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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., (Published 2020 by the International Association for Food Protection.)

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.

• 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]