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.

Phenotypic and genomic characterizations of Klebsiella pneumoniae ssp. pneumoniae and Rahnella inusitata strains reveal no clear association between genetic content and ropy phenotype.

Ropy defect of pasteurized fluid milk is a type of spoilage which manifests itself by an increased viscosity, slimy body, and string-like flow during pouring. This defect has, among other causes, been attributed to the growth, proliferation and exopolysaccharide production by coliform bacteria, which are most commonly introduced in milk as post-pasteurization contaminants. As we identified both Klebsiella pneumoniae ssp. pneumoniae and Rahnella inusitata that were linked to a ropy defect, the goal of this study was to characterize 3 K. pneumoniae ssp. pneumoniae strains and 2 R. inusitata for (1) their ability to grow and cause ropy defect in milk at 6°C and 21°C and to (2) probe the genetic basis for observed ropy phenotype. Although all K. pneumoniae ssp. pneumoniae and R. inusitata strains showed net growth of >4 log10 over 48 h in UHT milk at 21°C, only R. inusitata strains displayed growth during 28-d incubation period at 6°C (>6 log10). Two out of 3 K. pneumoniae ssp. pneumoniae strains were capable of causing the ropy defect in milk at 21°C, as supported by an increase in the viscosity of milk and string-like flow during pouring; these 2 strains were originally isolated from raw milk. Only one R. inusitata strains was able to cause the ropy defect in milk; this strain was able to cause the defect at both 6°C and 21°C, and was originally isolated from a pasteurized milk. These findings suggest that the potential of K. pneumoniae ssp. pneumoniae and R. inusitata to cause ropy defect in milk is a strain-dependent characteristic. Comparative genomics provided no definitive answer on genetic basis for the ropy phenotype. However, for K. pneumoniae ssp. pneumoniae, genes rffG, rffH, rfbD, and rfbC involved in biosynthesis and secretion of enterobacterial common antigen (ECA) could only be found in the 2 strains that produced ropy defect, and for R. inusitata a set of 2 glycosyltransferase- and flippase genes involved in nucleotide sugar biosynthesis and export could only be identified in the ropy strain. Although these results provide some initial information for potential markers for strains that can cause ropy milk, the relationship between genetic content and ropiness in milk remains poorly understood and merits further investigation.

Development of a database and standardized approach for rpoB sequence-based subtyping and identification of aerobic spore-forming Bacillales.

Aerobic spore-forming Bacillales are a highly diverse and ubiquitous group that includes organisms that cause foodborne illnesses and food spoilage. Classical microbiological and biochemical identification of members of the order Bacillales represents a challenge due to the diversity of organisms in this group as well as the fact that the phenotypic-based taxonomic assignment of some named species in this group is not consistent with their phylogenomic characteristics. DNA-sequencing-based tools, on the other hand, can be fast and cost-effective, and can provide for a more reliable identification and characterization of Bacillales isolates. In comparison to 16S rDNA, rpoB was shown to better discriminate between Bacillales isolates and to allow for improved taxonomic assignment to the species level. However, the lack of a publicly accessible rpoB database, as well as the lack of standardized protocols for rpoB-based typing and strain identification, is a major challenge. Here, we report (i) the curation of a DNA sequence database for rpoB-based subtype classification of Bacillales isolates; (ii) the development of standardized protocols for generating rpoB sequence data, and a scheme for rpoB-based initial taxonomic identification of Bacillales isolates at the species level; and (iii) the integration of the database in a publicly accessible online platform that allows for the analysis of rpoB sequence data from uncharacterized Bacillales isolates. Specifically, we curated a database of DNA sequences for a 632-nt internal variable region within the rpoB gene from representative Bacillales reference type strains and a large number of isolates that we have previously isolated and characterized through multiple projects. As of May 21, 2021, the rpoB database contained more than 8350 rpoB sequences representing 1902 distinct rpoB allelic types that can be classified into 160 different genera. The database also includes 1129 rpoB sequences for representative Bacillales reference type strains as available on May 21, 2021 in the NCBI database. The rpoB database is integrated into the online Food Microbe Tracker platform (www.foodmicrobetracker.com) and can be queried using the integrated BLAST tool to initially subtype and taxonomically identify aerobic and facultative anaerobic spore-formers. While whole-genome sequencing is increasingly used in bacterial taxonomy, the rpoB sequence-based identification scheme described here provides a valuable tool as it allows for rapid and cost-effective initial isolate characterization, which can help to identify and characterize foodborne pathogens and food spoilage bacteria. In addition, the database and primers described here can also be adopted for metagenomics approaches that include rpoB as a target, improving discriminatory power and identification over what can be achieved using 16S rDNA as a target.

Food Safety and Employee Health Implications of COVID-19: A Review.

The COVID-19 pandemic has greatly impacted the U.S. food supply and consumer behavior. Food production and processing are being disrupted as illnesses, proactive quarantines, and government-mandated movement restrictions cause labor shortages. In this environment, the food industry has been required to adopt new, additional practices to minimize the risk of COVID-19 cases and outbreaks among its workforce. Successfully overcoming these challenges requires a comprehensive approach that addresses COVID-19 transmission both within and outside the facility. Possible interventions include strategies (i) to vaccinate employees, (ii) to assure that employees practice social distancing, (iii) to assure that employees wear face coverings, (iv) to screen employees for COVID-19, (v) to assure that employees practice frequent hand washing and avoid touching their faces, (vi) to clean frequently touched surfaces, and (vii) to assure proper ventilation. Compliance with these control strategies must be verified, and an overall COVID-19 control culture must be established to implement an effective program. Despite some public misperceptions about the health risk of severe acute respiratory syndrome coronavirus 2 on foods or food packaging, both the virus biology and epidemiological data clearly support a negligible risk of COVID-19 transmission through food and food packing. However, COVID-19 pandemic-related supply chain and workforce disruptions and the shift in resources to protect food industry employees from COVID-19 may increase the actual food safety risks. The goal of this review was to describe the COVID-19 mitigation practices adopted by the food industry and the potential impact of these practices and COVID-19-related disruptions on the industry's food safety mission. A review of these impacts is necessary to ensure that the food industry is prepared to maintain a safe and nutritious food supply in the face of future global disruptions.

The occurrence, growth, and biocontrol of Listeria monocytogenes in fresh and surface-ripened soft and semisoft cheeses.

Listeria monocytogenes continues to pose a food safety risk in ready-to-eat foods, including fresh and soft/semisoft cheeses. Despite L. monocytogenes being detected regularly along the cheese production continuum, variations in cheese style and intrinsic/extrinsic factors throughout the production process (e.g., pH, water activity, and temperature) affect the potential for L. monocytogenes survival and growth. As novel preservation strategies against the growth of L. monocytogenes in susceptible cheeses, researchers have investigated the use of various biocontrol strategies, including bacteriocins and bacteriocin-producing cultures, bacteriophages, and competition with native microbiota. Bacteriocins produced by lactic acid bacteria (LAB) are of particular interest to the dairy industry since they are often effective against Gram-positive organisms such as L. monocytogenes, and because many LAB are granted Generally Regarded as Safe (GRAS) status by global food safety authorities. Similarly, bacteriophages are also considered a safe form of biocontrol since they have high specificity for their target bacterium. Both bacteriocins and bacteriophages have shown success in reducing L. monocytogenes populations in cheeses in the short term, but regrowth of surviving cells can commonly occur in the finished cheeses. Competition with native microbiota, not mediated by bacteriocin production, has also shown potential to inhibit the growth of L. monocytogenes in cheeses, but the mechanisms are still unclear. Here, we have reviewed the current knowledge on the growth of L. monocytogenes in fresh and surface-ripened soft and semisoft cheeses, as well as the various methods used for biocontrol of this common foodborne pathogen.

Escherichia coli O121 outbreak associated with raw milk Gouda-like cheese in British Columbia, Canada, 2018.

BACKGROUND: In 2018, a Shiga toxin-producing Escherichia coli O121 outbreak that affected seven individuals was associated with raw milk Gouda-like cheese produced in British Columbia, Canada. OBJECTIVES: To describe the E. coli O121 outbreak investigation and recommend greater control measures for raw milk Gouda-like cheese. METHODS: Cases of E. coli O121 were identified through laboratory testing results and epidemiologic surveillance data. The cases were interviewed on exposures of interest, which were analyzed against Foodbook Report values for British Columbia. Environmental inspection of the dairy plant and the cheese products was conducted to ascertain a source of contamination. Whole genome multi-locus sequence typing (wgMLST) was performed on all positive E. coli O121 clinical and food isolates at the provincial laboratory. RESULTS: Four out of the seven cases consumed the same raw milk Gouda-like cheese between August and October 2018. The implicated cheese was aged longer than the required minimum of 60 days, and no production deficiencies were noted. One sample of the implicated cheese tested positive for E. coli O121. The seven clinical isolates and one cheese isolate matched by wgMLST within 6.5 alleles. CONCLUSION: Raw milk Gouda and Gouda-like cheese has been implicated in three previous Shiga toxin-producing E. coli outbreaks in North America. It was recommended product labelling to increase consumer awareness and thermization of milk to decrease the risk of illness associated with raw milk Gouda and Gouda-like cheese.

Evaluation of non-traditional visualization methods to detect surface attachment of biofilms.

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.

Extended Enrichment Procedures Can Be Used To Define False-Negative Probabilities for Cultural Gold Standard Methods for Salmonella Detection, Facilitating Comparisons between Gold Standard and Alternative Methods.

ABSTRACT: Evaluation of alternative detection methods for foodborne pathogens typically involves comparisons against a "gold standard" culture method, which may produce false-negative (FN) results, particularly under worst-case scenarios such as low contamination levels, difficult-to-detect strains, and challenging food matrices (e.g., matrices with a water activity of <0.6). We used extended enrichment times (up to 72 h for both primary and secondary enrichments) to evaluate a gold standard method for Salmonella detection (the U.S. Food and Drug Administration Bacteriological Analytical Manual [BAM] method) in two low-water-activity foods (dry pet food and chocolate) inoculated at low contamination levels (most probable number ca. 1/25 g) with five Salmonella strains. Strains were selected to include those with a poor ability to grow in enrichment media. Among the 100 pet food and 100 chocolate samples tested, 53 and 50, respectively, were positive with the standard BAM method, and 57 and 59, respectively, were positive with the extended BAM method. Thus, the FN probabilities for the standard BAM method were 7% for pet food and 15% for chocolate. An alternative enzyme immunoassay method for detection of Salmonella in chocolate produced FN probabilities of 6 and 20% when compared against the standard and extended BAM methods, respectively. Detection of Salmonella Mississippi was significantly reduced with the alternative method (P = 0.023) compared with the extended BAM method. We calculated a composite reference standard to further define FN probabilities based on variable results from multiple assays (the standard BAM, extended BAM, and alternative methods). Based on this standard, the enzyme immunoassay for Salmonella detection in chocolate had a 28% FN probability and the standard and extended BAM methods had 23 and 9% FN probabilities, respectively. These results provide a framework for how inclusion of extended enrichment times can facilitate evaluation of alternative detection methods.

A Survey of Raw Frozen Breaded Chicken Products for Salmonella in British Columbia, Canada, and Phylogenetically Associated Illnesses.

ABSTRACT: The incidence of Salmonella enterica infection resulting from consumption of chicken products has historically been elevated in British Columbia compared with the rest of Canada. Raw frozen breaded chicken products are often implicated as the source of infection as there is a potential for consumers to not cook these products adequately. This occurs because the production process for these foods involves par-frying, a step which lends a cooked appearance to the product surface without reaching the internal temperatures required to fully inactivate potential pathogens. A survey of frozen chicken products from 10 retail stores of various sizes was conducted in order to determine the type and source of frozen chicken products that are available for purchase in British Columbia. Information on 391 individual products was collected and 50 were sampled for microbiological testing. Raw frozen breaded chicken products represented 59% of the frozen chicken products available to consumers at retail; 34% of these raw products were made by a single processor. The same processor was also found to have the highest proportion (33%) of samples testing positive for Salmonella. Whole genome sequencing of isolates obtained during this study revealed that majority of these isolates were phylogenetically related to clinical isolates of Salmonella. A substantial reduction of risk and increased consumer protection may be achieved by implementing a kill step (e.g., cook process that has been validated to achieve a 7-log reduction) during production of these products.

Farm-to-fork profiling of bacterial communities associated with an artisan cheese production facility.

The various stages of the cheese-making process harbor distinct bacterial communities which may influence the sensory characteristics of artisanal cheeses. The objective of this study was to investigate the microbiota from dairy farm to final cheeses along an artisanal cheese-making continuum. Environmental and food samples were collected from 21 sites, including the dairy farm, milk, cheese plant, and finished cheeses. The microbiota of these samples were analyzed using 16S rRNA amplicon sequencing, with sequences grouped into operational taxonomic units (OTUs) by phylotype at the genus level. Alpha diversity decreased from dairy farm to finished cheese. Firmicutes was the dominant phylum, ranging from 31% to 92% between the dairy farm and finished cheeses, respectively, with Proteobacteria, Actinobacteria, and Bacteroides also present (25%, 11%, and 9% overall relative abundance, respectively). Of the 37 core OTUs (>5 reads in >80% of site replicates) observed in cheese, 32 were shared with the dairy farm. Starter-related genera (i.e., Lactococcus, Lactobacillus, Streptococcus, and Leuconostoc) represented between 69% and 98% relative abundance in final cheeses depending on style, with the remainder likely acquired from various environmental sources on the farm and during the cheese-making process.

Biofilm-Forming Capacity of Five Salmonella Strains and Their Fate on Postharvest Mini Cucumbers.

Salmonella enterica is one of the pathogens that is frequently identified as the cause of fresh produce-related outbreaks. Biofilm formation is a factor that can contribute to pathogen survival on produce surface. The goal of our current research was to investigate the survival of five S. enterica strains representing different serotypes (i.e., Typhimurium, Enteritidis, Daytona, Poona, and Newport) on whole mini cucumbers stored at refrigeration (4°C) and room temperature (22°C). We also determined the strains survival on glass slides and in phosphate-buffered saline at 4 and 22°C, as well as the ability to form biofilms on a solid-liquid interphase. A rapid decrease in cell density (>4-log reduction over 8 days) of all five tested strains was observed on glass slides, while a slower die-off (<1-log reduction in 8 days) was observed in PBS. No significant difference in the die-off rate was observed among the five strains at 4 or 22°C. The die-off rate on the surface of mini cucumbers at 4°C was significantly slower ( P < 0.02) for Salmonella Enteritidis LMFS-S-JF-005 compared with the remaining four strains. At 22°C, Salmonella Poona S306 was able to grow by more than 1.5 log units on whole mini cucumbers over a period of 8 days, while the cell density of the other four strains remained at the same level compared with day 0. At this temperature, Salmonella Poona S306 was also able to form significantly stronger biofilms on a solid-liquid interphase ( P < 0.01) and was the only strain that presented a red, dry, and rough morphotype on Congo red agar plates, indicating the formation of both curli fimbriae and cellulose. These results revealed that the fate of Salmonella on mini cucumbers is strain specific, which highlighted the need for tailored mitigation strategies, such as the effective control of temperature and moisture for limiting the survival or growth of high-risk Salmonella strains between harvest and consumption of fresh produce.

The Evolving Role of Coliforms As Indicators of Unhygienic Processing Conditions in Dairy Foods.

Testing for coliforms has a long history in the dairy industry and has helped to identify raw milk and dairy products that may have been exposed to unsanitary conditions. Coliform standards are included in a number of regulatory documents (e.g., the U.S. Food and Drug Administration's Grade "A" Pasteurized Milk Ordinance). As a consequence, detection above a threshold of members of this method-defined, but diverse, group of bacteria can result in a wide range of regulatory outcomes. Coliforms are defined as aerobic or facultatively anaerobic, Gram negative, non-sporeforming rods capable of fermenting lactose to produce gas and acid within 48 h at 32-35°C; 19 genera currently include at least some strains that represent coliforms. Most bacterial genera that comprise the coliform group (e.g., Escherichia, Klebsiella, and Serratia) are within the family Enterobacteriaceae, while at least one genus with strains recognized as coliforms, Aeromonas, is in the family Aeromonadaceae. The presence of coliforms has long been thought to indicate fecal contamination, however, recent discoveries regarding this diverse group of bacteria indicates that only a fraction are fecal in origin, while the majority are environmental contaminants. In the US dairy industry in particular, testing for coliforms as indicators of unsanitary conditions and post-processing contamination is widespread. While coliforms are easily and rapidly detected, and are not found in pasteurized dairy products that have not been exposed to post-processing contamination, advances in knowledge of bacterial populations most commonly associated with post-processing contamination in dairy foods has led to questions regarding the utility of coliforms as indicators of unsanitary conditions for dairy products. For example, Pseudomonas spp. frequently contaminate dairy products after pasteurization, yet they are not detected by coliform tests. This review will address the role that coliforms play in raw and finished dairy products, their sources and the future of this diverse group as indicator organisms in dairy products.

Improved Draft Genome Sequence of Probiotic Strain Lactobacillus gasseri K7.

Lactobacillus gasseri K7 is an isolate from infant feces and has in vitro and in vivo established probiotic properties. Here, we report the improved version of the draft genome sequence, which comprises 8 scaffolds (13 contigs), a total length of 1.99 Mb, and 1,841 predicted protein-coding sequences.

Detection and quantification of probiotic strain Lactobacillus gasseri K7 in faecal samples by targeting bacteriocin genes.

Lactobacillus gasseri K7 is a probiotic strain that produces bacteriocins gassericin K7 A and K7 B. In order to develop a real-time quantitative PCR assay for the detection of L. gasseri K7, 18 reference strains of the Lactobacillus acidophilus group and 45 faecal samples of adults who have never consumed strain K7 were tested with PCR using 14 pairs of primers specific for gassericin K7 A and K7 B gene determinants. Incomplete gassericin K7 A or K7 B gene clusters were found to be dispersed in different lactobacilli strains as well as in faecal microbiota. One pair of primers was found to be specific for the total gene cluster of gassericin K7A and one for gassericin K7B. The real-time PCR analysis of faecal samples spiked with K7 strain revealed that primers specific for the gene cluster of the gassericin K7 A were more suitable for quantitative determination than those for gassericin K7 B, due to the lower detection level. Targeting of the gassericin K7 A or K7 B gene cluster with specific primers could be used for detection and quantification of L. gasseri K7 in human faecal samples without prior cultivation. The results of this study also present new insights into the prevalence of bacteriocin-encoding genes in gastrointestinal tract.