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Small- and medium-sized dairy processing facilities (SMDFs) may face unique challenges with respect to controlling Listeria in their processing environments, e.g., due to limited resources. The aim of this study was to implement and evaluate environmental monitoring programs (EMPs) for Listeria control in eight SMDFs in a ∼1-year longitudinal study; this included a comparison of pre-operation (i.e., after cleaning and sanitation and prior to production) and mid-operation (i.e., at least 4 h into production) sampling strategies. Among 2,072 environmental sponge samples collected across all facilities, 272 (13%) were positive for Listeria. Listeria prevalence among pre- and mid-operation samples (15% and 17%, respectively), was not significantly different. Whole genome sequencing (WGS) performed on select isolates to characterize Listeria persistence patterns revealed repeated isolation of closely related Listeria isolates (i.e., ≤20 high-quality single nucleotide polymorphism [hqSNP] differences) in 5/8 facilities over >6 months, suggesting Listeria persistence and/or reintroduction was relatively common among the SMDFs evaluated here. WGS furthermore showed that for 41 sites where samples collected pre- and mid-operation were positive for Listeria, Listeria isolates obtained were highly related (i.e., ≤10 hqSNP differences), suggesting that pre-operation sampling alone may be sufficient and more effective for detecting sites of Listeria persistence. Importantly, our data also showed that only 1/8 of facilities showed a significant decrease in Listeria prevalence over 1 year, indicating continued challenges with Listeria control in at least some SMDFs. We conclude that options for simplified Listeria EMPs (e.g., with a focus on pre-operation sampling, which allows for more rapid identification of likely persistence sites) may be valuable for improved Listeria control in SMDFs., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Martin Wiedmann is a paid consultant for Neogen Corporation. All other coauthors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

In food safety and food quality, biofilm research is of great importance for mitigating food-borne pathogens in food processing environments. To supplement the traditional staining techniques for biofilm characterization, we introduce several non-traditional imaging methods for detecting biofilm attachment to the solid-liquid and air-liquid interfaces. For strains of Pseudomonas aeruginosa (the positive control), Acinetobacter baumanii, Listeria monocytogenes and Salmonella enterica, the traditional crystal violet assay showed evidence of biofilm attachment to the well plate base as well as inferred the presence of an air-liquid biofilm attached on the upper well walls where the meniscus was present. However, air-liquid biofilms and solid-surface-attached biofilms were not detected for all of these strains using the non-traditional imaging methods. For L. monocytogenes, we were unable to detect biofilms at a particle-laden, air-liquid interface as evidenced through microscopy, which contradicts the meniscus staining test and suggests that the coffee-ring effect may lead to false positives when using meniscus staining. Furthermore, when L. monocytogenes was cultivated in a pendant droplet in air, only microbial sediment at the droplet apex was observed without any apparent bacterial colonization of the droplet surface. All other strains showed clear evidence of air-liquid biofilms at the air-liquid interface of a pendant droplet. To non-invasively detect if and when air-liquid pellicles form in a well plate, we also present a novel in situ reflection assay that demonstrates the capacity to do this quantitatively., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Farmstead dairy processing facilities may be particularly susceptible to Listeria spp. contamination due to the close physical proximity of their processing environments (PE) to associated dairy farm environments (FE). In this case study, we supported the implementation of interventions focused on improving (1) cleaning and sanitation efficacy, (2) hygienic zoning, and (3) sanitary equipment and facility design and maintenance in a farmstead dairy processing facility, and evaluated their effect on Listeria spp. detection in the farmstead's PE over 1 yr. Detection of Listeria spp. in the farmstead's PE was numerically reduced from 50% to 7.5% after 1 yr of intervention implementation, suggesting that these interventions were effective at improving Listeria spp. control. In addition, environmental samples were also collected from the farmstead's FE to evaluate the risk of the FE as a potential source of Listeria spp. in the PE. Overall, detection of Listeria spp. was higher in samples collected from the FE (75%, 27/36) compared with samples collected from the PE (24%, 29/120). Whole-genome sequencing performed on select isolates collected from the PE and FE supported the identification of 6 clusters (range of 3 to 15 isolates per cluster) that showed ≤ 50 high-quality single nucleotide polymorphism differences. Of these 6 clusters, 3 (i.e., clusters 2, 4, and 5) contained isolates that were collected from both the PE and FE, suggesting that transmission between these 2 environments was likely. Moreover, all cluster 2 isolates represented a clonal complex of L. monocytogenes commonly associated with dairy farm environmental reservoirs (i.e., CC666), which may support that the farmstead's FE represented an upstream source of the cluster 2 isolates that were found in the PE. Overall, our data underscore that although the FE can represent a potential upstream source of Listeria spp. contamination in a farmstead dairy processing facility, implementation of targeted interventions can help effectively minimize Listeria spp. contamination in the PE. The list of standard abbreviations for JDS is available at adsa.org/jds-abbreviations-24. Nonstandard abbreviations are available in the Notes. [ABSTRACT FROM AUTHOR]