3M Food Safety Technical Bulletin

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1 3M Petrifilm Aqua Enterobacteriaceae Count Plates Performance Summary 3M Petrifi lm Aqua Enterobacteriaceae (AQEB) Count Plates are sample ready media plates used in the microbial testing of bottled water. Each plate contains a water-soluble gelling agent, nutrients and indicators in a dry, shelf-stable format. This technical bulletin summarizes data 3M Food Safety collected during performance testing of 3M Petrifi lm Aqua AQEB Plates. Table of Contents 3M Petrifilm Aqua Enterobacteriaceae Count Plate Comparative Method Study Results... p. 2 Inclusivity/Exclusivity Study Results... p M All rights reserved. TB Page 1 of 5

2 Comparative Method Study A comparative method study was conducted at an external reference laboratory 1 to compare results of the 3M Petrifi lm Aqua AQEB Plate method to Violet Red Bile Glucose agar (VRBG) for the detection of Enterobacteriaceae in bottled water. Matrices: Fifteen brands of water were tested, with two lots per brand to equal a total of 30 samples. Table 1: Brands of bottled water tested Water Type Purifi ed Regional Spring Natural Spring Natural Spring (Carbonated) Brand, Country of Manufacture Nestle Pure Life, United States Aquafi na, United States Dasani, United States Trauth Dairy, United States Ice Mountain, United States Kroger, United States Evian, France Jana, Croatia Fiji, Fiji Ty Nant, Wales Voss Flat, Norway Voss Sparkling, Norway Gerolsteiner, Germany Apollinaris, Germany Perrier, France Organisms and Stress Method Overnight broth cultures were washed and inoculated into bottled water samples at a low/medium inoculum level (targeting 25 cfu/sample) and a medium/high inoculum level (targeting 75 cfu/sample). A non-inoculated control was also prepared for each water sample. The following three organisms were randomly assigned to the 30 different water types: Enterobacter aerogenes ATCC 35029, Escherichia coli ATCC 25922, and Klebsiella pneumonia ATCC Inoculated water was left at room temperature in the dark for 2 weeks to stress the organisms before testing. 1 Q Laboratories, Inc., Cincinnati, OH, study comparing 3M Petrifi lm Aqua Plate performance vs. reference methods. Study presented at 2011 International Association for Food Protection (IAFP) M All rights reserved. TB Page 2 of 5

3 Method Comparison The following methods were compared: 3M Petrifi lm Aqua AQEB Plates vs. VRBG agar (100 ml fi ltered.) 3M Petrifi lm Aqua AQEB Plates were incubated at 35 ± 1 C for 24 ± 2h. VRBG plates were incubated at 35 ± 1 C for 24 ± 2h.. A mixed cellulose ester fi lter was used for the method comparison. Samples were plated in duplicate for each level of inoculum. After incubation, typical colonies were counted on plates: for 3M Petrifi lm Aqua AQEB Plates, red colonies with gas, red colonies with acid zones, or red colonies with both gas and acid zones were counted. For VRBG agar, pink to dark purple colonies were counted. Statistical Analysis Counts were converted to log 10 counts. For each level of inoculum, a paired t-test was used to compare differences in counts between the 3M Petrifi lm Aqua AQEB Plate method and the reference method. A p-value of <0.05 was taken to indicate a signifi cant difference. Results Chart 1: Enterobacteriaceae Comparison: Average Log Counts Average Log Counts Enterobacteriaceae Comparison N = 30 per level Average Log Counts No statistical difference No statistical difference 1 Petrifilm AQEB Plate Low VRBG agar 35 C Petrifilm AQEB Plate High VRBG agar There was no statistical difference between the mean log Enterobacteriaceae counts per fi lter on the 3M Petrifi lm Aqua AQEB Plate method and the VRBG agar method at either the low inoculation level (p-value=0.844) or the high inoculation level (p-value=0.47) M All rights reserved. TB Page 3 of 5

4 Inclusivity/Exclusivity Study Inclusivity and exclusivity studies were performed using pure cultures of 25 Enterobacteriaceae strains and 22 non- Enterobacteriaceae strains. The strains were obtained from the American Type Culture Collection (), the National Collection of Industrial, Food and Marine Bacteria (NCIMB, UK), 3M Tecra Culture Collection (TICC, Australia), and isolates from naturally contaminated samples. Enterobacteriaceae organisms were diluted and inoculated into bottled water targeting cfu/fi lter; non- Enterobacteriaceae strains were diluted and inoculated, targeting approximately 1x105 cfu/fi lter. Inoculated samples were fi ltered through a mixed cellulose ester fi lter and the fi lters plated onto pre-hydrated 3M Petrifi lm Aqua AQEB Plates and VRBG agar and incubated at 36 ± 2 C for 24 ± 2h. For a positive control, non-enterobacteriaceae strains were fi ltered and the fi lters plated onto plate count agar (or, for Lactobacillus planarum ATCC 10241, onto Lactobacillus MRS agar) and incubated for 36 ± 2 C for 48 ± 2h. Inclusivity Strains Citrobacter brakii ATCC Citrobacter TICC 542 Citrobacter gillenii SWW4* Enterobacter aerogenes ATCC Enterobacter cancerogenus SWW2* Escherichia coli ATCC Escherichia coli ATCC Escherichia coli PWW4* Escherichia coli SWW1* Hafnia alvei ATCC Klebsiella pneumoniae ATCC Klebsiella oxytoca ATCC Leclericia adecarboxylata ATCC Morganella morganii TICC 4467 Proteus mirabilis ATCC Proteus vulgaris ATCC Providencia TICC 4346 Salmonella Typhimurium TICC 1280 Salmonella enterica ATCC Salmonella Typhimurium ATCC Salmonella Krefeld TICC T440 Salmonella enterica ATCC Serratia plymuthica ATCC 4475 Shigella sonnei ATCC Yersinia ruckeri TICC 4346 Yersinia entercolitica ssp entercolitica ATCC 9610 *Strain isolated from waste water and identifi ed by API20E or by MicroSEQ ID identifi cation system. Exclusivity Strains Acinetobacter ssp ATCC Acinetobacter baumanii NCIMB Aeromonas caviae SWW12* Bacillus atrophaeus ATCC Bacillus/Alicyclobacillus/Brevibacillus 8 A1b** Burkholderia cepacia ATCC Delftia acidovorans 5A4** Delftia acidovorans ATCC Enterococcus faecalis ATCC Enterococcus faecium ATCC Enterococcus faecalis ATCC Pseudomonas aeruginosa ATCC Pseudomonas aeruginosa ATCC 7700 Pseudomonas aeruginosa ATCC Pseudomonas putida ATCC Staphylococcus aureus ATCC 6538 Staphylococcus aureus ATCC * Strain isolated from waste water and identifi ed by API20E or by MicroSEQ ID identifi cation system. ** Strain isolated from bottled water and identifi ed by MicroSeq ID identifi cation system M All rights reserved. TB Page 4 of 5

5 Inclusivity/Exclusivity Study (continued) Results Inclusivity Results 3M Petrifi lm Aqua AQEB Plates VRBG Agar 26/26 strains (100%) had typical growth 26/26 strains (100%) had typical growth Exclusivity Results 3M Petrifi lm Aqua AQEB Plates VRBG Agar 9/22 strains (41%) had typical growth 13/22 strains (59%) had typical growth Strains with typical colonies: Aeromonas caviae SWW12 Enterococcus faecalis ATCC Enterococcus faecium ATCC Enterococcus faecalis ATCC Strains with typical colonies: Acinetobacter ssp ATCC Acinetobacter baumanii NCIMB Aeromonas caviae SWW12 Burkholderia cepacia ATCC Pseudomonas aeruginosa ATCC Pseudomonas aeruginosa ATCC 7700 Pseudomonas aeruginosa ATCC Pseudomonas putida ATCC Conclusion: For inclusivity testing, the 3M Petrifi lm Aqua AQEB Plate method compared well to the VRBG agar method. For exclusivity testing, the 3M Petrifi lm Aqua AQEB Plate method showed less breakthrough of non-enterobacteriaceae compared to the VRBG agar method. 3M and Petrifi lm are trademarks of 3M M All rights reserved. TB Page 5 of 5

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