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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 solidliquid 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. [ABSTRACT FROM AUTHOR]

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. • The rpoB is sufficient for accurate identification of the majority of aerobic spore-formers. • The standardized protocols facilitate the generation of rpoB sequence data from health and food-related Bacillales isolates. • The curation of an online rpoB sequence database and analysis tool allows for the identification of Bacillales spp. [ABSTRACT FROM AUTHOR]

• Crystal violet staining for air–liquid biofilms can show false positives. • Novel reflection-based method detects pellicle formation in a well plate. • Pendant droplet tests shown to have significant potential for biofilm studies. 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. [ABSTRACT FROM AUTHOR]

The sanitary status of reusable plastic containers (RPCs) in fresh produce packing/grower operations located in Ontario, British Columbia, and Quebec was evaluated. Visibly soiled, Ready-to-Use, RPCs and those visually clean, were considered sanitary using a criterion of <3 log relative light units (RLU)/100 cm2 in ATP testing, < 4 log colony forming units (CFU)/crate Total Aerobic Count (TAC), <3 log CFU/crate for yeast & mould, and <3 log CFU/crate Enterobacteriaceae, and coliforms along with the absence of Escherichia coli. Collectively, 162 RPCs (48 visibly soiled and 114 visually clean) were sampled with 10% (5/48) of the visibly soiled crates exceeding ATP RLU readings that compares to 81% of clean crates sampled. All visibly soiled crates exceeded the acceptable limits for TAC that compares to 83% of visually clean RPCs. The fail percentage of visibly soiled crates, with respect to Enterobacteriaceae and coliform counts, was 96% (23/24) and 25% (12/48) respectively, compared to 69% (48/70) and 3% (4.4/114) for clean crates. E. coli was recovered from five (3.5%; 5/114) of visually clean RPCs. It was concluded that visual assessment and ATP readings are poor metrics to assess the sanitary status of RPCs. In laboratory trials, Salmonella inoculated onto RPCs declined at a greater rate under low (55%) compared to high (88%) relative humidity (RH). However, the rate of decline of Salmonella was lower if cucumber homogenates were periodically introduced onto the surface of inoculated RPCs with growth occurring under high RH. Transfer of Salmonella from RPC to cucumbers occurred in a diphasic manner with an initial rapid phase followed by a slower rate of attachment with time. Sanitation cycles that included a caustic rinse followed by a peracetic acid treatment supported a 1.0–1.8 log reduction of Salmonella although residual survivors could be transferred from RPCs to cucumbers. The study demonstrated that Salmonella can persist for extended periods on RPCs and then be transferred to produce can occur if crates are inadequately sanitized. This result, coupled with the unsanitary condition of RPCs sampled in the field, reinforces the need to adequately sanitize crates between uses. • The sanitary status of RPCs in the field remains a key knowledge gap. • The study illustrated poor sanitary status of RPCs with associated food safety risk. • Salmonella can persist on RPCs over long periods with nutrients from produce exudates. • Salmonella can rapidly transfer between RPCs and fresh produces. • Current sanitation is insufficient to prevent Salmonella from surviving and transferring. [ABSTRACT FROM AUTHOR]

In February and March of 2024, an unusual illness began affecting dairy herds primarily in Texas and neighboring states. The causative agent of this illness was ultimately confirmed in late March 2024 to be a strain of highly pathogenic avian influenza H5N1 belonging to clade 2.3.4.4b. In the months following the emergence of this viral disease in cattle, infections have spread to at least 191 herds in 13 states at the time of this writing in August 2024, primarily through cattle and human movement. Surprisingly, early examination of raw milk samples from clinically affected animals indicated that the virus had an affinity for the mammary tissue, and viral shedding into raw milk occurred at high levels, exceeding 10 8 log 10 50% tissue culture infectious dose (TCID 50 ) in some cases. These high viral loads coupled with evidence that farm cats who consumed raw milk from clinically ill animals were infected and exhibited high mortality rates, raised concerns about the safety of the US milk supply for human consumption. To date, 4 cow-associated human infections have been reported, all from farm employees with direct contact with infected animals. Several parameters ultimately affect the theoretical public health risk from consumption of dairy products manufactured from a milk supply containing H5N1, namely (1) initial viral load, (2) persistence of H5N1 in raw milk, (3) viral inactivation through processing practices including pasteurization, and (4) human susceptibility and infectious dose. In the short period since the emergence of this disease in dairy cattle in the United States, research has begun to answer these critical questions, although our knowledge is still quite limited at this time. Here we review the literature available from the current H5N1 outbreak in US dairy cattle, as well as selected relevant literature from previous research in other animal agriculture sectors, that affect our current understanding of the parameters associated with the food safety risk of this disease in the US dairy supply chain., (© 2024.)

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., Competing Interests: Competing interests: None.

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., (Copyright ©, International Association for Food Protection.)