Location: Produce Safety and Microbiology Research2013 Annual Report
1a. Objectives (from AD-416):
1. Determine the effect of SO2 fumigation on the survival of common foodborne pathogens under in vitro conditions. 2. Determine the best inoculum concentration of bacterial pathogens and their survival on table grapes. 3. Examine the effect of SO2 on the survival of foodborne pathogens on freshly harvested California table grapes.
1b. Approach (from AD-416):
Procedure to accomplish Objective 1: Strains of bacterial pathogens will be cultured in Luria-Bertani broth at room temperature (25ºC) with rotation (150 rpm) to stationary phase. In order to determine the appropriate inoculum concentration that will allow us to detect a change in the cells population size on the grapes under the test conditions, 10 µl of cell suspension at a concentration of 105, and 107 CFU/ml will be spotted onto a 0.2 µm-pore Durapore filter placed on a water agar plate. For each inoculation concentration, three treatments with a dose of SO2 of 100, 200, and 300 ppm for one hour, along with a control (no SO2 treatment) will be conducted. The bacteria deposited on the filter will be stored at 32ºF and with 95% RH for at least one month. Fumigation will be carried out on the day starting the experiment, followed by once a week for a total of four weeks. The bacterial population sizes on the filter will be monitored weekly. Cells on the filter will be released to the KP solution by sonication and strong vortexing. Bacterial concentrations will be measured by counting colony forming units. Statistical analysis will be applied to test if SO2 treatment is a significant factor in survival of the bacterial pathogens under our test conditions.Procedure to accomplish Objective 2: To monitor the population of bacterial pathogens on surface of grapes during the storage period, we will use a spontaneous rifampin-resistant strain of each bacterial pathogen. This will enable us to recover from grapes onto LB agar containing rifampin, only the pathogen cells from the inoculum and select against all the indigenous bacteria on the grapes. To inoculate the grapes, 10 µl of bacterial cell suspension in KP buffer will be deposited onto the surface of individual grapes fruits. The plate will then be moved to the incubator and stored at 32ºF with 95% RH for at least one month. To process the grapes for measurement of pathogen population sizes, the inoculated grapes will be placed in a Whirl-PaK® sample collection bag (2.0 oz). Two ml of KP buffer will be added in the bag prior to sealing. The inoculated bacteria will be washed off the grapes with by gently massaging their surface, followed by sonication and agitation of the bag. A time course of survival of the pathogens will be determined using the initial inoculum on the grapes to infer if the pathogen population remained stable, increased, or declined on the grapes during the one-month storage period. Procedure to accomplish Objective 3: Freshly-harvested California table grapes will be shipped directly from the harvesting site to the USDA-ARS laboratory located in Albany, California, on ice overnight. Each bacterial pathogen (Escherichia coli, Salmonella, and Listeria monocytogens) will be inoculated onto the grapes with an initial inoculum size that was determined under Objective 2.
3. Progress Report:
The specified incubation conditions (at 32ºF with 95% humidity) for pathogens following fumigation treatments were established and successfully stably maintained. The sulfur dioxide fumigation system that delivers the specified sulfur dioxide dosages (100 ppm-hr, 200 ppm-hr, and 300 ppm-hr) was established. The in vitro survival of Escherichia coli O157:H7, Salmonella enterica Thompson, and Listeria monocytogenes following weekly exposure of 100 ppm-hr, 200 ppm-hr, and 300 ppm-hr sulfur dioxide were measured. A Rifampin-resistant strain of Listeria monocytogens was isolated. Currently, we are determining the recovery efficiencies of the three pathogens inoculated onto freshly-harvested California table grapes. This research relates to Objective 1: Identify and characterize the microbial genes that are involved in the attachment, colonization and survival of enteric pathogens on produce.