Non invasive imaging approaches to evaluate potential infusion of pathogens during vacuum cooling of lettuce leaves and real time dynamics of microbes on leaf tissues as a function of moisture content.

The proposed research is aimed at developing non-invasive imaging methods to characterize interactions of pathogens with leafy green vegetables and evaluate the potential risk of infiltration/infusion upon vacuum cooling of leafy greens. The proposed study uses lettuce as a model system to demonstrate the potential of non-invasive imaging methods to detect changes in structure of pathogens both on surface and in side of leafy green vegetables as a function of moisture content. With vacuum processing, effect of different varieties of lettuce, role of process conditions (spray water, vacuum pressure and release rate of vacuum) and differences in laboratory and field inoculated lettuce on localization will be compared using non-invasive imaging methods. Results of this study will provide a comprehensive assessment of potential risks of infiltration during a vacuum cooling process and practical approaches to address the risk factors.

There is an unmet need to characterize in-situ interactions of pathogens on fresh produce as a function of environmental factors and post harvest processing. Non-invasive imaging methods can map real time dynamics of pathogens of fresh produce and can complement the scientific information obtained using microbiological and gene expression studies. The significant limitations of current confocal and widefield imaging methods are limited depth of detection (30-50 microns) and lack of sensitivity to detect precise location and limit contributions from leaf autofluorescence. The proposed research plan proposes to address these significant limitations using a combination of multiphoton and confocal reflectance imaging. This imaging approach provides simultaneous information on localization of pathogens in a leaf matrix and morphological structures of a leaf. The overall aim of the proposed research is to characterize distribution of microbes (on surface and inside) of a fresh produce as a function of environmental and processing conditions during vacuum cooling. The specific research aims of the proposed research are: (a) Develop a non-invasive imaging approach for in-situ measurement of localization of microbes in a plant matrix and characterize changes in distribution of microbes as a function of moisture conditions; (b) Evaluate potential of infusion/infiltration of microbes in selected leafy green vegetables and determine the role of process control variables in controlling surface localization of microbes during a vacuum cooling process. The proposed research plan contributes to two critical needs: a) develop tools to understand organization and physiology of pathogens on leafy greens; b) understand changes in microbial localization with processing particularly vacuum cooling process and develop practical solutions to address potential risks of infusion/infiltration during vacuum cooling. The proposed study will use lettuce as a model system. To understand differences in laboratory and field conditions, the proposed research will use a combination of both field and laboratory inoculated lettuce leaves. These studies will be conducted in collaboration with Dr. Linda Harris. Laboratory inoculated samples will be inoculated using an attenuated strain of E. coli with a constitutive expression of GFP, while field grown lettuce will be inoculated using the same strain of E. coli stained with vital dyes. This model leaf-pathogen system will be characterized to detect distribution of pathogens in a leaf matrix as a function of moisture content and vacuum processing. Population dynamics and changes in distribution of pathogens on surface will be characterized as a function of moisture content (ranging from 20% to 90%) using non-invasive imaging approaches. With vacuum processing, effect of different varieties of lettuce, role of process conditions (spray water, vacuum pressure and release rate of vacuum) and differences in laboratory and field inoculated lettuce on localization will be compared using non-invasive imaging methods. Results of this study will establish a novel non-invasive approach to characterize in-situ distribution of microbes on intact leafy vegetables, characterize changes in localization of microbes on surface of produce as a function of moisture content and a comprehensive assessment of potential risks of infiltration during a vacuum cooling process and practical approaches to address the risk factors.

1. Develop a non-invasive imaging approach for in-situ measurement of localization of microbes in a plant matrix and characterize changes in distribution of microbes as a function of moisture conditions 

2. Evaluate potential of infusion/infiltration of microbes in selected leafy green vegetables and determine the role of process control variables in controlling surface localization of microbes during a vacuum cooling process

• Results of multiphoton imaging highlight the significant advantages in spatial mapping of the distribution of microbes on surface and inside of intact lettuce leaves as compared to confocal microscopy. 

• Results of microscopic measurements demonstrate no significant increase in infiltration of microbes during vacuum cooling process. These results were validated using fresh lettuce from green house as well as lettuce leaves from the market. 

• Results also highlight a slight increase in association of microbes with stomata with vacuum cooling as compared to control lettuce samples. However, statistical analysis of the data highlight that the increase was not significant for all conditions except high inoculation level of microbes on lettuce leaves (6 log CFU/ leaf disk) under low moisture conditions. 

• Further studies are needed to quantify if dispersion of microbes during vacuum cooling process is responsible for the increase observed in association of stomata with microbes. In addition to direct inoculation of microbes on surface of lettuce leaves as used in this study, further analysis may also include analysis of pre-formed biofilms on the surface of produce to mimic the environmental conditions.

• Further studies are required to compare these measurements based on attenuated strain of E. coli 0157:H7 with the measurements using pathogenic strains. These measurements may also include following the fate of microbes over an extended period of time after vacuum cooling process. This is particularly important for the population of microbes that show an increase in association with stomata following vacuum cooling process.