Validation of the efficiency of a decontamination process
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Validation of the efficiency of a decontamination process
23/11/2010 -1- Validation of the efficiency of a decontamination process using ozone for EURO MVP Study performed By 2010 September to November Laboratoire SILLIKER Laboratoire de Microbiologie - Unité R&D 10, rue les Châteaux Saint Sylvère 95011 CERGY Cedex FRANCE Technical contact Aurélie PERNOT Research engineer Phone : 33 (0)1 30 75 61 56 E-mail : [email protected] Joël CROCIANI Expert on microbiology Project Phone : 33 (0) 1 34 41 13 36 E-mail : [email protected] Customer Euro MVP 2, Chemin des Cossins 60750 Choisy au Bac FRANCE http://www.euromvp.com Mr Olivier VANCOILLIE Phone : 06 38 84 53 58 [email protected] 23/11/2010 -2- CONTENTS I. SUMMARY................................................................................................................................................... 3 II. MATERIAL AND METHODS............................................................................................................... 3 II.1. MEASURE OF THE BACTERIAL DESTRUCTION ON A SURFACE .................................................................. 3 II.1.1. Surface .......................................................................................................................................... 3 II.1.2. Ozone system................................................................................................................................. 3 II.1.3. Choice of strains ........................................................................................................................... 3 II.1.4. Analysis method ............................................................................................................................ 4 II.1.5. Protocol ........................................................................................................................................ 4 II.1.6. Calculation of the percentage of bacterial reduction ................................................................... 5 II.2. MEASURE OF BACTERIAL REMOVAL OF LEGIONELLA PNEUMOPHILA...................................................... 6 II.3. STUDY OF THE EFFECT OF THE TREATMENT ON A BIOFILM ...................................................................... 6 II.3.1. III. Choice of the strain....................................................................................................................... 6 RESULTS ................................................................................................................................................. 7 III.1. MEASURE OF THE BACTERIAL DESTRUCTION ON A SURFACE .................................................................. 7 III.1.1. Escherichia coli ............................................................................................................................ 7 III.1.2. Listeria monocytogenes ................................................................................................................ 7 III.1.3. Bacillus cereus.............................................................................................................................. 8 III.1.4. Staphylococcus aureus.................................................................................................................. 9 III.1.5. Yeast.............................................................................................................................................. 9 III.1.6. Mould.......................................................................................................................................... 10 III.2. INTERPRETATION OF RESULTS .............................................................................................................. 10 III.3. MEASURE OF THE BACTERIAL REDUCTION OF LEGIONELLA PNEUMOPHILA ...................................... 11 III.4. STUDY OF THE EFFECT OF THE TREATMENT ON A BIOFILM .................................................................... 12 23/11/2010 I. -3- SUMMARY This report presents the protocol details and the results of validation study performed by SILLIKER for Euro MVP. This study consists in validating a decontamination process using ozone. The process is estimated for different germs on a plastic surface. The study consists in contaminating artificially the surface with a germ. The contamination allows to calculate the microbial reduction for each germs. II. MATERIAL AND METHODS II.1. MEASURE OF THE BACTERIAL DESTRUCTION ON A SURFACE II.1.1. SURFACE A plane surface in plastic of 1000 cm² (25 x 40 cm) is used. II.1.2. OZONE SYSTEM The ozone system model WMS (Brand GO3) is used. II.1.3. CHOICE OF STRAINS The selected germs are bacteria linked to food-borne illness and/or responsible of spoilage. Yeasts and moulds represent a spoilage flora for fruit and vegetables so they will be studied too. The selected germs are: Escherichia coli Listeria monocytogenes Bacillus cereus Staphylococcus aureus Yeast : Saccharomyces cerevisiae Mould : Aspergillus niger 23/11/2010 -4- II.1.4. ANALYSIS METHOD Analyses are performed according to methods in the table below: Parameters Methods Escherichia coli NF ISO 16649-2 Listeria monocytogenes RAPID'L mono (BRD-07/05-09/01) Bacillus cereus (spore) ISO 7932 Bacillus cereus (cell) ISO 7932 Staphylococcus aureus NF V 08-057-1 Yeast NF V08-059 Mould NF V08-059 II.1.5. PROTOCOL The surface is artificially contaminated by microbial suspension obtained by transplanting a colony on a tube of culture media. The required inoculation level is between 105 and 107 CFU/1000cm². The inoculation is performed in order to contaminate the surface homogeneously. A plastic surface of 1000 cm² is used. The suspension is spread on the surface and allowed to dry 30 minutes in a sterile atmosphere thanks to Bunsen burner. After inoculation, germs remaining on the surface are removed with a sterile wipe which is plunge into diluent. The suspension is spread on plates for enumeration. This enumeration indicates the contamination level before treatment (called Control). For each germ, 3 controls are performed. a. Treatment with Ozoned water The inoculated surface is treated with ozone: ozoned water is applied perpendicularly at about 20 cm of the surface during 30 seconds, then the inoculated germ remaining on the surface is enumerated: this enumeration indicates the level of residual contamination. For each germ, 3 repetitions of the test are performed. In order to check if the bacterial removal is due to ozone or if it is due to the pressure of the ozoned water, a test is carried out using tap water with E. coli, L. monocytogenes, S. aureus and S. cerevisiae. The final pipe of the equipment is connected to the faucet (so the water is not ozoned). As the water goes through the same pipe (same length, same diameter) the flow of tap water is the same that the flow of ozoned water. The tap water is applied during 30 seconds on the inoculated surface. 23/11/2010 -5- b. Treatment with a disinfectant 3 repetitions of the test are carried out treating the surface with a disinfectant: Amphospray 41 (disinfectant to be spray) of Laboratoires ANIOS, in order to compared the efficacy of the process to a disinfectant. In the same way, the suspension is spread on the surface and allowed to dry 30 minutes in a sterile atmosphere thanks to Bunsen burner. The disinfectant (4 mL) is sprayed uniformly on the surface and allowed to dry 5 minutes. Germs remaining on the surface are enumerated. For each germ, 3 repetitions are performed. Amphospray 41: Composition: Denatured ethanol (41 % v/v), polyhexamethylene biguanide hydrochloride, N(3-aminopropyl)-N-dodecylpropane-1,3-diamine, didecyldimethylammonium chloride, excipients. Instructions for use: Ready-to-use solution for professional use. Spray uniformly to obtain an even antimicrobial film. Allow to dry, do not rinse. Used 30 à 40 mL/m² Microbiological properties: Bactericidal: EN 1040, T 72-301, NF T 72-171, NF T 72-190. Active against MRSA (EN 13727) and Mycobacter tuberculosis (B.K). Fungicidal activity (C. albicans): EN 1275. Active against HIV-1, HBV and Rotavirus. II.1.6. CALCULATION OF THE PERCENTAGE OF BACTERIAL REDUCTION Percentage of bacterial reduction = ((Median of Controls – residual concentration) / Median of Controls) x100 23/11/2010 -6- II.2. MEASURE OF BACTERIAL REMOVAL OF LEGIONELLA PNEUMOPHILA The study is performed with Legionella pneumophila. The microbial suspension is obtained by transplanting a colony on a tube of culture media. The suspension is ½ diluted with ozoned water and allow in contact during 1 hour. The Control is perfomed with tap water instead of ozoned water. Three enumerations are performed after 1 minute, 30 minutes and 1 hour of contact. The concentration of Legionella pneumophila before and after treatment is indicated. These results allow to calculate logarithmic reduction due to the decontamination treatment. Parameter Method Legionella pneumophila NF T90-431 II.3. STUDY OF THE EFFECT OF THE TREATMENT ON A BIOFILM II.3.1. CHOICE OF THE STRAIN The study concerns the strain which resists the most at the ozone treatment, here spores of Bacillus cereus. The surface is artificially contaminated overnight with spores of Bacillus cereus. The required inoculation level is between 105 and 107 CFU/1000cm². A plastic surface of 1000 cm² is used. The surface is washed with ozoned water during 30 seconds. Germs remaining on the surface are removed with a sterile wipe which is plunge into diluent. The suspension is spread on plates for enumeration. This enumeration indicates the contamination level after treatment. The control (before treatment) is performed in the same way but without the ozoned water. The concentration of the germ before and after treatment allows to calculate logarithmic reduction due to the decontamination treatment. 23/11/2010 -7- III. RESULTS III.1. MEASURE OF THE BACTERIAL DESTRUCTION ON A SURFACE III.1.1. ESCHERICHIA COLI Result Residual flora cfu/1000cm² Log cfu/1000cm² Control 1 140 000 000 8.15 144 000 000 8.16 Control 2 Control 3 148 000 000 8.17 Tap water 2900 3.46 1400 3.15 2200 3.34 Ozoned water 2600 3.41 190 2.28 Disinfectant 260 2.41 Amphospray 41 300 2.48 The Median of the 3 repetitions is in bold. Protocol % of bacterial reduction 99.99 99.99 99.99 99.99 99.99 99.99 99.99 Median of Controls = 144 000 000 cfu/1000 cm² Average of bacterial reduction: 99.99% for Ozoned water and Disinfectant. The residual flora after using the disinfectant is 1 Log cfu/1000cm² lower than the residual flora after treatment with ozoned water. There is no difference between the results of residual flora for tap water and ozoned water. III.1.2. LISTERIA MONOCYTOGENES Result Residual flora cfu/1000cm² Log cfu/1000cm² 120 000 000 8.08 Control 1 Control 2 123 000 000 8.09 Control 3 102 000 000 8.01 Tap water 3700 3.57 200 2.30 Ozoned water ~40 1.60 710 2.85 28 000 4.45 Disinfectant ~20 1.30 Amphospray 41 ~30 1.48 The Median of the 3 repetitions is in bold Protocol % of bacterial reduction 99.99 99.99 99.99 99.99 99.98 99.99 99.99 Median of Controls = 120 000 000 cfu/1000 cm² Average of bacterial reduction: 99.99% for Ozoned water and Disinfectant. The residual flora after treatment with ozoned water is 1 Log cfu/1000cm² lower than residual flora after treatment with tap water. There is no difference between the results of residual flora for disinfectant and ozoned water. 23/11/2010 -8- III.1.3. BACILLUS CEREUS Cells of Bacillus cereus: Result Residual flora cfu/1000cm² Log cfu/1000cm² Control 1 ~13 000 4.11 Control 2 ~15 000 4.18 ~14 000 4.15 Control 3 ~70 1.85 ~30 1.48 Ozoned water ~30 1.48 <10 1 Disinfectant <10 1 Amphospray 41 <10 1 The Median of the 3 repetitions is in bold Protocol % of bacterial reduction 99.50 99.79 99.79 99.93 99.93 99.93 Median of Controls = 14 000 cfu/1000 cm² Average of bacterial reduction: 99.69% for Ozoned water and 99.93% for Disinfectant. The residual flora after using disinfectant is lower than residual flora after treatment with ozoned water. Spores of Bacillus cereus: Result Residual spore cfu/1000cm² Log cfu/1000cm² Control 1 18 000 4.26 ~12 000 4.08 Control 2 Control 3 ~7000 3.85 220 2.34 Ozoned water 210 2.32 310 2.49 510 2.71 Disinfectant 94 1.97 Amphospray 41 790 2.90 The Median of the 3 repetitions is in bold Protocol % of bacterial reduction 98.17 98.25 97.42 95.75 99.22 93.42 Median of Controls = 12 000 cfu/1000 cm² Average of bacterial reduction: 97.95% for Ozoned water and 96.13% for Disinfectant. There is no difference between the results of residual spore obtained with the disinfectant and the ozoned water (reduction about 2 Log cfu/1000cm² in comparison to the control). 23/11/2010 -9- III.1.4. STAPHYLOCOCCUS AUREUS Result Residual flora cfu/1000cm² Log cfu/1000cm² Control 1 580 000 5.76 650 000 5.81 Control 2 Control 3 710 000 5.85 Tap water 2700 3.43 290 2.46 Ozoned water 220 2.34 350 2.54 ~60 1.78 Disinfectant ~80 1.90 Amphospray 41 ~60 1.78 The Median of the 3 repetitions is in bold Protocol % of bacterial reduction 99.59 99.96 99.97 99.95 99.99 99.99 99.99 Median of Controls = 650 000 cfu/1000 cm² Average of bacterial reduction: 99.96% for Ozoned water and 99.99% for Disinfectant. The residual flora after using the disinfectant is 1 Log cfu/1000cm² lower than residual flora after treatment with ozoned water. The residual flora after treatment with ozoned water is 1 Log cfu/1000cm² lower than residual flora after treatment with tap water. III.1.5. YEAST Result Residual flora cfu/1000cm² Log cfu/1000cm² ~5000 Control 1 3.70 ~2000 Control 2 3.30 ~4000 3.60 Control 3 80 Tap water 1.90 <10 1 Ozoned water <10 1 <10 1 ~150 2.18 Disinfectant <10 1 Amphospray 41 <10 1 The Median of the 3 repetitions is in bold Protocol % of bacterial reduction 98.00 99.75 99.75 99.75 96.25 99.75 99.75 Median of Controls = 4000 cfu/1000 cm² Average of bacterial reduction: 99.75% for Ozoned water and 98.58% for Disinfectant. The residual flora after treatment with ozoned water is lower than residual flora after treatment with tap water. 23/11/2010 - 10 - III.1.6. MOULD Result Residual flora cfu/1000cm² Log cfu/1000cm² 34 000 Control 1 4.53 28 000 Control 2 4.45 29 000 4.46 Control 3 210 2.32 Ozoned water 350 2.54 250 2.40 ~90 1.95 Disinfectant 180 2.26 Amphospray 41 ~110 2.04 The Median of the 3 repetitions is in bold Protocol % of bacterial reduction 99.28 98.79 99.14 99.69 99.38 99.62 Median of Controls = 29 000 cfu/1000 cm² Average of bacterial reduction: 99.07% for Ozoned water and 99.56% for Disinfectant There is no difference between the results of residual flora obtained with the disinfectant and with ozoned water. III.2. INTERPRETATION OF RESULTS The treatment of the surface with ozoned water during 30 seconds allows a bacterial reduction of 99.99% for Escherichia coli and Listeria monocytogenes. The same bacterial reduction is obtained with the disinfectant Amphospray 41. However, the results obtained with ozoned water should be discussed because the tests with tap water during 30 secondes show also a bacterial reduction of 99.99%. So germs are removed of the plastic surface with the pressure of the water and suspended in the air. A treatment of the surface with ozoned water during 30 seconds allows a bacterial reduction of 99.96% for Staphylococcus aureus, 99.75% for Saccharomyces cerevisiae and 99.07% for Aspergillus niger. The cells of Bacillus cereus are reduced to 99.69% and the spores of Bacillus cereus are reduced to 97.95% thanks to the treatment with ozoned water during 30 seconds. 23/11/2010 - 11 - III.3. MEASURE OF THE BACTERIAL REDUCTION OF LEGIONELLA PNEUMOPHILA Enumeration of the Legionella pneumophila suspension: 4 400 000 cfu/ml Analysis Enumeration of Legionella pneumophila in the mix « suspension of Legionella pneumophila + Tap water » Enumeration of Legionella pneumophila in the mix « suspension of Legionella pneumophila + Ozoned water » Contact time 1 minute 30 minutes 1 hour 1 minute 30 minutes 1 hour cfu/ml Log cfu/ml 2 400 000 2 600 000 2 100 000 2 500 000 2 600 000 2 300 000 2 300 000 1 900 000 2 500 000 1 900 000 2 600 000 2 700 000 2 800 000 3 200 000 1 500 000 2 300 000 1 600 000 700 000 6.38 6.41 6.32 6.40 6.41 6.36 6.36 6.28 6.40 6.28 6.41 6.43 6.45 6.51 6.18 6.36 6.20 5.85 Median after 1 hour (Log cfu/ml) Logreduction (Log cfu/ml) 6.36 - 6.20 0.16 The Median of the 3 repetitions is in bold. The mix at equal volume of Legionella pneumophila suspension and tap water has a constant concentration during the hour of testing (Median: 2 400 000 cfu/ml). So tap water has no effect on the concentration of Legionella pneumophila There is no difference between the mix « suspension and ozoned water » and the mix « suspension and tap water » for 1 and 30 minutes of contact. After one hour of contact between the suspension and ozoned water, a decrease is detected: the log reduction is 0.16 log cfu/ml compared with the mix “suspension and tap water”. This log reduction is not enough to ensure the total destruction of Legionella pneumophila. 23/11/2010 - 12 - III.4. STUDY OF THE EFFECT OF THE TREATMENT ON A BIOFILM Result Residual flora cfu/1000cm² Log cfu/1000cm² 360 000 Control 1 5.56 440 000 Control 2 5.64 390 000 5.59 Control 3 27 000 4.43 37 000 4.57 Ozoned water 78 000 4.89 The Median of the 3 repetitions is in bold Protocol % of bacterial reduction 93.08 90.51 80.00 Median of Controls = 390 000 cfu/1000 cm² A treatment with ozoned water during 30 seconds allows to reduce about 1 Log cfu/1000cm² the biofilm of Bacillus cereus spores. Without biofilm, ozoned water allows a reduction of 2 Log cfu/1000cm² of Bacillus cereus spores (Results page 8). So the effect of ozoned water is less efficient on biofilms.