Jan. 1, 2017 - Dec. 31, 2017Award Number
Center for Produce SafetyAmount Awarded
Keith Warriner, Ph.D.
University of Guelph
Dr. Suresh Neethirajan, University of GuelphSummary
Shiga Toxin producing Escherichia coli (STEC) are potentially highly virulent and can cause illness at levels of 10 cells if ingested by a susceptible host. Manure is a significant source of STEC and consequently when applied to land there is an interval of 90 – 120 days before harvest to permit any pathogens to die-off. In field trials it has been demonstrated that STEC die-off rapidly within the first weeks of being incorporated into soil but a sub-population persist and can be recovered beyond 120 days. This led to speculation that there is a persistent sub-population of STEC that have enhanced tolerance to stress encountered in the field and possibly post-harvest. In the proposed study the persistent (dormant) state will be studied in STEC. Specifically, the culture conditions that induce the dormant state will be elucidated along with potential genes implicated. Studies will then determine the extent to which dormancy contributes to persistence in soil and resistance to sanitizers. Finally, the virulence of STEC in the dormant state will be determined. The main benefit of the research relates to providing data for risk assessment and also to develop novel methods to make STEC more susceptible to pre-along with post-harvest interventions.
There has been an increase in the number of outbreaks linked to fresh produce contaminated with Shiga Toxin producing Escherichia coli (STEC). In a bid to enhance food safety there is a need to further understand the survival, resistance and interaction of the pathogenic group with produce. In recently completed studies the applicant has found that STEC introduced into soil harbor a persistent sub-population that can survive over extended periods. Further research has also found that certain STEC strains have the ability to acquire or lose virulence factors. Collectively, the results indicate that there are sub-populations within STEC cultures with different virulence and/or persistence. The persister state has been documented since the 1940’s although has not been studied in STEC. Persister or dormant state can be confused with Viable but Non-Culturable state (VBNC). There are commonalities but a distinct difference is that the persister state is caused by a metabolic switch that down regulates metabolism to enter a stage of dormancy thereby enhancing stress resistance. Moreover the persister state can be broken by metabolic triggers which may then initiate growth and become susceptible to stress as with the parent population. The proposed research will investigate the perister cells found within STEC isolated from different sources that include produce related foodborne illness outbreaks. The genomic sequences of the STEC strains has been published thereby enabling the correlation between persistence and genomic composition. Methods based on flow cytometry will be used to recover persister cells from the main population thereby enabling the persistence in soil and resistance to hypochlorite to be determined. An additional part of the study will establish metabolic triggers induce or break the dormant state in STEC. The triggers to be investigated will be relevant to fresh produce- specifically, plant root exudates, soil extracts, leafy green extracts, in addition to metabolic products. Subsequent work will further probe the interaction of dormant STEC with growing plants. Finally, the virulence of STEC cells derived from the dormant/persister state will be assessed. This will establish if virulence is lost for the price of persistence or if retained within dormant populations and subsequent progeny.
The significance of the persistent or dormant state in STEC has not been studied to any great extent although has far reaching implications with respect to food safety. The outcomes of the research could be that the persister population exists in STEC although virulent stains harbor a low proportion suggesting persistence is at the cost of virulence. Alternatively, it could be found that dormancy is induced by exudates in soil thereby resulting in enhanced persistence. Moreover, plant extracts could break dormancy thereby become virulent and hence, pathogenic to the consumer. In this event the persister state must be taken into account when developing detection and enumeration protocols, in addition to policy relating to manure management or post-harvest wash guidelines. The research will also form a foundation for future studies that will reduce persistence and resistance of STEC through determining approaches to induce or prevent breakage of the dormant state.
I) Determine the proportion of dormant cells within populations of the Top 7 STEC including isolates implicated in produce outbreaks.
II) Assess the role of different soil types and lettuce root exudates in the induction or breaking of the dormant state.
III) Establish the resistance of dormant STEC to free chlorine.
IV) Evaluate the virulence of dormant STEC with respect to shiga toxin production and attachment.