Persistence and detection of norovirus, Salmonella, and pathogenic Escherichia coli on basil and leafy greens.

The questions addressed in this proposal are applicable to major priority area 2 of the CPSBARD program: Survival and growth requirements for human pathogens on produce, as well as minor priority area 4: Definition of human pathogen reservoirs and vectors and quantification of transference. In the U.S. it is estimated that there are approximately three million cases a year of illness caused by fecally contaminated fruits and vegetables; in Israel at least 20% of foodborne illnesses are attributed to fruits and vegetables. Noroviruses, Salmonella and pathogenic Escherichiacoli are the three main groups of enteric pathogens associated with fresh produce and selected for study by the three participating laboratories. The overall objective of this study is to assess the persistence and transfer of norovirus, Salmonella, Escherichia coli O157:H7 and avian pathogenic E. coli (APEC) on leafy greens (lettuce and spinach) and basil, to gain information to better enhance the safety of produce and reduce transmission of these pathogens in the field. Technion‐Israel will use basil and spinach to examine persistence and mechanisms of attachment of Salmonella employing mutant strains for study of factors affecting attachment to foliar surfaces. The University of Delaware and USDA‐EMFSL in Beltsville will evaluate lettuce, spinach and basil in their studies of norovirus and bacteria; the University of Delaware will investigate persistence of noroviruses, APEC and nonpathogenic E. coli on plant surfaces in a comparison study using spot inoculation, while USDA‐EMFSL will evaluate E. coli O157:H7, APEC, nonpathogenic E. coli, and Salmonella simulating field conditions using lower, more realistic population levels and different irrigation regimes. Methodology will include enumerating of pathogens by culture methods and RT‐PCR and localization by microscopy. USDA‐EMFSL and the University of Delaware will share detection and enumeration protocols for the shared varieties of E. coli under study. In addition to RT‐PCR, norovirus will also be quantified by plaque assay. Completion of the project incorporating three different plant environments from the three laboratories will generate information that better characterizes and predicts the survival and transmission of these pathogens at more realistic levels of contamination in plants in the field.

Overall: To investigate the transmission and persistence of norovirus, Salmonella, and Escherichia coli O157:H7 (and other pathogenic E. coli) introduced to leafy green foliar surfaces in water using more realistic lower population levels, different irrigation regimes and other related factors. University of Delaware: To investigate the persistence of norovirus on lettuce with viral detection and counting by plaque assay and RT-PCR, and determine the sites of adherence on produce using confocal microscopy. Technion – Israel Institute of Technology: To determine the effect of the irrigation regime on transfer and survival of Salmonella in plants by dripping vs. spraying, day vs. night, summer vs. winter crops, daily vs. multiple short irrigations and other associated factors. USDA, ARS‐EMFSL (Beltsville): To determine the fate of enterohemorrhagic, avian pathogenic (APEC) and nonpathogenic E. coli introduced to leafy green foliar surfaces in irrigation water at levels stated in the California Leafy Green Marketing Agreement.

University of Delaware: MNV-1 was taken up by Romaine lettuce through the roots via contaminated irrigation water and reached edible leaf tissue. The internalization of human enteric viruses into produce during irrigation is possible under favorable conditions, and the fact that some internalized virus remained infectious poses a food safety threat. Furthermore, the virus may be taken up in a passive manner by transpiration. The exact method of virus internalization under different environmental conditions, such as soil water content and environmental relative humidity, is still unclear. With regard to unexpected outcomes, as noted on page 4, results were inconsistent among published studies that have been conducted assessing the impact of internalization of human enteric viruses or bacteriophage into produce during hydroponic or traditional growth conditions. As noted, these conflicting results may be a result of variable plant properties affecting virus penetration and uptake, as well as influence from different experimental parameters of produce growth, irrigation, and soil conditions. In a concluding collaborative study between the University of Delaware and the USDA Beltsville, the detection of low populations of strains of APEC and E. coli O104:H4 ten days postinoculation indicated that APEC and E. coli O104:H4 may be better adapted to environmental conditions than strains of E. coli O157:H7. This is the first reported study of E. coli O104:H4 on a produce commodity. These results suggest a variety of pathotypes of E. coli harbor potential for environmental transfer to foods. Technion – Israel Institute of Technology: Microbiological and molecular-based methods were developed and shown to precisely quantify different amounts at low levels of Salmonella enterica serovar Typhimurium artificially inoculated on parsley leaves. These methods can be applied to study transfer of Salmonella from contaminated water or soil to plants using low and more reasonable levels of contamination. Higher levels of Salmonella were detected in the phyllosphere when plants were irrigated at night compared to irrigation during the morning, and during winter when compared to the other seasons. Further elucidation of the mechanisms underlying the transfer of Salmonella from contaminated water to crops, as well as its persistence over time, will enable the implementation of effective irrigation and control strategies. Susceptibility experiments demonstrated that S. Senftenberg was resistant to basil oil and its antimicrobial compounds and grew well on basil. This strain of S. Senftenberg may have adapted to the basil environment by developing resistance to components naturally found in basil oil and thus harbor increased risk to human safety. The emergence of resistant pathogens to naturally occurring antibacterial substances may have significant potential to alter the ecology of foods and enhance the ability for pathogens to survive in new niches in the environment, such as basil and other plants. USDA, ARS‐EMFSL: APEC generally survived at population levels ~ 1 log MPN/g higher than E. coli O157:H7 on lettuce. Whether this difference in survival is related to an enhanced environmental fitness of APEC strains compared to E. coli O157:H7 is unclear. Since the APEC inoculum consisted of four strains compared to one strain for the E. coli O157:H7 inoculum, it is possible that the strain diversity in the APEC inoculum contained one or more strains which were more persistent than the E. coli O157:H7 outbreak strain used in this work. APEC strain 07-1707 is an E. coli O157 serotype and was used in both individual inoculation studies on basil and in simultaneous inoculation studies on spinach and lettuce. The potential survival of this strain compared to E. coli O157:H7 on all three commodities may indicate that persistence on foliar surfaces is less a function of serotype and more dependent on source of isolation or previous environmental exposure of the strain (i.e., adaptation). Previous studies demonstrated that when co-inoculated on to spinach foliar surfaces, non-pathogenic E. coli isolates from produce commodities survived at higher populations for up to 28 days, compared to E. coli O157:H7 strains from produce outbreaks, which only survived for 7 days. The specific geospatial origin of an isolate has been suggested to affect environmental fitness. Our findings indicated that E. coli O157:H7 strains from produce outbreaks may not survive as well in nonhost environments (e.g., foliar surfaces, soil, water) as E. coli isolated from environmental sources, where a greater opportunity exists for adaption to stresses in pre-harvest, leafy greengrowing environments. These findings support the hypothesis that E. coli O157:H7 outbreak strains may not possess the environmental fitness of other environmentally-isolated E. coli isolates.