Utilizing whole genome sequencing to characterize Listeria spp. persistence and transmission patterns in a farmstead dairy processing facility and its associated farm environment.

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 (i) cleaning and sanitation efficacy, (ii) hygienic zoning, and (iii) sanitary equipment/facility design and maintenance in a farmstead dairy processing facility, and evaluated their impact on Listeria spp. detection in the farmstead's PE over 1 year. Detection of Listeria spp. in the farmstead's PE was numerically reduced from 50% to 7.5% after 1 year 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 (WGS) 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 (hqSNP) 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 (CC) 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, while 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.

Intensive Environmental Sampling and Whole Genome Sequence-based Characterization of Listeria in Small- and Medium-sized Dairy Facilities Reveal Opportunities for Simplified and Size-appropriate Environmental Monitoring Strategies.

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.

Development and Validation of Pathogen Environmental Monitoring Programs for Small Cheese Processing Facilities.

Pathogen environmental monitoring programs (EMPs) are essential for food processing facilities of all sizes that produce ready-to-eat food products exposed to the processing environment. We developed, implemented, and evaluated EMPs targeting Listeria spp. and Salmonella in nine small cheese processing facilities, including seven farmstead facilities. Individual EMPs with monthly sample collection protocols were designed specifically for each facility. Salmonella was detected in only one facility, with likely introduction from the adjacent farm indicated by pulsed-field gel electrophoresis data. Listeria spp. were isolated from all nine facilities during routine sampling. The overall Listeria spp. (other than Listeria monocytogenes ) and L. monocytogenes prevalences in the 4,430 environmental samples collected were 6.03 and 1.35%, respectively. Molecular characterization and subtyping data suggested persistence of a given Listeria spp. strain in seven facilities and persistence of L. monocytogenes in four facilities. To assess routine sampling plans, validation sampling for Listeria spp. was performed in seven facilities after at least 6 months of routine sampling. This validation sampling was performed by independent individuals and included collection of 50 to 150 samples per facility, based on statistical sample size calculations. Two of the facilities had a significantly higher frequency of detection of Listeria spp. during the validation sampling than during routine sampling, whereas two other facilities had significantly lower frequencies of detection. This study provides a model for a science- and statistics-based approach to developing and validating pathogen EMPs.

Molecular subtyping and characterization of psychrotolerant endospore-forming bacteria in two New York State fluid milk processing systems.

Psychrotolerant endospore-forming bacteria Bacillus and Paenibacillus spp. are important spoilage organisms in fluid milk. A recently developed rpoB subtyping method was applied to characterize the diversity and phylogenetic relationships among Bacillus and related sporeformers associated with milk processing systems. Milk samples representing the processing continuum from raw milk to pasteurized products were collected from two fluid milk processing plants, held at 6 degrees C up to the code date that had been established by each processing plant (i.e., either 18 or 21 days), and plated for bacterial enumeration throughout storage. Bacterial colonies selected to represent the visible diversity in colony morphology on enumeration plates were examined further. Among 385 bacterial isolates characterized, 35% were Bacillus spp., and 65% were Paenibacillus spp. A total of 92 rpoB allelic types were identified among these isolates, indicating considerable diversity among endospore-forming spoilage organisms present in fluid milk systems. Of the 92 allelic types identified, 19 were isolated from samples collected from both processing plants. The same rpoB allelic types were frequently identified in paired raw milk and packaged product samples, indicating that Bacillus and Paenibacillus spp. can enter dairy processing systems through raw milk. Certain subtypes were found exclusively in pasteurized samples, including those that were temporally independent, suggesting the possibility of in-plant sources for these spoilage organisms, including through the persistence of selected subtypes in processing plants. Development of effective control strategies for the diverse array of psychrotolerant endospore-forming organisms that currently limit the shelf lives of high-temperature short-time fluid milk products will require comprehensive, integrated efforts along the entire milk processing continuum.