Public Health and OAFP were interested in the research, and recommended publication for IAFP's Food Protection Trends, as information gained from this study would be of great value to the food industry and for the Conference for Food Protection. What do you plan to do during the next reporting period to accomplish the goals? A publication is currently a work-in-progress, and will be submitted to either Journal of Food Protection or Food Protection Trends. Final statistical analyses will be performed by end of November/early December 2014. The final report will be submitted to NIFA no later than December 31, 2014.
Impacts
What was accomplished under these goals?
Data collection for objectives 1, 2, and 3 are complete. We evaluated manual and mechanical warewashing procedures on ceramic plates inoculated with contaminated cream cheese (Salmonella Typhimurium, Tulane virus [TV], Listeria innocua, and murine norovirus [MNV-1]). Manual warewashing was performed in a 3-compartment sink, with each compartment designated for washing, rinsing, and sanitizing. Each compartment held 28 L of 100 ppm Monsoon detergent at 44 + 0.5°C (wash), 23 + 1°C tap water (rinsing), and sanitizing solution at 22 + 2°C. Sanitizers tested included tap water, 200 ppm chlorine, acidic electrolyzed water (AEW; pH 3.30 + 0.26,248 + 12 ppm chlorine, and an oxidation reduction potential ranging from +1120.1 to +1173.2 mV ), neutral electrolyzed water (NEW; pH 7.06 + 0.06, 199 + 1 ppm chlorine, and an ORP ranging from +873.2 to +882.3 mV), and basic electrolyzed water (BEW; pH 11.75 + 0.03, ORP -841.7 to -884.7 mV). 3 g of inoculated cream cheese were pasted on the surface of sterile ceramic plates and allowed to air-dry for 1 hr. After drying, plates were washed in the washing compartment with a sponge attached to a spring-loaded device to deliver an average force of 0.8 + 0.22 kg. Plates were washed for 30 sec, using 15 clockwise and 15 counterclockwise strokes. After washing, plates were immersed in the rinsing compartment for 10 sec. After rinsing, plates were immersed in the sanitizing compartment for 30 sec. Plates were air-dried for 1 hr. Mechanical warewashing was performed in a Hobart LxiC Dishwasher. The dishwasher tank held approximately 11.4 L of water. 3,000 ppm of Ecotemp Ultra Klene detergent was used to wash the plates. During the wash cycle, water (49°C) and detergent were sprayed onto the plates for 76.5 seconds at 138 kPa. After wash, the rinse/sanitize cycle sprayed sanitizing solution for 10 seconds. Sanitizers tested included tap water (control) and 208 + 8 ppm of chlorine. Unfortunately, due to the design of the dishwasher, we were unable to successfully incorporate the electrolyzed waters into the mechanical warewashing procedures. Microbial enumeration was performed by swabbing the surface of the plates with2 sterile swabs moistened with phosphate buffered saline (PBS). Swabs were vortexed in 2 ml of PBS, serially diluted, and applied to tryptic soy agar (TSA, non-selective medium) andselective agars(XLD, a selective medium for Salmonella; MOX, a selective medium for Listeria) pour plates, or to viral plaque assays (6-well plates seeded with LLC-MK2 cells for TV; RAW 264.7 cells for MNV). Bacterial pour plates were incubated at 37°C for 48 hours, while viral plaque assays were incubated at 37°C + 5% CO2for 72 hours. Experiments were replicated three times. For manual warewashing, Salmonella was appeared to be the most susceptible to washing and sanitization out of all the test organisms. Using tap water, 200 ppm of chlorine, or AEW provided a < 5 log reduction of Salmonella populations on the contaminated plates. The NEW and BEW sanitizers were the most effective, providing a 5.7 to 6.9 log reduction of the bacteria on both TSA and XLD. Listeria was more resistant to the manual warewashing procedures than Salmonella (3.4 to 4.7 log reduction for all sanitizer types). All 5 of the sanitizers failed to provide a 5 log reduction of the bacteria. Both foodborne virus surrogates were also resistant to washing and sanitization. All 5 sanitizers only provided a 3.3 to 4.2 log reduction of TV. For MNV, only BEW produced a 5.1 log reduction of the virus, however virus was still present on the plates. Tap water, 200 ppm chlorine, AEW, and NEW only produced a 4.1 to 4.3 log reduction of MNV. For mechanical washing, Salmonella was again the most susceptible organism. Washing and sanitizing with tap water provided a 6.7 log reduction of the bacteria, while 200 ppm chlorine produced a > 7 log reduction. However, the two sanitizers tested do not appear to be significantly different from each other. Tap water and chlorine sanitization only provided a 4.5 to 4.9 log reduction of Listeria. For the viruses, TV was resistant to washing and sanitization, producing only a 4.1 to 4.2 log reduction when using tap water or 200 ppm chlorine, respectively. MNV experienced a 5.1 log reduction in titer after washing and sanitization with tap water and 200 ppm chlorine. When comparing bacterial populations recovered from warewashing/sanitization plated on nonselective and selective agars, there was a difference of 0.5 to 1 log reduction of Salmonella and a < 0.5 log reduction of Listeria on the plates. This indicates that warewashing and sanitization causes sublethal injury to the bacteria, with injured populations being unable to recover on XLD or MOX agars. In general, the results generated from this study indicated that manual washing and sanitization procedures were effective against Salmonella, but were ineffective against Listeria, TV,and MNV. The use of tap water, chlorine, AEW, and NEW as sanitizers did not produce a > 5 log reduction of the resistant microorganisms. BEW, which provided the highest log reduction for all organisms, may not be sufficient for use as an alternative sanitizer because it acts as more of a surfactant (rather than microbicidal) and left a residue on the plates after drying. Mechanical warewashing and sanitization procedures produced higher log reductions of the organisms, but again, Listeria, TV, and MNV proved to be resistant to the process. The higher log reductions observed with mechanical warewashing may be due to the use of higher water temperature (43°C for manual vs. 49°C for mechanical) as well as the application of a pressurized water spray. Even though washing and sanitization (tap water and 200 ppm chlorine) produced a 5.1 log reduction of MNV, the presence of both MNV and TV on plates after washing is still a safety concern. The human norovirus infectious dose is as low as 10 virus particles required to cause illness. Although the human norovirus is currently noncultivable in the laboratory, the use of surrogates such as MNV or TV may give insight to how the human strain would survive on contaminated tableware after washing or sanitization. Based on the results of this study, changes to the Food Code may be required in order to enhance proper cleaning and sanitization of tableware in food establishments, as well as address the issue of foodborne viruses.
Publications
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Progress 08/15/12 to 08/14/13
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals? Objective 1: Continue manual warewashing studies with Listeria innocua, Escherichia coli K12, and murine norovirus 1 (MNV-1) Manual warewashing studies will also include stainless steel forks (contaminated with 0.5 g cream cheese) and drinking glasses (contaminated with 0.5 ml of contaminated milk) Mechanical dishwashing and sanitization studies (with plates, forks, and drinking glasses using the above food soils and organisms) will also be conducted to compare how well the two washing procedures will remove microorganisms from food utensils Objective 3: Continue evaluating the use of AEW and NEW as an alternative sanitizer against bacteria and viruses Attempt to use existing ozone-generators at the OSU Food Science Department to generate ozonated water for use as another alternative sanitizer Objective 4: One to two manuscripts will be prepared for submission to journals such as Applied and Environmental Microbiology, Journal of Food Protection, Food Control, International Journal of Food Microbiology, or Journal of Food Science The project director will also plan to attend conferences to disseminate research findings to peers (academics, industry professionals) via poster or oral presentation at the International Association for Food Protection Annual Meeting (August 3-6, Indianapolis, IN) and/or the Institute of Food Technologists Annual Meeting and Food Expo (June 21-24, New Orleans, LA). Abstracts for these conferences will be submitted in December 2013 or January 2014. The project director will also share their research findings at the Conference for Food Protection Biennial Meeting (May 1-7, Orlando, FL). Objective 5: The project director will provide lectures about the Food Code and their research findings to the Food Regulations class, taught at OSU. Unfortunately, the Food Safety and Public Health and Advanced Food Microbiology 2 courses are not being offered for the upcoming semester. The project director will work with his mentors and department faculty to find alternate courses in which to present these lectures.
Impacts
What was accomplished under these goals?
Progress has been made under objectives 1, 2 and 3. We evaluated manual warewashing procedures on ceramic plates inoculated with contaminated cream cheese (Salmonella Typhimurium and Tulane virus [TV]). Manual warewashing was performed in a 3-compartment sink, with each compartment designated for washing, rinsing, and sanitizing. Each compartment held 28 L of 100 ppm Monsoon detergent at 44 + 0.5°C (wash), 23 + 1°C tap water (rinsing), and sanitizing solution at 22 + 2°C. Sanitizers tested included tap water, 200 ppm chlorine, acidic electrolyzed water (AEW; pH 3.30 + 0.26, 248 + 12 ppm chlorine, and an oxidation reduction potential ranging from +1120.1 to +1173.2 mV ), and neutral electrolyzed water (NEW; 7.06 + 0.06, 199 + 1 ppm chlorine, and an ORP ranging from +873.2 to +882.3 mV). 3 g of inoculated cream cheese were pasted on the surface of sterile ceramic plates and allowed to air-dry for 1 hr. After drying, plates were washed in the washing compartment with a sponge attached to a spring-loaded device to deliver an average force of 0.8 + 0.22 kg. Plates were washed for 30 sec, using 15 clockwise and 15 counterclockwise strokes. After washing, plates were immersed in the rinsing compartment for 10 sec. After rinsing, plates were immersed in the sanitizing compartment for 30 sec. Plates were air-dried for 1 hr. Microbial enumeration was performed by swabbing the surface of the plates with a sterile swab moistened with phosphate buffered saline (PBS). Swabs were vortexed in 2 ml of PBS, serially diluted, and applied to either tryptic soy agar (TSA, non-selective medium) and xylose lysine deoxycholate agar (XLD, a selective medium) pour plates for Salmonella, or to viral plaque assays (6-well plates seeded with LLC-MK2 cells) for Tulane virus. Bacterial pour plates were incubated at 37°C for 48 hours, while viral plaque assays were incubated at 37°C + 5% CO2 for 72 hours. Experiments were replicated three times. For Salmonella, the mean reduction in bacterial concentration on plates before and after the 1 hr drying period ranged from 0.3 to 0.5 log CFU/ceramic plate. Salmonella populations on plates after drying were 8.93 + 0.23 (TSA) and 8.63 + 0.21 (XLD) log CFU/ceramic plate. Washing, rinsing, and sanitizing with tap water of contaminated plates provided a 4.6 and 5.2 log reduction in Salmonella populations pour plated to TSA and XLD, respectively. The 200 ppm sanitizing treatment was not more effective than tap water, resulting in a 4.5 and 4.9 log reduction in Salmonella on TSA and XLD, respectively. Using AEW as an alternative sanitizer, Salmonella reductions were 4.8 and 5.2 log on TSA and XLD, respectively. NEW appeared to provide the greatest log reductions in Salmonella populations (5.6 log reduction when plated on TSA, 6.2 log reduction on XLD) of all the sanitizers tested thus far. Statistical analyses will be conducted in the future to determine if these differences are significant. These results indicate that warewashing techniques provide a 4.5 or greater log reduction in Salmonella populations on cream cheese contaminated ceramic plates. Of the four sanitizers tested, NEW provided the greatest reduction (5.6 to 6.2 log reduction) in Salmonella populations, while tap water, 200 ppm chlorine, and AEW were less effective. When comparing recovered Salmonella populations on nonselective and selective media, the results suggest that warewashing and sanitizing procedures may also cause injury or damage to the bacterial cells. TV, a human norovirus surrogate, proved to be more resistant to warewashing and sanitization than Salmonella. The mean reduction of viral titer on plates before and after the 1 hr drying period was 0.5 log PFU/ceramic plate. TV populations on plates after drying were 6.59 + 0.27 log PFU/ceramic plate. Washing, rinsing, and sanitizing with tap water provided only a 3.3 log reduction in viral titer. 200 ppm chlorine, AEW, and NEW provided a slightly higher reduction of viral titer (4.0, 3.8, and 3.8 log reductions, respectively) than tap water. The results provided by chlorine, AEW, and NEW do not appear to be significantly different from each other, however this will be determined with future statistical testing. Based on the data obtained, chlorine, AEW, and NEW were better sanitizers than tap water but no one sanitizer stood out amongst the others. Warewashing and sanitization produced a much lower log reduction for TV than for Salmonella, suggesting that viruses are more difficult to remove from contaminated ceramic plates.
Publications
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