Understanding foodborne pathogens and indicators from field to fork

Abstract

The global food supply is a complex network of interconnected industries, and opportunities for the introduction of foodborne pathogens are abundant throughout. While there is a fair amount of information available for some sectors of the food industry surrounding risk and mitigation techniques such as cleaning and sanitation standards, there is a lack of knowledge within industries that utilize or yield minimally-processed products. The purpose of this work is to generate a better understanding of potential routes that foodborne pathogens can take throughout the field-to-fork continuum as well as how different factors such as surface type, environmental conditions, and sanitizer type can impact how pathogen populations are able to survive. In order to better understand resident bacteria within from Midwest feed mills, a multi-season survey of swine feed mills identified 121 resident isolates within the Enterobacteriaceae, Pseudomonadaceae, and Enterococcoccaceae families; a majority of isolates were identified in environmental samples from feed contact surfaces (32%, 37/121), non-feed contact surfaces (41%, 48/121), transient surfaces like brooms or workers’ shoes (17%, 20/121), with the remaining 10% (12/121) identified directly from finished feed. The isolates were also analyzed for the presence of antimicrobial resistance genes (ARG) and metal tolerance genes (MTG). To evaluate the efficacy of multiple commercially available sanitizers when used for the control of mature biofilms on surfaces commonly found within the tree-fruit harvesting process, multi-strain E. coli or L. monocytogenes biofilms were grown for 96 hours in a Centers for Disease Control (CDC) reactor at 25± 2˚C on high density polyethylene (HDPE), wood, or nylon. Bacteria were exposed to chlorine (500 ppm), peroxyacetic acid (PAA) (500 ppm), steam (75 psi), or silver-dihydrogen citrate (SDC) (4%) for 1 or 2 minutes; or chlorine dioxide gas (ClO₂) (100 ppm) for 24 hours. Results were considered significant at P < 0.05 for both E. coli and L. monocytogenes Interactions between sanitizer, surface type, and exposure time were found to be significant, and all sanitizers with the exception of SDC were able to reduce population on all surfaces. ClO₂ was overall the most effective method of managing biofilm populations grown on all three surfaces. After laboratory testing, a better understanding of how differing environmental conditions can impact sanitizer efficacy was needed. To do this, SDC and ClO₂ gas were tested against E. coli and Listeria innocua that was experimentally inoculated harvesting equipment at commercial orchards within the Midwest and Pacific Northwest (PNW) regions of the United States. Rifampicin resistant E. coli and Listeria were grown for either 24-hours (sessile form) or 96-hours (biofilm form) at 25± 2˚C on 10 cm² HDPE, wood, or nylon coupons. Surfaces were allowed to dry for one hour and then exposed to ClO₂ (100 ppm) for 24-hours or SDC (4%) for 2 minutes. Results were significant at P < 0.05. ClO₂ was the most effective treatment (P < 0.05) in controlling sessile E. coli and Listeria on HDPE and nylon in the Midwest and PNW. A lower level of inactivation was observed for biofilms grown on wood after ClO₂ treatment (P < 0.05). SDC did not reduce the population of sessile E. coli on HDPE in the PNW region. Neither E. coli bacterial forms were significantly reduced (P > 0.05) by SDS when grown on nylon, independently of the region. Conversely, biofilm population was reduced on HDPE after SDS exposure in both regions and bacteria tested (P < 0.05). Bacterial populations grown on wood surfaces were not reduced after exposure to SDC, regardless of form or bacteria type (P > 0.05). Since differences in sanitizer impact on surviving biofilm populations were observed in the previous studies, and in order to better understand the relationship between sanitizer-induced stress and gene expression, analysis was performed on differentially expressed genes (DEGs) in multi-strain L. monocytogenes biofilms after exposure to sub-lethal levels of chlorine, PAA, SDC, and lactic acid (LA) for 15 seconds. Biofilm-dwelling L. monocytogenes cells exhibited different transcriptome profiles in response to various sanitizers when compared to untreated biofilms. Few DEGs were noted in either the biofilms exposed to SDC and LA, indicating there was almost no change in the L. monocytogenes transcriptome. Exposure to PAA and chlorine caused relatively high levels of DEGs in L. monocytogenes, with 65 DEGs and 100 DEGs , respectively. Functional enrichment analysis found a 13-gene functional annotation cluster involved in the regulation of DNA-templated transcription was significantly regulated (Q-value = 0.0049) after exposure to chlorine. Both PAA and chlorine, which are categorized as oxidative sanitizers, had limited overlap in gene response. Only 10% (1/10) of the DEGs that were downregulated and 22% (28/126) of the upregulated DEGs were shared between the biofilms exposed to either. The results obtained from these data underscore the importance of understanding how different surface types, sanitizers, and environments all should be considered when attempting to characterize risk within the food supply chain. The presence of resident bacteria within facilities could enable further transfer of ARGs and MTGs, which subsequently can impact both medical treatments for disease caused by pathogenic bacteria, as well as future developments with antimicrobial agents and sanitizers. Surface type, in addition to sanitizer type, can result in significant differences in how effective an antimicrobial is at managing bacterial populations. Further research is needed to better understand how differences with bacterial form, surface type, and how exposure to stress from sanitizer applications can impact bacterial populations within facilities that are harvesting and handling minimally processed products.