Semester

Fall

Date of Graduation

2023

Document Type

Dissertation

Degree Type

PhD

College

Davis College of Agriculture, Natural Resources and Design

Department

Division of Animal and Nutritional Sciences

Committee Chair

Cangliang Shen

Committee Co-Chair

Annette Freshour

Committee Member

Kristen Matak

Committee Member

Janet Tou

Committee Member

Timothy Boltz

Abstract

Fresh produce and poultry have been associated with several foodborne illness outbreaks. Strategies to mitigate these risks are a much-needed research priority; therefore, five experiments were conducted to analyze pathogen cross-contamination capabilities on produce and poultry. Microbial contamination prevention and reduction strategies were also studied.

In Chapter Two, three antimicrobial delivery methods in combination with increasing concentrations of hydrogen peroxide-peroxyacetic acid (H2O2-PAA) (0.05-0.25 ml/dL) were compared to evaluate their ability to reduce and prevent cross-contamination of Listeria monocytogenes (L. monocytogenes) and Listeria innocua (L. innocua) on inoculated apples. Apples have been identified as a source of L. monocytogenes contamination and were responsible for several multistate outbreaks in the United States. Additionally, the surrogate L. innocua is well-established as an acceptable surrogate for L. monocytogenes in thermal inactivation studies, however, few studies have compared the surrogate to the pathogen using antimicrobial parameters. The objectives of this study were to: 1) compare the three antimicrobial delivery methods, conventional garden spray (GS), electrostatic spray (ES), and a single immersion dip using a H2O2-PAA sanitizer to mitigate microbial cross-contamination of L. monocytogenes on apples; 2) determine whether L. innocua is an appropriate surrogate for L. monocytogenes for use in antimicrobial challenge studies. The antimicrobial used in this study was SaniDate® 5.0 (H2O2-PAA), which is recommended by the wholesale distribution company (Appalachian Harvest). Appalachian Harvest is a buyer of produce from several small-quantity produce growers in WV. Based on the results of this study, the dip method caused the highest reductions (P < 0.05) of both the pathogen and surrogate bacteria (> 3 log10MPN/ml); whereas the GS method caused the lowest L. monocytogenes (-0.35 log10MPN/ml) and L. innocua (0.19 log10MPN/ml) (P < 0.05) cross-contamination on to uninoculated apples. The concentration 0.25 ml/dL was the most effective at reducing bacteria and preventing cross-contamination. This study concluded that since L. innocua behaved similarly (P > 0.05) to L. monocytogenes, L. innocua is an acceptable surrogate for L. monocytogenes for H2O2-PAA antimicrobial studies. Results from this challenge study also indicated that the dip and GS methods were more effective at reducing and preventing cross-contamination of bacteria on apples, respectively.

In addition to apples, tomatoes were another culprit of several multi-state outbreaks in the United States. Nationally, and locally, tomatoes were associated with Salmonella contamination. Therefore, in Chapter Three, the triple wash methods (TWM) water+antimicrobial+water (WAW) or water+water+antimicrobial (WWA) were analyzed to determine their efficacy in reducing and preventing cross-contamination of Salmonella Typhimurium (S. Typhimurium) and the surrogate Enterococcus faecium (E. faecium) on tomatoes. The TWM was used in this study because it is the washing method recommended by the WV Small Farm Center (WVU-SFC) to control pathogens on locally grown fruits and vegetables. The first objective of this study was to evaluate the efficacy of reducing and preventing cross-contamination of S. Typhimurium and E. faecium on tomatoes using two TWMs with different concentrations of a H2O2-PAA sanitizer. The second objective of this study was to evaluate the feasibility of E. faecium as a surrogate for future antimicrobial challenge studies. The results showed that the application of 0.25 ml/dL of H2O2-PAA in WAW or WWA TWM resulted in the lowest (P < 0.05) cell counts compared to lower concentration treatments for S. Typhimurium (0.16-0.69 log10MPN/g) and E. faecium (-0.41 log10MPN/g). There were no significant differences (P > 0.05) in survival between S. Typhimurium and E. faecium after TWM treatments. It was concluded that E. faecium is an acceptable surrogate for S. Typhimurium in antimicrobial challenge studies. It was also concluded that although 0.25 ml/dL significantly (P < 0.05) reduced bacteria, it did not completely prevent cross-contamination of the pathogen. Therefore, future studies are needed to analyze what concentrations of H2O2-PAA could significantly reduce and prevent cross-contamination of bacteria on tomatoes. (0, 0.0064, 0.1, and 0.25 ml/dL) of

Previous concentrations analyzed in Chapter Three (0-0.25 mL/ dL) did not completely inactivate the surrogate or pathogen on tomatoes; therefore, in Chapter Four, a follow-up study to the experiment described in Chapter Three was conducted. Higher, off-label concentrations (0.5-0.7ml/dL) of the sanitizer SaniDate® 5.0 (H2O2-PAA) were tested to determine their efficacy in reducing and preventing the cross-contamination of S. Typhimurium and E. faecium on tomatoes. This study aimed to find the minimal concentrations of H2O2-PAA required to prevent microbial cross-contamination of S. Typhimurium and surrogate E. faecium on tomatoes. The sanitizer was applied to inoculated tomatoes by washing fruits for 45 s using TWM methods (WWA or WWA). Significant reductions (P < 0.05) of Salmonella were only achieved when the off-label concentration of 0.7% H2O2-PAA was applied using the WWA strategy. Results showed cross-contamination of S. Typhimurium was prevented at 0.7% of H2O2-PAA using the WWA strategy. Additionally, E. faecium was prevented at and above 0.50% of H2O2-PAA, regardless of wash strategy. Though 0.7% H2O2-PAA is effective at mitigating cross-contamination and achieving more than a 4-log reduction of S. Typhimurium on tomatoes, future research is needed to determine the safety of these greater antimicrobial concentrations. Additionally, approval by the federal government is required before changing the recommendations for the use of these concentrations. Similar to the previous study, results of this study also suggested that E. faecium could be an acceptable surrogate for S. Typhimurium when validating antimicrobial washing systems on tomatoes.

In the previous Chapter, we found that the TWM can be an effective washing strategy to reduce pathogen growth and cross-contamination on produce. However, it is unknown if local growers are aware of the WVU-SFC recommended TWM or how much they know about produce cross-contamination. In Chapter Five, a survey was conducted at Farmers’ Markets (FMs) in WV to analyze small and very small-quantity produce growers’ knowledge of food safety. Their awareness of the WVU-SFC recommended TWM and willingness to implement the washing method was also assessed. The information obtained in these surveys were important because previous studies have found that produce at local FMs have been infected with pathogens. Surveys filled out by consumers revealed that most FM consumers consider it the vendors’ responsibility to wash the produce and they generally view FM produce as safe. However, few surveys have analyzed the FM vendors’ perception of food safety on their products. The survey results revealed knowledge gaps about food safety among FM produce vendors. Additionally, 53.7% of FM vendors did not wash their produce due to increased spoilage (61.4%; P < 0.05). Among participants who washed produce, only 28.2% were familiar with the TWM. Most respondents who washed produce (65.3%; P < 0.05) stated they would be interested in attending good agricultural practices/ Food Safety Modernization Act (GAP/FSMA) and TWM training. Understanding FM vendors’ perceptions and current wash methods allows local state government agencies to make better-informed decisions regarding food safety policies to ensure produce safety. Using the information from the findings of this study, the authors’ team plans to work closely with the WVU-SFC to develop outreach activities on training WV small to very small-quantity produce growers on the application of the TWM.

In addition to produce being a cross-contamination risk, chicken is a risk both nationally and locally because it is the number one meat commodity associated with foodborne illness. Poultry is also one of the most harvested meats in WV. The risk of pathogen contamination on chickens in the state has only increased since the rise in popularity of mobile poultry units (MPPUs) in WV. Therefore, the objectives of Chapter Six were to characterize the microbial quality of broilers (total aerobic plate counts and generic E. coli), and the presence/absence of Salmonella and Campylobacter after processing carcasses in a pilot commercial MPPU at the West Virginia University Poultry Farm. Aerobic plate counts (APCs), Escherichia coli (E. coli), and the presence/absence of Salmonella and Campylobacter on 96 broiler carcasses after processing in the MPPU were assessed. Samples were taken after the processing steps, immersion scalding, manual evisceration, and immersion chilling in water or water with 5ppm chlorine (Cl2). Results showed that the highest number of bacteria recovered from carcasses were APCs (4.21 log10CFU/mL) and E. coli (3.77 log10CFU/mL). Overall, results show that E. coli, Salmonella, and Campylobacter were significantly reduced (P < 0.05) from the initial scalding to the chilling step. However, Salmonella frequency doubled (15.63-34.38%) after the evisceration step, indicating evisceration may be a critical control point in preventing cross-contamination of Salmonella. Results also show that proper chilling after evisceration is an important microbial mitigation step in MPPU processing. Campylobacter (18.75% positive) was more resistant (P < 0.05) to water chilling than Salmonella (0% positive). Additionally, Campylobacter was not completely inactivated (0/16) until carcasses were chilled in CW, whereas W chilling was sufficient to completely inactivate Salmonella (0/16) on carcasses. Although the addition of 5ppm Cl2 in chill water was significant in reducing bacteria (P < 0.05) on chilled broiler carcasses, it did not necessarily cause a more significant reduction than water chilling alone. Therefore, more MPPU processing studies are needed to research what post- evisceration wash methods and chill-water sanitizers are most effective at preventing cross-contamination, while preserving the integrity of the final product.

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