Summary
More fresh fruits and vegetables are grown, sold and eaten today than at any other time in history. Unfortunately, outbreaks of food poisoning caused by pathogenic bacteria in fresh produce are also more common than in the past. Products are routinely analyzed to ensure that they are free of such bacteria, but some are very difficult to find because they do not grow on media used by quality control laboratories. These are known as “viable but non-culturable” (VBNC) bacteria. We will develop a new, inexpensive and easy-to-use method based on the well-known PCR reaction to make it possible for quality control laboratories to detect two important pathogens in fresh produce, Salmonella and E. coli, even when they are in the difficult-to-find VBNC form. The performance of the new method will be checked through field trials and pilot plant experiments with leaf lettuce. Information from these experiments will be used to reveal how likely fresh produce is to be contaminated with VBNC pathogens during production and after harvest. Therefore, this research will contribute to an important new laboratory method and key information to support on-going efforts by the industry to improve the safety of fresh fruits and vegetables.
Technical Abstract
Comprehensive data about the prevalence of bacterial pathogens is essential for the development of accurate risk assessments and relevant quality control systems for fresh produce. None of the methods commonly used for the microbiological analysis of fresh produce can account for bacterial pathogens in dormant physiological states that preclude detection, notably the viable but non-culturable state (VBNC). We will develop and validate a new diagnostic assay by modification of the loop-mediated isothermal amplification (LAMP) method through incorporation of an intercalating dye to enable the rapid and sensitive detection of both viable and VBNC Salmonella and Shiga toxigenic E. coli (STEC) in fresh produce. The method will be used to examine the survival of VBNC Salmonella and STEC in lettuce in the field and during simulated fresh-cut processing studies and the data applied in a semi-quantitative assessment of the attendant risks. Hence, this research will provide a rapid, low-cost, and easily interpreted diagnostic assay to improve produce safety. The acquired knowledge of risk implied by VBNC Salmonella and STEC on fresh produce will improve fresh produce safety by 1) ensuring produce grown in California and the USA meets or exceeds industry and regulatory food safety standards and 2) reducing foodborne illness caused by the consumption of contaminated fresh produce.
Research Objectives
The central research objective is to modify loop-mediated isothermal amplification (LAMP) methods by incorporation of intercalating dye to enable rapid and sensitive detection of both viable and VBNC Salmonella and Shiga toxigenic E. coli (STEC). Assay performance will be validated and field/processing trials conducted to provide data to support an analysis of the risk implied by the survival of VBNC Salmonella and STEC in lettuce. To achieve this central research objective, we propose the following individual research objectives:
1. Induce the VBNC state in relevant bacterial pathogens. The effect of low nutrient and temperature on VBNC induction will be examined in Salmonella and STEC by measurement of total culturable and VBNC cells over time.
2. Assess intercalating dye performance and optimize treatment condition (dye concentration, treatment time, etc.) of intercalating dye. The ability of ethidium monoazide and propidium monoazide to penetrate viable cells will be compared to select the dye less likely to cause consequent false positive results.
3. Develop a LAMP assay, optimize assay conditions for both Salmonella and STEC, and verify assay performance in vitro.
4. Develop and optimize a PCR assay for both Salmonella and STEC and verify assay performance in vitro.
5. Optimize conditions for the detection of pathogens in spiked produce (lettuce, tomato, cantaloupe and spinach) with the intercalating dye-LAMP assay and verify assay performance in planta.
6. Determine the survival of VBNC Salmonella and STEC on lettuce in the field and during simulated processing.
7. Risk assessment. Perform semi-quantitative assessment of the risk implied by Salmonella and STEC in the VBNC state using data from field trials and simulated processing.
Findings & Recommendations
In the current study, the PMA-LAMP assay was validated to be rapid (from 15 to 45 min), specific (100% inclusivity and 100% exclusivity), sensitive (1–20 CFU/reaction in pure bacterial culture), quantitative (R2 from 0.896 to 0.996), and robust. Qualitative risk assessment was performed based upon the field study conducted in Summerland, BC, Canada. There were relatively high risks related to the consumption of romaine lettuce contaminated with non-VBNC and VBNC E. coli O157:H7 and Salmonella. The major findings in the current study were summarized as follows:
1. The VBNC state of E. coli O157:H7 and S. Enteritidis was successfully induced by using 7% NaCl, and the cells lost culturability in 5 and 14 days, respectively.
2. PMA-LAMP assay was validated to be a rapid (from 15 to 45 min), sensitive (1–20 CFU/ reaction in pure bacterial culture), specific (100% inclusivity and 100% exclusivity) and quantitative (R2 from 0.896 to 0.996) technique to determine the viable and VBNC state of E. coli O157:H7 and Salmonella.
3. In the field study, the behaviour of S. Typhimurium on romaine lettuce was different from that observed by using E. coli O157:H7; S. Typhimurium showed a slower decline rate within 3 weeks of inoculation than that of E. coli O157:H7, indicating that S. Typhimurium is more resistant to the environmental conditions used as compared to E. coli O157:H7. Furthermore, the significant difference between viable cell counts and culturable cell counts recovered from lettuce leaves suggested the successful induction of both bacteria into the VBNC state.
4. In the simulated sanitization process, we identified that there was potential survival and temperature-dependent resuscitation of VBNC bacterial cells in cut lettuce harvested from the contaminated crop. The appropriate storage temperature and effective sanitization step were essential to reduce the risks related to the viable and VBNC state of E. coli O157:H7 and Salmonella.
5. In the qualitative risk assessment study, we have demonstrated that the risk associated with the consumption of romaine lettuce contaminated by VBNC E. coli O157:H7 and Salmonella, which has been subjected to a final sanitizing step of 100-ppm free chlorinated wash for 5 min, is serious for E. coli O157:H7 and moderate to serious for Salmonella. Our distinctive, quantifiable and measurable outcomes provide the first comprehensive data on the extent and fate of the VBNC state of Salmonella and E. coli O157:H7 in fresh produce, which supports realistic assessment of potential risks and a critical first step in the development of future mitigation strategies to address the consequence of produce contaminated by pathogens in physiological states leading to variable survival characteristics. In addition, the availability of the PMA-LAMP assay provides a reliable method to verify the efficacy of post-harvest treatments meant to reduce contamination by both viable and VBNC pathogens in fresh produce. Considering all the findings in the current project, we recommend using the PMA-LAMP assay to determine the viable and VBNC state of enteric bacterial pathogens in pure culture and sanitized fresh produce samples. However, PMA-qPCR is more feasible to quantify viable and VBNC bacteria in environmental samples such as field-grown lettuce. When growing romaine lettuce, one should avoid harsh and stressful growing conditions and use warm chlorinated water along with UV light. After an effective sanitization step, using an appropriate storage temperature is essential for post-harvest processing to reduce the risks derived from the viable and resuscitated VBNC state of pathogens in fresh produce. Accidental consumption of romaine lettuce contaminated by non-VBNC and VBNC E. coli O157:H7 and Salmonella can lead to moderate to serious consequences.