Screening and Identification Methods for Official Control of Banned Antibiotics and Growth Promoters in Feedingstuffs

Screening and Identification Methods for official control of Banned Antibiotics and Growth promoters in Feedingstuffs SIMBAG FEED Competitive and sustainable growth programme (GROWTH) project G6RD-CT-2000-00413 Coordinator: Jacob de Jong RIKILT – Institute of Food Safety Wageningen, The Netherlands Method protocol for screening on (banned) antibiotics in complete feedingstuffs and milk replacers by the microbiological plate test Author : Harry van Egmond RIKILT – Institute of Food Safety, Wageningen, The Netherlands Workpackage : Microbiological screening system (WP 2) Workpackage leader: Harry van Egmond Date of deliverable : 14-07-2005 Harry van Egmond, 14-7-2005 1/10 Method protocol for screening on (banned) antibiotics in complete feedingstuffs and milk replacers by the microbiological plate test 1 SCOPE AND FIELD OF APPLICATION This method describes the screening on banned antibiotics in complete feeding stuffs and milk replacers by a microbiological 3-plate test. To recognize interference of (carry over) of veterinary medicines or high concentrations of avilamycin, three additional test plates (mentioned in appendix 1) are necessary. Screening on banned antibiotics (virginiamycin, tylosin, spiramycin, zinc bacitracin, avoparcin) in the presence of aminoglycosides, macrolides, furazolidon, tetracyclines or quinolones is impossible with the microbiological plate test. Furthermore high concentrations of metals (Cu, Zn) might interfere. The limit of detection of the method is 1 mg/kg for avoparcin, tylosin, spiramycin, virginiamycin and 3-5 mg/kg for zinc bacitracin. 2 PRINCIPLE The sample is extracted at low pH with a mixture of acetone, hydrochloric-acid (HCl) and water. After addition of phosphate buffer pH 6.5, and further extraction, the feed is centrifuged. The supernatant is adjusted to pH 6.0-6.5 and dispensed into wells in three different agar media. The agars differ in inoculated bacterial strains and pH. After incubation, presence of antibiotics is shown by the formation of zones of inhibition. An indication of the antibiotic present is possible by observing with which micro- organisms zones of inhibition are produced and comparison of inhibition patterns (appendix 2) with known standards. 3 MICRO-ORGANISM 3.1 Teststrains • Micrococcus luteus ATCC 10240 • Bacillus megatherium ATCC 10778 • Micrococcus luteus ATCC 9341 3.2 Preparation of the bacterial suspensions (1) Inoculate tubes containing slopes of culture medium (pH 6.5, 4.1.2) with the test strains (3.1) and incubate for 18-24 hours at 30°C (5.7). Harvest the growth with 2-3 ml of sodium chloride solution (4.8). Use this suspension to inoculate agarplates (pH 6.5, 4.1.2) and incubate for 18-20 hours at 30°C (5.7). Harvest the growth in 25 ml of sodium chloride solution (4.16) and mix. Add 12.5 ml sterile glycerol (4.14) and 12.5 ml sterile full-cream milk (4.15). Mix and distribute the bacterial suspension in suitable containers/ampoules. This suspension may be kept for at least one year at <-60°C. Determine (before use) the amount of viable bacterial cells conform ISO 4833. Suspensions with approximately 109 cells/ml are fit for use. 4 CULTURE MEDIA AND REAGENTS 4.1 Assay media 4.1.1 Assay medium M.luteus ATCC 9341(Antib. medium no. 1 + phosphate) (2) meat peptone 6 g tryptone 4 g yeast extract 3 g meat extract 1.5 g glucose 1 g agar 10-20 g dipotassium hydrogen phosphate (K2HPO4)(4.7) 20 g (addition direct after sterilisation) demineralised water 1000 ml Sterilise. Add the di-potassium hydrogen phosphate and cool to approx. 50°C. Adjust the pH to 8.0 ± 0.1. (1) Any other method of preparation of bacterial suspensions, giving the same, results may be used. (2) This medium from Difco gives good results, but any commercial culture medium of similar composition and giving the same results may be used. (3) mg/kg microbiological activity, 1 mg spiramycin-base = 3200 IU and 1 mg zinc bacitracin = 42 IU. Harry van Egmond, 14-7-2005 2/10 4.1.2: Assay medium M.luteus ATCC 10240 (Antib. medium no. 1) (2) meat peptone 6 g tryptone 4 g yeast extract 3 g meat extract 1.5 g glucose 1 g agar 10-20 g demineralised water 1000 ml Sterilise. Cool to approx. 50°C and adjust the pH to 6.5 ± 0.1 4.1.3: Assay medium B.megatherium ATCC 10778 (Plate count agar) (2) pancreatic digest of casein 5 g yeast extract 2.5 g glucose 1 g agar 15 g demineralised water 1000 ml Sterilise. Cool to approx. 50°C and adjust the pH to 6.0 ± 0.1. 4.2 Sodium chloride (NaCl) 4.3 Acetone p.a. 4.4 Hydrochloric acid (p20= 1.18) 4.5 Acetone (4.3)/hydrochloric acid (4.4)/water mixture (475/25/500)(v/v) 4.6 Potassium dihydrogen phosphate (KH2PO4) 4.7 diPotassium hydrogen phosphate (K2HPO4) 4.8 Sodium hydroxide (NaOH) 4.9 Methanol p.a. 4.10 Phosphate buffer pH 6.5 Dissolve 18.6 g potassium dihydrogen phosphate (4.6) and 14.8 g dipotassium hydrogen phosphate (4.7) in water. Adjust pH to 6.5 ± 0.1and make up to 1000 ml with water. 4.11 Standard neomycin of known activity (in neomycin-base) 4.11.1 Neomycin stock solution (500 µg/ml). Dissolve a quantity of neomycin-sulphate (4.11) corresponding to 50 mg neomycin-base in water, mix and make up to 100 ml. This solution is stable for one month at 4°C. 4.12 Standard tylosin of known activity (in tylosin-base) 4.12.1 Tylosin stock solution (50 µg/ml). Dissolve a quantity of tylosin (4.11) corresponding to 5 mg tylosin-base in 5 ml methanol (4.9) and make up to 100 ml with phosphate buffer pH 6.5 (4.10). This solution is stable for one month at 4°C. 4.12.2 Tylosin working solution (1 µg/ml) Dilute 2 ml tylosin stock solution to 100 ml final volume with water. 4.13 Penicillinase solution 10.000.000 IU/ml 4.14 Glycerol 4.15 Sterile full-cream milk (2) Any commercial culture medium of similar composition and giving the same results may be used. Harry van Egmond, 14-7-2005 3/10 4.16 Sodium chloride solution 0.85% (w/v) Dissolve 8.5 g sodium chloride (4.2) in 1000 ml water and autoclave. 4.17 Sodium hydroxide solution 5 mol/l Dissolve 20 g sodium hydroxide (4.8) in water and make up to 100 ml with water. 5 APPARATUS 5.1 Volumetric glassware 5.2 Analytical balance 5.3 Autoclave 5.4 Laboratory shaker or magnetic stirrer 5.5 Vernier caliper or equivalent equipment 5.6 Centrifuge (±3000xg) 5.7 Incubators at 25 ± 1°C and 30 ± 1°C 5.8 pH meter 5.9 Micropipets 5.10 Cork borer (diameter of 12 ± 0.5 mm) 5.11 Plastic (disposable) Petri dishes with a flat surface 6 PROCEDURE In each analysis run at least one positive control sample (= spike sample) should be included, to check the procedure. In Appendix 3, the procedure for preparation of these positive control samples is described. 6.1 Preparation of the extract Weigh out 10 g of sample, add 20 ml Acetone/hydrochloric acid/water mixture (4.5) and shake (5.4) for 15 minutes. Add 10 ml phosphate buffer pH 6.5 (4.10) and shake for another 15 minutes(1). Centrifuge (5.6) and take a suitable aliquot of the supernatant. Adjust the pH to 6.0 - 6.5 (5.8) by means of a 5 M sodium hydroxide solution (4.17). (2) 6.2 Inoculation of the assay media Inoculate the assay media (4.1.1 – 4.1.3) with the bacterial suspension (3.1) at about 50°C, to give approximately 105-106 colony forming units/ml agar. The following combinations of bacteria and pH are necessary: Bacterial suspension Medium pH assay medium Inoculum (cfu/ml agar) M.luteus ATCC 10240 AB 1 (*) 6.5 106 + neomycin (1 mg/l agar) M.luteus ATCC 9341 AB1+ phosp (**) 8 106 +tylosin (7.5 µg/l agar) B.megatherium ATCC 10778 PCA 6 105 (*) Add 0.2 ml of neomycin stock solution (4.11.1) per 100 ml of assay medium (1) If lumps/clods occur in (milk replacer) samples, use a spatula to completely wet the samples. (2) For the last part of this pH adjustment a diluted NaOH solution may be used. Harry van Egmond, 14-7-2005 4/10 (**) Add 0.75 ml of tylosin working solution (4.12.2) per 100 ml of assay medium 6.3 Preparation of the plates Diffusion through agar is carried out in plates (5.11). Pour into different plates a quantity of every medium inoculated with the different bacterial strains (6.2) to give a layer of about 2.5 mm thick. Allow to set in a level position and bore holes with a diameter of approx. 12 mm (minimal of 30 mm between centres) (5.10). 6.4 Filling of the holes Place (5.9) in every of the three plates 0,20 ml feed extract per hole. Use remaining wells on the plates for positive control samples or other sample extracts. 6.5 Incubation Incubate the plates for 16-18 hours at 30 ± 1°C except the B.megatherium ATCC 10778 testplate at 25 ± 1°C. 7 INTERPRETATION The presence of antibiotics is shown by the formation of zones of inhibition. Measure the diameter of the zones of inhibition to the nearest 0.1 mm, by direct measurement or projection (5.5). Small inhibition zones (smaller than “holes + 2 mm”) on the M.luteus ATCC 10240 and B.megatherium ATCC 10778 plates have to be interpreted as negative.

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