Summary of Awards to Date

Significance of sanitizers on induction of viable but non-cultivable (VBNC) foodborne bacteria and their survival and resuscitation in fresh produce

Date

Jan. 1, 2019 - Dec. 31, 2020

Funding Agency

Center for Produce Safety

Amount Awarded

$258,974.62

Investigator

Ana Allende, Ph.D.
CEBAS-CSIC Campus de Espinardo, Spain

Co-Investigator(s)

Maria I. Gil, Ph.D.

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.