- Email: [email protected]
Quantitative Assay for Antibiotics Used Commonly in Treatment of Bovine Infections1
Quantitative Assay for Antibiotics Used Commonly in Treatment of Bovine Infections I J. R. BISHOP, 2 A. B. B O D I N E , G. D. O ' D E L L , and J. J. J A N Z E N Department of Dairy Science
Clemson University Clemson, SC 29631
ABSTRACT
uses have led to contamination of milk and milk products. The presence o f antibiotics in milk in even minute quantities has created problems in the dairy industry including: a) inadequate curdling of milk and improper ripening of cheeses during their manufacture; b) decreased acid and flavor production during the manufacture of buttermilk and similar products; c) diminished starter culture growth when propagated in reconstituted skim milk; and d) validity of certain quality control tests (9). In addition, residues in milk should be avoided because residues are illegal, and they may be biologically active metabolites of drugs in milk (6). A major problem facing the dairy industry in the area o f antibiotic treatment is the rapid, accurate detection of low amounts of antibiotic residues in milk. The objectives of this study were to compare the more recently adopted and approved detection assays and to modify the Bacillus stearotbermopbilus Difco disc assay so as to make it possible to quantitate antibiotics used commonly in treatment of bovine infections.
Delvotest-P and Bacillus stearothermophilus Difco disc assay procedures were utilized, and assay sensitivity for detecting various antibiotics was tested. Bacillus stearotberrnopbilus spores used in the disc assay were purchased from two laboratories and compared. The dyes methylene blue, 2,3,5-triphenyltetrazolium chloride, orange-O, and bromcresol purple were incorporated separately into agar used for the disc assay to determine if sharper, clearer zones could be produced. None of the dyes had an observable effect on zone size or clarity. Using the Difco disc assay, quantitation of penicillin, cephapirin, and oxytetracycline was accomplished with reliable precision by relating the size of the zone of inhibition to the log of the antibiotic concentration. The Delvotest-P and Difco disc assays were equal in sensitivity, as were spore suspensions used from the two laboratories. INTRODUCTION
Antibiotics have been used in dairy cattle for more than three decades. They are administered to cattle by four general methods: a) infusion into the udder for treatment and prevention of mastitis; b) injection (intramuscular, intravenous, or subcutaneous) for treatment of numerous diseases; c) orally or as a dietary supplement; and d) reproductive "flush" for uterine, cervical, and vaginal infections. Such
Received February 1, 1985. Technical Contribution No. 2410. Published with the approval of the Director, South Carolina Experiment Station. 2Present address: Diversey Corp., Wyandotte, MI. 1985 J Dairy Sci 68:3031-3036
MATERIALS AND METHODS
The two antibiotic detection assays used in this study were the De]votest-P and Difco disc assay. These procedures were used because of their relative rapidity, sensitivity, and simplicity. The Delvotest-P (14) is an agar diffusion test. Tablets containing nutrients and bromcresol purple are added to ampules containing plain agar with spores of B. stearotbermopbilus var. calidolactics. After adding a .1-ml milk sample, the ampule is incubated 2.5 h at 63 to 66°C. In the absence of antimicrobial substances, the whole of the solid medium turns yellow; it remains purple in the presence of sufficiently high concentrations of antibiotics. At intermediate concentrations of antibiotic
3031
3032
BISHOP ET AL.
the solid medium turns partly yellow (doubtful result). The test method has a high sensitivity for penicillin. Concentrations of .002 units/ml or lower give negative results in all samples, and .003, .004, and .005 units/ml give predominantly negative, doubtful, and positive results, respectively. These results were obtained in bulk milk samples and in individual cow milk samples. This method is also sensitive to most of the other antibiotics administered to lactating cows. The Difco disc assay (7) employs Antibiotic Medium #4 (Difco, Detroit, MI) seeded with B. stearotbermophilus (Difco). This Antibiotic Medium #4 is the recommended medium of choice, as illustrated by Peeler et al. (11). Six milliliters of seeded agar are put into 15 x 100-ram plastic petri dishes (Fisher Scientific, Norcross, GA). Plates must be used within 7 d of preparation. Thirteen-millimeter paper discs (Schleicher and Schuell, Keene, NH) were used to absorb milk by capillary action and discs then placed on agar surface. Plates were incubated for 2.5 h at 64°C. An alternate method is for incubation to proceed for 4 h at 55°C. After incubation, zones of inhibition are measured in millimeters. A positive result was designated by a zone of 15 ram, which is a compromise between 14 and 16-ram zones suggested for a positive result (1). This assay detects as little as .005 units peniciUin/ml of milk. Certain questions remained from earlier work (5,8,10,11); therefore, the initial objective of this study was to determine which assay was the most accurate and precise. This was accomplished by conducting both assays each time milk was analyzed for antibiotic residues during this study and a r.etention data study (2). Another objective was to compare B. stearotbermopbilus spores purchased from Difco Laboratories and from Penicillin Assays, Inc. (Boston, MA) since they are the two major distributors of the spore suspension. Penicillin (Penicillin G Procaine, Veticare, Baltimore, MD) concentrations (U/ml) of .050, .010, .005, and control were prepared in raw milk. Eight seeded agar plates were prepared with each of the spore suspensions. Thirteen-millimeter paper discs were used to absorb each penicillin concentration and the four discs placed on each plate. Plates were incubated and zones of Journal of Dairy Science Vol. 68, No. 11, 1985
inhibition measured. This experiment was replicated three times. The experimental design was a randomized complete block with subunits, and results were analyzed by analysis of variance utilizing the F-test and single degree of freedom contrasts (13). Each suspension was also plated on Antibiotic Medium #4 to compare concentrations. The third objective was to produce more distinct zones of inhibition by incorporating various dyes into the Difco disc assay to increase the accuracy of reading the assay. Dyes used were orange-O, 2,3,5-triphenyltetrazolium chloride, methylene blue, and bromcresol purple, along with a control containing no dye. For each dye, 1.25 mg/100 ml agar was incorporated into the medium immediately before the agar was seeded with spore suspension. Penicillin concentrations (units/ml) of .100, .050, .010, .005, and control were prepared in raw milk by serial dilution. Thirteen-millimeter discs were used to absorb by capillary action an aliquot of each antibiotic concentration; discs were then placed on each of 8 spore-seeded, dye-incorporated, and control agar plates. Plates were incubated 2.5 h at 64°C, and zones of inhibition measured in milliliters. The experiment was replicated four times. The experimental design was a randomized complete block with split units. Analysis of variance was conducted utilizing the F-test to compare each dye to the control (13). The fourth objective was to quantitate by Difco disc assay some antibiotics, namely, penicillin, cephapirin, and oxytetracycline, used commonly in treatment of bovine infections. For penicillin, concentrations (U/ml) o f 0, .100, .050, .010, and .005 were prepared in raw milk by serial dilution. An aliquot of each antibiotic concentration was absorbed onto 13-mm paper discs by capillary action and then placed on each of 8 spore-seeded agar plates. Plates were incubated 2.5 h at 64°C, and zones of inhibition measured in milliliters. The experiment was replicated 20 times. For cephapirin (cephapirin sodium, BristolMyers Co.) and oxytetracycline (Oxytetracycline HCI, Bio-Ceutic, St. Joseph, MO), concentrations for each of 1.00, .50, .10, .05, and 0 /ag/ml were prepared in raw milk. The reason for the increased concentrations, compared with that used to test for penicillin, was the decreased sensitivity o f the assay for these
ANTIBIOTIC QUANTITATION antibiotics, partly due to the natural cationic inhibition of antibiotics in milk (11). For each antibiotic tested, an aliquot of each concentration was allowed to absorb onto 13-ram paper discs by capillary action, and was then plated onto each o f 8 spore-seeded agar plates. Plates were incubated 2.5 h at 64°C, and zones of inhibition measured in millimeters. Both experiments were replicated 10 times. All three quantitation trials were set up with a randomized complete block design with split units. Correlation coefficients were determined and linear and quadratic contrasts performed• RESULTS A N D DISCUSSION
The sensitivities of Delvotest-P and Difco disc assays were essentially equal at .050 U penicillin/ml. Table 1 illustrates data obtained from conducting both assays for detection of selected concentrations of penicillin in milk, and individual cow milk samples obtained during an antibiotic retention study (2). The only apparent difference was that with the Delvotest-P, a " d o u b t f u l " result was possible to record; but because the Difco disc assay possesses measurable zones, the zone must be /> 15 mm to indicate a positive result. Of the 11 " d o u b t f u l " results from the Delvotest-P, 6 were positive and 5 were negative by the Difco disc assay. All 91 positives and 206 negatives by the Delvotest-P were the same with the Difco disc assay. When B. s t e a r o t b e r m o p b i l u s spore suspensions were seeded into agar used for the Difco
3033
disc assay, no significant differences in zone sizes of inhibition were detected (Table 2). However, zones from Penicillin Assays' spore suspensions were consistently larger than those from Difco spore suspensions. Spore concentrations were not significantly different between the two sources• No differences were noted in zone size when comparing Difco disc assays conducted with dye-incorporated agar to control agar containing no dye (Table 3). When incubation time was extended to approximately 5 h, agar containing bromcresol purple produced more distinct zones due to the contrast in color between the purple zone of inhibition and the yellow lawn of growth; therefore a slightly larger zone was produced. Quantitation of penicillin residues in milk by the Difco disc assay was accomplished. Zones of inhibition produced by selected concentrations of penicillin were significantly different (P<.001) (Table 4). The linear correlation of penicillin concentration and zone of inhibition was .939. This relationship was linear (P<.0001) and quadratic (P<.001). When the linear relationship of zone of inhibition vs. log10 (penicillin concentration) was tested, the correlation was .999. Using this relationship, the following equation was formulated: Log10 (penicillin concentration) = (zone--35.587)~8.628 where penicillin concentration is in units per
TABLE 1. Comparison of Delvotest-P and Difco disc assay for known concentrations of penicillin in milk and unknown individual cow samples of milk obtained during an antibiotic retention study (2). Penicillin
Delvotest-P
(U/ml)
Difco disc assay (% positive)
.050 .010
100 100
100 100
•0 0 5
100
100
Control Milk samples (n = 308) l + +/---
0
0
91
97
11
NA
206
2
211
t + = Positive (+), doubtful (+/-), or negative (-) detection of antiobiotic residues. 2Not applicable. Journal of Dairy Science Vol. 68, No. 11, 1985
3034
BISHOP ET AL.
TABLE 2. Comparison of spore suspensions purchased from Difco Laboratories and Penicillin Assays, Inc., used in the Difco disc assay. Zone of inhibition Penicillin
Difco
Penicillin Assays
(U/ml)
SE
(mm)
.050 .010 .005 Control
24.58 18.62 15.63 13.001
24.92 19.23 15.89 13.00 ~
.26 .26 .26 0
Diameter of disc.
milliliter and zone is measured in millimeters. F r o m this equation, a 15-mm zone (the minim u m zone for positive detection) w o u l d represent a penicillin c o n c e n t r a t i o n of .004 U/ml. To validate this, a penicillin concentration o f .004 U / m l was prepared in milk, and 40 Difco disc assays were c o n d u c t e d (10 plates with 4 discs/plate). All 40 zones o f inhibition measured precisely 15 m m . This value o f .004 U / m l m i n i m u m d e t e c t i o n agrees exactly with results obtained b y Britt (3). This e q u a t i o n is m o r e quantitative than the one introduced by Ginn et al. (4), which only c o m p u t e s quantitative estimates above or below the reference c o n c e n t r a t i o n o f antibiotic used (.016 U/ml). Q u a n t i t a t i o n of cephapirin residues in milk by the Difco disc assay was also accomplished. Zones o f inhibition p r o d u c e d by selected concentrations of cephapirin were significantly different ( P < . 0 0 0 1 ) (Table 5). The linear correlation o f cephapirin c o n c e n t r a t i o n and
zone o f inhibition was .823. This relationship was linear ( P < . 0 0 0 1 ) and quadratic (P<.0001). When the linear relationship o f zone o f inhibition and log10 (cephapirin concentration) was tested, the following e q u a t i o n was f o r m u l a t e d : Log10 (cephapirin c o n c e n t r a t i o n ) = (zone--31.823)/7.674 where cephapirin c o n c e n t r a t i o n was in micrograms per milliliter and z o n e measured in millimeters. F r o m this equation, a 15-mm zone would represent a c o n c e n t r a t i o n o f . 0 0 6 / l g / m l . To validate this, a cephapirin c o n c e n t r a t i o n of .006 /~g/ml was prepared in milk, and 40 Difco disc assays were c o n d u c t e d . All 40 zones of inhibition measured precisely 15 mm. The .006 /~g/ml m i n i m u m d e t e c t i o n is e x t r e m e l y close to the . 0 0 5 / l g / m l obtained by Katz (6). In addition, o x y t e t r a c y c t i n e residues were quantitated. Zones o f inhibition p r o d u c e d by
TABLE 3. Zones of inhibition from selected concentrations of peniciIIin obtained when incorporating various dyes into agar used in the Difco disc assay.
Dye
.100
Orange-O Methylene blue TTC 3 Bromcresol purple No dye (control)
27.16 t 26.91
26.97 26.97 26.94
Penicillin, U/ml .050 .O10
.005
Control
SE
24.41 24.03 23.94 24.03 23.94
15.56 15.41 15.65 15.59 15.53
13.002 13.00 13.00 13.00 13.00
.14 .09 .15 .08 .16
Mean value of four replicates. 2 Diameter of disc. 32,3,5-Triphenyltetrazolium chloride. Journal of Dairy Science Vol. 68, No. 11, 1985
18.75 18.34 18.38 18.37 18.75
ANTIBIOTIC QUANTITATION
3035
TABLE 4. Zones of inhibition for concentrations of penicillin used to produce a standard curve for quantitation of the Difco disc assay. Penicillin
Zone of inhibition
(U/ml)
(mm)
.100 .050 .010 .005 Con~ol
27.08 24.14 18.57 15.61 13.001
SE
.05 .05 .05 .05 0
I Diameter of disc.
TABLE 5. Zones of inhibition for concentrations of cephapirin used to produce a standard curve for quantitation of the Difco disc assay. Cephalosporin
Zone of inhibition
(tzg/ml)
(mm)
1.00 .50 .10 .05 Control
31.78 29.46 24.54 21.56 13.00 ~
SE
.14 .14 .14 .14 0
1 Diameter of disc.
selected c o n c e n t r a t i o n s o f o x y t e t r a c y c l i n e were d i f f e r e n t ( P < . 0 0 0 1 ) (Table 6). T h e linear c o r r e l a t i o n of o x y t e t r a c y c l i n e c o n c e n t r a t i o n a n d z o n e o f i n h i b i t i o n was . 9 6 1 . This relationship was linear ( P < . 0 0 0 1 ) a n d q u a d r a t i c ( P < . 0 0 0 1 ) . Using this r e l a t i o n s h i p , t h e following e q u a t i o n was f o r m u l a t e d : Log10 ( o x y t e t r a c y c l i n e c o n c e n t r a t i o n ) = (zone--22.282)~7.638
w h e r e o x y t e t r a c y c l i n e c o n c e n t r a t i o n was in m i c r o g r a m s per milliliter a n d z o n e o f i n h i b i t i o n was m e a s u r e d in millimeters. F r o m this equat i o n , a 1 5 - m m z o n e w o u l d r e p r e s e n t an o x y t e t r a c y c l i n e c o n c e n t r a t i o n o f .111 /ag/ml. T o validate this, a c o n c e n t r a t i o n o f . 111 # g / m l was p r e p a r e d in milk, a n d 4 0 Difco disc assays were c o n d u c t e d . T h i r t y - n i n e o f 4 0 z o n e s o f inhibit i o n m e a s u r e d e x a c t l y 15 ram. This . 1 1 1 / ~ g / m l is c o n s i d e r a b l y l o w e r t h a n t h e .30 /ag/ml
TABLE 6. Zones of inhibition for concentration of oxytetracycline used to produce a standard curve for quantitation of the Difco disc assay. Oxytetracycline
Zone of inhibition
(~g/ml)
(mm)
1.00 .50 .10 .10 .05
20.33 20.20 13.74 13.001 13.00
SE
.10 .10 .10 .00 0
t Diameter of disc. Journal of Dairy Science Vol. 68, No. 11, 1985
3036
BISHOP ET AL.
m i n i m u m d e t e c t i o n o b t a i n e d b y Britt (3). The cationic a n t i b i o t i c i n h i b i t i o n e n c o u n t e r e d in milk (12) s e e m s to have an e f f e c t o n sensitivity o f the Difco disc assay t o o x y t e t r a c y c l i n e b u t n o t cephapirin. CONCLUSIONS
The Difco disc assay and t h e Delvotest-P were similar in sensitivity. The o n l y d i f f e r e n c e was t h e possibility o f a " d o u b t f u l " result w i t h the Delvotest-P, w h i c h is n o t possible with t h e Difco disc assay. No d i f f e r e n c e s were d e t e c t e d with B. s t e a r o t b e r m o p b i l u s s p o r e s u s p e n s i o n s p u r c h a s e d f r o m Difco L a b o r a t o r i e s and Penicillin Assays, Inc., or in i n c o r p o r a t e d d y e s as far as z o n e size or clarity in the Difco disc assay. Q u a n t i t a t i o n o f a n t i b i o t i c s used c o m m o n l y in t r e a t m e n t o f m a s t i t i s - i . e . , penicillin, cephapirin, and o x y t e t r a c y c l i n e - w a s derived f r o m t h e Difco disc assay b y establishing a relationship b e t w e e n the log o f t h e a n t i b i o t i c c o n c e n t r a t i o n a n d t h e respective z o n e o f i n h i b i t i o n p r o d u c e d . T h e y are as follows: LOgl0 (penicillin c o n c e n t r a t i o n ) -- ( z o n e - 3 5 . 5 8 7 ) / 8 . 6 2 8 Log10 ( c e p h a p i r i n c o n c e n t r a t i o n ) = (zone-31.823)/7.674 Log10 ( o x y t e t r a c y c l i n e c o n c e n t r a t i o n ) = (zone--22.282)/7.638 REFERENCES
1 Anonymous. 1982. Uniform antibiotics test OKd for Pasteurized Milk Ordinance. Dairy Rec. 83:25. 2 Bishop, J. R., A. B. Bodine, G. D. O'Dell, and J. J. Janzen. 1984. Retention data for antibiotics
Journal of Dairy Science Vol. 68, No. 11, 1985
commonly used for bovine infections. J. Dairy Sci. 67:437. 3 Britt, J. 1981. Detection of Milk residue problems. Neb. Ext. Newslett. 10:59. 4 Ginn, R. E., R. Case, V. S. Packard, and S. Tatini. 1982. Quantitative assay of beta-lactam residues in raw milk using a disc assay method. J. Food Prot. 45:571. 5 Johnson, M. E., J. H. Martin, R. J. Baker, and J. G. Parsons. 1977. A comparison of several assay procedures to detect penicillin residues in milk. J. Food Prot. 40:785. 6 Katz, S. E. 1982. Beta-lactam residues in m i l k Methods of measurement and significance of residues. N. J. Agric. Exp. Stn. Publ. No. J-011121-82. 7 Kaufman, O. W. 1977. A practical sensitive test to detect penicillin in milk. J. Food Prot. 40:250. 8 MacCauley, D. M., and V. S. Packard. 1981. Evaluation of methods used to detect antibiotic residues in milk. J. Food Prot. 44".696. 9 Marth, E. H., and B. E. Ellickson. 1959. Problems created by the presence of antibiotics in milk and milk products-A review. J. Milk Food Technol. 22:266. 10 Packard, V. S., S. Tatini, and R. E. Ginn. 1975. An evaluation of methods for detecting and comparative incidence of penicillin residues in different types of raw milk samples. J. Milk Food Technol. 38:601. 11 Peeler, J. T., J. E. Leslie, J. E. Barnett, G. A. Houghtby, and J. W. Messer. 1983. Detectability and precision of the AOAC Bacillus stearotbermopbilus disc assay demonstrated in the 1981 FDA split milk sample testing program. J. Food Prot. 46:84. 12 Read, R. B., J. G. Bradshaw, A. A. Swartzentruber, and A. R. Brazis. 1971. Detection of sulfa drugs and antibiotics in milk. Appl. Microbiol. 21:806. 13 SAS Institute, Inc. 1982. SAS User's guide: Statistics. Cary, NC. 14 Van Os, J. L., S. A. Lameris, J. Doodewaard, and J. G. Oostendorp. 1975. Diffusion test for the detection of antibiotic residues in milk. Neth. Milk Dairy J. 29:16.
Clemson University Clemson, SC 29631
ABSTRACT
uses have led to contamination of milk and milk products. The presence o f antibiotics in milk in even minute quantities has created problems in the dairy industry including: a) inadequate curdling of milk and improper ripening of cheeses during their manufacture; b) decreased acid and flavor production during the manufacture of buttermilk and similar products; c) diminished starter culture growth when propagated in reconstituted skim milk; and d) validity of certain quality control tests (9). In addition, residues in milk should be avoided because residues are illegal, and they may be biologically active metabolites of drugs in milk (6). A major problem facing the dairy industry in the area o f antibiotic treatment is the rapid, accurate detection of low amounts of antibiotic residues in milk. The objectives of this study were to compare the more recently adopted and approved detection assays and to modify the Bacillus stearotbermopbilus Difco disc assay so as to make it possible to quantitate antibiotics used commonly in treatment of bovine infections.
Delvotest-P and Bacillus stearothermophilus Difco disc assay procedures were utilized, and assay sensitivity for detecting various antibiotics was tested. Bacillus stearotberrnopbilus spores used in the disc assay were purchased from two laboratories and compared. The dyes methylene blue, 2,3,5-triphenyltetrazolium chloride, orange-O, and bromcresol purple were incorporated separately into agar used for the disc assay to determine if sharper, clearer zones could be produced. None of the dyes had an observable effect on zone size or clarity. Using the Difco disc assay, quantitation of penicillin, cephapirin, and oxytetracycline was accomplished with reliable precision by relating the size of the zone of inhibition to the log of the antibiotic concentration. The Delvotest-P and Difco disc assays were equal in sensitivity, as were spore suspensions used from the two laboratories. INTRODUCTION
Antibiotics have been used in dairy cattle for more than three decades. They are administered to cattle by four general methods: a) infusion into the udder for treatment and prevention of mastitis; b) injection (intramuscular, intravenous, or subcutaneous) for treatment of numerous diseases; c) orally or as a dietary supplement; and d) reproductive "flush" for uterine, cervical, and vaginal infections. Such
Received February 1, 1985. Technical Contribution No. 2410. Published with the approval of the Director, South Carolina Experiment Station. 2Present address: Diversey Corp., Wyandotte, MI. 1985 J Dairy Sci 68:3031-3036
MATERIALS AND METHODS
The two antibiotic detection assays used in this study were the De]votest-P and Difco disc assay. These procedures were used because of their relative rapidity, sensitivity, and simplicity. The Delvotest-P (14) is an agar diffusion test. Tablets containing nutrients and bromcresol purple are added to ampules containing plain agar with spores of B. stearotbermopbilus var. calidolactics. After adding a .1-ml milk sample, the ampule is incubated 2.5 h at 63 to 66°C. In the absence of antimicrobial substances, the whole of the solid medium turns yellow; it remains purple in the presence of sufficiently high concentrations of antibiotics. At intermediate concentrations of antibiotic
3031
3032
BISHOP ET AL.
the solid medium turns partly yellow (doubtful result). The test method has a high sensitivity for penicillin. Concentrations of .002 units/ml or lower give negative results in all samples, and .003, .004, and .005 units/ml give predominantly negative, doubtful, and positive results, respectively. These results were obtained in bulk milk samples and in individual cow milk samples. This method is also sensitive to most of the other antibiotics administered to lactating cows. The Difco disc assay (7) employs Antibiotic Medium #4 (Difco, Detroit, MI) seeded with B. stearotbermophilus (Difco). This Antibiotic Medium #4 is the recommended medium of choice, as illustrated by Peeler et al. (11). Six milliliters of seeded agar are put into 15 x 100-ram plastic petri dishes (Fisher Scientific, Norcross, GA). Plates must be used within 7 d of preparation. Thirteen-millimeter paper discs (Schleicher and Schuell, Keene, NH) were used to absorb milk by capillary action and discs then placed on agar surface. Plates were incubated for 2.5 h at 64°C. An alternate method is for incubation to proceed for 4 h at 55°C. After incubation, zones of inhibition are measured in millimeters. A positive result was designated by a zone of 15 ram, which is a compromise between 14 and 16-ram zones suggested for a positive result (1). This assay detects as little as .005 units peniciUin/ml of milk. Certain questions remained from earlier work (5,8,10,11); therefore, the initial objective of this study was to determine which assay was the most accurate and precise. This was accomplished by conducting both assays each time milk was analyzed for antibiotic residues during this study and a r.etention data study (2). Another objective was to compare B. stearotbermopbilus spores purchased from Difco Laboratories and from Penicillin Assays, Inc. (Boston, MA) since they are the two major distributors of the spore suspension. Penicillin (Penicillin G Procaine, Veticare, Baltimore, MD) concentrations (U/ml) of .050, .010, .005, and control were prepared in raw milk. Eight seeded agar plates were prepared with each of the spore suspensions. Thirteen-millimeter paper discs were used to absorb each penicillin concentration and the four discs placed on each plate. Plates were incubated and zones of Journal of Dairy Science Vol. 68, No. 11, 1985
inhibition measured. This experiment was replicated three times. The experimental design was a randomized complete block with subunits, and results were analyzed by analysis of variance utilizing the F-test and single degree of freedom contrasts (13). Each suspension was also plated on Antibiotic Medium #4 to compare concentrations. The third objective was to produce more distinct zones of inhibition by incorporating various dyes into the Difco disc assay to increase the accuracy of reading the assay. Dyes used were orange-O, 2,3,5-triphenyltetrazolium chloride, methylene blue, and bromcresol purple, along with a control containing no dye. For each dye, 1.25 mg/100 ml agar was incorporated into the medium immediately before the agar was seeded with spore suspension. Penicillin concentrations (units/ml) of .100, .050, .010, .005, and control were prepared in raw milk by serial dilution. Thirteen-millimeter discs were used to absorb by capillary action an aliquot of each antibiotic concentration; discs were then placed on each of 8 spore-seeded, dye-incorporated, and control agar plates. Plates were incubated 2.5 h at 64°C, and zones of inhibition measured in milliliters. The experiment was replicated four times. The experimental design was a randomized complete block with split units. Analysis of variance was conducted utilizing the F-test to compare each dye to the control (13). The fourth objective was to quantitate by Difco disc assay some antibiotics, namely, penicillin, cephapirin, and oxytetracycline, used commonly in treatment of bovine infections. For penicillin, concentrations (U/ml) o f 0, .100, .050, .010, and .005 were prepared in raw milk by serial dilution. An aliquot of each antibiotic concentration was absorbed onto 13-mm paper discs by capillary action and then placed on each of 8 spore-seeded agar plates. Plates were incubated 2.5 h at 64°C, and zones of inhibition measured in milliliters. The experiment was replicated 20 times. For cephapirin (cephapirin sodium, BristolMyers Co.) and oxytetracycline (Oxytetracycline HCI, Bio-Ceutic, St. Joseph, MO), concentrations for each of 1.00, .50, .10, .05, and 0 /ag/ml were prepared in raw milk. The reason for the increased concentrations, compared with that used to test for penicillin, was the decreased sensitivity o f the assay for these
ANTIBIOTIC QUANTITATION antibiotics, partly due to the natural cationic inhibition of antibiotics in milk (11). For each antibiotic tested, an aliquot of each concentration was allowed to absorb onto 13-ram paper discs by capillary action, and was then plated onto each o f 8 spore-seeded agar plates. Plates were incubated 2.5 h at 64°C, and zones of inhibition measured in millimeters. Both experiments were replicated 10 times. All three quantitation trials were set up with a randomized complete block design with split units. Correlation coefficients were determined and linear and quadratic contrasts performed• RESULTS A N D DISCUSSION
The sensitivities of Delvotest-P and Difco disc assays were essentially equal at .050 U penicillin/ml. Table 1 illustrates data obtained from conducting both assays for detection of selected concentrations of penicillin in milk, and individual cow milk samples obtained during an antibiotic retention study (2). The only apparent difference was that with the Delvotest-P, a " d o u b t f u l " result was possible to record; but because the Difco disc assay possesses measurable zones, the zone must be /> 15 mm to indicate a positive result. Of the 11 " d o u b t f u l " results from the Delvotest-P, 6 were positive and 5 were negative by the Difco disc assay. All 91 positives and 206 negatives by the Delvotest-P were the same with the Difco disc assay. When B. s t e a r o t b e r m o p b i l u s spore suspensions were seeded into agar used for the Difco
3033
disc assay, no significant differences in zone sizes of inhibition were detected (Table 2). However, zones from Penicillin Assays' spore suspensions were consistently larger than those from Difco spore suspensions. Spore concentrations were not significantly different between the two sources• No differences were noted in zone size when comparing Difco disc assays conducted with dye-incorporated agar to control agar containing no dye (Table 3). When incubation time was extended to approximately 5 h, agar containing bromcresol purple produced more distinct zones due to the contrast in color between the purple zone of inhibition and the yellow lawn of growth; therefore a slightly larger zone was produced. Quantitation of penicillin residues in milk by the Difco disc assay was accomplished. Zones of inhibition produced by selected concentrations of penicillin were significantly different (P<.001) (Table 4). The linear correlation of penicillin concentration and zone of inhibition was .939. This relationship was linear (P<.0001) and quadratic (P<.001). When the linear relationship of zone of inhibition vs. log10 (penicillin concentration) was tested, the correlation was .999. Using this relationship, the following equation was formulated: Log10 (penicillin concentration) = (zone--35.587)~8.628 where penicillin concentration is in units per
TABLE 1. Comparison of Delvotest-P and Difco disc assay for known concentrations of penicillin in milk and unknown individual cow samples of milk obtained during an antibiotic retention study (2). Penicillin
Delvotest-P
(U/ml)
Difco disc assay (% positive)
.050 .010
100 100
100 100
•0 0 5
100
100
Control Milk samples (n = 308) l + +/---
0
0
91
97
11
NA
206
2
211
t + = Positive (+), doubtful (+/-), or negative (-) detection of antiobiotic residues. 2Not applicable. Journal of Dairy Science Vol. 68, No. 11, 1985
3034
BISHOP ET AL.
TABLE 2. Comparison of spore suspensions purchased from Difco Laboratories and Penicillin Assays, Inc., used in the Difco disc assay. Zone of inhibition Penicillin
Difco
Penicillin Assays
(U/ml)
SE
(mm)
.050 .010 .005 Control
24.58 18.62 15.63 13.001
24.92 19.23 15.89 13.00 ~
.26 .26 .26 0
Diameter of disc.
milliliter and zone is measured in millimeters. F r o m this equation, a 15-mm zone (the minim u m zone for positive detection) w o u l d represent a penicillin c o n c e n t r a t i o n of .004 U/ml. To validate this, a penicillin concentration o f .004 U / m l was prepared in milk, and 40 Difco disc assays were c o n d u c t e d (10 plates with 4 discs/plate). All 40 zones o f inhibition measured precisely 15 m m . This value o f .004 U / m l m i n i m u m d e t e c t i o n agrees exactly with results obtained b y Britt (3). This e q u a t i o n is m o r e quantitative than the one introduced by Ginn et al. (4), which only c o m p u t e s quantitative estimates above or below the reference c o n c e n t r a t i o n o f antibiotic used (.016 U/ml). Q u a n t i t a t i o n of cephapirin residues in milk by the Difco disc assay was also accomplished. Zones o f inhibition p r o d u c e d by selected concentrations of cephapirin were significantly different ( P < . 0 0 0 1 ) (Table 5). The linear correlation o f cephapirin c o n c e n t r a t i o n and
zone o f inhibition was .823. This relationship was linear ( P < . 0 0 0 1 ) and quadratic (P<.0001). When the linear relationship o f zone o f inhibition and log10 (cephapirin concentration) was tested, the following e q u a t i o n was f o r m u l a t e d : Log10 (cephapirin c o n c e n t r a t i o n ) = (zone--31.823)/7.674 where cephapirin c o n c e n t r a t i o n was in micrograms per milliliter and z o n e measured in millimeters. F r o m this equation, a 15-mm zone would represent a c o n c e n t r a t i o n o f . 0 0 6 / l g / m l . To validate this, a cephapirin c o n c e n t r a t i o n of .006 /~g/ml was prepared in milk, and 40 Difco disc assays were c o n d u c t e d . All 40 zones of inhibition measured precisely 15 mm. The .006 /~g/ml m i n i m u m d e t e c t i o n is e x t r e m e l y close to the . 0 0 5 / l g / m l obtained by Katz (6). In addition, o x y t e t r a c y c t i n e residues were quantitated. Zones o f inhibition p r o d u c e d by
TABLE 3. Zones of inhibition from selected concentrations of peniciIIin obtained when incorporating various dyes into agar used in the Difco disc assay.
Dye
.100
Orange-O Methylene blue TTC 3 Bromcresol purple No dye (control)
27.16 t 26.91
26.97 26.97 26.94
Penicillin, U/ml .050 .O10
.005
Control
SE
24.41 24.03 23.94 24.03 23.94
15.56 15.41 15.65 15.59 15.53
13.002 13.00 13.00 13.00 13.00
.14 .09 .15 .08 .16
Mean value of four replicates. 2 Diameter of disc. 32,3,5-Triphenyltetrazolium chloride. Journal of Dairy Science Vol. 68, No. 11, 1985
18.75 18.34 18.38 18.37 18.75
ANTIBIOTIC QUANTITATION
3035
TABLE 4. Zones of inhibition for concentrations of penicillin used to produce a standard curve for quantitation of the Difco disc assay. Penicillin
Zone of inhibition
(U/ml)
(mm)
.100 .050 .010 .005 Con~ol
27.08 24.14 18.57 15.61 13.001
SE
.05 .05 .05 .05 0
I Diameter of disc.
TABLE 5. Zones of inhibition for concentrations of cephapirin used to produce a standard curve for quantitation of the Difco disc assay. Cephalosporin
Zone of inhibition
(tzg/ml)
(mm)
1.00 .50 .10 .05 Control
31.78 29.46 24.54 21.56 13.00 ~
SE
.14 .14 .14 .14 0
1 Diameter of disc.
selected c o n c e n t r a t i o n s o f o x y t e t r a c y c l i n e were d i f f e r e n t ( P < . 0 0 0 1 ) (Table 6). T h e linear c o r r e l a t i o n of o x y t e t r a c y c l i n e c o n c e n t r a t i o n a n d z o n e o f i n h i b i t i o n was . 9 6 1 . This relationship was linear ( P < . 0 0 0 1 ) a n d q u a d r a t i c ( P < . 0 0 0 1 ) . Using this r e l a t i o n s h i p , t h e following e q u a t i o n was f o r m u l a t e d : Log10 ( o x y t e t r a c y c l i n e c o n c e n t r a t i o n ) = (zone--22.282)~7.638
w h e r e o x y t e t r a c y c l i n e c o n c e n t r a t i o n was in m i c r o g r a m s per milliliter a n d z o n e o f i n h i b i t i o n was m e a s u r e d in millimeters. F r o m this equat i o n , a 1 5 - m m z o n e w o u l d r e p r e s e n t an o x y t e t r a c y c l i n e c o n c e n t r a t i o n o f .111 /ag/ml. T o validate this, a c o n c e n t r a t i o n o f . 111 # g / m l was p r e p a r e d in milk, a n d 4 0 Difco disc assays were c o n d u c t e d . T h i r t y - n i n e o f 4 0 z o n e s o f inhibit i o n m e a s u r e d e x a c t l y 15 ram. This . 1 1 1 / ~ g / m l is c o n s i d e r a b l y l o w e r t h a n t h e .30 /ag/ml
TABLE 6. Zones of inhibition for concentration of oxytetracycline used to produce a standard curve for quantitation of the Difco disc assay. Oxytetracycline
Zone of inhibition
(~g/ml)
(mm)
1.00 .50 .10 .10 .05
20.33 20.20 13.74 13.001 13.00
SE
.10 .10 .10 .00 0
t Diameter of disc. Journal of Dairy Science Vol. 68, No. 11, 1985
3036
BISHOP ET AL.
m i n i m u m d e t e c t i o n o b t a i n e d b y Britt (3). The cationic a n t i b i o t i c i n h i b i t i o n e n c o u n t e r e d in milk (12) s e e m s to have an e f f e c t o n sensitivity o f the Difco disc assay t o o x y t e t r a c y c l i n e b u t n o t cephapirin. CONCLUSIONS
The Difco disc assay and t h e Delvotest-P were similar in sensitivity. The o n l y d i f f e r e n c e was t h e possibility o f a " d o u b t f u l " result w i t h the Delvotest-P, w h i c h is n o t possible with t h e Difco disc assay. No d i f f e r e n c e s were d e t e c t e d with B. s t e a r o t b e r m o p b i l u s s p o r e s u s p e n s i o n s p u r c h a s e d f r o m Difco L a b o r a t o r i e s and Penicillin Assays, Inc., or in i n c o r p o r a t e d d y e s as far as z o n e size or clarity in the Difco disc assay. Q u a n t i t a t i o n o f a n t i b i o t i c s used c o m m o n l y in t r e a t m e n t o f m a s t i t i s - i . e . , penicillin, cephapirin, and o x y t e t r a c y c l i n e - w a s derived f r o m t h e Difco disc assay b y establishing a relationship b e t w e e n the log o f t h e a n t i b i o t i c c o n c e n t r a t i o n a n d t h e respective z o n e o f i n h i b i t i o n p r o d u c e d . T h e y are as follows: LOgl0 (penicillin c o n c e n t r a t i o n ) -- ( z o n e - 3 5 . 5 8 7 ) / 8 . 6 2 8 Log10 ( c e p h a p i r i n c o n c e n t r a t i o n ) = (zone-31.823)/7.674 Log10 ( o x y t e t r a c y c l i n e c o n c e n t r a t i o n ) = (zone--22.282)/7.638 REFERENCES
1 Anonymous. 1982. Uniform antibiotics test OKd for Pasteurized Milk Ordinance. Dairy Rec. 83:25. 2 Bishop, J. R., A. B. Bodine, G. D. O'Dell, and J. J. Janzen. 1984. Retention data for antibiotics
Journal of Dairy Science Vol. 68, No. 11, 1985
commonly used for bovine infections. J. Dairy Sci. 67:437. 3 Britt, J. 1981. Detection of Milk residue problems. Neb. Ext. Newslett. 10:59. 4 Ginn, R. E., R. Case, V. S. Packard, and S. Tatini. 1982. Quantitative assay of beta-lactam residues in raw milk using a disc assay method. J. Food Prot. 45:571. 5 Johnson, M. E., J. H. Martin, R. J. Baker, and J. G. Parsons. 1977. A comparison of several assay procedures to detect penicillin residues in milk. J. Food Prot. 40:785. 6 Katz, S. E. 1982. Beta-lactam residues in m i l k Methods of measurement and significance of residues. N. J. Agric. Exp. Stn. Publ. No. J-011121-82. 7 Kaufman, O. W. 1977. A practical sensitive test to detect penicillin in milk. J. Food Prot. 40:250. 8 MacCauley, D. M., and V. S. Packard. 1981. Evaluation of methods used to detect antibiotic residues in milk. J. Food Prot. 44".696. 9 Marth, E. H., and B. E. Ellickson. 1959. Problems created by the presence of antibiotics in milk and milk products-A review. J. Milk Food Technol. 22:266. 10 Packard, V. S., S. Tatini, and R. E. Ginn. 1975. An evaluation of methods for detecting and comparative incidence of penicillin residues in different types of raw milk samples. J. Milk Food Technol. 38:601. 11 Peeler, J. T., J. E. Leslie, J. E. Barnett, G. A. Houghtby, and J. W. Messer. 1983. Detectability and precision of the AOAC Bacillus stearotbermopbilus disc assay demonstrated in the 1981 FDA split milk sample testing program. J. Food Prot. 46:84. 12 Read, R. B., J. G. Bradshaw, A. A. Swartzentruber, and A. R. Brazis. 1971. Detection of sulfa drugs and antibiotics in milk. Appl. Microbiol. 21:806. 13 SAS Institute, Inc. 1982. SAS User's guide: Statistics. Cary, NC. 14 Van Os, J. L., S. A. Lameris, J. Doodewaard, and J. G. Oostendorp. 1975. Diffusion test for the detection of antibiotic residues in milk. Neth. Milk Dairy J. 29:16.