Significance of the dormant state in the persistence, interaction with growing plants and virulence of Shiga Toxin producing Escherichia coli

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.

1. Determine the proportion of dormant cells within populations of the top 7 Shiga toxin– producing Escherichia coli (STEC), including isolates implicated in produce outbreaks. 

2. Assess the role of different soil types and lettuce root exudates in the induction or breaking of the dormant state. 

3. Establish the resistance of dormant STEC to free chlorine. 4. Evaluate the virulence of dormant STEC with respect to Shiga toxin production and attachment.

The persister state in bacteria was first documented in the 1940s, although interest has been primarily in clinical microbiology with respect to antibiotic resistance and infectivity. Within the food sector, the main interest in dormancy has been with respect to viable but nonculturable (VBNC). Although still debated, the persister state can be differentiated from VBNC by the fact the former is induced in a population of the cells and ultimately the bacteria can be cultured under appropriate conditions. In contrast, VBNC represents a state that cells undertake by a progressive loss of viability to the point of nonculturability. It is likely that text referring to VBNC was actually observing persisters, given that it was possible to eventually culture cells. The current study was one of the first to explore the significance of the persister state in STEC. Importantly, the study was placed into the context of how the persister state can influence the activity of STEC within the fresh produce chain. Baseline studies evaluated the extent to which the persister state was encountered within STEC, especially those belonging among the Big 7 serotypes. Although persisters could be recovered from all strains tested, it was evident that E. coli O157:H7 and O103 produced a higher proportion compared to the other serotypes tested. Given that only a single strain of a serotype was screened, it would be premature to assume that no intra-strain differences in the perister yield exist. Yet, it is clear that different E. coli types have a range of abilities to enter the persister state. Through use of spectroscopic techniques, it was illustrated that persister cells could be differentiated from the main population. Moreover, it was illustrated that the induction of the persister state occurs in the lag phase and a more rapid adaption compared to the VBNC state that requires an extended time to enter. From the range of inducing agents, only indole was found to increase the proportion of persisters within the STEC strains tested. Indole is encountered in soil in varying amounts depending on the presence of producing and utilizing microbes, in addition to exogenous sources in waste. It is possible that the antimicrobial activity of indole causes the induction of the persister state, or effects could be more indirect as a physiological regulator. It was interesting to note that the other agents tested did not lead to increased persisters but the majority decreased the numbers recovered. Bile salts (or more correctly the breakdown products of bile metabolism) are antimicrobial in a similar way to indole, yet inclusion in the growth medium reduced the levels of persisters. Enteric bacteria typically use bile and bile breakdown products as metabolic regulators to sense the status of the gastrointestinal tract. In this respect, the presence of bile could have provided a metabolic signal for E. coli to initiate growth as opposed to entering dormancy. It may have been expected that dormancy would be promoted in the soil environment, given that the environment would increase the need for survival as opposed to growth. However, it is more likely that the soil environment selected for persisters rather than inducing the dormant state. This may reflect that the history of the STEC bacteria could be more relevant, be it manure or irrigation water, which in turn would suggest the induction of the persister state within water or gastrointestinal tract could be more relevant. Similar to soil, seed exudates from sprouting seeds (mung beans) depressed the formation of persisters relative to controls. This result may have been expected, given that the release of nutrient-rich exudates would be conducive to growth of STEC as opposed to entering a dormant state. The composition of root exudates changes during plant development, so it is possible that in maturing plants, constituents may result in a greater proportion of persisters. However, this will need to be demonstrated experimentally. The higher tolerance of persisters to stresses, such as survival in soil or to hypochlorite, was confirmed in the current study. By down-regulating metabolism, cells become tolerant to stressful environments and hence provide long-term survival. Indeed, it is thought that the dormant state evolved to ensure that a proportion of a bacterial population survived in the event of a sudden imposition of stress. The main significance of the findings in the current study is that one could expect STEC to be primarily in the persister state in the natural environment and hence more stress tolerant than laboratory-prepared cultures. This is one likely reason why validation studies (e.g., with post-harvest washes) do not tend to reflect observations made in commercial practice. This is the case for both predictive modelling for pathogens in soil along with the efficacy of post-harvest washing. Although still to be studied, it could also apply to tolerance of pathogens to stresses encountered within the food industry. With respect to pathogen control, there is interest in identifying agents that could be used to activate dormant cells, thereby making them more susceptible to stress and amenable to culturing. In the current study, it was noteworthy that inclusion of lettuce extract in recovery media enhanced the recovery of persisters. It is unclear at this time if there is a specific constituent within lettuce that activates the cell or is a combination of compounds. Using nutrient rich media (e.g., tryptic soy or brain heart infusion) did not stimulate recovery of persisters, thereby suggesting there are key components that alleviate the persister state. In the current study, the extract from leafy green lettuce was used and it is unclear if different varieties or other produce types would have the same effect. In a commercial context, it can be envisioned that processed lettuce could increase the risk of STEC persisters being activated and then undergoing outgrowth if the product is temperature abused. Yet, the finding could have positive effects when recovering STEC from produce and environmental samples. In this respect, it is noteworthy that culturing STEC from PCR-positive samples still represents a challenge, with a high frequency of false positives recorded. However, all the aforementioned are purely speculative at the current time and will require further research. Recommendations: It can be envisaged that the perister state is frequently encountered with pathogens associated with the fresh produce chain. Despite the significance, there are many aspects of the persister state in STEC and other foodborne pathogens that remain unknown. 

Such knowledge gaps need to be addressed, given the impact the persister state has on pathogen survival, resistance and culturability. Based on the results of the study, further research is recommended to: 

• Assess the extent to which persisters are generated by a diverse range of STEC strains and serotypes. 

• Identify regulatory genes within STEC that control induction and breaking the persister state. 

• Establish if indole concentration in soil promotes persister formation and thereby can be used as an additional marker to identify high (food safety) risk fields. 

• Evaluate the significance of the persister state on the survival of STEC in the rhizosphere, phyllosphere and internalized populations. 

• Evaluate the resistance of STEC persister cells to a diverse range of sanitizers encountered within the fresh-cut chain. 

• Establish if amending fields with lettuce extracts would decrease the persistence of STEC within the environment. 

• Identify constituents within lettuce that can reactivate persisters thereby potentially leading to lower survival in the environment, increased susceptibility to sanitizers and increasing the culturability of STEC.