Comparative genomics analysis and physiological assessment of the avirulent Salmonella surrogate relevant to produce safety

Coliforms and generic E. coli are poor predictors of the behavior of human pathogens (like Salmonella, pathogenic E. coli and Listeria) in the crop production environment. Mounting evidence suggests that accurate models of Salmonella behavior in the production environment will have to be built based on the experiments conducted with Salmonella, and not based on data from distantly related surrogates like generic E. coli. This, however, necessitates availability and careful characterization of “disarmed” strains of Salmonella that could be used for on-site research. Upon completion of this study we will have developed robust tools for modeling behavior of these outbreak strains in the pre- and post-harvest production environments. The purpose of this project is to carry out comparative genomic and physiological characterization of the outbreak strains under production conditions and to compare them with the nonvirulent strain of Salmonella that we have developed. We will also have tested two key hypotheses aimed at understanding why only a dozen out of over 2,500 Salmonella serovars are associated with produce-linked outbreaks of illness. With previous CPS funding we engineered and verified the first nonvirulent, nontransgenic strain of Salmonella suitable for on-site studies as an indicator organism.

Outbreaks of produce-borne salmonellosis have been disproportionately associated with only a few of the 2,500 Salmonella serovars. Several hypotheses could explain this observation. From a practical point of view, however, it is important to define what makes these strains more likely to occur and persist in the fruit and vegetable production chain in order to reasonably accurately model their behavior under the conditions relevant to produce industry, with the aim to further reduce the occurrence and persistence of pathogens in the production environment or disrupt their virulence. Our comparative genomics analysis of the outbreak strains will reveal features and potential physiological adaptations that may be responsible for the persistence of these serovars in the production chain (soil, water, plant surfaces and tissues). This information will be integrated with the on-going characterization of the avirulent surrogate strain of Salmonella Typhimurium developed by our group to determine to what extent it could be used to model behaviors of the outbreak strains. Furthermore, once relevant features in the outbreak strains are identified, an additional surrogate strain may be constructed. To accomplish the goals of this project, the following objectives will be completed: 

Objective 1: Characterization of the outbreak S. enterica serovars under the conditions that simulate pre- and post-harvest environments, and comparison to the avirulent S. enterica Typhimurium surrogate. Biofilm formation, desiccation, survival in soil and water, colonization of leafy greens, tomatoes and cantaloupes by Salmonella serovars Saintpaul, Newport, Braenderup, Javiana, Montevideo and Heidelberg will be tested and compared to that of the avirulent surrogate to determine to what extent serovar-level differences are responsible for these phenotypes. These experiments will determine how well the already constructed avirulent surrogate mimics behaviors of the outbreak strains under production-relevant conditions. 

Objective 2: Identification of the core and accessory genomes of the serovars associated with produce-related outbreaks and comparison to the avirulent surrogate. In parallel with identifying serovar-level differences (Obj. 1), we will define the core genome that is composed of the genes shared by all strains of the selected serovars. The core genome represents genes and physiological functions that are present in all tested Salmonella serovars. The “accessory genome,” which includes genes unique to each serovar and strain, also will be identified. It is most likely that physiological functions associated with increased persistence in the crop production environment of some strains are encoded within the accessory genome. Therefore, characterization of the accessory genome will help further optimize the avirulent indicator strain or engineer additional surrogates. 

Objective 3: Functional characterization of the core and accessory genomes of the serovars associated with produce-linked outbreaks. The main goal is to connect behavior of the outbreak strains (defined in Obj. 1) with the comparative genomics data (Obj. 2) to determine genetic and physiological determinants of the serovars’ differential fitness under pre- and post-harvest conditions. To this end, specific genomic regions unique to the outbreak strains will be deleted and the resulting mutants will be tested under the same conditions that approximate the environment commonly found in the produce industry.

1. Characterization of the outbreak S. enterica serovars under the conditions that simulate preand post-harvest environments and comparison to the avirulent S. enterica Typhimurium surrogate. 

2. Identification of the core and accessory genomes of the serovars associated with producerelated outbreaks and comparison to the avirulent surrogate. 

3. Functional characterization of the core and accessory genomes of the serovars associated with produce-linked outbreaks.

Previous funding from CPS supported the construction of a non-virulent surrogate strain of Salmonella that is suitable for on-site experiments. The current project demonstrated that this avirulent surrogate behaves like the parental virulent strain during growth in produce, persistence in soil and water, and in its sensitivity to heat and disinfectants. Thus, we have validated the use of this fully avirulent, non-immunogenic, non-transgenic, and antibiotic sensitive surrogate as a new tool in addressing FSMA regulations. In addition, the pangenome analysis of over 2,000 serovars of Salmonella revealed high diversity in the genomic composition across strains. Genomes of the Salmonella serovar Newport, which has been associated with several produce-linked foodborne illness outbreaks, had a set of unique core genes that may be involved in adaptation to growth in plants. A massive parallel screening of mutants revealed that growth of Salmonella serovars Typhimurium and Newport required the production of lipopolysaccharide and nucleotide biosynthesis, regardless of the host. However, growth in plants required genes involved in the biosynthesis of amino acids, due to the low availability of amino acids in plant tissues. In addition, the novel gene papA (for plant associated protein A) was identified in Salmonella Newport and contributed to fitness of the strain in tomatoes. While the function of papA is currently unknown, homologues of this gene were found in Enterobacteriaceae capable of growth in both plants and animals. Collectively, the pangenome analysis supports the idea that only a handful of Salmonella serovars can grow in produce because their genotype contains genes for adaptation to growth in plants. Furthermore, the discovery of this novel plant-associated Salmonella gene could be used as a marker gene to detect Salmonella that flourish in both plants and animals.