Summary
Washing is a key intervention step for fresh fruit and vegetables to remove dirt, foreign materials, tissue exudates from cut surfaces and microorganisms. Sanitizing agents are needed to maintain the microbial quality of the water and prevent cross-contamination of the product. Several CPS projects have been focused on the efficacy of disinfection treatments using standard plate count procedures to determine the bacterial inactivation. However, foodborne pathogens are able to develop a stress resistance mechanism that enables them to enter into a temporary state of low metabolic activity in which cells can persist for extended periods without division, called viable but non-cultivable (VBNC) state. There is a pending need to determine the capability of water disinfection treatments to induce the VBNC state of foodborne pathogens in process wash water particularly when optimizing the sanitizer dose. The ability of foodborne bacteria in the VBNC state, present in the process wash water, to attach to the surface of fresh produce during washing as well as the conditions needed to survive and recover from VBNC to culturable state during storage will be also evaluated. Data obtained will provide us with better understanding on the role of sanitizers in the induction of VBNC foodborne bacteria.
Technical Abstract
Most fresh produce are subjected to minimal postharvest handling, which in most of the cases only includes washing, drying and packaging. Washing is a key intervention step for fresh fruit and vegetables to remove dirt, foreign materials, tissue exudates from cut surfaces and microorganisms. Sanitizing agents are needed to maintain the microbial quality of the water and prevent cross-contamination of the product. Optimal operational conditions for the use of sanitizers in process wash water have been established to reduce cross-contamination of fresh produce. However, target recommended concentrations of sanitizers are difficult to maintain and fluctuations are usually observed during the production day, which can compromise the safety of the product. Recent studies have evidenced that chemical sanitizers can inflict sublethal injuries to many bacterial species that have acquired multiple complex mechanisms of stress resistance as a temporary state called viable by non-cultivable (VBNC) state. This state is characterized for a low metabolic activity in which cells can persist for extended periods without division. Bacteria in the VBNC state cannot be detected by culture-based techniques. These findings indicate that the use of conventional plate counting methods might lead to an overestimation of the efficacy of the water disinfection treatments. Moreover, several studies have shown that resuscitation of foodborne bacteria from the VBNC state to the culturable state is possible under appropriate conditions after adhesion, survival and resuscitation. Consequently, there is an urgent need for the assessment of the real impact of chemical disinfection treatments on foodborne pathogens and the conditions needed for resuscitation in an industry setting. This project is specifically designed to generate standardized data that will provide foundational evidence for determining the significance of VBNC foodborne bacteria in process wash water when sanitizers are used to prevent cross-contamination and the conditions needed for the VBNC foodborne bacteria to recover from VBNC to culturable state. Our objectives are to: (1) Estimate the microbial inactivation and the induction of VBNC state of foodborne pathogens in process wash water due to the action of commercial chemical sanitizers (sodium hypochlorite, calcium hypochlorite, chlorine dioxide and peroxyacetic acid); (2) establish the ability of foodborne bacteria in the VBNC state, present in the process wash water, to attach to the surface of fresh produce during washing; (3) evaluate the conditions needed for VBNC foodborne bacteria to survive and recover from VBNC to culturable state during storage and distribution mimicking the conditions of the cold chain; and (4) assess the growth potential of L. monocytogenes in fresh produce under foreseeable conditions of transportation, distribution and storage using molecular techniques able to differentiate between VC and VBNC. As a result we expect to provide scientific evidence to demonstrate the relevance of human pathogens to remain non-culturable and the concerns about the overestimation of the anti-microbial activity of commercial sanitizers. The use of standardized protocols to determine the attachment and survival of foodborne bacteria in fresh produce mimicking the industry conditions will generate valuable data for the industry collaborators as well as for the fresh produce industry in general.
Research Objectives
1. Estimation of the microbial inactivation and the induction of VBNC state of foodborne pathogens in process wash water due to the action of commercial chemical sanitizers (sodium hypochlorite, calcium hypochlorite, chlorine dioxide and peroxyacetic acid) in the processing facilities of industry collaborators.
2. Establishment of the ability of foodborne bacteria in the VBNC state, present in the process wash water, to attach to the surface of fresh produce during washing.
3. Evaluation of the conditions needed for VBNC foodborne bacteria, attached to fresh produce, to survive and recover from VBNC to culturable state during storage and distribution mimicking the conditions of the cold chain.
4. Performance of challenge tests to assess the growth potential of L. monocytogenes in fresh produce under foreseeable conditions of transportation, distribution and storage using molecular techniques able to differentiate between viable-culturable (VC) and VBNC.
Findings & Recommendations
• Taking into account the complex composition of PWW, with high organic matter content and interfering compounds, fluorescent dyes were not able to differentiate among the physiological stages of the different bacteria species. Based on the results obtained, the flow cytometry was not a suitable methodology to distinguish between viable and dead cells in PWW.
• The combination of two photoreactive dyes (PMAxx and EMA) reduced amplification of dead cells after chlorine treatment. Based on these results, the combination of 75 µM of PMAxx and 10 µM of EMA followed by incubation at 40 ºC was the methodology selected for enumeration of viable L. monocytogenes using qPCR. This method is defined as qPCR EMA+PMA.
• This is the first study demonstrating the induction of the VBNC state in bacterial cells present in PWW when water is disinfected with chlorine in an industrial setting.
• Free chlorine: Operational limits established by the fresh-cut processing industry for free chlorine (20 mg/L) are effective in inactivating L. monocytogenes and E. coli O157:H7 cells present in PWW in all the vegetable matrixes evaluated, including shredded lettuce, cabbage, diced onions and baby spinach.
• PAA: A concentration of 80 mg/L of PAA was not able to inactivate the pathogenic bacteria inoculated in any of the PWWs studied. Cultivable as well as VBNC cells were detected in all the cases when PAA was used. Based on these results, PAA is not a suitable sanitizer to maintain the microbiological quality of PWW.
• ClO2: Depending on the type of PWW, ClO2 showed different behavior. For instance, in the case of shredded lettuce PWW, 3 mg/L ClO2 could inactivate part of the inoculum but induced the VBNC stage.
• Resuscitation of VBNC L. monocytogenes and E. coli O157:H7 cells present in PWW treated with 20 mg/L free chlorine was not observed. However, resuscitation of VBNC cells of L. monocytogenes present in PWW from shredded lettuce and diced onion was observed when 3 mg/L of ClO2 was used.
• Based on these results, VBNC cells present in PWW could probably attach to fresh produce. However, under commercial storage conditions, the probability of resuscitation and multiplication of VBNC bacterial cells in fresh produce is very low. This indicates that the presence of VBNC bacteria in PWW can be associated with a low risk for public health.
• VBNC cells artificially inoculated on fresh produce survive during storage but, in most cases, they were not able to recover to the culturable state. Only in the case of diced onions, L. monocytogenes was able to resuscitate, showing levels between 1–2 logs cfu/g at the end of storage. More research should be performed to fully understand the specific characteristics of diced onions that allow the recovery of the injured L. monocytogenes cells.