Genomic elucidation of the physiological state of enteric pathogens on pre-harvest lettuce

Agents of foodborne disease present in the pre-harvest environment can contaminate produce and enter into the food supply. The methods used by these pathogens to survive on pre-harvest produce have the potential to impact survival of the pathogen during post-harvest processing. The purpose of this work is to determine how the enteric pathogens EHEC and Salmonella respond to the pre-harvest environment by monitoring changes in gene expression under growth conditions representing those occuring during different harvest seasons. Changes in gene expression will be used as an indicator of the physiological state of the pathogen under different conditions. The physiological state of the pathogen plays a significant role in its ability to survive subsequent stresses, such as decontamination treatments and those stresses encountered during distribution. Understanding how these pathogens survive on plants is the first step in mitigating the presence of these pathogens in the food supply. Data resulting from this research determining how pre-harvest conditions impact the ability of EHEC and Salmonella to survive in the food chain will provide insights for developing effective post-harvest treatments to reduce consumer exposure to these pathogens via produce.

Contamination of produce with enteric pathogens can occur in the pre-harvest environment, and many produce associated outbreaks have been traced back to the pre-harvest environment. Once contamination of produce in the field occurs, the enteric foodborne pathogens EHEC and Salmonella are capable of surviving on the plant surface over long periods of time, yet little is known about the physiological state of the pathogen in this environment, and how pre-harvest environmental factors influence the physiological state of the pathogen. Transcriptional profiling techniques have tremendous potential for improving our ability to understand the physiological state of pathogens in the pre-harvest environment. Here we propose to directly assess the physiological state of EHEC and Salmonella associated with lettuce leaves under pre-harvest conditions by quantifying genome-wide changes in gene expression. This comprehensive transcriptional assessment of the physiological state of pathogens associated with lettuce will indicate how the pathogens are responding to this non-host environment. Changes in gene expression under different lettuce cultivation conditions will be used to predict which stress responses are activated by the pathogens. As exposure to environmental stress has the potential to influence how these pathogens survive subsequent stresses, the proposed research directly assesses how the physiological state of the pathogens on lettuce plants impacts the ability of the pathogens to survive post-processing decontamination procedures, such as a chlorine wash, as well as the ability of the pathogen to survive gastric acidity, through phenotypic survival assays. The physiological state of enteric pathogens on pre-harvest produce can also significantly impact the ability to detect these pathogens in this environment. Therefore, it is critical to understand the physiological state of enteric pathogens on pre-harvest produce for development and assessment of appropriate detection methods. The goal of this proposed research is to determine how pre-harvest conditions impact the ability of EHEC and Salmonella to survive in the food chain, providing insights for developing effective post-harvest treatments to reduce consumer exposure to these pathogens on produce. The researchers will determine how pre-harvest conditions impact the ability of EHEC and Salmonella to survive in the food chain. This data will be used to develop effective post-harvest treatments to reduce consumer exposure to these pathogens on produce.

1) Identify the physiological state of Salmonella and EHEC on lettuce plants. 

2) Determine if pre-harvest environmental conditions affect the physiological state of Salmonella and EHEC on the surface of lettuce plants. 

3) Determine if the physiological state of Salmonella and EHEC on lettuce plants influences subsequent survival during decontamination washes as well as during gastric passage.

• Different strains within the same EHEC or Salmonella serotype can show considerable variation in survival on lettuce plants 

• Our data suggest that validation studies on post-harvest interventions and specifically chlorine washes may not correctly estimate the effectiveness of these washes, unless challenge strains have not been adapted to lettuce for at least 3 days. 

• EHEC and Salmonella associated with pre-harvest lettuce are experiencing osmotic and oxidative stresses, as indicated by the changes in gene expression during incubation on lettuce. This may suggest changes in the susceptibility of these pathogens to osmotic and oxidative stresses after adaptation to lettuce surfaces in the pre-harvest environment.Recommendations 

• Evaluation and validation of post-harvest pathogen interventions for leafy greens should use pathogen strains that have been adapted to the leafy green environment for at least 3 days 

• Our data show that both strain and environment can have a significant impact on physiological state, suggesting that further work is needed to determine how strains and pre-growth conditions should be selected for challenges studies and evaluation of interventions 

• Genes that show consistent high transcript levels on lettuce should be further pursued as potential targets for mRNA-based detection systems, which may provide significant advantages over traditional DNA-based methods as (i) mRNA is found at a higher level than DNA in a given cell, thus allowing for more sensitive detection in a shorter time frame and (ii) as mRNA is rapidly degraded in a dead bacterial cell. Our data suggest that osmB, osmC, sodC, and outer membrane protein encoding genes such as yiaD, yadM, and cmeC are likely candidates for mRNA-based detection, and should be evaluated further.