Laura K. Strawn

Microbial Quality of Agricultural Water Used in Produce Preharvest Production on the Eastern Shore of Virginia

Several produce-borne outbreaks have been associated with the use of contaminated water during preharvest applications. Salmonella has been implicated in a number of these outbreaks. The purpose of this study was to evaluate the microbial quality of agricultural surface water used in preharvest production on the Eastern Shore of Virginia in accordance with the Food Safety Modernization Acts Produce Safety Rule water standards. The study also examined the prevalence, concentration, and diversity of Salmonella in those water sources. Water samples (1 L) from 20 agricultural ponds were collected during the 2015 and 2016 growing seasons ( n = 400). Total aerobic bacteria, total coliforms, and Escherichia coli were enumerated for each sample. Population levels of each microorganism were calculated per 100-mL sample and log transformed, when necessary. Samples (250 mL) were also enriched for Salmonella. Presumptive Salmonella isolates were confirmed by PCR ( invA gene) and were serotyped. In 2016, the concentration of Salmonella in each sample was also estimated by most probable number (MPN). Indicator bacteria and environmental and meteorological factors were analyzed for their association with the detection of a Salmonella-positive water sample by using logistic regression analysis. Seventeen of the 20 ponds met the Food Safety Modernization Acts Produce Safety Rule standards for production agricultural water. Three ponds did not meet the standards because the statistical threshold value exceeded the limit. Salmonella was detected in 19% of water samples in each year (38 of 200 in 2015 and 38 of 200 in 2016). Of the 118 Salmonella isolates serotyped, 14 serotypes were identified with the most prevalent being Salmonella Newport. E. coli concentration, farm, and total aerobic bacteria concentration were significantly associated with the likelihood of detecting a Salmonella-positive sample The average concentration of Salmonella in all samples was 4.44 MPN/100 mL, with the limit of detection being 3.00 MPN/100 mL. The highest concentration of Salmonella was 93.0 MPN/100 mL. These data will assist in a better understanding of the risks that production water poses to produce contamination events.

Cavitation Bubbles Remove and Inactivate Listeria and Salmonella on the Surface of Fresh Roma Tomatoes and Cantaloupes

Raw produce has frequently been identified as the source of bacterial pathogens that can cause human illnesses, including listeriosis and salmonellosis. Microbial pathogens may attach and form biofilms on raw fruit surfaces and can be difficult to remove. A cavitation process (injection of bubbles into water) was studied for its effectiveness for removal and inactivation of Listeria monocytogenes and Salmonella Newport from the surfaces of fresh Roma tomatoes and cantaloupes. Individual fruit were separately inoculated with each pathogen, then submerged in a water tank and treated with a bubble flow through an air stone using one airflow rate (0 – 14 liters/min.) for up to 60 sec. As airflow increased, pathogen reduction increased up to 1.2 log CFU/fruit greater than with water alone (no bubbles). Additional pathogen reduction in the tank water (organisms detached from the fruit) was observed with the bubble treatments. Therefore, these bubble streams can be used to enhance the detachment of bacteria from fruit surfaces and to inactivate a proportion of these detached microorganisms. Additionally, recoveries of Salmonella from inoculated Roma tomatoes and cantaloupe were determined for treatment water that contained 50 or 150 ppm sodium hypochlorite. Combining both cavitating bubbles and chlorine in the tank water resulted in greater efficacy of removing or inactivating S. Newport from the surface of Roma tomatoes and cantaloupe than with cavitation or chlorine alone. The physical force of a bubble stream on raw produce can effectively detach and inactivate surface bacteria, and has the potential to reduce antimicrobial chemical use and water use in post-harvest packing operations.

Agricultural Practices Influence Salmonella Contamination and Survival in Pre-harvest Tomato Production

Between 2000 and 2010 the Eastern Shore of Virginia was implicated in four Salmonella outbreaks associated with tomato. Therefore, a multi-year study (2012-2015) was performed to investigate presumptive factors associated with the contamination of Salmonella within tomato fields at Virginia Techs Eastern Shore Agricultural Research and Extension Center. Factors including irrigation water sources (pond and well), type of soil amendment: fresh poultry litter (PL), PL ash, and a conventional fertilizer (triple superphosphate - TSP), and production practices: staked with plastic mulch (SP), staked without plastic mulch (SW), and non-staked without plastic mulch (NW), were evaluated by split-plot or complete-block design. All field experiments relied on naturally occurring Salmonella contamination, except one follow up experiment (worst-case scenario) which examined the potential for contamination in tomato fruits when Salmonella was applied through drip irrigation. Samples were collected from pond and well water; PL, PL ash, and TSP; and the rhizosphere, leaves, and fruits of tomato plants. Salmonella was quantified using a most probable number method and contamination ratios were calculated for each treatment. Salmonella serovar was determined by molecular serotyping. Salmonella populations varied significantly by year; however, similar trends were evident each year. Findings showed use of untreated pond water and raw PL amendment increased the likelihood of Salmonella detection in tomato plots. Salmonella Newport and Typhimurium were the most frequently detected serovars in pond water and PL amendment samples, respectively. Interestingly, while these factors increased the likelihood of Salmonella detection in tomato plots (rhizosphere and leaves), all tomato fruits sampled (n = 4800) from these plots were Salmonella negative. Contamination of tomato fruits was extremely low (< 1%) even when tomato plots were artificially inoculated with an attenuated Salmonella Newport strain (104 CFU/mL). Furthermore, Salmonella was not detected in tomato plots irrigated using well water and amended with PL ash or TSP. Production practices also influenced the likelihood of Salmonella detection in tomato plots. Salmonella detection was higher in tomato leaf samples for NW plots, compared to SP and SW plots. This study provides evidence that attention to agricultural inputs and production practices may help reduce the likelihood of Salmonella contamination in tomato fields.

Bioinformatics Aspects of Foodborne Pathogen Research

This chapter functions as an introduction to the field of bioinformatics and current sequencing technologies as it pertains to the research of foodborne pathogens. As the field of bioinformatics and the field of whole genome sequencing are rapidly evolving, the chapter focuses on general principles in bioinformatics analyses of microbial genomes and microbiome, such as genome assembly, comparative genomics, population genetics and phylogenetics. The last part of the chapter discusses microbiome research and it’s applications to foodborne pathogen research.