1a. Objectives (from AD-416):
Objective 1: Characterize pathogenic E. coli and Salmonella cell surface structures (fimbriae, pili, flagella) and elucidate their functions in interacting with abiotic environmental matrices and plant surfaces. Sub-objective 1.1. Develop methods for profiling and characterizing bacterial cell surface structures. Sub-objective 1.2. Determine the effects of environmental factors on the expression of various surface components of E. coli and Salmonella. Sub-objective 1.3. Determine the role of pathogenic E. coli and Salmonella surface structures in attachment to plant surfaces and to abiotic surfaces, and in biofilm formation and persistence. Objective 2: Elucidate survival strategies of E. coli and Salmonella strains under produce production, processing, and storage conditions. Sub-objective 2.1. Determine if produce sanitation and fresh-cut preparation environments promote rpoS related adaptive mutations in enteric foodborne pathogens. Sub-objective 2.2. Determine the role of periplasmic components of pathogenic E. coli and Salmonella in cell survival in low nutrient and low osmolarity environments.
1b. Approach (from AD-416):
Objective 1: A proteomic approach will be applied for developing the surface profiling technologies. Various cell surface proteins will be harvested using sheering or enzymatic shaving techniques or membrane- impermeable biotin mediated affinity purification. Proteins and pipetides will be identified using MALDI-TOF mass spectrometry and various liquid chromatography (LC) coupled MS detection technology. Besides the proteomic approach, antibody and micelle glycoprotein libraries will be tested in collaboration with CRADA partners. Similar approaches wil be used to determine the effects of environmental factors on the expression of surface proteins. Selected genes for targeted cell surface proteins will be mutated using site directed allelic change procedures and the effect of mutation on cell interacting with plant and environments will be studied using genetic and proteomic tools. Objective 2: Short-term and long-term nutrient starvation studies using Salmonella and E. coli O157:H7 under varying physiological conditions will be applied to determine the role of rpoS mediated adaptive mutations. In vitro growth conditions such as nutrient limited chemostat cultures, or vegetable wash waters in batch cultures will be utilized. Induction of acid tolerance by EHEC during different packaging conditions on various acidic and non-acidic produce during storage will be characterized. In a recent collaboration natural Salmonella and E. coli O157:H7 isolates undergone minimal subculturing (>3) in the laboratory media will be used to determine rpoS heterogeneity. Genes encoding for osmoregulated cytoplasmic glucans (OPGs) will be cloned and characterized using site directed mutagenesis. Functions of OPGs in cell surface and cytoplasmic protein expression, cell motility, biofilm formation and survival in adverse environments will be studied using genetics and proteomic approaches.
3. Progress Report:
Several techniques for preparing bacterial cell surface proteins have been tested. Cell surface protein sheering and concentration protocols have been optimized using ultrasonic blending, high frequency vortex, and microfiltration. A membrane-impermeable cleavable biotinylation reagent (Sulfo-NHS-SS-Biotin) was used to selectively label bacterial cell surface proteins. Labeled proteins are concentrated using neutoavidin affinity binding. Improvement to minimize cytoplasmic protein contamination is continuing. The interaction of E. coli O157:H7 with bacterial strains isolated from fresh produce processing plants, resulting in the formation of heterospecies biofilms, was assessed. Certain Gram-negative phytopathogenic bacteria, such as Burkholderia caryophylli and Rhizobium radiobacter, enhanced E. coli O157:H7 presence in dual-species biofilms. Two soil bacteria, Ralstonia insidiosa and Sphingomonas rhizophila, were able to significantly promote the presence of E. coli O157:H7 with a total increase of 0.7 log CFU/cm2 compared to its monoculture biofilm. Research is being conducted to understand this apparent synergetic process and the implications to sanitization practices. The effect of a new wash aid (T128) in combination with chlorine on Salmonella survival and infiltration in tomato was examined. As the organic load increased and free chlorine level decreases in chlorinated wash water, Salmonella were able to survive and infiltrate into the fruits through the stem scars. Application of T128 was found to significantly decrease this risk. The ability to survive under environmental stress conditions enables Salmonella spp. to successfully enter the food chain. Survival of human pathogens in vegetable wash waters is a persistent cause of food borne infections. We have identified two genes in the glucans gene family, opgB and opgC, which are essential to overcome detergent and sanitation agents which are used in produce wash steps. Genetic mutation in the corresponding gene rendered Salmonella strains incapable of rapid growth in the presence of detergents, however they remained fully virulent. Identification of genes essential for stress tolerance is crucial in designing specific sanitation agents to wash vegetables. The development of better cleaning agents to eliminate Salmonella spp. in food would be extremely beneficial to the food processing industry.