Research Note Vip For Ehec

Research Note Evaluation of a Modified Buffered Peptone Water Enrichment  For use with the VIP for EHEC in Ground Beef Linda Mui, Andrew Lienau, Stephanie Leung and Robin Forgey BioControl Systems, Inc. , 12822 SE 32nd Street, Bellevue, WA 98005 Abstract A rapid method to detect EHEC in foods using the VIP for EHEC was previously reported ( 1,2). This method, which has been approved as an AOAC Official Method consists of an overgnight (18-28 hours) enrichment protocol using Modified Trypticase Soy Broth (mTSB) and Novobiocin. The ground beef industry has need for a faster preliminary screening enrichment protocol because of short shelf life and an accelerated distribution network found in the industry. Reported herein is an accelerated, 8 hour enrichment protocol with a detection limit of <1 CFU/g of E.coli O157:H7. This enrichment protocol represent a significant departure from the Official Method but affords the benefits of rapid determinations for time sensitive samples. Ground beef samples were inocalated with E.coli O157:H7 isolates at three different levels, low medium and high. The inoculated samples were enriched in modiffed Buffered Peptone Water (3.4) supplemented with acriflavin, cefsoludin and vancomycin (mBPW + acv) for eight hours at 42 ºC. After incubation 0.1 ml of each enriched sample was placed into the VIP unit and results were recorded after 10 minutes. A total of 65 samples were tested and confirmed with complete correlation between the VIP for EHEC using the accelerated, 8 hour enrichment and the reference cultural method. The AOAC Official Method for detection of E.coli O157:H7 using the VIP for EHEC consists of an 18-28h enrichment in mTSB and Novobiocin. The ojective of the study was to evaluate the effectiveness of a reduced enrichment time on the ability to detect E.coli O157: H7 in ground beef using the Vip for EHEC. The USDA cultural method (5) was chosen as the reference methodology. Materials and Methods Inoculation of Samples. A total of 65 ground beef samples were tested, 60 inoculated and 5 uninoculated control samples. The ground beef was purchased at the wholesale level. The samples were inoculated with E.coli O157:H7 that had previously been isolated from ground beef. The culture was enriched overnight in Brain Heart infusion at 35 ºC. Serial dilutions of the overnight enrichment were made in Butterfield´s Buffered Phosphate Diluent and the appropriate volume was mixed into the ground beef. The beef was inoculated at three different levels, low medium and high. Twenty samples were inoculated for each level. The low level was designed to approximate 1-10 cfu/25g, the medium level to approximate 10-75 cfu/25g. and the high level to approximate greater than 100 cfu/25g. The inoculated beef was refrigerated overnight prior to running the samples.

Contamination levels were determined by running MPNs using the United States Department of Agriculture (USDA) cultural methods as outlined in Figure1. For the VIP for EHEC, inoculated samples were enriched by weighing a 25g sample into 225ml of prewarmed mBPW+acv. Sample were incubated at 42 ºC without shaking for eight hours after mixing. Positive and negative media controls were also included. After incubation, 0,1 ml of each enriched sample was placed into the VIP unit according to the manufacturer´s directions for use. Results were recorded after ten minutes. Confirmation of Samples. Both positive and negative samples were confirmed via the USDA cultural method. Serial dilutions of the enriched samples were spread plated on MacConkey Sorbitol Agar with BCIG (MSA/BCIG) and incubated at 42 ºC overnight. Typical colonies were selected from each sample and tranferred to Eosin Methylene Blue Agar (EMB) and Phenol Red Sorbitol MUG Agar (PRS/MUG). Colonies that displayed the typical reactions on these two agars were then identifield using the API 20E.

(BioMerieux) and serotyped for O157:H7 agglutination (Difco). Results and Discussion, Results are summarized in Table 1. In the low level, the VIP identified seventeen presumptive positive samples and three negative samples, all of which verified by plating and cultural confirmation. The three negative samples did not exhibit O157 colonies on isolation agars, in the medium and high levels, all samples were positive the VIP and confirmed by plating. There were no false positive or false negative samples. The VIP for EHEC and the cultural method produced the same results on each sample. Table 1. Number of positive samples hour Enrichment Protocol Inoculation MPN/g Level Uninoculated <0.003 Low 0.38 Medium 2.4 High >11

5- Okrend, A,. and B.E. Rose. 1989. USDA-FSIS Laboratory Comunication No. 38, Revision 3, U.S. Department of Agriculture, Food Safety and inspection Service, Wasghington DC. Fig. 1. USDA Method for E.coli O157:H7

Add Sample to modified EC Broth with Novobicion incubate 20-24h at 35 ºC

on VIP for EHEC using 8Positive by VIP 0 17 20 20

Confirmed Positivo 0 17 20 20

The VIP for EHEC has been previously studied using a different enrichment protocol that requires an 18-hour enrichment (1,2). Based on these studies, the VIP for EHEC was granted AOAC Official Method status (methods 996.09). The 8-hour enrichment protocol mentioned above is a significant deviation from the AOAC Official Method, and therefore, is not considered an Official Method. Howerer, it may be useful as a preliminary screening procedure for time sensintive samples where immediate determinations are necessary. The data from this study indicates that the 8-hour enrichment may detect E.coli O157:H7 in ground beef

Spread plate serial dilutions Onto MSA/B-CIG Incubate 18-24h at 42 ºC

Pick at least 12 Typical Colonies to EMB & PRS/MUG Incubate 18-24h at 35 ºC Modified Buffered Peptone (mBPW)

References

1- Feldsine, P.T Falbo-Nelson , M.T., S. Brunelle and Forgey, R.L, 1997. Visual. Immunoprecipitase Assay (VIP) for Detection Enterohemohagic Escherichia coli (EHEC) O157:H7 in Selected Foods: Collaborative Study. J. AOAC Int´l. 80 517-529. 2- Feldsine, P.T Forgey, R.L. Falbo-Nelson, M.T. and S.Brunelle. 1997. Escherichia coli O157:H7 Visual Immunoprecipitate Assay: A Comparative Validation Study. J. AOAC Int´l. 80:43:83. 3- Restaino, L 1996. Accentuate the positive. Food Quality 2.68-70 4- Restaino, L, Castillo, H.J.; Stewart, D., and M.L. Tortorelio. 1996. Antibody direct apiflourescent filter technique and immunomagnetic separation for 10h screening and 24h confirmation of Escherichia coli O157:H7 in beef. J. Food Safety Prot. 59:1072-1075.

Run biochemicals Using API20E

Modified Buffered Peptone (mBPW) Peptone NaCI Na2 PO4 KH2PO4 Casamino Acids Yeast Extarct Lactose Distilled water*

10g 5g 3.6g 1.5g 5g 6g 10g 1000ml

* pH 7.2 ± 0.2 prior to addition of the above ingredients

Add all of the above ingredients to one liter of purified water. Mix well to dissolve. Dispense in the desired aliquots and autoclave at 121 ºC for 15 minutes. After cooling, add antibiotic stock solutions in the following amounts per liter of media: Acriflavin Cefsulodin Vancomycin

0.1ml 0.1ml 0.08ml

Preparation of Antibiotic Stock Solutions: Prepare a stock solution of each antibiotic (acriflavin, cefsulodin and vancomycin) by dissolving 1000mg of each antibiotic in a separate tube containing 10ml of distilled water. Filter sterilize the solution can be kept for several months in foil wrapped tubes at 4 ºC.