Progress 09/01/08 to 08/31/12
Outputs
OUTPUTS: 1. Internalization and interaction of hepatitis A virus (HAV) with plants. HAV was inoculated into soil matrices or into two hydroponic systems, floating and nutrient film technique (NFT) systems. HAV was not detected in spinach and green onion plants grown in contaminated soil systems, with the exception of one spinach plant positive for HAV. In hydroponic systems, HAV was internalized to higher titers and frequencies compared to other viruses. HAV was internalized in all tissues of the spinach and green onions grown by NFT, including edible tissues. In the NFT, oasis cubes holding the plants served as a reservoir for HAV. 2. Plant defense response to E. coli O157:H7. The population of wild-type E. coli O157:H7 strain was lower on wild-type Arabidopsis plants compared with that of the flagella-deficient mutant. A curli-deficient E. coli O157:H7 strain survived better on wild-type Arabidopsis plants than the curli-producing wild-type strain. 3. Persistence of Salmonella and E. coli O157:H7 on plants. Lettuce, spinach, green onions, and scallions were grown to maturity and then spray or surface irrigated once with water containing E. coli O157:H7 or Salmonella Stanley. Surface irrigated crops had lower counts of both pathogens than the spray irrigated ones. 4. Effect of water, soil, or manure on survival/colonization of E. coli O157:H7 on plants. E. coli O157:H7 exposed to water, soil, and manure showed higher colonization on the wild-type plant compared with non-exposed E. coli O157:H7. E. coli O157:H7 exposed in water and manure showed 3-fold less GUS activity compared with non-exposed cells. 5. Pressure inactivation of E. coli O157:H7 and Salmonella internalized within green onions. Green onions were grown hydroponically in water containing either of E. coli O157:H7 or Salmonella. After 15 days, the plants were harvested and pressure treated. Treatment at 450 MPa resulted in the complete decontamination of green onions from both pathogens. 6. Use of aerosolized antimicrobials to inactivate E. coli O157:H7 on baby spinach. Baby spinach inoculated with E. coli O157:H7 were washed with 3% H2O2 and then treated with aerosolized allyl isothiocyanate (2%). During the subsequent 10 days storage at 4C, a 3.1 log reduction of E. coli O157:H7 was achieved. 7. UV inactivation of E. coli O157:H7 on green onions and baby spinach. Combined treatments of UV and acidified sodium hypochlorite (ASC) at 50C reduced E. coli O157:H7 populations by > 5 log and 2.2 log, respectively on spot and dip-inoculated green onions. On spot and dip-inoculated baby spinach, the combined treatment of UV and ASC at 20C reduced E. coli O157:H7 populations by 2.8 log CFU/spot and 2.6 log CFU/g, respectively. 8. Decontamination of green onions and baby spinach by vaporized ethyl pyruvate (EP). Baby spinach leaves and green onions were inoculated with E. coli O157:H7 using the dip-inoculation method and treated with vaporized EP. The samples were then stored at 4C for 7 days. EP (420 mg/L) reduced the population of E. coli O157:H7 by > 4.7 log CFU/g and 4.3 log on green onions and baby spinach, respectively. PARTICIPANTS: This project provided educational opportunities and training for undergraduate and graduate students involved with this project. It also provided high school students an opportunity to work in the laboratory processing samples for microbial analysis and learned about production of spinach and green onions. In addition, the photomicrographs of bacterial capsule and photos of plates demonstrating curli production by E. coli O157:H7 were used as educational tools to teach visiting high school science students about bacterial cell surface moieties. TARGET AUDIENCES: Using the findings of the research from the collaborative research project, the findings are being incorporated into existing Good Agricultural Practices, produce processing, and produce specific Good Manufacturing Practices courses. The highlights of the research component are incorporated to stress prevention of contamination at the production, harvesting, and processing level. The training materials have been disseminated through the National GAPs Program, Better Process Control School, and the Food Venture Center. The final survey of participants will be conducted in the following year (2013) to determine attitude and knowledge changes. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Here are the highlights of our outcomes/impacts: 1. Hepatitis A virus (HAV) was not detected in spinach and green onion plants grown in contaminated soil systems, with the exception of one spinach plant positive for HAV. In hydroponic systems, HAV was internalized to higher titers. 2. Cell surface moieties including curli and flagella on E. coli O157:H7 influence survival of the enteric pathogen on plant tissue. Bacterial surface structures recognized by the plant defense system negatively influence the ability of the pathogen to survive and colonize plant tissue. 3. E. coli O157:H7 and Salmonella survival was most limited on scallions and spinach, respectively, following spray irrigation. 4. Physiological changes of E. coli O157:H7 encountered in water, soil, or manure may influence plant defense response, and consequently impacting survival and colonization of the human pathogen on the plants. 5. Green onions grown in soil and hydroponic media contaminated with E. coli O157:H7 and Salmonella were found to uptake the pathogens in their roots, bulbs, stems and leaves. High pressure treatment could eliminate both pathogens that were internalized within green onions during plant growth. 6. The wetness state of green onions affected pressure inactivation of E. coli O157:H7. Pressure had minimal impact on the quality of chopped green onions. 7. H2O2 washing followed by aerosolized Allyl isothiocyanate (AIT) combined with lactic acid (LA) is an effective decontamination method for inactivating E. coli O157:H7 on baby spinach. 8. Washing with 1% LA at 40C was more effective than chlorine washing on inactivation of E. coli O157:H7 on baby spinach. Mild heat enhanced the efficacy of washing solutions on the inactivation of the pathogen. 9. Combined treatments of UV, acidified sodium hypochlorite (ASC), and mild heat treatment were capable of achieving a greater than 5-log reduction in E. coli O157:H7 surface contaminated spinach and green onions. Internalized E. coli O157:H7 on spinach and green onions only resulted in a 2.2 log reduction. 10. Ethyl pyruvate (EP), a GRAS status food additive, was shown to be effective for the decontamination of E. coli O157:H7 on spinach and green onions without any deleterious effects on sensory properties of the produce.
Publications
- Durak, M.Z., Churey, J.J., and Worobo, R.W. 2011. Decontamination of Green Onions and Spinach Using Gaseous Ethyl Pyruvate. International Association for Food Protection Annual Meeting, Milwaukee, WI
- Durak, M.Z., Churey, J.J., and Worobo, R.W. 2011. Postharvest Intervention Methods and Combined Treatments to Decontaminate Spinach. International Association for Food Protection Annual Meeting, Milwaukee, WI
- Durak, M. Z., Churey, J.J., Gates, M., Sacks, G.L., and Worobo, R.W. 2012. Decontamination of green onions and baby spinach by vaporized ethyl pyruvate. J. Food Prot. 75: 1012-22.
- Durak, M.Z., Churey, J.J., and Worobo, R.W. 2012. Efficacy of UV, acidified sodium hypochlorite, and mild heat for decontamination of surface and infiltrated Escherichia coli O157:H7 on green onions and baby spinach. J. Food Prot.75: 1198-1206.
- Hirneisen, K.A. and Kniel, K.E. 2012. Comparative Uptake of Enteric Viruses into Spinach and Green Onions. Food. Environ. Virol. In press.
- Hirneisen, K.A., Sharma, M., and Kniel, K.E. 2012. Human Enteric Pathogen Internalization by Root Uptake into Food Crops. Foodborne Path. Dis. 9: 396-405.
- Hirneisen, K.A. and Kniel, K.E. 2012. Mechanisms of inactivation affect the relationship between viral attachment and infectivity. International Association for Food Protection Annual Meeting, Providence, RI.
- Neetoo, H., Lu, Y., Wu, C., Chen, H. 2012. Use of high hydrostatic pressure to inactivate E. coli O157:H7 and Salmonella enterica internalized within and adhered to pre-harvest contaminated green onions. Appl. Environ. Microbiol. 78: 2063-2065.
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Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: Experiments were conducted to determine the influence of cell surface structures of E. coli O157:H7, such as flagella, curli fimbriae, lipopolysaccharides or exopolysaccharides on their survival or colonization on plants. E. coli O157:H7 ATCC 43895 wild-type strain showed significantly lower colonization on wild-type Arabisopsis plant compared with flagella-deficient 43895 mutant. Curli-deficient E. coli O157:H7 86-24 strain highly colonized wild-type Arabidopsis plants compared with the curli-producing 86-24 wild-type strain. Internalization of hepatitis A virus (HAV) and murine norovirus (MNV) through root uptake into spinach and green onions was assessed through both soil and hydroponic growth systems. Despite survival of both HAV and MNV in soil for over 30d, neither virus was recovered from internal tissues spinach or green onions when soil was contaminated with virus. Both a nutrient flow hydroponic system and a static uptake hydroponic system were tested for virus internalization. In both systems, HAV and MNV were detected in all tissues of spinach and green onions, leaves, stems and roots. The individual and combined efficacies of UV, acidified sodium chlorite (ASC), and mild heat for decontaminating green onions and baby spinach infected with E. coli O157:H7 were determined. The individual efficacies of UV, ASC, and mild heat treatments varied based on the produce type and contamination method. A combined treatment with 125 mJ/cm2 UV and 200 ppm ASC at 50C resulted in a >5 log reduction for spot-inoculated green onions with an initial contamination level of 7.2 log CFU/spot. A combined treatment of 500 mJ/cm2 UV and 200 ppm ASC at 50C selected for the decontamination of dip-inoculated green onions resulted in a 2.2 log CFU/g reduction. For spot and dip-inoculated baby spinach, a combined treatment of 125 mJ/cm2 UV and 200 ppm ASC at 20C reduced E. coli O157:H7 populations by 2.8 log CFU/spot and 2.6 log CFU/g, respectively. The effect of high pressure on the inactivation of E. coli O157:H7 and Salmonella inoculated on un-wetted, wetted (briefly dipped in water) or soaked (immersed in water for 30 min) green onions was determined. The extent of pressure inactivation increased in the order of soaked > wetted > un-wetted state. Overall, after pressure treatment at 400-450 MPa (soaked) or 450-500 MPa (wetted) for 2 min at 20-40C, wild-type and antibiotic-resistant mutant strains of E. coli O157:H7 and Salmonella on green onions were undetectable immediately after treatment and throughout the 15-day storage at 4C. The effect of aerosolized antimicrobials on inactivating E. coli O157:H7 on baby spinach was also determined. In a glass jar system, washing with 3% H2O2 followed by a 2-min treatment of aerosolized 2.5% Lactic Acid (LA) + 1% Allyl Isothiocyanate (AIT) or 2.5% LA + 2% AIT reduced E. coli O157:H7 population by 4.7 and > 5 log CFU/g, respectively, after 10 days refrigerated storage. In a scale-up system, up to 4-log reduction of bacterial population was achieved for the same treatments without causing noticeable adverse effect on the appearance of leaves. PARTICIPANTS: In addition to training the graduate students involved with this project, the photomicrographs of bacterial capsule and photos of plates demonstrating curli production by E. coli O157:H7 were used as educational tools to teach visiting high school science students about bacterial cell surface moieties. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Results of the persistence of E. coli O157:H7 on plants study suggested that curli, a component of cell surface structure found in many pathogenic E. coli O157:H7 strains, affected bacterial colonization. Greater emphasis must be placed on understanding characteristics associated with bacteria being used in research directed at the interaction of human enteric bacteria with plant tissue. The methods by which bacteria are cultured, stage of growth, and method of processing may all influence cell surface characteristics and subsequently attachment and persistence. Results of the virus internalization study indicated that enteric viruses such as hepatitis A virus and murine norovirus were very unlikely to internalize spinach and green onions through root uptake using the soil system. However, they could internalize the plants using the hydroponic growth systems. The findings of the UV study suggest that when foodborne pathogens contaminate produce and subsequently infiltrate, attach or become localized into protected areas, the individual or combined applications of UV, ASC, and mild heat treatments have limited decontamination efficacies on both green onions and baby spinach (< 3 log). However, treatments combining UV, ASC, and mild heat could be a promising application for reducing pathogen populations (> 5 log) on E. coli O157:H7 surface-contaminated green onions. This study also highlights the importance of developing and optimizing produce-specific decontamination methods to ensure the safety of fresh produce commodities. The pressure treatments had minimal adverse impact on most sensorial characteristics as well as on the instrumental color of chopped green onions, demonstrating the promising applications of high pressure to minimally process green onions in order to alleviate the risks of E. coli O157:H7 and Salmonella infections associated with the consumption of this commodity. Our research also highlights the potential application of aerosolized AIT + LA as a new post-washing intervention strategy to control E. coli O157:H7 on baby spinach during refrigeration storage. The findings for all the collaborators work will be collectively used to synthesize best practice recommendations for produce growers and processors. These recommendations will be incorporated into updated GAPs training materials and disseminated to all GAPs train the trainers and trainers thru the National GAPs Program hosted at Cornell University. The extension materials will be evaluated by all grant collaborators and GAPs national experts prior to incorporation into GAPs training materials.
Publications
- Huang, Y. and Chen H. 2011. Effect of organic acids, hydrogen peroxide and mild heat on inactivation of Escherichia coli O157:H7 on baby spinach. Food Control. 22, 1178 - 1183.
- Durak, M.Z., J.J. Churey, and R. W. Worobo. Inactivation of E. coli O157:H7 on green onions. 2010. IFT Annual Conference. Chicago, IL.
- Huang, Y., Ye, M. and Chen H. 2011. Efficacy of washing with hydrogen peroxide followed by aerosolized antimicrobials as a novel sanitizing process to inactivate Escherichia coli O157:H7 on baby spinach. Int. J. Food Microbiol. In press.
- Neetoo, H., Nekoozadeh, S., Jiang, Z., Chen, H. 2011. Application of high hydrostatic pressure to decontaminate green onions from Salmonella and Escherichia coli O157:H7. Food Microbiol. 28, 1275-1283.
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: A study was conducted to determine whether plant age would have an effect on colonization and persistence of E. coli O157:H7. Green onion was harvested at approximately 60 days and therefore considered a scallion. Plants were harvested at 1, 5, and 10 days post-exposure to E. coli O157:H7 (Spray or surface irrigation). For green onions no surface irrigated surface sterilized samples were positive for E. coli O157:H7 following direct plating of serial dilutions or enrichment. Spray irrigated samples harvested one-day post-challenge had 2 to 3-log E. coli O157:H7. At day 10 post-challenge 50% of spray irrigated plants, surface sterilized or direct plated, were positive for E. coli O157:H7 based enrichment. For scallions, similar to green onions, no surface irrigated surface sterilized samples were positive for E. coli O157:H7 following direct plating of serial dilutions or enrichment. At one-day post challenge 8 of 9 spray irrigated plants were positive for E. coli O157:H7; detectable levels ranged from 102 to 104 CFU/g) based on direct plating. By day 10 post-challenge 30% of spray irrigated plants were positive for E. coli O157:H7. Unlike soil grown spinach and green onions, enteric viruses were detected within the tissue of plants grown in contaminated hydroponic systems. Spinach and green onion seedlings at 10 days of age were placed in 10mL of Hoagland's solution contaminated with hepatitis A virus (HAV) or murine norovirus (MNV) for 1, 3 and 5 days. Internalized HAV and MNV were detected by both infectivity assays and qPCR. Overall, both HAV and MNV were shown to internalize into plant tissues via root uptake. HAV titers in green onions increased over the 5 days to reach an average of 5 log genomic copies by the 5th day. MNV showed similar trends compared to HAV in green onions. Similar results were also obtained in spinach whereby HAV and MNV were detected in all parts of the plant after 5 days of growth in contaminated hydroponic solution. UV light (500 mJ/cm2) was shown to reduce E. coli O157:H7 populations by 1.0 log CFU/g with dip-inoculated samples, the population was reduced by 1.7 log at 90 mJ/cm2 and 2.7 log at 1000 mJ/cm2 level for spot-inoculated green onions. The combination of selected UV exposure (90 mJ/cm2) and chlorine (200 ppm at 50C) treatments showed a total of 4.7 log reduction with a five-strain cocktail of E. coli O157:H7 spot-inoculated green onions. Additional UV exposure (500 mJ/cm2), yielded a 2.1 log CFU/g for dip-inoculated samples. Baby spinach dip-inoculated with E. coli O157:H7 was washed with a variety of organic acids, chlorine and hydrogen peroxide. Chlorinated water (200 ppm free chlorine) decreased the population of E. coli O157:H7 on baby spinach by only 1.2-1.6 log CFU/g. Washing with 1% lactic acid at 40C for 5 min was the most effective treatment achieving a 2.7 log reduction of E. coli O157:H7 which is significantly higher than chlorine washing. Washing with lactic acid + citric acid or lactic acid + hydrogen peroxide at 40C for 5 min was equally effective against E. coli O157:H7. PARTICIPANTS: In addition to the graduate students working on this project, two undergraduate students had the opportunity to participate in the project. The undergraduate students assisted with care of the plants, harvesting, and processing for microbiological analysis. Last summer two SEED students sponsored through the American Chemical Society worked on the project. Two new SEED students participated in the project this past summer. Recall, the students are gifted students from inner city high schools. The students had an opportunity to work in the laboratory processing samples for microbial analysis and learned about production of spinach and green onions. The educational opportunities afforded to students (high school, undergraduate and graduate) are unique and would not have been possible without the funding received. The students gained knowledge on the commercial production of scallions and green onions and foodborne pathogens and on food processing methods (high pressure, UV and washing with sanitizers). TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Results of the scallion and green onion cultivation study suggest that under the conditions evaluated E. coli O157:H7 is as likely to persist on scallions as green onions. They also demonstrate that E. coli O157:H7 can persist on plants for at least 10 days under conditions studied. There exists a need to develop effective post-harvest sanitizing method (s). The UV results indicate that dip inoculated green onions requires significantly higher levels of UV exposure to achieve the same reduction as for spot inoculated green onions. Combination treatments on produce showed additional inactivation compared to the cumulative reductions for individual treatments. Infiltration of pathogens into fresh produce may occur via overhead irrigation or produce wash water, and these results suggest that water quality is of vital importance for the safety of fresh green onions. The application of mild heat significantly enhanced the efficacy of washing solutions on the inactivation of E. coli O157:H7. The results suggested that the use of organic acids in combination with mild heat can be a potential intervention to control E. coli O157:H7 on spinach.
Publications
- K.A. Hirneisen and K.E. Kniel. 2010. Hydroponic internalization of enteric viruses into green onions and spinach. IAFP Annual Meeting, Anaheim, CA.
- Y. Huang, H. Neetoo, M. Ye and H. Chen. 2010. Effect of organic acids and hydrogen peroxide against Escherichia coli O157: H7 on baby spinach. IFT Annual Meeting. Chicago, IL.
- H. Neetoo, S. Nekoozadeh, J. Zheng and H. Chen. 2010. Use of high hydrostatic pressure (hhp) to eliminate Escherichia coli O157:H7 and Salmonella on scallions (green onions). IAFP Annual Meeting, Anaheim, CA.
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Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: Spinach plants were harvested at 1, 5, and 10 days post-exposure to E. coli O157:H7 (Spray or surface irrigation). Following harvest spinach leaves were surface sterilized or processed directly for presence of E. coli O157:H7. A greater number of samples of spinach leaves from plants spray irrigated were positive at each sample day compared to leaves from soil irrigated plants. A similar set of experiments have been completed for green onion. No surface irrigated surface sterilized samples were positive for E. coli O157:H7 following direct plating of serial dilutions or enrichment. Spray irrigated samples harvested one-day post-challenge had 2 to 3-log E. coli O157:H7. At day 10 post-challenge all spray irrigated plants were negative for E. coli O157:H7. Spinach and green onion seeds were planted and grown for 10 days before being transferred to growing pots with soil or water contaminated with 2 x 10^7 log TCID50/g Hepatitis A virus (HAV). Days 5, 10 and 20 post-inoculation plant samples were collected and analyzed. Internalized HAV was detected by qPCR on day 5 post-inoculation in one spinach plant contaminated through inoculated soil; however, HAV was not detected in this spinach plant through TCID50 analysis. HAV was undetectable in all other plant and soil samples between days 5 and 20. Spinach and green onions grown under natural conditions exposed to a single inoculation of contaminated water did not internalize into plants using current evaluation techniques. The ability of HAV to survive in plant tissue was assessed using plant homogenates. Over a 10 day period only ~1.3 log TCID50/g was lost sitting in green onion homogenate at 22C. HAV also survives in spinach homogenate; however, there is a cytotoxic effect on the cells making this data more difficult to determine. Green onions were inoculated by either spot or infiltration method. While UV light (500 mJ/cm^2) was able to reduce the E. coli O157:H7 population only by 1.2 log CFU/g in infiltration inoculated samples, the population was reduced by 2.8 log at 90 mJ/cm^2 and by 3.5 log at 1040 mJ/cm^2 level for spot inoculated green onions. The combination of selected UV (84 mJ/cm^2) and chlorine (200 ppm at 50C) treatments demonstrated a total of 4.7 log reduction on five-strain cocktail of E. coli O157:H7 spot inoculated green onions. Even with an increase in UV power (500 mJ/cm^2), this reduction fell to 2.1 log CFU/g for infiltration inoculated samples. Green onions inoculated with a cocktail of E. coli O157:H7 or Salmonella were stored at 4 and 21C for 14 and 7 days respectively. The bacterial populations underwent a significant increase when the green onions were held at 21C and were fairly constant when stored at 4C. Inoculated green onions were also subjected to pressure treatment in a dry, wet or soaked state. The decontamination efficacy of high pressure increased in the order of soaked > wet > dry states for both pathogens. Pre-soaking of green onions and pressurization (450 MPa for 2 min at 20C) reduced a 5-log population of E. coli O157:H7 and Salmonella to below detectable levels. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Providing educational opportunities and training for undergraduate and graduate students is an important component of the grant proposal. A first year Master's candidate has taken on the responsibility of cultivating green onions and spinach in the Rutgers greenhouse facilities. She has shared the experience with other students in the laboratory. During this past summer two SEED students sponsored through the American Chemical Society worked on the project. The students are gifted students from inner city high schools. The students had an opportunity to work in the laboratory processing samples for microbial analysis and learned about production of spinach and green onions. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Similar to previous studies with lettuce, results of the present study indicate that surface irrigation minimizes the likelihood that the edible portion of a plant will become contaminated following irrigation with contaminated water. In addition, under conditions of the present study, exposure of green onions to contaminated irrigation water greater than 10 days prior to harvest represented no risk of harvesting contaminated onions. The educational opportunities afforded to students (high school and graduate) are unique and would not have been possible without the funding received. The students gained knowledge on the commercial production of spinach and green onions and foodborne pathogens. The students are now positioned to study the interaction of enteric foodborne pathogens with plant tissue. Results from this study will elucidate the ability of enteric virus to survive within green onions and spinach. We will gain an appreciation into how human calicivirus and picornavirus compare in terms of survival on fresh produce. The lower reduction of pathogens due to infiltration suggest that water quality and safety is a key factor for produce production. Since compromised water quality could allow for pathogens to be infiltrated and the reduced effectiveness of pathogen decontamination methods would pose a higher risk for pathogen survival in fresh and cut produce. Results from the stability study underscore the influence of storage temperature on the persistence or growth potential of E. coli O157:H7 and Salmonella on green onions. Moreover, the study highlights the potential application of high pressure on green onions to alleviate the risks of E. coli O157:H7 and Salmonella infections associated with consumption of green onions.
Publications
- Matthews, K.R. 2009. Foodborne and commensal bacteria associated with produce: Illness, antibiotic resistance, mitigation strategies. Session S1606. P. 108. In: Book of Abstracts 11th ASEAN Food conference. Bandar Seri Begawan, Brunei.
- K. A. Hirneisen, H. Chen, K. Matthews, R. Worobo and K.E. Kniel. 2009. Internalization of Enteric Viruses in Spinach and Green Onions. IAFP Annual Meeting, Grapevine, TX. P3-17.
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