AOAC-OMA/MicroVal Harmonized Validation of Peel PlateTM EB (Enterobacteriaceae Bacteria), First Action 2018.05.

BACKGROUND: Peel PlateTM  Enterobacteriaceae Bacteria (EB) is dried selective media on a 47 mm plastic plate that produces enzyme substrate colored colonies on rehydration and incubation for 24 h and up to 48 h at 37 ± 1°C. PURPOSE: The method validation compared quantification of EB to reference methods ISO 21528:2017 Parts 1 and 2. METHODS: Matrixes compared were whole milk, skim powdered milk, vanilla ice cream, butter, infant formulas (soy- and dairy-based), infant cereals ± probiotic, environmental sponge swab of stainless steel surface, and poultry carcass rinse with two different peptone buffers. RESULTS: In inclusivity and exclusivity studies, the method detected 54 of 54 EB strains and did not detect 30 of 30 non-EB strains. In matrix studies, the claimed foods were tested at three contamination levels using paired analysis between the reference and Peel Plate EB methods. Colony-forming units per gram or mL [CFU/g (mL)] were log10 transformed for statistical analysis. The candidate method and reference method were shown to be equivalent by the performance requirement of all 95% confidence intervals on mean difference falling between -0.5 and +0.5 log10 CFU/g (mL). An international collaborative study with dried infant formula spiked with Cronobacter sakazakii at log10 CFU/g (mL) 1.05, 2.31, and 3.21 levels, produced method differences -0.16, 0.15, and 0.18 log10 CFU/g (mL) with repeatabilities (r) = 0.33, 0.20, and 0.12 log10 CFU/g (mL) and reproducibilities (R) = 0.45, 0.26, and 0.18 log10 CFU/g (mL). CONCLUSIONS: Based on these evaluations, the candidate method is considered equivalent to the reference methods at both the 24 h and 48 h incubation periods at 37 ± 1°C. HIGHLIGHTS: Ready to use Enterobacteriaceae method equivalent to ISO-21528:2017 Parts 1 and 2; EB test colored colonies at 37°C for 24 h are equivalent at 48 h incubation; Singlet determined CFU/mL are statistically the same as duplicate average results; EB test validated for infant formula and dairy products including with probiotics; EB test for environmental surfaces and poultry carcass rinses using peptone buffers.

Validation of the Peel Plate™ AC for Detection of Total Aerobic Bacteria in Dairy and Nondairy Products.

Peel Plate™ AC (aerobic count) is a low-profile plastic 47 mm culture dish with adhesive top that contains a dried standard plate count medium with oxidation/reduction indicator triphenyl tetrazolium chloride (TTC) that turns red with dehydrogenase enzyme activity of growing aerobic bacteria. The method provides a conventional quantitative count with simple rehydration and incubation for 48 ± 3 h at 35 ± 1°C for most food matrixes and 32 ± 1°C for 48 ± 3 h for dairy products. Dairy matrixes claimed and supported with total aerobic count data are whole milk, skim milk, chocolate milk (2% fat), light cream (20% fat), pasteurized whole goat milk, ultra-high temperature pasteurized milk, nonfat dried milk, lactose-reduced milk, strawberry milk, raw cow milk, raw goat milk, raw sheep milk, condensed skim milk, and vanilla ice cream. Food matrixes claimed for aerobic count detection are raw ground beef, environmental sponge of stainless steel, raw ground turkey, dry dog food, liquid whole pasteurized eggs, milk chocolate, poultry carcass rinse, and large animal carcass sponge. The method has been independently evaluated for aerobic count in dairy products: whole milk, skim milk, chocolate milk, and light cream. The method was also independently evaluated for aerobic count in food matrixes: ground beef and sponge rinse from stainless steel surfaces. In the matrix study, each matrix was assessed separately at each contamination level in comparison to an appropriate reference method. Colony counts were determined for each level and then log10-transformed. The transformed data were evaluated for repeatability, mean comparison between methods with 95% confidence interval (CI), and r(2). A CI range of (-0.5, 0.5) on the mean difference was used as the acceptance criterion to establish significant statistical differences between methods. The evaluations demonstrate that the Peel Plate AC provides no statistical differences across most of the matrixes with r(2) > 0.96. In the case of skim milk, there were significant differences that may be explained by a matrix-related stress on the spiked organisms but were not repeated in subsequent experiments. Within method repeatability of Peel Plate AC was similar to reference method with relative standard deviations in the ranges of 2 to 5% when log10 means were ≥1.5. Quality control data support that Peel Plate AC is stable for at least 1 year refrigerated. Incubation temperature ranges 30-36°C and times 45 -51 h were not significantly different.

Evaluation of Peel Plate™ EC for Determination of E. coli and Coliform or Total Coliform in Dairy Products.

Peel Plate™ EC is a low-profile plastic, 47 mm culture dish with an adhesive top that contains a dried medium with Gram-negative selective agents and with enzyme substrate indicators for ß-galactosidase (coliform) and ß-glucuronidase (Escherichia coli). The method provides a conventional quantitative coliform (red) and E. coli (blue/purple/black) count with simple rehydration and incubation for 24 ± 2 h at 35 ± 1°C, while providing a total coliform result, sum of E. coli, and coliform without color differential in dairy products at 32 ± 1°C for 24 ± 2 h. Dairy matrixes claimed and supported with total coliform data are whole milk, skim milk, chocolate milk (2% fat), heavy cream (35% fat), pasteurized whole goat milk, ultra-high-temperature pasteurized milk, powdered milk, lactose-reduced milk, strawberry milk, shredded cheddar cheese, raw cow milk, raw goat milk, raw sheep milk, sour cream, condensed milk, eggnog, vanilla ice cream, condensed whey, yogurt, and cottage cheese. Matrixes claimed for E. coli and total coliform detection are raw ground beef, mixed cellulose 0.45 µm filtered bottled water, environmental sponge of stainless steel, raw ground turkey, dry dog food, liquid whole pasteurized eggs, milk chocolate, leafy green (mixed greens) rinse/flume water, irrigation water, poultry carcass rinse, and large animal carcass sponge. The method has been independently evaluated for total coliform in whole milk, skim milk, chocolate milk, and heavy cream. The method was also independently evaluated for E. coli and coliform in ground beef, filtered bottled water, and sponge rinse from stainless steel surfaces. In inclusivity and exclusivity studies, the method detected 57 of 58 different strains of coliform and E. coli at 32 ± 1°C and 35 ± 1°C in and excluded 31 of 32 different noncoliform strains consisting of Gram-negative and Gram-positive bacteria. In the matrix study, each matrix was assessed separately at each contamination level in comparison to an appropriate reference method. Colony counts were determined for each level and then log10 transformed. The transformed data were evaluated for repeatability, log-mean comparison between methods with 95% confidence interval, and r(2). A 95% confidence interval range of -0.5 to 0.5 on the mean difference was used as the acceptance criterion to establish significant statistical difference between methods. The evaluations demonstrate that the Peel Plate EC method provides no statistical differences across most of the matrixes. The coliform r(2) values were greater than 0.9 except in the case of skim milk (r(2) = 0.77 and 0.69), sheep milk (0.84), and chocolate (0.81). In the case of skim milk, the three highest concentrations were significantly biased low compared with the reference method, whereas in the case of chocolate, the highest concentration was significantly biased high. The E. coli r(2) values were greater than 0.9 except in the case of hog rinse (0.89), flume water (0.82), and chocolate (0.77). The lower values were generally from only a 1 log difference between highest and lowest concentrations except in the case of chocolate, in which the highest concentration was biased high compared with the reference method. Within-method repeatability of Peel Plate EC was similar to the reference method, with relative SDs generally less than 5% when log10 means were ≥1.5. QC data support that the Peel Plate EC is stable for 1 year when refrigerated. Incubation temperature ranges, 30-36°C, and times, 22-26 and 48 h for yogurt, were not significantly different in paired t-test comparison. The method is selective without the need for confirmation, although confirmation of coliform and E. coli was performed as part of the validation work.

Proposed modifications of Environmental Protection Agency Method 1601 for detection of coliphages in drinking water, with same-day fluorescence-based detection and evaluation by the performance-based measurement system and alternative test protocol validation approaches.

Coliphages are microbial indicators specified in the Ground Water Rule that can be used to monitor for potential fecal contamination of drinking water. The Total Coliform Rule specifies coliform and Escherichia coli indicators for municipal water quality testing; thus, coliphage indicator use is less common and advances in detection methodology are less frequent. Coliphages are viral structures and, compared to bacterial indicators, are more resistant to disinfection and diffuse further distances from pollution sources. Therefore, coliphage presence may serve as a better predictor of groundwater quality. This study describes Fast Phage, a 16- to 24-h presence/absence modification of U.S. Environmental Protection Agency (EPA) Method 1601 for detection of coliphages in 100 ml water. The objective of the study is to demonstrate that the somatic and male-specific coliphage modifications provide results equivalent to those of Method 1601. Five laboratories compared the modifications, featuring same-day fluorescence-based prediction, to Method 1601 by using the performance-based measurement system (PBMS) criterion. This requires a minimum 50% positive response in 10 replicates of 100-ml water samples at coliphage contamination levels of 1.3 to 1.5 PFU/100 ml. The laboratories showed that Fast Phage meets PBMS criteria with 83.5 to 92.1% correlation of the same-day rapid fluorescence-based prediction with the next-day result. Somatic coliphage PBMS data are compared to manufacturer development data that followed the EPA alternative test protocol (ATP) validation approach. Statistical analysis of the data sets indicates that PBMS utilizes fewer samples than does the ATP approach but with similar conclusions. Results support testing the coliphage modifications by using an EPA-approved national PBMS approach with collaboratively shared samples.

Comparative analysis of a modified ecolite method, the colicomplete method, and a most-probable-number method for detecting Escherichia coli in orange juice.

The Ecolite High Volume Juice (HVJ) presence-absence method for a 10-ml juice sample was compared with the U.S. Food and Drug Administration Bacteriological Analytical Manual most-probable-number (MPN) method for analysis of artificially contaminated orange juices. Samples were added to Ecolite-HVJ medium and incubated at 35 degrees C for 24 to 48 h. Fluorescent blue results were positive for glucuronidase- and galactosidase-producing microorganisms, specifically indicative of about 94% of Escherichia coli strains. Four strains of E. coli were added to juices at concentrations of 0.21 to 6.8 CFU/ ml. Mixtures of enteric bacteria (Enterobacter plus Klebsiella, Citrobacter plus Proteus, or Hafnia plus Citrobacter plus Enterobacter) were added to simulate background flora. Three orange juice types were evaluated (n = 10) with and without the addition of the E. coli strains. Ecolite-HVJ produced 90 of 90 (10 of 10 samples of three juice types, each inoculated with three different E. coli strains) positive (blue-fluorescent) results with artificially contaminated E. coli that had MPN concentrations of <0.3 to 9.3 CFU/ml. Ten of 30 E. coli ATCC 11229 samples with MPN concentrations of <0.3 CFU/ml were identified as positive with Ecolite-HVJ. Isolated colonies recovered from positive Ecolite-HVJ samples were confirmed biochemically as E. coli. Thirty (10 samples each of three juice types) negative (not fluorescent) results were obtained for samples contaminated with only enteric bacteria and for uninoculated control samples. A juice manufacturer evaluated citrus juice production with both the Ecolite-HVJ and Colicomplete methods and recorded identical negative results for 95 20-ml samples and identical positive results for 5 20-ml samples artificially contaminated with E. coli. The Ecolite-HVJ method requires no preenrichment and subsequent transfer steps, which makes it a simple and easy method for use by juice producers.

Comparative Study of a Colorimetric DNA Hybridization Method and Conventional Culture Procedures for the Detection of Listeria spp. in Foods.

A colorimetric DNA hybridization-based assay has been evaluated against two conventional culture methods (FDA and USDA) for detection of Listeria spp. in dairy, Meat, and seafood products. A total of 1,300 samples representing 15 food types were analyzed in parallel by both the DNA hybridization (DNAH) and culture methods (FDA for dairy products and seafoods, USDA for meats). Samples included inoculated and naturally contaminated products and uninoculated controls. Fifteen strains representing five species of Listeria were used as inocula. Of 660 dairy and seafood samples tested, the FDA culture method detected 354 positives and the DNAH method detected 393 positives, 391 of which were confirmed. The DNAH method was statistically equivalent to the FDA method for eight of the nine products tested. In some trials, the DNAH method detected more positives than the FDA method for cheddar cheese and in some cases these differences were statistically significant. Of 540 meat samples tested, the USDA culture method detected 261 positives and the DNAH method detected 378 positives, all of which were confirmed. The DNAH method was statistically equivalent to the culture method for three of the six products tested. In some trials, the DNAH method detected more positives than the USDA method for roast beef, hot dogs, and fermented sausage. In some cases, these differences were statistically significant. Of 100 naturally contaminated products tested, the DNAH method detected 86 positives and the culture methods detected 84 positives. The DNAH method was statistically equivalent to the culture methods for these samples. The DNAH method gives a negative result 48 h after the start of sample enrichment, whereas the FDA and USDA methods require 3 to 4 days or longer. It is concluded that the DNAH assay is a rapid, accurate, and objective alternative to the culture procedures for the detection of Listeria spp. in foods.