Summary
Prevalence studies on L. monocytogenes in foods and food processing environments have been performed primarily in meat and dairy production chains. In the fresh produce processing industry, collaborative research between the industry and academia is still needed to generate practical knowledge and solutions for the implementation of Environmental Monitoring (EM) programs in fresh produce processing facilities as different industrial practices and processing environments may account for different contamination patterns. Within this aim, this project will generate empirical data on the performance of EM programs in different fresh produce processing facilities. This valuable information will be used to improve the management of the food processing environment, highlighting the need for adjustments of the intervention strategies. Achieving this goal will be possible thanks to the collaboration of the CEBAS-CSIC with three major fresh produce companies, who expressed their interest in this project. The current project aims to have an impact on the fresh produce processing industry in several ways including a reduction on the L. monocytogenes contamination in fresh produce processing facilities, establishment of common contamination patters within processing plants and among different industrial practices, and selection of the most efficient sanitizing treatments to eliminate L. monocytogenes from the processing environment.
Technical Abstract
Environmental contamination sources have been widely studied but most of the research has not been focused on fresh produce. Well-establish routine Environmental Monitoring (EM) programs should be designed on a risk-based approach, considering the nature and size of the food operation and reflecting aspects related to the raw materials, the production processes and the final product but they also need to be regularly revised based on trend analysis. Current approach to designing environmental monitoring includes zoning and sanitary design but it has been demonstrated that it should also consider location connectivity and ranking with respect to the expected length and level of contamination of a surface. The current proposal aims to contribute generating practical knowledge and solutions for the implementation of EM programs in fresh produce processing facilities as different industrial practices and processing environments may account for different contamination patterns. Three objectives have been identified. The first objective will improve understanding on how different factors interact and affect the probability of contamination in different fresh produce processing facilities by means of a systematic sampling through an EM plan including zoning, sanitary design, location connectivity, and ranking with respect to the length and level of contamination of Listeria spp. In the second objective, the application of whole genome sequencing (WGS) in food processing environments (FPEs) will be used as a tool to enhance the understanding of the origin, cross-contamination, reservoir, and possible persistence of specific Listeria spp./L. monocytogenes isolates. Establishment of the genetic correlations of the Listeria spp./L. monocytogenes isolates will be necessary to understand the distribution patterns across different compartments within the same processing plant and among different industrial practices. Data will be obtained on the distribution of identical or similar Listeria spp./L. monocytogenes WGS sequence types in different locations and times for each processing plant and among facilities. The third objective will focus on the evaluation of control measures currently implemented in the produce processing facilities of the industry collaborators. Observational studies in different processing lines will help to determine the efficacy of different sanitizing procedures against different types of contamination scenarios (transient and persistent). These results will allow the identification of potential strain adaptation to common sanitizers and its impact on the tolerance to different environmental stresses. The current project aims to impact the fresh produce processing industry in several ways including: i) Development of an improved set of guidance based on empirical data for the industry, ii) A reduction on the L. monocytogenes contamination in fresh produce processing facilities, iii) Establishment of common contamination patters within one processing plant and among different processing plants, and iv) Recommendations on the most efficient sanitizing treatments to eliminate L. monocytogenes from the processing environment.
Research Objectives
1. Assessment of the environmental contamination by Listeria spp. in fresh produce processing plants.
2. Establishment of the genetic correlations of the Listeria spp./L. monocytogenes isolates to understand the distribution patterns across different compartments within the same processing plant and among different processing facilities. Identification of persistent and transient Listeria spp. strains.
3. Evaluation of the efficacy of control measures currently implemented in commercial fresh produce processing plants against transient and persistent Listeria spp./L. monocytogenes contamination.
Findings & Recommendations
Objective 1
• A modified protocol of the ISO method was performed to enhance the detection of L. monocytogenes in the environmental monitoring samples. The improvements in the protocol included the transport of the swaps from the plant to the lab in the pre-enrichment broth (half Fraser broth) instead of buffered peptone water (BPW) and the use of filtration of 100 mL of the pre-enrichment broth instead of 1 mL of the pre-enrichment to the enrichment step to decrease the detection limit (detected in 100 mL).
• Two types of environmental samplings were performed: 1) after processing, just before cleaning; and 2) after cleaning and disinfection. The information obtained from the sampling of the processing environment after processing was very valuable to obtain information about the routes for the entrance of contamination in the processing environment.
• In all the EM samplings, samples were also taken from FCS (Zone 1). It was found that the prevalence of L. monocytogenes in these samples was, in many cases, higher than in Zone 2 samples. Therefore, additional information can be obtained including Zone 1 samples, mostly for the identification of hotspots of contamination.
• After processing, just before cleaning, the highest L. monocytogenes prevalence was observed in Zone 3 (61%, 160/264), followed by Zone 1 (25%, 48/195) and Zone 2 (21%, 27/132).
• Relationship between Listeria spp. counts and L. monocytogenes detection: Detection of L. monocytogenes was associated with variable Listeria spp. counts. In samples taken after processing, L. monocytogenes was detected in about 80% of the sampling points that showed counts of Listeria spp. Therefore, analyses of Listeria spp. in all the sampling zones represent a valuable strategy and an appropriate approach for an EM sampling program.
• The EM sampling performed after the cleaning and disinfection operations demonstrated that L. monocytogenes was still detected mostly in sites of Zone 3, but also in one site of Zone 1.
• The high prevalence of L. monocytogenes found in Zone 3 indicates the difficulties of the cleaning and disinfection of these areas and the need for improving the sampling frequency and the number of test points to ensure that the corrective actions have been undertaken.
Objective 2
• The collection of multiple isolates (up to five confirmed positive colonies from filtration and enrichment) per sample point helped to capture the diversity of L. monocytogenes in the processing facilities. In the two sampling approaches, only two different serotypes were observed.
• WGS was performed on isolates obtained from the cut lettuce and cut fruit facilities. The genetic characterization of the L. monocytogenes isolates (lineage groups and serotypes) evidenced the low diversity within the different zones of the processing environment as well as different processing facilities.
• Two L. monocytogenes serotypes, ST155 and ST6, were identified in samples taken after processing. Each strain belonged to a different core-genome type (CT), with CT155 and CT6 for ST155 and ST6, respectively. ST155 belongs to lineage groups 1/2a, 3a while ST6 belongs to lineage 4b, 4d, 4e.
• Among the 100 isolates included in the WG, 19 corresponded to the ST6 and 78 to the ST155. Most of the isolates belonging to the ST6 (n=15) were found in the cut lettuce facility, while only four ST6 isolates were in the cut fruit facility. These results could indicate potential reservoir sites in the cut lettuce facility and the potential risks of decontamination.
• All the ST6 isolates corresponded to samples obtained from Zone 3 (e.g., wheels and floors), indicating the potential presence of Listeria niches in these areas mostly due to inadequate cleaning and disinfection procedures.
• The same serotypes were found in the cut facilities after the cleaning and disinfection activities.
• Our results indicate that the diversity of L. monocytogenes serotypes was very low. This reduced diversity could be due to the fact that all the isolates were obtained after conventional culture methods. The use of a metagenomic approach could open the possibility that less adapted serotypes could have been identified.
• Interventions after the sampling events changed contamination scenarios substantially.
Objective 3
• Isolates obtained from the EM performed after the cleaning and disinfection activities were tested against the biocides used by the industry for the cleaning and disinfection activities.
• All the isolates were sensitive to concentrations of the biocides much lower than the doses recommended by the manufacturer.
• The results obtained highlight the need for validating the cleaning activities because even if there are no persister cells in the processing environment if the cleaning activities are not well performed, contamination niches might occur.
• Intensive cleaning and sanitation protocols, and maintenance procedures and repair strategies for drains, floor cracks and boot washers were some of the corrective actions that the company implemented.