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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]

Management of Shiga toxin–producing Escherichia coli (STEC), including E. coli O157:H7, in food products is a major challenge for the food industry. Several interventions, such as irradiation, chemical disinfection, and pasteurization, have had variable success controlling STEC contamination. However, these interventions also indiscriminately kill beneficial bacteria in foods, may impact organoleptic properties of foods, and are not always environmentally friendly. Biocontrol using bacteriophage-based products to reduce or eliminate specific foodborne pathogens in food products has been gaining attention due to the specificity, safety, and environmentally friendly properties of lytic bacteriophages. We developed EcoShield PX, a cocktail of lytic bacteriophages, that specifically targets STEC. This study was conducted to examine the efficacy of this bacteriophage cocktail for reducing the levels of E. coli O157:H7 in eight food products: beef chuck roast, ground beef, chicken breast, cooked chicken, salmon, cheese, cantaloupe, and romaine lettuce. The food products were challenged with E. coli O157:H7 at ca. 3.0 log CFU/g and treated with the bacteriophage preparation at ca. 1 × 106, 5 × 106, or 1 × 107 PFU/g. Application of 5 × 106 and 1 × 107 PFU/g resulted in significant reductions (P < 0.05) in E. coli O157:H7 levels of up to 97% in all foods. When bacteriophages (ca. 1 × 106 PFU/g) were used to treat lower levels of E. coli O157:H7 (ca. 1 to 10 CFU/10 g) on beef chuck roast samples, mimicking the levels of STEC found under real-life conditions in food processing plants, the prevalence of STEC in the samples was significantly reduced (P < 0.05) by ≥80%. Our results suggest that this STEC-targeting bacteriophage preparation can result in significant reduction of both the levels and prevalence of STEC in various foods and, therefore, may help improve the safety and reduce the risk of recalls of foods at high risk for STEC contamination. Shiga toxin–producing E. coli is a major food safety challenge for the food industry. EcoShield PX bacteriophage cocktail reduced E. coli O157:H7 levels in foods by up to 97%. The bacteriophage cocktail reduced the prevalence of E. coli O157:H7 in foods by ≥80%. [ABSTRACT FROM AUTHOR]

Diets rich in minimally processed foods are associated with numerous health benefits, in part, due to their diverse, natural microbiota. However, antimicrobials, such as chlorine and peracetic acid (PAA), that are used to address food safety concerns may damage the natural microflora of fresh produce. One promising approach for targeting pathogenic bacteria in foods without impacting the normal food microbiota are bacteriophages. In this study, we observed that combinational treatment of conventional antimicrobials (PAA and chlorine) and bacteriophages, specifically the Salmonella‐targeted preparation SalmoFresh, retained the bactericidal effectiveness of individual interventions, and in some cases, achieved substantially increased efficacy. Additionally, the bacterial microbiomes of farm fresh and organic produce were less affected after phage treatment compared to PAA and chlorine. Finally, our study revealed that resistance rates against SalmoFresh were relatively minor and unaffected by the stresses introduced after chemical washes and/or bacteriophage treatment. [ABSTRACT FROM AUTHOR]

Results of previous studies revealed that (i) splash can transfer microbes from in-field feces to preharvest produce and (ii) wildlife can be vectors for the introduction of foodborne pathogens into produce fields. However, few peer-reviewed studies have been conducted to examine pathogen transfer from wildlife feces to in-field produce via splash during irrigation. Although two previous studies found a significant relationship between distance and Escherichia coli transfer via splash, the studies sampled produce <1 m from the feces. The present study was conducted to refine our understanding of the impact of distance on E. coli splash. Two trials were conducted 1 month apart. For each trial, fecal pellets inoculated with a three-strain E. coli cocktail were placed in a lettuce field 2.5 h before irrigation. After irrigation, E. coli levels on lettuce heads 0 to 6 m from the pellets were determined. Although E. coli was not detected in any of the heads ≥2 m from the fecal pellets (n = 39), 39% of heads (13 of 33) <2 m from the pellets tested positive for E. coli. According to logistic regression, the odds of harvesting a head that tested positive for E. coli decreased by a factor of 50 (odds ratio, 0.02; 95% confidence interval, <0.01, 0.28; P = 0.004) for each meter increase in the distance between the lettuce and the feces. Thus, the likelihood of E. coli transfer from feces to produce should be minimal at a given distance from the feces. Our model can be used to predict the probability of harvesting a microbially contaminated lettuce head following implementation of a no-harvest buffer around in-field feces. For example, our model suggests that the probability of harvesting a contaminated head was 0.1% at 3 m from the feces. Although the approaches utilized in this study provide a conceptual framework that can be used to help define appropriate no-harvest buffers, delineation of appropriate buffer zones requires additional information (e.g., acceptable risk and regional data). [ABSTRACT FROM AUTHOR]

Although wildlife intrusion and untreated manure have been associated with microbial contamination of produce, relatively few studies have examined the survival of Escherichia coli on produce under field conditions following contamination (e.g., via splash from wildlife feces). This experimental study was performed to estimate the die-off rate of E. coli on preharvest lettuce following contamination with a fecal slurry. During August 2015, field-grown lettuce was inoculated via pipette with a fecal slurry that was spiked with a three-strain cocktail of rifampin-resistant nonpathogenic E. coli. Ten lettuce heads were harvested at each of 13 time points following inoculation (0, 2.5, 5, and 24 h after inoculation and every 24 h thereafter until day 10). The most probable number (MPN) of E. coli on each lettuce head was determined, and die-off rates were estimated. The relationship between sample time and the log MPN of E. coli per head was modeled using a segmented linear model. This model had a breakpoint at 106 h (95% confidence interval = 69, 142 h) after inoculation, with a daily decrease of 0.70 and 0.19 log MPN for 0 to 106 h and 106 to 240 h following inoculation, respectively. These findings are consistent with die-off rates obtained in similar studies that assessed E. coli survival on produce following irrigation. Overall, these findings provide die-off rates for E. coli on lettuce that can be used in future quantitative risk assessments. [ABSTRACT FROM AUTHOR]

Listeria-infecting phages are currently being used to control and detect the important foodborne pathogen Listeria monocytogenes; however, the influence of environmental conditions on the interactions between L. monocytogenes and its phages has not been explored in depth. Here, we examined the infective potential of four Listeria phages (two each from the P70-like and P100-like phages of Listeria) against five strains of L. monocytogenes (representing serotypes 1/2a, 1/2b, 4a, and 4b) grown under a range of temperatures (7-37°C). We show that the plaquing efficiencies for all four phages were significantly affected by temperature. Interestingly, no plaques were observed for any of the four phages at 37°C. Adsorption assays performed with the P100-like phages, LP-048 and LP-125, showed that LP-048 had a severely reduced adsorption efficiency against susceptible strains at 37°C as compared to 30°C, suggesting that there is considerably less accessible rhamnose (LP-048's putative phage receptor) on the host at 37°C than at 30°C. LP-125 adsorbed to host cells at 37°C, indicating that the inability for LP-125 to plaque at 37°C is not due to adsorption inhibition. LP-048 showed significantly higher adsorption efficiency against a mutant strain lacking N-acetylglucosamine in its wall teichoic acids (WTA) than the parental strain at both 30 and 37°C, suggesting that N-acetylglucosamine competes with rhamnose for glycosylation sites on the WTA. The data presented here clearly shows that L. monocytogenes can gain physiological refuge from phage infection, which should be carefully considered for both the design and implementation of phage-based control and detection applications. [ABSTRACT FROM AUTHOR]

Listeria-infecting phages are currently being used to control and detect the important foodborne pathogen Listeria monocytogenes; however, the influence of environmental conditions on the interactions between L. monocytogenes and its phages has not been explored in depth. Here, we examined the infective potential of four Listeria phages (two each from the P70-like and P100-like phages of Listeria) against five strains of L. monocytogenes (representing serotypes 1/2a, 1/2b, 4a, and 4b) grown under a range of temperatures (7-37°C). We show that the plaquing efficiencies for all four phages were significantly affected by temperature. Interestingly, no plaques were observed for any of the four phages at 37°C. Adsorption assays performed with the P100-like phages, LP-048 and LP-125, showed that LP-048 had a severely reduced adsorption efficiency against susceptible strains at 37°C as compared to 30°C, suggesting that there is considerably less accessible rhamnose (LP-048's putative phage receptor) on the host at 37°C than at 30°C. LP-125 adsorbed to host cells at 37°C, indicating that the inability for LP-125 to plaque at 37°C is not due to adsorption inhibition. LP-048 showed significantly higher adsorption efficiency against a mutant strain lacking N-acetylglucosamine in its wall teichoic acids (WTA) than the parental strain at both 30 and 37°C, suggesting that N-acetylglucosamine competes with rhamnose for glycosylation sites on the WTA. The data presented here clearly shows that L. monocytogenes can gain physiological refuge from phage infection, which should be carefully considered for both the design and implementation of phage-based control and detection applications. [ABSTRACT FROM AUTHOR]