Pre-harvest and Post-harvest Decontamination Strategies for Reducing Foodborne Pathogens on Cantaloupes

<p>NON-TECHNICAL SUMMARY:<br/> During the last few years, cantaloupe has been increasingly linked to large food-borne disease outbreaks in the US, indicating the emerging role of this fruit as a vehicle of foodborne pathogens. A variety of FDA-approved disinfectants, including chlorine have been evaluated for cantaloupe washing, but are found to be ineffective in reducing pathogens on the fruit surface. . Octenidine di hydrochloride (OH) is an antimicrobial used in mouth rinses in Europe, with a high degree of safety. The objectives of this proposal are to determine the efficacy of OH (1) for reducing L. monocytogenes and E. coli O157:H7 on cantaloupes in the field when applied as a spray, (2) as a post-harvest wash for reducing L. monocytogenes, Salmonella and E. coli O157:H7 on cantaloupes, and (3) as an antimicrobial coating for reducing the pathogens on cantaloupes. This
proposal would potentially identify an effective and safe antimicrobial agent that can used to improve the microbiological safety of cantaloupes.
<p>APPROACH: <br/>Objective 1: Investigate the efficacy of OH for reducing L. monocytogenes and E. coli O157:H7 applied as a spray on cantaloupes in the field: Soil Preparation, seedling and planting: Soil beds (6-8 inch) will be created at the Beltsville Agricultural Research Center (BARC) farm, Beltsville, Maryland. Cantaloupe seed will be planted indoors in peat pots (2 inch x 2 inch) or other biodegradable containers in mid April, about 2 to 4 weeks prior to the transplant date. Subsequently once the transplants develop 2-3 mature leaves and a well-developed root system, they will be moved to the field. Seedlings will be planted 2 feet apart. Alternatively, seeds will be seeded during optimal germination temperature (25-35ºC, during the month of May in Maryland) ½ to 1 inch deep in soil. Two to 3 seeds will be planted in groups 18 to 24 inches apart within
the row and will be later thinned to the best plant per group. Cantaloupe inoculation: Plants with mature fruits will be artificially contaminated by nalidixic acid (NA) resistant Listeria innocua or a non-pathogenic Escherichia coli O157:H12 (Ingram et al., 2011) using overhead spraying. Octenidine di hydrochloride (OH) treatment: Bacterial suspensions on cantaloupe will be allowed to air dry for up to 2 h, and then treated with OH (0, 0.1 and 0.2%) using the sprayer as described above. Controls will be sprayed with 1% ethanol since ethanol will be used to dissolve OH. At 0, 15 and 30 days following inoculation, cantaloupes will be sampled for surviving populations of inoculated bacteria. Cored rind samples of cantaloupes will be collected using a sterilized stainless steel core borer (10 cm2 area), and appropriate dilutions of homogenized rind samples will be spiral-platted on Oxford
agar (OX) plus NA (OX-NA) and Sorbitol MacConkey Agar supplemented with 0.1% 4-methylumbelliferyl-b-D-glucuronide (MUG) and NA (SMAC-MUG-NA) for L. innocua and E. coli O157:H12, respectively, followed by 24-48 h incubation at 37ºC. Randomly selected colonies on OX and SMA-NA will be confirmed by Microgen Listeria Latex assay and Dryspot O157 agglutination assays, respectively. Objective 2. To determine the efficacy of OH as a post-harvest wash treatment for rapid inactivation of L. monocytogenes, Salmonella and E. coli O157:H7 on whole cantaloupes. The efficacy of 0, 0.05, 0.1, and 0.2% OH as an antimicrobial wash for inactivating L. monocytogenes Salmonella, and E. coli O157:H7 will be investigated at 25°C on whole, fresh cantaloupes. Preparation and inoculation of whole cantaloupes: Cantaloupes tempered at 25°C will be submerged in 3 liters of PBS containing a 5 strain
mixture of NA resistant L. monocytogenes, 5-serotype mixture Salmonella spp. (S. Typhimurium, S. Newport, S. Poona, S. Saintpaul, S. Montevideo) or 5-strain mixture of E. coli O157:H7 at (8 log CFU/ml) for 10 min, followed by drying for 2 h in a laminar flow hood without UV light. Inoculated cantaloupes, but not subjected to any wash treatment will serve as baseline to determine the efficiency of bacterial inoculation. OH treatment of whole cantaloupes: OH will be dissolved in 95% ethanol and subsequently added to sterile deionized water. Each inoculated cantaloupe will be subjected to OH wash treatment at 3 time points (1, 3, or 5 min) in 3 liters of sterile deionized water (control) or water containing 200 ppm chlorine, or 0.05, 0.1 or 0.2% OH in a water bath shaker. After treatment, the whole cantaloupe will be aseptically transferred to a second stomacher bag containing 3 liters of
Dey-Engley neutralizing broth and vigorously surface massaged for 2 min. The broth from stomacher bag will be serially diluted (1:10 in PBS) and plated (100 ?l) on duplicate Oxford+NA (OX), XLD+NA (XLD) or SMA+MUG+NA (SMA-NA) plates, followed by incubation at 37°C for 48 h for bacterial enumeration. In addition, 10 ml of the broth will be added to 100 ml tryptic soy broth and incubated at 37°C for 24 h. Following enrichment, the culture will be streaked on the aforementioned agars for the respective pathogens, and incubated at 37°C for 48 h. The detection limit for the experiment will be 200 bacterial cells/cantaloupe. Objective 3. To determine the efficacy of OH as an antimicrobial coating treatment for reducing L. monocytogenes, Salmonella and E. coli O157:H7 on whole cantaloupes. Preparation and application of OH coating on whole cantaloupes: Briefly, 2 g of low
molecular weight chitosan will be dissolved in sterile deionized water containing 1% acetic acid and stirred at room temperature for 12 h to obtain a final concentration of 2% (pH 4.6). Subsequently, OH will be added to the chitosan solution at the required concentrations and the solution will be stirred for another 6 h to facilitate proper mixing of OH. After the coating treatment, the cantaloupes will be inoculated with each pathogen as before, packed in sterile stomacher bags and stored at 23°C for 7 days. Enumeration of each inoculated pathogen on cantaloupe will be done as previously described on days 1, 3, 5, and 7 of storage.</p>