ASSESSING THE DYNAMICS OF SALMONELLA NEWPORT IN TOMATO PRE-HARVEST ECOSYSTEMS: INSIGHTS FROM ENVIRONMENTAL SURVEYS AND PHENOTYPIC INVESTIGATIONS

Abstract

Illnesses caused by Salmonella have been associated with a variety of food commodities, such as nuts, spices, fresh produce, low moisture packaged foods, seafood, poultry, and other meat. This diversity highlights the adaptability of this pathogen to persist in various environments and demonstrates the genetic diversity of this microbe. Epidemiological trends reveal that infections attributed to this pathogen exhibit peak incidences during the summer months, a period that coincides with the peak harvest of fresh produce in agricultural regions across the United States. Investigation and trace-back of many fresh produce-related foodborne outbreaks often implicate the growing environment or surrounding areas as the source of contamination. Salmonella is capable of surviving for prolonged periods in soil, water and sediment while maintaining the ability to contaminate produce plants and cause human illness. To gain a better understanding of the ecology, distribution and persistence of Salmonella in the Atlantic region environmental sampling of surface waters and sediments were conducted. Collectively, over 1,600 samples were tested yielding 1,420 Salmonella isolates. Whole genome sequencing (WGS) identified the most common serovar as S. Newport sequence type (ST) 118 and suggests that Newport(JJPX01.0061) may be geographically sequestered to the Virginia Eastern Shore (VES), a major producer of tomatoes and where outbreaks linked to the consumption of them has occurred. Using five distinct S. Newport isolates, characterized by varying pulsed-field gel electrophoresis (PFGE) patterns, viability assays using the nematode Caenorhabditis elegans were conducted to investigate differences in the virulence potential of environmentally acquired isolates. Phylogenetic analysis revealed the related nature of ST118 Newports and provided a comprehensive diversity profile of environmental isolates found on the VES. After closing the genomes of Newport isolates with PFGE patterns significant to foods, we focused on the nearly genetically identical Newport-61 and the Newport-1015 isolates which differ by a ~1.7 Mbp genomic inversion. Viability assays with these isolates demonstrated that C. elegans exposed to Newport-61 had a significantly shortened lifespan compared to other strains tested said for one of the Newport-1015 which caused greater mortality. These findings enhance our understanding of the pathogenic potential of environmental S. Newport and highlighted the need to understand the regulatory mechanisms that contribute to virulence capacity. To clarify the potential mechanisms behind Newport's persistence and colonization within the tomato plant and its pre-harvest environment, a comparative analysis including both Newport and non-Newport strains was conducted using a phenotypic microarray approach. Under controlled conditions the difference in utilization capacity of various carbon, nitrogen, phosphorus and sulfur sources as well as nutrient supplements, osmolytes and various pHs were assessed. The results demonstrated that S. Newport exhibited distinct phenotypic traits in the presence of certain metabolites that may enhance their survival in agricultural settings. To understand the specific genetic and regulatory mechanisms that facilitate Salmonella’s adaptation to the agricultural environment, transposon mutant libraires were constructed. The transposon library will be used, as independent isolates, in tomato plants to identify the genes necessary for colonization and proliferation of this organism. Additionally, RNA expression studies in tomato intracellular fluid will highlight secondary regulation mechanisms that may also be contributing to the adaptive phenotype. This collective body of research demonstrates Salmonella’s pervasiveness throughout the pre-harvest environment as well as evidence to suggest geographical confinement of particular serovars, such as serovar Newport (JJPX01.0061). It further presents evidence that though serovars may be highly related, their phenotypic profiles and virulence capacity may not be predictable by their genetic sequence alone. Future research into the expression profile of these highly related strains in various environments as well as genes necessary for adaptation within them, will contribute to the understanding of Salmonella ecology in the tomato production environment, and highlight potential pathways for reducing the risk of foodborne outbreaks associated with contaminated produce.