Validation of the 3M™ Molecular Detection Assay 2-STEC Gene Screen (stx) for Detection of Shiga Toxin Gene (stx1 and/or stx2) in Dried Cannabis Flower and Dried Hemp Flower: AOAC Performance Tested MethodSM 071903.

BACKGROUND: The 3M™ Molecular Detection Assay 2-STEC Gene Screen (stx) method is based on gene amplification by the use of real-time loop-mediated isothermal amplification when used with the 3M Molecular Detection System for the rapid and specific detection of Shiga toxin gene (stx1 and/or stx2) from Shiga toxin-producing Escherichia coli (STEC) in enriched products. The 3M Molecular Detection Assay 2-STEC Gene Screen (stx) was approved as AOAC Performance Tested MethodSM Certificate No. 071903. OBJECTIVE: This matrix extension study evaluated the 3M Molecular Detection Assay 2-STEC Gene Screen (stx) method for detection of STECs in dried cannabis flower [>0.3% delta 9-tetrahydrocannabinol (THC)] and dried hemp flower (≤0.3% THC) at a 10 g test portion size. METHOD: Testing followed procedures outlined in 3M Molecular Detection Assay 2-STEC Gene Screen (stx) product instructions and Standard Method Performance Requirement (SMPR®) for Detection of Shiga Toxin-Producing Escherichia coli in Cannabis and Cannabis Products (AOAC SMPR 2020.012). The method was evaluated at low, high, and non-inoculated levels. RESULTS: Results showed no statistically significant difference between the presumptive positive 3M Molecular Detection Assay 2-STEC Gene Screen (stx) results and the SMPR 2020.012 recommended cultural confirmations. CONCLUSIONS: This study provides data that demonstrate the 3M Molecular Detection Assay 2-STEC Gene Screen (stx) is a reliable method for the rapid and specific detection of STECs in dried cannabis flower and dried hemp flower. HIGHLIGHTS: The 3M Molecular Detection Assay 2-STEC Gene Screen (stx) method is suitable for the rapid and specific detection of STECs in dried cannabis flower and dried hemp flower.

Validation of the 3M™ Molecular Detection Assay 2-STEC Gene Screen (stx and eae) for Detection of Shiga Toxin Gene (stx1 and/or stx2) and Intimin Gene (eae) in Dried Cannabis Flower and Dried Hemp Flower: AOAC Performance Tested MethodSM 071902.

BACKGROUND: The 3M™ Molecular Detection Assay 2-STEC Gene Screen (stx and eae) method is based on gene amplification by the use of real time loop-mediated isothermal amplification when used with the 3M Molecular Detection System for the rapid and specific detection of Shiga toxin gene (stx1 and/or stx2) and intimin gene (eae) from Shiga toxin-producing Escherichia coli (STEC) in enriched products. The 3M Molecular Detection Assay 2-STEC Gene Screen (stx and eae) was approved as AOAC Performance Tested MethodSM Certificate No. 071902. OBJECTIVE: This matrix extension study evaluated the 3M Molecular Detection Assay 2-STEC Gene Screen (stx and eae) method for detection of STECs in dried cannabis flower [>0.3% delta 9-tetrahydrocannabinol (THC)] and dried hemp flower (≤0.3% THC) at a 10 g test portion size. METHOD: Testing followed procedures outlined in 3M Molecular Detection Assay 2-STEC Gene Screen (stx and eae) product instructions and Standard Method Performance Requirements (SMPR®) for Detection of Shiga Toxin-Producing Escherichia coli in Cannabis and Cannabis Products (AOAC SMPR 2020.012). The method was evaluated at low, high, and non-inoculated levels. RESULTS: Results showed no statistically significant difference between the presumptive positive 3M Molecular Detection Assay 2-STEC Gene Screen (stx and eae) results and the SMPR 2020.012 recommended cultural confirmations. CONCLUSIONS: This study provides data that demonstrate the 3M Molecular Detection Assay 2-STEC Gene Screen (stx and eae) is a reliable method for the rapid and specific detection of STEC organisms in dried cannabis flower and dried hemp flower. HIGHLIGHTS: The 3M Molecular Detection Assay 2-STEC Gene Screen (stx and eae) method is suitable for the rapid and specific detection of STEC organisms in dried cannabis flower and dried hemp flower.

Validation of the 3MTM Molecular Detection Assay 2-Salmonella for detection of Salmonella in Dried Cannabis Flower and Dried Hemp Flower: AOAC Performance Tested MethodsSM 091501.

BACKGROUND: The 3M™ Molecular Detection Assay 2-Salmonella method is based on real-time loop-mediated isothermal amplification when used with the 3M Molecular Detection System for the rapid and specific detection of Salmonella in enriched products. The 3M Molecular Detection Assay 2-Salmonella was approved as AOAC INTERNATIONAL (AOAC) Performance Tested MethodSM (PTM) Certificate No. 091501 and as AOAC Official Method of AnalysisSM2016.01. OBJECTIVE: This matrix extension study evaluated the 3M Molecular Detection Assay 2-Salmonella for detection of Salmonella in dried cannabis flower [>0.3% delta 9-tetrahydrocannabinol (THC)] and dried hemp flower (≤0.3% THC) at a 10 g test portion size. METHOD: Matrix studies in dried cannabis and hemp flowers followed procedures outlined in 3M Molecular Detection Assay 2-Salmonella product instructions and Standard Method Performance Requirement (SMPR®) for Detection of Salmonella species in Cannabis and Cannabis Products (AOAC SMPR 2020.002). The method was evaluated at low, high, and non-contaminated levels. RESULTS: Results showed no statistically significant difference between the presumptive positive 3M Molecular Detection Assay 2-Salmonella results and the SMPR 2020.002 recommended cultural confirmations. CONCLUSIONS: This study demonstrates that the 3M Molecular Detection Assay 2-Salmonella is a reliable method for the rapid and specific detection of Salmonella in dried cannabis flower and dried hemp flower. HIGHLIGHTS: The 3M Molecular Detection Assay 2-Salmonella method is suitable for the rapid and specific detection of Salmonella in dried cannabis flower and dried hemp flower.

Implementation of ATP and Microbial Indicator Testing for Hygiene Monitoring in a Tofu Production Facility Improves Product Quality and Hygienic Conditions of Food Contact Surfaces: A Case Study.

Rapid ATP testing and microbiological enumeration are two common methods to monitor the effectiveness of cleaning and sanitation in the food industry. In this study, ATP testing and microbiological enumeration were implemented at a tofu production facility with the goal of improving cleaning practices and overall plant hygiene. Results from ATP monitoring were used to target areas of the production environment needing additional cleaning; ATP results were verified by microbiological enumeration of aerobic microorganisms, lactic acid bacteria, and yeasts and molds. Products from the production line were enumerated for the same microorganisms to determine if there was an impact on product quality. After the implementation of ATP monitoring and targeted cleaning, there was a statistically lower proportion of swabs that failed to meet established sanitary requirements for ATP, aerobic microorganisms, and lactic acid bacteria (p < 0.05), but not for yeasts and molds. ATP swabs and microbiological enumeration agreed on site hygiene 75.1% (72.3-77.7%, 95% CI) of the time. Product data indicated that unpasteurized finished products contained a statistically lower microbial load of the three groups of organisms following implementation of the practices (p < 0.05).ImportanceCleaning and sanitation are critical to maintaining safe and high-quality food production. Monitoring these activities is important to ensure proper execution of procedure and to assure compliance with regulatory guidelines. The results from monitoring activities can direct targeted cleaning of areas with higher risk of contamination from foodstuffs and microorganisms. The results of this study show that ATP monitoring and microbiological enumeration are useful tools to verify and improve the efficacy of cleaning and sanitation practices, which can have a positive impact on both plant hygiene and product quality. However, testing regimes and critical parameters will vary based on the product and facility.

Effect of proximity to a cattle feedlot on Escherichia coli O157:H7 contamination of leafy greens and evaluation of the potential for airborne transmission.

The impact of proximity to a beef cattle feedlot on Escherichia coli O157:H7 contamination of leafy greens was examined. In each of 2 years, leafy greens were planted in nine plots located 60, 120, and 180 m from a cattle feedlot (3 plots at each distance). Leafy greens (270) and feedlot manure samples (100) were collected six different times from June to September in each year. Both E. coli O157:H7 and total E. coli bacteria were recovered from leafy greens at all plot distances. E. coli O157:H7 was recovered from 3.5% of leafy green samples per plot at 60 m, which was higher (P < 0.05) than the 1.8% of positive samples per plot at 180 m, indicating a decrease in contamination as distance from the feedlot was increased. Although E. coli O157:H7 was not recovered from air samples at any distance, total E. coli was recovered from air samples at the feedlot edge and all plot distances, indicating that airborne transport of the pathogen can occur. Results suggest that risk for airborne transport of E. coli O157:H7 from cattle production is increased when cattle pen surfaces are very dry and when this situation is combined with cattle management or cattle behaviors that generate airborne dust. Current leafy green field distance guidelines of 120 m (400 feet) may not be adequate to limit the transmission of E. coli O157:H7 to produce crops planted near concentrated animal feeding operations. Additional research is needed to determine safe set-back distances between cattle feedlots and crop production that will reduce fresh produce contamination.

Diversity of the spinach (Spinacia oleracea) spermosphere and phyllosphere bacterial communities.

The bacterial diversity of seeds, transmission of bacteria from seed to phyllosphere, and fate of seed-transmitted bacteria on mature plants are poorly characterized. Understanding the dynamics of microbial communities is important for finding bio-control or mitigation strategies for human and plant pathogens. Bacterial populations colonizing spermosphere and phyllosphere of spinach (Spinacia oleracea) seedlings and plants were characterized using pyrosequencing of 16S rRNA gene amplicons. Spinach seed microbiota was composed of three bacterial phyla: Proteobacteria, Firmicutes and Actinobacteria, belonging to > 250 different operational taxonomic units (OTUs). Seed and cotyledon bacterial communities were similar in richness and diversity. Richness of 3-4 leaf-stage of development plants increased markedly to > 850 OTUs classified within 11 phyla. Although some bacterial OTUs were detected on seeds, cotyledons and plants, the breadth of new sequences indicates the importance of multiple sources outside the seed in shaping phyllosphere community. Most classified sequences were from previously undescribed taxa, highlighting the benefits of pyrosequencing in describing seed diversity and phyllosphere bacterial communities. Bacterial community richness increased from 250 different OTUs for spinach seeds and cotyledons, to 800 OTUs for seedlings. To our knowledge this is the first comprehensive characterization of the spinach microbiome, complementing previous culture-based and clone library studies.

Helicobacter pylori colonization ameliorates glucose homeostasis in mice through a PPAR γ-dependent mechanism.

BACKGROUND: There is an inverse secular trend between the incidence of obesity and gastric colonization with Helicobacter pylori, a bacterium that can affect the secretion of gastric hormones that relate to energy homeostasis. H. pylori strains that carry the cag pathogenicity island (PAI) interact more intimately with gastric epithelial cells and trigger more extensive host responses than cag(-) strains. We hypothesized that gastric colonization with H. pylori strains differing in cag PAI status exert distinct effects on metabolic and inflammatory phenotypes. METHODOLOGY/PRINCIPAL FINDINGS: To test this hypothesis, we examined metabolic and inflammatory markers in db/db mice and mice with diet-induced obesity experimentally infected with isogenic forms of H. pylori strain 26695: the cag PAI wild-type and its cag PAI mutant strain 99-305. H. pylori colonization decreased fasting blood glucose levels, increased levels of leptin, improved glucose tolerance, and suppressed weight gain. A response found in both wild-type and mutant H. pylori strain-infected mice included decreased white adipose tissue macrophages (ATM) and increased adipose tissue regulatory T cells (Treg) cells. Gene expression analyses demonstrated upregulation of gastric PPAR γ-responsive genes (i.e., CD36 and FABP4) in H. pylori-infected mice. The loss of PPAR γ in immune and epithelial cells in mice impaired the ability of H. pylori to favorably modulate glucose homeostasis and ATM infiltration during high fat feeding. CONCLUSIONS/SIGNIFICANCE: Gastric infection with some commensal strains of H. pylori ameliorates glucose homeostasis in mice through a PPAR γ-dependent mechanism and modulates macrophage and Treg cell infiltration into the abdominal white adipose tissue.

Assessment of root uptake and systemic vine-transport of Salmonella enterica sv. Typhimurium by melon (Cucumis melo) during field production.

Among melons, cantaloupes are most frequently implicated in outbreaks and surveillance-based recalls due to Salmonella enterica. There is limited but compelling evidence that associates irrigation water quality as a significant risk of preharvest contamination of melons. However, the potential for root uptake from water and soil and subsequent systemic transport of Salmonella into melon fruit is uncharacterized. The aim of this work was to determine whether root uptake of S. enterica results in systemic transport to fruit at high doses of applied inoculum through sub-surface drip and furrow irrigation during field production of melons. Cantaloupe and honeydew were grown under field conditions, in a silt clay loam soil using standard agronomic practices for California. An attenuated S. enterica sv. Typhimurium strain was applied during furrow irrigation and, in separate plots, buried drip-emitter lines delivered the inoculum directly into the established root zone. Contamination of the water resulted in soil contamination within furrows however Salmonella was not detected on top of the beds or around melon roots of furrow-irrigated rows demonstrating absence of detectable lateral transfer across the soil profile. In contrast, positive detection of the applied isolate occurred in soil and the rhizosphere in drip injected plots; survival of Salmonella was at least 41 days. Despite high populations of the applied bacteria in the rhizosphere, after surface disinfection, internalized Salmonella was not detected in mature melon fruit (n=485). Contamination of the applied Salmonella was detected on the rind surface of melons if fruit developed in contact with soil on the sides of the inoculated furrows. Following an unusual and heavy rain event during fruit maturation, melons collected from the central area of the beds, were shown to harbor the furrow-applied Salmonella. Delivery of Salmonella directly into the peduncle, after minor puncture wounding, resulted in detection of applied Salmonella in the sub-rind tissue below the fruit abscission zone. Results indicate that Salmonella internalization from soil and vascular systemic transport to fruit is unlikely to occur from irrigation water in CA production regions, even if substantially above normal presumptive levels of contamination. Although contaminated irrigation water and subsequently soil in contact with fruit remains a concern for contamination of the external rind, results suggest an acceptable microbial indicator threshold and critical limit for the presence of Salmonella in applied water may be possible by defining appropriate microbiological standards for melon irrigation in California and regions with similar climate, soil texture, and crop management practices.

Evaluation of current operating standards for chlorine dioxide in disinfection of dump tank and flume for fresh tomatoes.

Standard postharvest unit operations that rely on copious water contact, such as fruit unloading and washing, approach the criteria for a true critical control point in fresh tomato production. Performance data for approved sanitizers that reflect commercial systems are needed to set standards for audit compliance. This study was conducted to evaluate the efficacy of chlorine dioxide (ClO(2)) for water disinfection as an objective assessment of recent industry-adopted standards for dump tank and flume management in fresh tomato packing operations. On-site assessments were conducted during eight temporally distinct shifts in two Florida packinghouses and one California packinghouse. Microbiological analyses of incoming and washed fruit and dump and flume system water were evaluated. Water temperature, pH, turbidity, conductivity, and oxidation-reduction potential (ORP) were monitored. Reduction in populations of mesophilic and coliform bacteria on fruit was not significant, and populations were significantly higher (P < 0.05) after washing. Escherichia coli was near the limit of detection in dump tanks but consistently below the detection limit in flumes. Turbidity and conductivity increased with loads of incoming tomatoes. Water temperature varied during daily operations, but pH and ORP mostly remained constant. The industry standard positive temperature differential of 5.5°C between water and fruit pulp was not maintained in tanks during the full daily operation. ORP values were significantly higher in the flume than in the dump tank. A positive correlation was found between ORP and temperature, and negative correlations were found between ORP and turbidity, total mesophilic bacteria, and coliforms. This study provides in-plant data indicating that ClO(2) can be an effective sanitizer in flume and spray-wash systems, but current operational limitations restrict its performance in dump tanks. Under current conditions, ClO(2) alone is unlikely to allow the fresh tomato industry to meet its microbiological quality goals under typical commercial conditions.

Acidified sodium chlorite optimisation assessment to improve quality of fresh-cut tatsoi baby leaves.

BACKGROUND: The fresh-cut industry commonly uses sodium hypochlorite (NaClO) for disinfection. However, there are certain problems related to its use, and acidified sodium chlorite (ASC) could be an alternative sanitiser to replace it. There is limited research evaluating the effect of ASC on the overall quality of fresh-cut produce, especially sensory quality. In this study the decontamination efficacy and quality attribute effects of ASC on fresh-cut tatsoi after application and during storage were investigated. RESULTS: Tatsoi baby leaves were minimally processed at 8 °C and stored under passive modified atmosphere packaging for up to 11 days at 5 and 10 °C. Low to moderate doses of ASC (100-500 mg L⁻¹) showed an initial antimicrobial efficacy on natural microflora and Escherichia coli as effective as that of NaClO. Regarding contact time, ASC was effective in reducting the E. coli population during the first 30 s of washing, and an increase in contact time did not improve the antimicrobial effect. Sensory quality attributes were well kept for up to 11 days at 5 °C but for only 5 days at the abusive temperature of 10 °C. CONCLUSION: ASC provides an alternative sanitising technique to NaClO for maintaining the quality and safety of fresh-cut tatsoi baby leaves for up to 11 days at 5 °C.

Characterization of interactions between Escherichia coli O157:H7 with epiphytic bacteria in vitro and on spinach leaf surfaces.

This study characterized the types of interactions between Escherichia coli O157:H7 and spinach phylloepiphytic bacteria and identified those that influence persistence of E. coli O157:H7 on edible plants. A total of 1512 phylloepiphytic bacterial isolates were screened for their ability to inhibit or to enhance the growth of E. coli O157:H7 in vitro and on spinach leaf surfaces. Fifteen different genera, the majority belonging to Firmicutes and Enterobacteriaceae, reduced growth rates of E. coli O157:H7 in vitro by either nutrient competition or acid production. Reduced numbers of E. coli O157:H7 were recovered from detached spinach leaves that were co-inoculated with epiphytic isolates belonging to five genera. A 1.8 log reduction in E. coli O157:H7 was achieved when co-inoculated with Erwinina perscinia and 20% cellobiose, a carbon source used by the phylloepiphytes but not E. coli O157:H7. The reduction on leaves was significantly less than reduction measured in vitro. Phylloepiphytic bacteria belonging to eight different genera, increased numbers of E. coli O157:H7 when co-cultured in vitro on spent medium and when co-cultured on detached spinach leaves. The results, showing reduction of E. coli O157:H7 numbers by natural epiphytic bacteria, support the hypothesis that native plant microbiota can be used for bio-control of foodborne pathogens, however, other epiphytes may promote the persistence of enteric pathogens on the phyllosphere.

Survival and distribution of Escherichia coli on diverse fresh-cut baby leafy greens under preharvest through postharvest conditions.

Escherichia coli O157:H7 has been associated in multiple outbreaks linked to the consumption of whole produce and fresh-cut leafy vegetables. However, plant-based foods had not been traditionally recognized as a host for enteric pathogens until the elevated incidence of produce-related outbreaks became apparent. The survival dynamics of two cocktails of generic E. coli (environmental water, plant and soil isolates) and E. coli O157:H7 within the phyllosphere of Mizuna, Red Chard and Tatsoi during their production, harvest, minimal processing, packaging and storage over two greenhouse production cycles were studied. Genotyping of applied generic E. coli strains to evaluate their comparative survival and relative abundance in the phyllosphere by REP-PCR is also reported. The Mizuna, Red Chard and Tatsoi shoots were grown under standard greenhouse conditions and fertility management. Both E. coli cocktails were spray-inoculated separately and determined to result in an initial mean population density of log 4.2 CFU/cm². Leaves were harvested as mini-greens approximating commercial maturity, minimally processed in a model washing system treated with 3 mg/L of ClO2 and stored for 7 days at 5 °C. Rapid decline of generic E. coli and E. coli O157:H7 populations was observed for all plant types regardless of the leaf age at the time of inoculation and the irrigation type across both seasonal growth cycle trials. The decline rate of the surviving populations for the fall season was slower than for the summer season. The minimal processing with 3 mg/L of ClO2 was not sufficient to fully disinfect the inoculated leaves prior to packaging and refrigerated storage. Viable populations of E. coli and E. coli O157:H7 were confirmed throughout storage, including the final time point at the end of acceptable visual leaf quality. In this study, the ability of low populations of E. coli to survive during production and postharvest operations in selected mini-greens has been demonstrated. However, further field-based trials are needed to expand understanding of the post-contamination fate of enteric bacterial pathogens on leafy vegetables. In summary, this research work provides baseline data upon which to develop food safety preventive control guidance during the production and minimal processing of these crops.

Alterations of the phylloepiphytic bacterial community associated with interactions of Escherichia coli O157:H7 during storage of packaged spinach at refrigeration temperatures.

This study investigated the effects of packaging and storage temperature on the spinach phylloepiphytic bacterial community and fate of Escherichia coli O157:H7. Freshly harvested spinach was rinsed and/or disinfected, packaged and stored under typical retail conditions (4 degrees C) or under temperature abuse conditions (10 degrees C) for a period of 15 days. The final population size of culturable epiphytic bacteria after 15 days of storage was not affected by the temperature of storage or the presence of E. coli O157:H7. However, analysis of the bacterial community using denaturing gradient gel electrophoresis of 16s rDNA revealed changes with time of storage and the presence of E. coli O157:H7. Excision and sequencing of prominent DGGE bands identified that the majority of sequences belonged to the phyla Actinobacteria, Bacteroidetes, Firmicutes and Alphaprotebacteria. After 10 days of storage at 4 degrees C or 10 degrees C the population became more dominated by psychrotrophic bacteria. Removal of the epiphytic bacteria resulted in significant increases in numbers of E coli O157:H7 at 10 degrees C and was associated with decreased expression of E. coli O157:H7 virulence (stxA, curli, eaeA) and stress response (rpoS, sodB) genes. In conclusion, storage temperature and time of storage of packaged spinach affected the diversity of the epiphytic spinach microbiota which influenced the growth, establishment, physiology and potentially virulence of E. coli O157:H7.