biomérieux France MicroVal Study 2014LR47 Summary report

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1 ACCREDITATION N PORTEE DISPONIBLE SUR biomérieux Chemin de l Orme MARCY L ETOILE France MicroVal Study 2014LR47 Summary report ISO part 2: validation study of a TEMPO method for Bacillus cereus enumeration in food samples and environmental samples This report includes 39 pages, with 2 appendixes. Only copies including the totality of this report are authorized. Expert laboratory competences are certified by COFRAC accreditation for all analysis marked with a symbol. Version 0 March 22, 2016 ADRIA DEVELOPPEMENT Creac h Gwen - F QUIMPER Cedex - Tél. (33) Fax (33) adria.developpement@adria.tm.fr - Site web : ASSOCIATION LOI DE N SIRET N EXISTENCE N TVA FR

2 1 INTRODUCTION 4 2 METHODS DESCRIPTION Reference method Alternative method 4 3 METHODS COMPARISON STUDY Relative trueness study Number and nature of the samples Artificial and natural contamination of the samples Calculation and interpretation Discordant results Data and interpretations using the lowest dilutions when possible TEMPO cards storage for 48 h at 2 8 C Conclusion Accuracy profile study Food matrices Calculation and interpretation Conclusion Quantification limits (LOQ) Inclusivity and exclusivity studies Protocol Results 27 4 INTER-LABORATORY STUDY ORGANISATION AND RESULTS Study organisation Verification of experimental parameters Strain stability during transport Logistic conditions Homogeneity of inoculation Calculations, summary and interpretation of data 31 5 CONCLUSION 37 Appendix 1 - Flow diagram of the ISO 7932 reference method test procedure - Horizontal method for the enumeration of presumptive Bacillus cereus: Colony-count technique at 30 C 38 Appendix 2 - Flow diagram of the alternative method 39 ADRIA Développement 2/39 March 22, 2016

3 Before comment Quality assurance documents related to this study can be consulted upon request from biomérieux. The technical protocol and the result interpretation were realized according to the EN ISO and the MicroVal technical rules. Company: biomérieux Chemin de l Orme F MARCY L ETOILE (France) Expert Laboratory: ADRIA Développement ZA Creac h Gwen F QUIMPER Cedex (France) Studied method: TEMPO method for Bacillus cereus group enumeration Validation standard: ISO/FDIS (2014) : Microbiology of the food chain Method validation Part 2: Protocol for the validation of alternative (proprietary) methods against a reference method Reference method : ISO 7932 (2004): horizontal method for the enumeration of presumptive Bacillus cereus - Colony-count technique at 30 C Scope: Food products and environmental samples Certification organization: MicroVal Analyses performed according to the COFRAC accreditation ADRIA Développement 3/39 March 22, 2016

4 1 INTRODUCTION This study provides data for an ISO approval of a TEMPO method for Bacillus cereus group enumeration. The proposed technical protocol fits with the ISO standard (FDIS version, 2014). Indeed, a minimum of 5 food categories has been tested to get a broad range of foods claim. The environmental samples are considered as an additional category. This method comparison study gathers: - The relative trueness study; - The accuracy profiles; - The limits of quantification (LOQ); - The inclusivity and exclusivity. 2 METHODS DESCRIPTION 2.1 Reference method The reference method will be the ISO 7932 (2004): horizontal method for the enumeration of presumptive Bacillus cereus - Colony-count technique at 30 C (See Appendix 1). 2.2 Alternative method TEMPO system is an automated method associating an innovative card with an adapted medium to ensure rapid enumeration of several quality indicators. The method is based on the MPN principle (Most Probably Number), with the TEMPO card. The TEMPO test consists in a culture medium associated to the TEMPO card. The culture medium is inoculated with the sample to be tested and transferred by the TEMPO Filler into the card. Analysis performed according to the COFRAC accreditation ADRIA Développement 4/39 March 22, 2016

5 Depending on the number and size of the positive wells, the TEMPO Reader calculates the Bacillus cereus group count. Card reading, interpretation and reporting are directly managed by the TEMPO system. The flow diagram is provided in Appendix 2. When the analyst doesn t know the contamination levels of the tested samples, it is advised to run the following dilutions (See Table 1). It is also possible to select another dilution which could be more appropriate to the contamination level, when this one is of course known. The calculations are presented using the usually done dilutions. An annex of the trueness study presents as well the calculations using the lowest available dilution. Note that the storage of the cards for 48 h at 2-8 C was also tested in this part of the study. Table 1 Dilutions usually done Products Dilution Baby food Milk powder Egg powder 1/40 Dusts from infant formula and baby food production units Infant formula Spices, inhibitory products (aromatic herbs...) 1/200, 1/400 Strongly colored products All other products 1/200 3 METHODS COMPARISON STUDY The method comparison study is a study, performed by the organizing laboratory to compare the alternative method with the reference method. 3.1 Relative trueness study The trueness is the closeness of agreement between the average of an infinite number of replicate measured quantity values and a reference quantity value. The trueness study is a comparative study between results obtained by the reference method and the results of the alternative method. This study is conducted using naturally or artificially contaminated samples. Different categories, types and items will be tested for this. ADRIA Développement 5/39 March 22, 2016

6 3.1.1 Number and nature of the samples Five food categories and environmental samples were tested. The repartition per tested category and type is provided in Table 2: Table 2 Categories and types to be tested Category Types Number of samples analysed Number of interpretable results 1 - Meat products, fish products and seafood 1 RTE foods: cooked shrimps, cooked delicatessen, surimi, terrines, etc RTE composite foods: sushi, deli-salads, etc Ready-to-cook and ready to reheat foods: meat preparations, seafood 7 5 Total Milk powders Dairy 2 Cream cheeses, cheese preparations (with dried tomatoes, spices, etc...) 8 5 products 3 Dairy desserts 7 5 Total Pasteurized liquid eggs Egg products 2 Egg powders Pastries, creams, desserts 7 7 Total Flours, spices, dehydrated vegetables Fruits and 2 Pasta, rices 13 7 vegetables 3 Deli salads, vegetables preparations 12 5 Total Infant formula and cereals, raw materials, without probiotics Baby food 2 Infant formula and cereals, raw materials, with probiotics Ready-to-reheat foods Environmental samples Total Process waters Wipes, sponges, swaps Dusts 6 6 TOTAL Total ADRIA Développement 6/39 March 22, 2016

7 164 samples were analyzed, leading to 101 exploitable results. The samples, which were not used in the calculations, did provide data as presented Table 3: Table 3 Number of samples Results below With the reference method 11 the detection limit With the alternative method 1 With the two methods 31 Results above With the reference method 1 the detection limit With the alternative method 1 Presence of high background microflora on 18 reference method plates TOTAL Artificial and natural contamination of the samples Artificial contaminations were realized by spiking or seeding protocols. Injury efficiency was evaluated by enumerating the pure culture on selective and non-selective agars. The observed injury measurements vary from 0.20 to more than 2.65 Log CFU/ml difference. 32 samples were naturally contaminated and 84 samples were artificially contaminated; 69 gave interpretable results.! Note that all the artificially contaminated samples are indicated in bold characters all among the report. ADRIA Développement 7/39 March 22, 2016

8 3.1.3 Calculation and interpretation The obtained data were analyzed using the scatter plot. The graphs are provided with the line of identity (y = x). The Figures 1a to 1f show the data plotted per category and for all products. The Figure 2 gathers all the categories, and two interpretations were done: - Figure 2a: All the substituted values are at first plotted as mentioned in the ISO standard. Indeed, for results (either reference or alternative method) below the quantification limit, the data are plotted using a substituted value of 1 log 10 unit less than the observed value in case of a lower than value. Any value greater than the upper limit is amended by adding 1 log 10 unit more. Indeed, values below the method quantification limit due to the dilution scheme are plotted, as well as values obtained with very low contamination levels. - Figure 2b: Only the true discordant results observed between the reference and the alternative methods are plotted, i.e. the values below the method quantification limit due to the dilution scheme are not plotted, as well as the values obtained with very low contamination levels. Note that many substituted values are observed with the reference method in comparison to the alternative method. ADRIA Développement 8/39 March 22, 2016

9 Alternative method (log cfu/g) Alternative method (log cfu/g) MicroVal Project 2014LR47 Figure 1 Scatter plot using substituted values for all the data below or upper the quantification limit - Usual dilutions: 22 h 8,00 Figure 1a Cat 1 : Meat products, fish products and seafood-22h 6,00 4,00 2,00 0,00 0,00 2,00 4,00 6,00 8,00 Reference method (log cfu/g) Type1 Type 2 Type 3 substituted values Figure 1b Cat 2 : Dairy products-22h 8,00 6,00 4,00 2,00 0,00 0,00 2,00 4,00 6,00 8,00 Reference method (log cfu/g) Type 1 Type 2 Type 3 substituted values ADRIA Développement 9/39 March 22, 2016

10 Alternative method (log cfu/g) Alternative method (log cfu/g) MicroVal Project 2014LR47 Figure 1c Cat 3 : Egg products-22h 8,00 6,00 4,00 2,00 0,00 0,00 2,00 4,00 6,00 8,00 Reference method (log cfu/g) Type 1 Type 2 Type 3 substituted values Figure 1d Cat 4 : Fruits and vegetables-22h 8,00 6,00 4,00 2,00 0,00 0,00 2,00 4,00 6,00 8,00 Reference method (log cfu/g) Type 1 Type 2 Type 3 substituted values ADRIA Développement 10/39 March 22, 2016

11 Alternative method (cfu/g) MicroVal Project 2014LR47 Figure 1e Cat 5 :Baby food-22h 8,00 6,00 4,00 2,00 0,00 0,00 2,00 4,00 6,00 8,00 Reference method (log cfu/g) Type 1 Type 2 Type 3 Figure 1f Cat 6: Environmental samples-22h 8,00 6,00 4,00 2,00 Type 1 Type 2 Type 3 Substituted values 0,00 0,00 2,00 4,00 6,00 8,00 ADRIA Développement 11/39 March 22, 2016

12 Alternative method (log CFU/g) Alternative method (log CFU/g) MicroVal Project 2014LR47 Figure 2 - Data plotted for all the categories Figure 2a - Data plotted with all the substituted values All categories-22h 8,00 7,00 6,00 5,00 4,00 3,00 2,00 1,00 0,00 0,00 2,00 4,00 6,00 8,00 Reference method (log CFU/g) Cat 1 Cat 2 Cat 3 Cat 4 Cat 5 Cat 6 Substituted values y=x Figure 2b - Data plotted with only the substituted values for the discordant results All categories-22h 8,00 7,00 6,00 5,00 4,00 3,00 2,00 1,00 0,00 0,00 2,00 4,00 6,00 8,00 Reference method (log CFU/g) Cat 1 Cat 2 Cat 3 Cat 4 Cat 5 Cat 6 Substituted values y=x ADRIA Développement 12/39 March 22, 2016

13 Difference Difference MicroVal Project 2014LR47 The Bland-Altman difference plot for all the categories is presented Figure 3, with as well two interpretations: - Figure 3a: Presentation with all the substituted values; - Figure 3b: Presentation with only the true discordant data. Figure 3 Bland-Altman difference plot for all the categories: 22 h Figure3a : Presentation with all the substituted values Bland-Altman difference plot for all categories-22h 3,00 2,00 1,00 0,00-1,00-2,00-3,00 0,00 1,00 2,00 3,00 4,00 5,00 6,00 Mean of log cfu/g Cat 1 Cat 2 Cat 3 Cat 4 Cat 5 Linear(95% upper limit) Linear (95% lower limit) substituted values Linear (Bias) Cat 6 Figure3b: Presentation with only the true discordant data Bland-Altman difference plot for all categories-22h 3,00 2,00 1,00 0,00-1,00-2,00-3,00 0,00 1,00 2,00 3,00 4,00 5,00 6,00 Mean of log cfu/g Cat 1 Cat 2 Cat 3 Cat 4 Cat 5 Cat 6 Substituded values Lower limit (95%) Upper limit (95% ) Linear bias ADRIA Développement 13/39 March 22, 2016

14 3.1.4 Discordant results We are used to highlight and provide additional explanations on biases higher than 0.5 Log CFU/g difference between the compared methods. A few samples show such positive or negative biases. Positive bias higher than 0.5 Log CFU/g Three results showing a higher enumeration with TEMPO BC method than with the ISO 7932 standard are observed. The TEMPO broths contained in the TEMPO cards were streaked on Mossel selective medium, and confirmation tests were run on characteristic colonies. The presence of B. cereus isolates were confirmed in all the cases (Table 4). Table 4 Discordant results with a positive bias Bias Sample Identification on TEMPO Products log Alt - log Ref n BC (log CFU/g) 551 Ready to-reheat preparation (Seafood) - Garniture bretonne > + 1 Presence of B.cereus strain 1093 Whole egg product Presence of B. cereus strain 73 Dehydrated mushrooms > + 2 Presence of B.cereus strain Note that all these positive biases concern naturally contaminated samples. Negative bias higher than 0.5 Log CFU/g Eight results showing a higher enumeration with ISO 7932 standard than with the TEMPO BC method are observed (See Table 5). ADRIA Développement 14/39 March 22, 2016

15 Table 5 Discordant results with a negative bias Bias Sample log Ref - log Alt Products Contaminations n (log CFU/g) 22 h 1364 Deli salad Natural Ready to reheat meat product Natural Cream cheese with onion B. cereus Ad White egg powder B. weihenstephensis Ad 729 (spores) Fruit salad B. cereus Ad Raw material with probiotics B. cereus AER 400 (spores) Infant formula milk powder B. weihenstephenensis Ad 782 with probiotics (spores) Wipe ready to eat meal B. weihenstephenensis Ad All the observed negative biases are below 1 Log CFU/g. The artificial contaminations were run to mimic as much as possible the natural contaminations, but it is impossible to guaranty the equivalence between the cell physiology states. Indeed, note that the negative biases are mainly encountered on artificially contaminated samples with vegetative cells or spores (6 samples on 8), while the positive biases previously mentioned were only observed on naturally contaminated samples Data and interpretations using the lowest dilutions when possible As observed previously, only few biases are observed. The trueness of the TEMPO BC method is as well confirmed using this dilution scheme TEMPO cards storage for 48 h at 2 8 C The scatter plots are presented Figures 4a to 4f per category with all the substituted values, and Figure 5a for all the categories with only the true discordant data. The Bland-Altman graph for all the categories with the true discordant data is presented Figure 5b. ADRIA Développement 15/39 March 22, 2016

16 Alternative method (log cfu/g) Alternative method (log cfu/g) Alternative method (log cfu/g) MicroVal Project 2014LR47 Figure 4 Scatter plot using substituted values Cards storage for 48 h at 2 8 C (Presentation with all the substituted values) 8,00 6,00 Figure 4a Cat 1 : Meat products, fish products and seafood (2-8 C) 4,00 2,00 0,00 0,00 2,00 4,00 6,00 8,00 Reference method (log cfu/g) Type 1 Type 2 Type 3 substitued values Figure 4b 8,00 Cat 2 : Dairy products (2-8 C) 6,00 4,00 2,00 0,00 0,00 2,00 4,00 6,00 8,00 Reference method (log cfu/g) Type 1 Type 2 Type 3 substituted values Figure 4c 8,00 Cat 3 : Egg products (2-8 C) 6,00 4,00 2,00 0,00 0,00 2,00 4,00 6,00 8,00 Reference method (log cfu/g) Type 1 Type 2 Type 3 substituted values ADRIA Développement 16/39 March 22, 2016

17 Alternative method (log cfu/g) Alternative method (log cfu/g) Alternative method (log cfu/g) MicroVal Project 2014LR47 8,00 Figure 4d Cat 4 : Fruits and vegetables (2-8 C) 6,00 Type 1 4,00 Type 2 2,00 Type 3 substituted values 0,00 0,00 2,00 4,00 6,00 8,00 Reference method (log cfu/g) 8,00 Figure 4e Cat 5 : Baby food (2-8 C) 6,00 4,00 2,00 Type 1 Type 2 Type 3 0,00 0,00 2,00 4,00 6,00 8,00 Reference method (log cfu/g) Figure 4f 8,00 Cat 6: Environmental samples (2-8 C) 6,00 4,00 2,00 0,00 0,00 2,00 4,00 6,00 8,00 Reference method (log cfu/g) Type 1 Type 2 Type 3 Substituded values ADRIA Développement 17/39 March 22, 2016

18 Difference Alternative method (log CFU/g) MicroVal Project 2014LR47 Figure 5 Scatter plot using substituted values Cards storage for 48 h at 2 8 C (Presentation with only the true discordant data) Figure 5a All categories-storage 48H at 2-8 C 8,00 7,00 6,00 5,00 4,00 3,00 2,00 1,00 0,00 0,00 2,00 4,00 6,00 8,00 Reference method (log CFU/g) Cat 1 Cat 2 Cat 3 Cat 4 Cat 5 Cat 6 Substituted values y=x Figure 5b Bland-Altman difference plot for all categories- Storage 48H at 2-8 C 4,00 3,00 2,00 1,00 0,00-1,00-2,00-3,00 0,00 1,00 2,00 3,00 4,00 5,00 6,00 Mean of log cfu/g Cat 1 Cat 2 Cat 3 Cat 4 Cat 5 Cat 6 Substituded values Lower limit (95%) Upper limit (95% ) All the cards were stored for 48 h at 2 8 C and very slight modifications were observed, except for two samples: 1104 and 1105 (milk powder). ADRIA Développement 18/39 March 22, 2016

19 A decrease of enumeration was observed for these two samples. Additional tests were realized on the matrices and strains used for inoculation; they are provided below (Table 6). Table 6 Inclusivity protocol on the inoculated B. cereus Ad 848 strain TEMPO 22 h Card storage for 48 h at 4 C 1ml (-4) 1ml (-5) 1ml (-4) 1ml (-5) B.cereus Ad 848 > > Analysis of blank matrices TEMPO 22H Card storage for 48 h at 4 C 1/40 1/200 1/40 1/200 Matrice 1104 <10 <50 <10 <50 Matrice 1105 <10 <50 <10 <50 Analysis of matrices inoculated with B. cereus Ad 840 TEMPO 22 h Card storage for 48 h at 4 C 1/40 1/200 1/40 1/200 Matrix Matrix Analyses of other matrices inoculated with B. cereus Ad 848 TEMPO 22 h Card storage for 48 h at 4 C 1/40 1/200 1/40 1/200 Cream cheese Milk powder The results observed on samples n 1104 and 1105 were not repeated by running an inclusivity testing, by analyzing other matrices inoculated with the same strain, or by analyzing the same matrices inoculated with another strain (B. cereus Ad 840). ADRIA Développement 19/39 March 22, 2016

20 3.1.7 Conclusion While TEMPO BC was able to provide quantitative data, the reference method failed in many cases. This was mainly due to the quantification limit of the reference method. Few differences higher than 0.5 Log CFU/g are observed between the alternative and the reference methods: 3 positive and 8 negative biases. Note that the negative biases mainly concerns artificially contaminated samples, which may not always mimic the natural conditions of contamination. Only two differences are observed when comparing a reading of the cards just after the incubation time to a reading of the cards after an additional storage at 2-8 C. We were not able to repeat the observed data on other similar matrices. The relative trueness study of the TEMPO BC method is satisfying, and the TEMPO Cards could be stored at 2-8 C for 48 h before reading, as no impact was observed. 3.2 Accuracy profile study The accuracy profile is a graphical representation of the capacity of measurement of the quantitative method, obtained by combining acceptability intervals and β-expectation tolerance intervals, both reported to different levels of the reference value. The accuracy profile study is a comparative study between the results obtained by the reference and the results of the alternative method. This study is conducted using artificially contaminated samples. One type per category is tested for this Food matrices Ten food matrices were tested with three contamination levels and five test portions per level. One environmental sample was tested with six contamination levels and five test portions per level. Note that an additional testing was done on the infant formula matrix using at least two different strains. The tested categories, types, items and inoculated strains are provided in the Table 7. ADRIA Développement 20/39 March 22, 2016

21 Table 7 - Categories, types and food items Category Type Item Strain Meat products, fish products and seafood RTE foods - Pâté Dairy products Dairy desserts - Panna cotta Egg products Fruits and vegetables Baby foods Environmental samples Egg products Deli salads, vegetables preparations Infant formula and cereals, raw materials with probiotics Dusts - Whole egg product - Liquid egg yolk - Deli salad (tabbouleh) - Purée - Infant formula milk powder - Infant cereals with probiotics - Dusts from dairy industry Bacillus cereus Ad 20 (group IV) Bacillus cereus Ad 1467 (group II) Bacillus weihenstephanensis Ad 730 (group VI) Bacillus cereus Ad 797 (group II) Bacillus cytotoxicus Ad 2031 (group VII) Bacillus cereus Ad 754 (group V) Bacillus weihenstephanensis Ad782 (group VI) (spores)* Bacillus cereus AER 400 (group IV) spores*** Bacillus cereus Ad 495 (group III) (spores)* Bacillus thuringiensis Ad 777 (group VI) (spores)* Target inoculation level (CFU/g) ** ** Sample dilution in the TEMPO BC sample preparation 1/200 1/200 1/200 1/200 1/200 1/200 1/40 1/40 1/200 * Spores from ADRIA collection were used. Spores are stored in sterile water and no difference of enumerations was observed by enumerating the spores and the total population (spores and vegetative cells): no heat treatment to kill the vegetative cells was required prior inoculation. A heat treatment was done on the freshly prepared spores suspensions of the Bacillus weihenstephanensis Ad 782 and Bacillus thuringiensis Ad 777 prior inoculation to kill the vegetative cells, as difference of enumerations was observed by enumerating the spores and the total population (spores and vegetative cells). ** An inversion was done between the second and third inoculation levels. *** As outlier data were observed with the (Infant formula / B. weihenstephanensis Ad782) pair, a second testing was done on this matrix using another strain from an appropriate origin, i.e. B. cereus AER 400. ADRIA Développement 21/39 March 22, 2016

22 3.2.2 Calculation and interpretation The statistical results and the accuracy profiles are provided Figure 6. For any of the upper or lower limits exceeded the limits and the standard deviation, additional evaluation procedure was followed, as described the ISO (FDIS, 16140): new acceptability limits as a function of the standard deviation AL 4 s were calculated. It was only the case for the s ref Deli-salads and vegetables preparations category. The AL were defined at ± instead of 0.5. ADRIA Développement 22/39 March 22, 2016

23 Bias Bias Bias Bias Bias Bias MicroVal Project 2014LR47 Figure 6 TEMPO BC Accuracy profile (Food) Category Meat & fish products (Food) Category Dairy products (Food) Type RTE foods (Food) Type Dairy desserts (Food) Category (Food) Type 0,800 0,600 0,600 0,400 RTE foods Meat and fish products RTE Foods RTE Foods 0,400 0,200 0,200 0,000 0,00 0,50 1,00 1,50 2,00 2,50 3,00 3,50 4,00 4,50 5,00-0,200 0,000 0,00 1,00 2,00 3,00 4,00 5,00 6,00-0,200-0,400-0,600-0,400 é Bias Bias AL = +/- 0.5 AL = +/ ,600 (Food) Category (Food) Type 0,400 0,200 0,600 Dairy desserts 0,000 0,400 0,00 0,50 1,00 1,50 2,00 2,50 3,00 3,50 4,00 4,50 Bias -0,200 0,200 AL = +/ ,400 0,000 0,00 0,50 1,00 1,50 2,00 2,50 3,00 3,50 4,00 4,50 5,00-0,600-0,200-0,800-0,800-0,400-0,600 Reference Median Reference Median Reference Median -0,600 Reference Median to Reference Reference compared to compared to compared to compared to Sample Name Sample Name Bias Lower Upper central value Bias Lower Upper Central value AL=±0.5 Median AL=±0.5 final AL final AL Reference compared Acceptable to compared Acceptable to Acceptable Acceptable Sample Name Bias Lower Upper -0,362 0, central 2,00 value -0,280-0,548-0,012 AL=±0.5 NO final AL ,20 0,000-0,299 0,299 YES YES NO Acceptable Acceptable ,73 2,64-0,010-0,300-0,309-0,662 0,289 0,062 YES NO YES NO ,57-0,370-0,638-0,102 NO NO ,00-0,280-0,511-0,049 NO NO ,20-0,050-0,349-0,412 0,249 0,312 YES YES (2015) 3,04 2,66-0,010-0,110-0,241-0,378 0,221 0,158 YES YES YES YES ,81 3,57-0,090-0,190-0,389-0,552 0,209 0,172 YES NO YES NO ,40 3,04-0,110-0,120-0,341-0,388 0,121 0,148 YES YES YES YES ,51-0,110-0,409 0,189 YES YES ,81 0,050-0,312 0,412 YES YES (2015) 3,98 3,60-0,190 0,170-0,421-0,098 0,041 0,438 YES YES YES YES ,81 0,130-0,169 0,429 YES YES ,98 0,170-0,061 0,401 YES YES ,72 0,150-0,212 0,512 NO NO ,18 0,200-0,068 0,468 YES YES (2015) 4,59 0,060-0,171 0,291 YES YES Reference Alternative SD repeatability of reference Final AL Reference method Alternative method SD repeatability method > of reference SD repeatability of reference Final AL Reference Alternative SD repeatability of reference Final AL method method method > method method method > Final AL method > SD Repeatability 0,103 0,207 NO +/-0,500 SD Repeatability 0,098 0,251 NO +/-0,500 SD SD Repeatability 0,121 0,082 0,160 0,186 NO NO +/-0,500 +/-0,500 Dairy products Dairy dessert Dairy dessert Bias AL = +/- 0.5 (Food) Category Egg products (Food) Category Fruits & vegetables (Food) Type Egg products (Food) Type Deli salads, vegetables Egg products Deli salads, vegetables preparations 0,600 0,800 0,400 0,600 0,200 0,400 0,000 0,00 0,50 1,00 1,50 2,00 2,50 3,00 3,50 4,00 4,50 5,00-0,200 Bias AL = +/ ,200 0,000 0,00 0,50 1,00 1,50 2,00 2,50 3,00 3,50 4,00 4,50-0,200 Bias AL = +/- 4SDr -0,400-0,400-0,600-0,600 Reference Median -0,800 Reference Median Sample Name Reference Median Bias Lower Upper compared to AL=±0.5 Acceptable compared to final AL Acceptable Sample Name Reference Median Bias Lower Upper compared to AL=±0.5 Acceptable ,23-0,190-0,455 0,075 YES YES ,78 0,220-0,101 0,541 NO YES ,43-0,230-0,495 0,035 YES YES ,65-0,190-0,511 0,131 NO YES ,26-0,060-0,325 0,205 YES YES ,98-0,150-0,471 0,171 YES YES ,51 0,050-0,215 0,315 YES YES ,45-0,170-0,491 0,151 YES YES ,32-0,090-0,355 0,175 YES YES ,67 0,010-0,311 0,331 YES YES ,51 0,140-0,125 0,405 YES YES ,11-0,260-0,581 0,061 NO YES Reference method Alternative method SD repeatability of reference method > Final AL SD Repeatability 0,110 0,184 NO +/-0,500 SD Repeatability 0,145 0,222 YES +/-0,582 Reference method Alternative method SD repeatability of reference method > compared to final AL Acceptable Final AL ADRIA Développement 23/39 March 22, 2016

24 Bias Bias Bias MicroVal Project 2014LR47 (Food) Category Baby food (Food) Type Infant formula and cereals ( 1,000 Infant formula and cereals ( Ad495 + Ad782) (Food) Category Baby food (Food) Type Infant formula and cereals 0,600 Infant formula and cereals (Ad495 + AER400) 0,500 0,400 0,000 0,00 0,50 1,00 1,50 2,00 2,50 3,00 3,50 4,00 4,50 Bias 0,200-0,500-1,000 AL = +/ ,000 0,00 0,50 1,00 1,50 2,00 2,50 3,00 3,50 4,00 4,50 5,00-0,200 Bias AL = +/ ,500-0,400-2,000 Reference Median -0,600 Reference Median Sample Name Reference Median Bias Lower Upper compared to AL=±0.5 Acceptable compared to final AL Acceptable ,95 0,050-0,319 0,419 YES YES ,15-1,150-1,519-0,781 NO NO ,70-0,140-0,509 0,229 NO NO ,40-0,440-0,809-0,071 NO NO ,65 0,000-0,369 0,369 YES YES ,63-0,400-0,769-0,031 NO NO Reference method Alternative method SD repeatability of reference method > Final AL SD Repeatability 0,108 0,255 NO +/-0,500 Sample Name Reference Central value Bias Lower Upper compared to AL=±0.5 Acceptable compared to final AL Acceptable ,95 0,050-0,153 0,253 YES YES ,45 0,070-0,133 0,273 YES YES ,70-0,140-0,343 0,063 YES YES ,56-0,030-0,233 0,173 YES YES ,65 0,000-0,203 0,203 YES YES ,43 0,050-0,153 0,253 YES YES Reference method Alternative method SD repeatability of reference method > Final AL SD Repeatability 0,096 0,141 NO +/-0,500 (Food) Category (Food) Type Environmental samples Dusts from dairy industry 0,600 Dusts from dairy industry 0,400 0,200 0,000 0,00 0,50 1,00 1,50 2,00 2,50 3,00 3,50 4,00 4,50 5,00-0,200 Bias AL = +/ ,400-0,600 Reference Median Sample Name Reference Median Bias Lower Upper compared to AL=±0.5 Acceptable compared to final AL Acceptable ,20 0,000-0,278 0,278 YES YES ,67 0,040-0,238 0,318 YES YES ,04 0,040-0,238 0,318 YES YES ,71 0,130-0,148 0,408 YES YES ,08 0,000-0,278 0,278 YES YES ,69-0,040-0,318 0,238 YES YES Reference method Alternative method SD repeatability of reference method > Final AL SD Repeatability 0,056 0,192 NO +/-0,500 ADRIA Développement 24/39 March 22, 2016

25 A heat treatment was done prior inoculation of Bacillus weihenstephanensis Ad 782 spores in infant formula: this may have impacted on the variability of spores behavior, particularly at low inoculation levels between 50 and 500 CFU/g. Indeed, when inoculating the infant formula with spores from the B. cereus AER 400 without running a heat treatment, the accuracy profile is fully satisfying. This may be as well simply due to the strain itself, which is one of the most probable reasons. Indeed, the observed profiles are comprised within the AL set at 0.5 Log CFU/g in the ISO standard or recalculated according to the procedure defined in the ISO (FDIS, 2014) Conclusion The observed profiles are comprised within the AL. All the accuracy profiles fulfill the performance criteria. 3.3 Quantification limits (LOQ) As TEMPO BC is not based counting visible colonies target microorganism, the LOQ was determined. One food matrix already tested in the accuracy profiles was selected by tested category. Blank samples were tested per tested matrix. 10 test portions from the same sample were analyzed by the alternative method. The threshold standard deviation s o was calculated as followed: where s 1 n 2 0 ( y j y ) n 1 j 1 n is the total number of test portions used; y j is the log 10 transformed result of test portion j; y is the average log 10 transformed result of all test portions. ADRIA Développement 25/39 March 22, 2016

26 The limit of quantification is calculated as LOQ = 10 s o. The results are summarized Table 8. Table 8 - Quantification limits per tested matrix Matrix So LOQ Salmon terrine 0 0 Infant formula with probiotics 0 0 Artichoke purée 0 0 Whole egg 0 0 Dairy dessert 0 0 Dusts from egg industry 0 0 Whatever the tested matrix, the quantification limit is 0. ADRIA Développement 26/39 March 22, 2016

27 3.4 Inclusivity and exclusivity studies The inclusivity study is a study involving pure target strains to be detected or enumerated by the alternative method. The exclusivity study is a study involving pure non-target strains, which can be potentially crossreactive, but are not expected to be detected or enumerated by the alternative method. Inclusivity and exclusivity testing is not required for general enumeration methods such as total plate count (TPC) and yeast & mould (Y&M) methods. It is required for enumeration methods designed to detect specific microorganisms (e.g. B. cereus group) Protocol After growth according to appropriate conditions, decimal dilutions were done and enumerated in duplicate by the TEMPO method and ISO 7932 standard. Target strains and non target strains dilutions were enumerated in parallel with a non selective agar (TSA). 50 target strains from different species, genotypes and origins, as well as 31 non target strains have to be tested Results 50 target strains were tested; all were enumerated by the TEMPO Bacillus cereus method. One of them (Bacillus mycoïdes Ad 746) showed lowest enumeration data than the reference method. The Bacillus weihenstephanensis Ad 854 strain shows as well slightly lower enumeration results with the TEMPO BC method than with the reference method. Bacillus cytotoxicus strains were not tested in the inclusivity study but gave satisfying results in the trueness study and accuracy profile study. Among the 31 non target tested strains, one was enumerated by the alternative method giving a very low result (2 log less than the control media), i.e. Lysinibacillus fusiformis Ad 842. For this strain, the test was repeated and an enumeration was obtained only for one replicate (3 log less than the control media). Note that two other Lysinibacillus fusiformis strains were tested (Ad 841 and Ad 828) and gave negative results as expected. ADRIA Développement 27/39 March 22, 2016

28 4 INTER-LABORATORY STUDY ORGANISATION AND RESULTS 4.1 Study organisation Samples were sent to 18 laboratories. The study was carried out on a scallop terrine contaminated with spores of Bacillus cereus Ad 825. Samples were inoculated on Monday, September 7 th below: 2015; as described - 8 blind coded samples (10 g) in TEMPO Stomacher bags for enumeration of Bacillus cereus with the reference ISO 7932 and alternative method TEMPO BC, - 1 sample (10 g labelled Sample for Total Count enumeration ) in stomacher bag for aerobic mesophilic flora enumeration by ISO method, - 1 water flask labelled Temperature Control with a temperature probe for temperature control during transport and storage in the laboratory until the beginning of the analyse. The targeted inoculation levels were the following: - Level 0: <10 CFU/ml, - Level 1: CFU/ml, - Level 2: CFU/ml, - Level 3: CFU/ml. Each laboratory received eight samples of 10 g, i.e. two samples per inoculation level. Blind coded samples were placed in isothermal boxes, which contained cooling blocks, and express-shipped to the different laboratories. A temperature control flask containing a sensor was added to the package in order to register the temperature profile during the transport, the package delivery and storage until analyses. ADRIA Développement 28/39 March 22, 2016

29 Samples were shipped in 24 h to 48 h to the involved laboratories. The temperature conditions had to stay lower or equal to 8 C during transport, and between 0 C 8 C in the labs. Collaborators and ADRIA Développement carried out the analyses with the alternative and reference methods at Day 2 (Wednesday, September 9 th 2015). The 1/200 dilution was run with the TEMPO BC method, as a RTE food (scallop terrine) was tested. The collaborative study instructions were sent to the collaborators on August 19 th Verification of experimental parameters Strain stability during transport In order to evaluate the Bacillus cereus strain viability during transport, bacterial count of samples were checked at different times, i.e. inoculation time, and after 48 h of storage at 2-8 C. Six samples (3 contamination levels x 2 samples) were enumerated. The results are reported Table 9. Table 9 Bacillus cereus count with reference method (in log CFU/g) Day of analysis D0 D2 Bacillus cereus enumeration (cfu/g) Reference method Alternative method Replicate 1 Replicate 2 Replicate 1 Replicate Aerobic mesophilic numeration(cfu/g) ISO No evolution of the inoculated strain was observed between Day 0 and Day 2. ADRIA Développement 29/39 March 22, 2016

30 4.2.2 Logistic conditions Temperature conditions are given below (See table 10). Laboratories Table 10 - Sample temperatures at receipt Temperature measured by the probe ( C) all along the delivery and receipt Temperature measured at receipt ( C) by the Lab itself Receipt date and time A /09/ h30 B /09/ h40 C /09/ h00 D /09/ h45 E /09/ h30 F /09/ h40 G /09/ h00 H /09/ h20 I /09/ h30 J /09/ h15 K /09/ h30 L /09/ h30 M /09/ h00 N /09/ h30 O /09/ h00 P /09/ h45 Q /09/ h45 T Not yet received /09/ h00 All the samples were delivered in appropriated conditions. Temperatures during shipment and at receipt were all correct Homogeneity of inoculation Homogeneity tests were conducted according to the ISO/TS Ten samples per inoculation level were analysed in duplicate by the reference method. The test concluded that the samples were sufficiently homogeneous for the three contamination levels. ADRIA Développement 30/39 March 22, 2016

31 4.3 Calculations, summary and interpretation of data As mentioned, the 1/200 dilution was run with the TEMPO BC method as a RTE food was analysed. One Lab (P) did not respect the instructions: they tested 1/40 and 1/400 dilution for the TEMPO method instead of 1/40 and 1/200. The results of this Lab were not taken into account for the interpretation. A summary of the test results is given Table 11 (CFU/g and log CFU/g). ADRIA Développement 31/39 March 22, 2016

32 Table 11 - Summary of data Raw data (cfu/g) Laboratory Reference method Alternative method Reference method Alternative method Reference method Alternative method Reference method Alternative method Duplicate 1 Duplicate 2 Duplicate 1 Duplicate 2 Duplicate 1 Duplicate 2 Duplicate 1 Duplicate 2 Duplicate 1 Duplicate 2 Duplicate 1 Duplicate 2 Duplicate 1 Duplicate 2 Duplicate 1 Duplicate 2 Blank Level Low Level Medium Level High Level Lab A <10 <10 <50 < Lab B <10 <10 <50 < Lab C <10 <10 <50 < Lab D <10 <10 <50 < Lab E <10 <10 <50 < Lab F <10 <10 <50 < Lab G <10 <10 <50 < Lab H <10 <10 <50 < Lab I <10 <10 <50 < Lab J <10 <10 <50 < Lab K <10 <10 <50 < Lab L <10 <10 <50 < Lab M <10 <10 <50 < Lab N <10 <10 <50 < Lab O <10 <10 <50 < Lab Q <10 <10 <50 < Lab T <10 <10 <50 < ADRIA Développement 32/39 March 22, 2016

33 Raw data (Log cfu/g) Laboratory Reference method Alternative method Reference method Alternative method Reference method Alternative method Reference method Alternative method Duplicate 1 Duplicate 2 Duplicate 1 Duplicate 2 Duplicate 1 Duplicate 2 Duplicate 1 Duplicate 2 Duplicate 1 Duplicate 2 Duplicate 1 Duplicate 2 Duplicate 1 Duplicate 2 Duplicate 1 Duplicate 2 Blank Level Low Level Medium Level High Level Lab A <1.00 <1.00 <1.70 < Lab B <1.00 <1.00 <1.70 < Lab C <1.00 <1.00 <1.70 < Lab D <1.00 <1.00 <1.70 < Lab E <1.00 <1.00 <1.70 < Lab F <1.00 <1.00 <1.70 < Lab G <1.00 <1.00 <1.70 < Lab H <1.00 <1.00 <1.70 < Lab I <1.00 <1.00 <1.70 < Lab J <1.00 <1.00 <1.70 < Lab K <1.00 <1.00 <1.70 < Lab L <1.00 <1.00 <1.70 < Lab M <1.00 <1.00 <1.70 < Lab N <1.00 <1.00 <1.70 < Lab O <1.00 <1.00 <1.70 < Lab Q <1.00 <1.00 <1.70 < Lab T <1.00 <1.00 <1.70 < ADRIA Développement 33/39 March 22, 2016

34 Log (Alternative) MicroVal Project 2014LR47 The figure 7 shows the data points after log 10 transformation. The visual inspection shows that the alternative method gives results, which are proportional to those of the reference method. The data are distributed closely to the first bisecting lines with a slope equal to 1. Figure 7 Visual linearity checking 5,5 5 4,5 4 3,5 3 2,5 2 1,5 1,5 2 2,5 3 3,5 4 4,5 5 5,5 Log (Reference) Statistical calculations were done according to the Excel spreadsheet available on A summary of the statistical test is provided Table 4. These values are collected in a graphical representation together with the acceptability limits (AL). This representation is given Figure 8 It is observed that for all the levels, the tolerance interval limits of the alternative method are within the acceptable limits of 0.5 log. The alternative method is equivalent to the reference method. ADRIA Développement 34/39 March 22, 2016

35 Table 12 Accuracy profile Study Name Tempo BC Date 07/09/2015 Coordinator Tolerance probability (beta) 80% 80% 80% Acceptability limit in log (lambda) MF Alternative method Reference method Levels Low Medium High Low Medium High Target value Number of participants (K) Average for alternative method Repeatability standard deviation (sr) Between-labs standard deviation (sl) Reproducibility standard deviation (sr) Corrected number of dof Coverage factor Interpolated Student t Tolerance interval standard deviation Lower TI limit Pooled repro standard dev Upper TI limit New acceptability limit (ALs) Bias Upper TI limit (beta = 80%) Lower TI limit (beta = 80%) Lower Acceptability limit Upper Acceptability limit ADRIA Développement 35/39 March 22, 2016

36 Accuracy (difference of Log) MicroVal Project 2014LR47 Figure 8 0,6 Bias Upper TI limit (beta = 80%) Lower TI limit (beta = 80%) Lower Acceptability limit Upper Acceptability limit 0,4 0,2 0,0 2,0 2,5 3,0 3,5 4,0 4,5 5,0-0,2-0,4-0,6 Levels Log(cfu/g) ADRIA Développement 36/39 March 22, 2016

37 5 CONCLUSION Method Comparison study The observed data and interpretation confirm the performances of the TEMPO BC method. Only few unexpected results are observed. - Many naturally contaminated samples were tested in the relative trueness study, which appears clearly satisfying; this study confirms as well the possibility to store the TEMPO BC cards for 48 h at cold temperature before reading. - The observed profiles are comprised within the AL actually set at 0.5 Log CFU/g in the ISO standard. - The inclusivity and exclusivity testing shows satisfying results. Inter-laboratory study The quality assurance parameters were verified (i.e. inoculation homogeneity, targeted levels, strain stability, logistic conditions, analyses), confirming that the inter-laboratory study was conducted in appropriated conditions. The data interpretations were done according to the ISO For the three contamination levels, the TEMPO method is accepted as equivalent to the ISO 7832 method. ADRIA Développement 37/39 March 22, 2016

38 Appendix 1 - Flow diagram of the ISO 7932 reference method test procedure Horizontal method for the enumeration of presumptive Bacillus cereus: Colony-count technique at 30 C Initial suspension xg sample + 9 x g appropriate suspension diluent Dilutions Surface plating on MYP medium 1 ml on 3 plates ( ) for low number estimation or 0.1 ml on 1 plate Incubation 18 to 24 h at 30 C If colonies are not visible, incubate 24h more Enumeration of characteristic colonies Streak 5 characteristic colonies on sheep blood agar Incubation 24 h 2 h at 30 C Haemolysis test result (Bacillus cereus: positive haemolysis test) ADRIA Développement 38/39 March 22, 2016

39 Appendix 2 - Flow diagram of the alternative method Sample preparation: 1/10 in TEMPO Stomacher bag Diluent: peptone-salt Dilution Protocol 1/40 1/200 1/ ml sterile distilled water (or equivalent) in a TEMPO flask + 1 ml suspension 1/ ml sterile distilled water (or equivalent) in a TEMPO flask ml suspension 1/10 Incubation 22 h 27 h Reading 3.9 ml sterile distilled water (or equivalent) in a TEMPO flask ml suspension 1/10 The 1/40 dilution allows 10 to cfu/g enumeration. The 1/200 dilution allows 50 to cfu/g enumeration. The 1/400 dilution allows 100 to cfu/g enumeration. ADRIA Développement 39/39 March 22, 2016