Illuminating the role of whole genome sequencing in produce safety

Whole genome sequencing (WGS) is rapidly becoming the gold standard for foodborne outbreak investigations by public health agencies around the world. However, as WGS continues to be developed as a tool it needs to be refined to maximize its potential, and thus reach the ultimate goal of speeding up investigations. Therefore, the goal of this project is to provide data to public health agencies like the FDA to improve the use of WGS as an outbreak investigation tool. Overall, this project will help to refine WGS as a tool for outbreak investigations for public health agencies to use during outbreaks. Furthermore, this project can be use by the produce industry to implement WGS for internal source tracking to identify “resident” versus “transient” pathogens, sources of contamination for either, and a better understanding of the breakdowns or gaps in prevention, thus improving produce safety by closing these gaps.

Due to its higher resolution, whole genome sequencing (WGS) is rapidly becoming the new gold standard for foodborne outbreak investigations. But, there is still a continued effort to refine the implementation of WGS thus improving accuracy while reducing investigation time. Foodborne pathogens often cycle around defined regions, which was a major factor behind the creation of the FDA’s GenomeTrakr network, because if you can match clinical cases to a defined region then the investigation can quickly narrow the search to that region. GenomeTrakr involves state, federal, commercial and international laboratories genome sequencing regional foodborne pathogens to create a public database of regional genomic pathogen profiles for investigations. However, as the pathogens continue to cycle through the environment in those regions the genomes may accrue mutations that could alter the regional genomic pathogen profile, which could limit the positive impact of GenomeTrakr on investigations. The goal of this project is to determine the mutation rates of Salmonella, Listeria, and Escherichia coli O157:H7 during long-term persistence in agricultural soil and irrigation water maintained under different geographical conditions. Understanding these mutational rates will help improve the development of GenomeTrakr for regional identification during an outbreak investigation. Furthermore, data from this project will assist the produce industry in developing WGS for internal source tracking to identify “resident” versus “transient” pathogens, sources of contamination for either, and better understand the breakdowns or gaps in prevention methods, thus improving produce safety by closing these gaps.

1. Determine mutational rates of pathogens during persistent colonization in different agricultural environments under distinctive geographical environmental conditions.

Unfortunately, in many cases, the pathogen-inoculated soil and irrigation water samples kept under Yuma-AZ conditions did not have high levels of pathogen DNA that could be used to accurately identify any mutations present in the pathogens. In fact, many of these samples did not have any pathogen DNA present in the sequence data after analysis, which is appropriate considering that the pathogens survived less than two weeks in any of the long-term samples or in most of the environmental cycling samples under the Yuma-AZ conditions; the lack of pathogen DNA present in the sample for sequencing resulted in the failure to get quality pathogen DNA for sequencing in these samples. However, this finding is a good indication to the produce industry that E. coli O157:H7, S. Typhimurium, and L. monocytogenes do not survive very well under Yuma-AZ conditions at least from September to November, but will survive slightly longer from December to mid-June, at least when cycling between environments. This was evident in the development of single mutations at week 42 in soil cycling for E. coli O157:H7 and L. monocytogenes. Salmonella was non-culturable in Yuma conditions at week 12 (November conditions), but WGS found mutations developed in the water cycling and soil/water cycling indicating that Salmonella might have been viable but non-culturable (VBNC) at that time point. However, further studies would be required to determine if VBNC pathogens, particularly Salmonella, can replicate and incur mutations in cycling through these environments. In contrast, pathogen-inoculated soil and irrigation water samples kept under Salinas-CA conditions did yield quality levels of pathogen DNA from the sequence data, which allowed for determination of any mutations that occurred during the long-term survival in these environments. Results from the long-term survival environments found that there were no mutations in the pathogen population even after 42 weeks, which indicates that the pathogens are surviving in these environments but not actively replicating. Therefore, if the pathogens are simply surviving in the environment in the long-term there is minimal risk of pathogens developing high numbers of mutations that would alter the data present in the GenomeTrakr database. However, the addition of cycling between new environments every few weeks or monthly during the study revealed a different result, as there were mutations detected in the pathogens in the different environments including both cycling through soil and irrigation water every two weeks. The goal of this project was to study the mutational rates of the pathogens in different agricultural environments to improve WGS for rapid outbreak identification and source tracking. The project found that no mutations occurred in E. coli O157:H7 during long-term colonization in soil or irrigation water under either condition studied. However, E. coli O157:H7 did have mutationrates from 1.24 to 2.48 mutations/yr in cycling through irrigation water and soil under the Salinas conditions, respectively. Under the Yuma conditions the only mutation rate was 1.24 mutations/yr during cycling through soil samples every few weeks. Like E. coli O157:H7, S. Typhimurium did not have any mutations develop during long-term colonization in soil or irrigation water under either condition. Interestingly, S. Typhimurium did have mutation rates from 1.24 to 2.48 mutations/yr in cycling through irrigation water and soil/irrigation water under Yuma conditions, respectively; in contrast, under the Salinas conditions the only mutation rate was 2.48 mutations/yr during cycling through soil samples every few weeks. L. monocytogenes also did not develop any mutations during long-term colonization of either soil or irrigation water under either of the conditions. However, L. monocytogenes did have the highest mutation rates of the three pathogens, ranging from 3.71 to 6.19 mutations/yr in water cycling and soil cycling under the Salinas conditions, respectively; under the Yuma conditions the mutation rate was 1.24 mutations/yr during cycling between soil samples every few weeks. Thus, these three pathogens do not appear to develop any mutations during long-term colonization of either agricultural soil or irrigation water regardless of the conditions that the pathogens are under. On the other hand, cycling between environments every few weeks or monthly, particularly different soils, results in mutations occurring in all three of the pathogens.