Control of Listeria monocytogenes in apple packing facilities
Open Access
- Author:
- Mendez Acevedo, Marysabel
- Graduate Program:
- Food Science
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- May 23, 2023
- Committee Members:
- Robert Roberts, Program Head/Chair
Jasna Kovac, Thesis Advisor/Co-Advisor
Luke F Laborde, Committee Member
Josephine Wee, Committee Member
Erika Ganda, Committee Member - Keywords:
- L. monocytogenes
Apple packing houses
Microbiota
Biocide resistance
Persistence
inlA - Abstract:
- L. monocytogenes is a foodborne pathogen that is known to persist in food packing and processing environments, such as tree fruit packing facilities. The application of ineffective cleaning and sanitizing procedures and resistance to sanitizers are two possible causes of L. monocytogenes persistence, which poses a food safety risk for consumers of tree fruit, such as apples. Therefore, tree fruit packing facilities should have preventive controls in place document as sanitation standard operating procedures (SSOP) in place. In this thesis, we conducted two studies focused on controlling L. monocytogenes in apple packing facilities. In the first study, we aimed to assess the efficacy of selected SSOPs in controlling L. monocytogenes and reducing the total aerobic mesophilic microbiota in three collaborating apple packing facilities. We further assessed the impact of applied SSOPs on the environmental microbiota composition. In the second study, we assessed the resistance, virulence, and evidence of persistence of L. monocytogenes isolates obtained from the apple packing facilities in the first study, as affected by two cleaning and sanitizing procedures. In the first study, we assessed the efficacy of a baseline cleaning and sanitizing procedure (treatment 1; T1) used by each of the participating apple packing facilities (F1, F2, F3) and three additional treatments (T2, T3, T4) that we developed in consultation with cleaning and sanitation industry representatives. Treatments were applied weekly in each of the three participating facilities twice: once in season 1 (2020-2021) and once in season 2 (2021-2022). T2 included an application of a wax remover, chlorinated alkaline cleaner, and a peracetic acid-based sanitizer. T3 was the same as T2, except for the addition of an acid cleaner used on stainless steel surfaces. T4 was the same as T2, except for the addition of a triple application of a biofilm remover and disinfectant. We compared the efficacy of each of the SSOPs by collecting five samples before and five samples after the treatments were applied. The collected samples were tested using enrichment and most probable number (MPN) methods to qualitatively and quantitatively detect L. monocytogenes. The total aerobic mesophilic microbial load was quantified using aerobic plate count (APC) Petrifilm. The microbiota composition was assessed by Illumina sequencing of the 16S rRNA V4 and ITS sequences. It was shown that T3 and T4 significantly reduced the aerobic plate count in all facilities across both years. T4 most effectively, but statistically insignificantly, reduced L. monocytogenes occurrence in all facilities in the first season but in F1 and F2 for the second season. However, T4 did not significantly reduce the occurrence of L. monocytogenes in F3 in the second season. None of the treatments had a significant effect on the composition of the bacterial or fungal microbiota in any of the participating facilities, possibly due to the inability of the applied sequencing method to distinguish between the DNA originating from live and dead cells. In the second study, we tested L. monocytogenes isolates obtained from each of the three apple packing facilities before and after the application of T2 and T4 to determine the minimum inhibitory concentrations (MIC) of peracetic acid sanitizer (PAA) and the biofilm remover and disinfectant. Isolates were also whole genome sequenced using Illumina MiSeq. Sequences were analyzed in GalaxyTrakr to assess potential presence of virulence and antimicrobial resistance genes, as well as for evidence of L. monocytogenes persistence. Persistence was assessed by analyzing genomic similarity among isolates obtained between 2020-2022, with historic isolates obtained in 2016-2017. Among the tested isolates, 57% had a PAA MIC of 250 ppm, while 80% of the isolates had a biofilm remover MIC of 0.133 ppm. The genomes of all 2020-2022 isolates carried 5 antimicrobial resistance genes (fosX, lin, mprF, norB, and mprL) and 9 virulent genes (inlA, inlB, plcA, plcB, prfA, hly, mpl, and iap). Single nucleotide polymorphism analysis identified 2 genotypes that belonged to multilocus sequence types ST 1003 and ST 554 that were highly similar and isolated from F2 at different times throughout 2016-2021, suggesting their persistence. Notably, these persistent genotypes were no longer detected in 2021-2022, after the repeated application of a combination of T2 and T4 in F2.