Antibiotics are widely used in dairy cattle management

Transcription

1 KUKUROVÁ & HOZOVÁ: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 3, FOOD BIOLOGICAL CONTAMINANTS Interactions of Antimicrobials in Milk and Their Detection by the Disk Diffusion Method and Delvotest SP IVETA KUKUROVÁ and BERNADETTA HOZOVÁ Slovak University of Technology, Faculty of Chemistry and Food Technology, Department of Nutrition and Food Evaluation, Radlinského 9, Bratislava, Slovak Republic The combination of more than 2 different microbials might show interactions with various effects (synergistic, additive, antagonistic, or indifferent) on target microorgnisms. An objective of this paper was to evaluate the possible interactions of several antimicrobials those used most frequently in the treatment of mastitis in clinical veterinary practice ( -lactam antibiotics, aminoglycosides, peptides, other antibiotics, and sulfonamides) and their consequences on detection limits. In the model experiment with milk artificially altered by means of Delvotest SP and the disk diffusion method with Bacillus stearothermophilus var. calidolactis C 953, we observed the synergistic effect between all the antimicrobials tested. The results show that Delvotest SP is more sensitive (approximately 7.5- to 40-fold) than the disk diffusion method in estimating the detection limits of cephalosporin antibiotics. Antibiotics are widely used in dairy cattle management for treatment of some diseases and as dietary supplements. The use of antibiotics may result in the presence of drug residues in milk, especially if the antibiotics are not used according to label directions. Antibiotic residues in milk may cause allergic reactions in sensitive individuals and interfere with starter cultures used in the production of various kinds of cheese and other fermented dairy products, resulting in economic loss. In addition, it is thought that the widespread use of antibiotics may be responsible for the promotion of resistant strains of bacteria (1). To overcome the problems of potential residues, some procedures for antibiotic detection have been developed for industrial use (2). The most important methods are simple, accurate, and rapid so that appropriate steps in production can be taken (1). However, in the laboratory diagnostics of inhibitory residues no universal method with sufficient sensitivity exists for the convenient detection of all antibacterial substances. According to STN (3), antibiotic residues and substances that inhibit the growth of dairy cultures in milk and Received August 21, Accepted by AH January 15, Corresponding author s kukurova@yahoo.com. dairy products may be detected by 6 methods, each having a different sensitivity and selectivity. Therefore, it is not unusual for a sample tested by 2 of the allowed methods to show different results (4). In general, methods for the detection of inhibitory substances in milk may be classified as microbiological, i.e., those using various testing organisms (e.g., Bacillus stearothermophilus var. calidolactis C 953 disk diffusion method), enzymatic (5), and special identification and detection methods (6, 7), which were described in more detail previously (8, 9). One of the microbiological methods used for the rapid detection of residues of inhibitory substances in milk is Delvotest SP. Because the synergistic effects between antimicrobials lead to decreases in detection limits, the levels of individual substances (MIC) arae below the detection limits maximum residue limit; (MRL) claimed by the European Union and the recommendations of the U.S. Food and Drug Administration (10). The effects of interactions among antimicrobials in model experiments have been reported previously by several researchers (11, 12). Luitz et al. (11) observed the synergistic effect among -lactam antibiotics in model experiments with Delvotest SP. Similar results were also obtained in the case of cephalosporin antibiotics (12). These results demonstrate the explicit synergistic effect. Determination of the sensitivity ranges of different accessible detection systems for 2- and 3-antibiotic combinations and their respective comparisons are of great practical significance. The purpose of the work described in this paper was to estimate the detection limits of 11 antimicrobials by means of Delvotest SP and of cephalosporins by means of 2 types of detection systems (Delvotest SP and the disk diffusion method with B. stearothermophilus var. calidolactis C 953), and also to estimate the detection limits of combinations of the antibiotics in milk. In the second stage of the work, the sensitivities of the 2 methods were verified. Experimental Bacterial Strain B. stearothermophilus var. calidolactis C 953 was obtained from the collection of microorganisms at the Research Institute of Veterinary Medicine in Bratislava (Slovak Republic); it was propagated on GTK agar before its application, and

2 530 KUKUROVÁ & HOZOVÁ: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 3, 2003 Table 1. Concentration ranges of the antimicrobials tested a Antimicrobial Producer Concentration range, g/ml Penicillin G b Biotika, Sl. L up a (Slovak Republic) c Ampicillin b Biotika, Sl. L up a (Slovak Republic) Cefotaxime b,d Roussel (France) Cefuroxime b,d Glaxo (England) Cefazolin b,d Lilly France SA (France) Cefoperazone b,d Pfizer (Germany) Streptomycin e Biotika, Sl. L up a (Slovak Republic) Neomycin e Biotika, Sl. L up a (Slovak Republic) Bacitracin f Biotika, Sl. L up a (Slovak Republic) Trimethoprim Biotika, Sl. L up a (Slovak Republic) Sulfathiazole g Interpharm (Slovak Republic) a n = 10. b c d e f g -Lactam. Expressed in IU/mL. Cephalosporin. Aminoglycoside. Peptide. Sulfonamide. subsequently in the liquid substrate of the same composition (producer Imuna, Šarišské Micha any, Slovak Republic). Antimicrobials Table 1 lists the concentration ranges of the 10 tested antibiotics and one sulfonamide. Deionized water was used to prepare the stock and working solutions. Model milk samples for estimating the detection limits of the tests were prepared from reconstituted skim milk (PROMIL-PML, Inc., Nový Byd ov, Czech Republic) and deionized water (1 + 9); the respective antimicrobials were added to the milk prepared in this way. According to the recommendations of the International Dairy Federation (13), the ph of the reconstituted milk was >6; natural inhibitors were subjected to thermal inactivation for 5 min at 80 C. The reconstituted dried milk without any antibiotics was used as a control. Delvotest SP These methods combine the principle of agar diffusion tests with a color change of the indicator resulting from the active metabolism of the testing microorganism in the absence of the inhibitor. The sample to be examined is batched into microtitration plates with wells filled with the agar nutrient medium containing B. stearothermophilus var. calidolactis. After incubation (64 1 C for h), during which the tested strain grows, the color of the indicator (bromcresol purple) changes from blue-violet to yellow. If the sample contains substances that inhibit the growth of the test strain, the color of the indicator remains blue-violet. Delvotest SP (Gist-Brocades, The Netherlands) has a sensitivity to penicillin G of IU/mL milk. In our experiment, the Delvotest SP was performed according to the instructions of the manufacturer. Inhibitor-free reconstituted milk was used as a control sample. Disk Diffusion Method The paper disk soaked with the sample is placed on the surface of the agar nutrient medium with B. stearothermophilus.after incubation (64 1 C for 4 5 h), during which the tested strain grows, the agar medium turns opaque. If the investigated sample contains substances that inhibit the growth of the test strain, clear zones are formed around the disk. Their size depends on the concentrations and types of antimicrobials and can be compared with the size of the zones created by the reference solutions of penicillin of known concentration (an inhibition zone of 1mm is considered positive). The disk diffusion method (3) has a sensitivity to penicillin G of IU/mL milk. Results and Discussion The influence of penicillin G (IU/mL) in combination with -lactams, cephalosporins, aminoglycosides, peptides, other antibiotics, and sulfonamides (that are used most frequently for the treatment of mastitis) on the detection limits ( g/ml) was observed. Two combinations of 2 cephalosporins were also tested. The synergistic effect can lead to a decrease in test detection limits through the cumulative effect from the content of the individual substances (11, 12, 14). A crucial criterion is also the sensitivity of the detection tests applied (11).

3 KUKUROVÁ & HOZOVÁ: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 3, Table 2. Detection limits of antimicrobial combinations obtained by Delvotest SP Table 3. Average values obtained by Delvotest SP (n = 3) for combinations of antimicrobials Antimicrobial Detection limit in combination, g/ml Combination of antimicrobials, g/ml Average values for Delvotest SP a Penicillin G a Ampicillin Cefotaxime Cefazolin Cefuroxime Cefazolin Penicillin G a Streptomycin Penicillin G a Neomycin Penicillin G a Bacitracin Penicillin G a Trimethoprim Penicillin G a Sulfathiazole a Expressed in IU/mL. Table 2 shows the estimated detection limits for the antimicrobial combinations, which were achieved by Delvotest SP in 2 parallel assays. In our measurement, the detection limits of the individual substances obtained by means of Delvotest SP were IU penicillin G/mL and g ampicillin/ml. Table 2 shows that the detection limit of penicillin G in combination with ampicillin at g/ml decreased to IU/mL. The detection limit of ampicillin in combination with penicillin G at IU/mL decreased to g/ml. The decrease in the detection limits of both antibiotics in combination demonstrates the synergistic effect. The detection limit of cefotaxime determined by Delvotest SP was 0.01 g/ml, and that of cefazolin was g/ml (Table 2). The detection limit of cefotaxime combined with cefazolin at g/ml decreased to g/ml. The detection limit of cefazolin combined with cefotaxime at g/ml decreased to g/ml. The detection limit of individually applied cefuroxime was 0.04 g/ml, but when cefuroxime was combined with defazolin at g/ml, its detection limit decreased g/ml. The sole application of cefazolin revealed a detection limit of g/ml. The combination of cefazolin with cefuroxime at 0.01 g/ml decreased the detection limit of cefazolin to g/ml. The detection limit of penicillin G was IU/mL, and that of streptomycin was 2.0 g/ml. Table 2 shows that the detection limit of penicillin G combined with streptomycin at 0.5 g/ml decreased to IU/mL. The detection limit of Penicillin G b Ampicillin ± ± ± ± Cefotaxime Cefazolin ± ± ± ± ± 0.01 Cefuroxime Cefazolin ± ± ± ± ± Penicillin G b Streptomycin ± Penicillin G b +0.9 Neomycin ± ± ± Penicillin G b Bacitracin ± ± ± ± ± Penicillin G b +0.9 Trimethoprim ± ± ± ± ± Penicillin G b Sulfathiazole ± ± ± ± a b, Negative reaction (yellow color); +, positive reaction (purple color); ±, intermediate reaction (bottom half, yellow; top half, purple) = synergistic effect. Express ea in IU/mL.

4 532 KUKUROVÁ & HOZOVÁ: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 3, 2003 Table 4. Results obtained by the disk diffusion method for cefuroxime and cefazolin used alone and in combination Cefuroxime alone Cefazolin alone Combination of cefuroxime + cefazolin Concn, g/ml Inhibition zone, mm Concn, g/ml Inhibition zone, mm Concn, g/ml Inhibition zone, mm streptomycin combined with penicillin G at IU/mL decreased to 1.5 g/ml. During the estimation of penicillin G and neomycin (as individual substances) the detection limits were IU/mL (penicillin G) and 0.5 g/ml (neomycin). The detection limit of penicillin G combined with neomycin at 0.15 g/ml decreased to IU/mL. The detection limit of neomycin combined with penicillin G at IU/mL decreased to 0.35 g/ml. The detection limit of individually applied penicillin G was IU/mL, but when penicillin G was combined with bacitracin at 0.1 g/ml, its detection limit decreased to IU/mL. The sole application of bacitracin revealed a detection limit of 0.4 g/ml. The combination of bacitracin with pencillin G at IU/mL decreased the detection limit of bacitracin to 0.3 g/ml. The detection limit of penicillin G determined by Delvotest SP was IU/mL, and that of trimethoprim was 0.6 g/ml (Table 2). The detection limit of penicillin G combined with trimethoprim at 0.2 g/ml decreased to IU/mL. The detection limit of trimethoprim combined with penicillin G at IU/mL decreased to 0.4 g/ml. The last combination applied was that of penicillin G with sulfathiazole. The detection limit of penicillin G was IU/mL, and that of sulfathiazole was 0.05 g/ml. The detection limit of penicillin G in combination with sulfathiazole at g/ml decreased to IU/mL. The detection limit of sulfathiazole in combination with penicillin G at IU/mL decreased to g/ml. Our results demonstrate the explicit synergistic effect with decreases in the detection limits of the substances used in combination, compared with the individual detection limits of the uncombined substances. No samples showed evidence of antagonism of the tested combinations resulting in increased detection limits. The estimated detection limits correspond to the requirements for MRL and the published data (11, 15 17). To determine the sensitivity of Delvotest SP, each concentration/combination (blind coded) was replicated exactly 3 times (n = 3). The results were evaluated visually step by step as negative ( ), positive (+), or intermediate reactions ( ). An intermediate reaction was evaluated as a synergistic effect. Six antimicrobials in concentrations above and below their individual detection limits in combination with penicillin G in concentrations below and above its detection limit showed negative, positive, or intermediate reactions. The results are shown in Table 3. The combinations of 2 cephalosporins in concentrations above and below their individual detection limits showed only intermediate reactions (Table 3). The total number of antimicrobial combinations at different concentration levels tested by Delvotest SP was 40. Table 3 lists the average values obtained by Delvotest SP for the combinations of antimicrobials. From the results summarized in Table 3, we concluded that the detection limit of the visual evaluation was defined as 10% positive results (+), 10% negative results ( ), and 80% intermediate results ( ). Positive re- Table 5. Results obtained by the disk diffusion method for cefotaxime and cefazolin used alone and in combination Cefotaxime alone Cefazolin alone Combination of cefotaxime + cefazolin Concn, g/ml Inhibition zone, mm Concn, g/ml Inhibition zone, mm Concn, g/ml Inhibition zone, mm

5 KUKUROVÁ & HOZOVÁ: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 3, sults were observed only for the combination of penicillin G with aminoglycosides (streptomycin and neomycin); negative results were observed for the combination of penicillin G with ampicillin, streptomycin, and sulfathiazole. Intermediate results with the synergistic effect were observed for each group of antimicrobials and sulfonamide tested (Table 3). The disk diffusion method with B. stearotermophilus var. calidolactis C 953 and Delvotest SP were both used in our experiment with cephalosporin antibiotics. By comparing both microbiological methods we found a difference between them. The sensitivity of Delvotest SP is as much as 40-fold higher than that of the disk diffusion method. To estimate the detection limits of 3 cephalosporin antibiotics, many results were obtained. Delvotest SP is more sensitive than the disk diffusion method. In the determination of cefazolin the sensitivity of Delvotest SP (0.007 g/ml) was as much as 11.4 times higher than that of the disk diffusion method (0.08 g/ml). When cefotaxim was tested by the disk diffusion method, the detection limit determined was 0.4 g/ml; the sensitivity of Delvotest SP for cefotaxim was 40-fold higher (0.01 g/ml). In the cefuroxime evaluation, the detection limit found by applying the disk diffusion method was 0.3 g/ml, and that found by applying Delvotest SP was 0.04 g/ml (7.5-fold higher sensitivity of Delvotest SP). The values obtained are comparable with those in the literature (14, 17) and vary within the range of the MRL values (18). The results obtained for cephalosporins by the disk diffusion method are shown in Tables 4 and 5. The results for the combination of cefuroxime and cefazolin are presented in Table 4, and those for the combination of cefotaxime and cefazolin are presented in Table 5. When the decrease in the detection limits is considered, the most suitable combination was that of cefotaxime and cefazolin when the value obtained was 1.43 mm. This value was produced by the combination of 0.4 g cefotaxime/ml g cefazolin/ml. In fact, these values represent the detection limits of the combined antibiotics. For all other values produced by the combination, the concentration of one or both of the combined substances was higher than its detection limit. From the results reported in this paper, it can be concluded that besides studying the effects of antibiotics that act individually on microorganisms, it is also worthwhile to study antibiotic concentrations at various research levels (e.g., the influence on new starter cultures) to achieve the safe production of dairy products. References (1) Hejzlar, M., Hylmar, B., & Teplý, M. (1980) Antibiotics and Their Use in Greengrocery and Agriculture, Slovak National Technical Literature, Prague, Czech Republic (2) Mitchell, J.M., Griffiths, M.W., McEwen, S.A., McNab, W.B., & Yee, A.J. (1998) J. Food Prot. 61, (3) STN Official Method of Determination of Antibiotic Residues and Substances Inhibiting the Growth of Dairy Metures in Milk and Milk Products (1995) VÚM, ilina, Slovak Republic, pp 1 16 (4) Kolinová, M. (2000) in New Methods for Detection of Antimicrobial Residues in Milk, ilina, Slovak Republic, pp 4 6 (5) Charm, S.E., & Ruth, G.P. (1993) Bull. Int. Dairy Fed. 283, (6) Barker, S.A., & Long, G.P. (1994) J. AOAC Int. 77, (7) Usleber, E., Hensler, E., Dötsch, K., Märtlbauer, E., & Terplan, G. (1993) Residues Vet. Drugs Food 2, (8) Hozová, B., Görner, F., & Sklenárová, Z. (1994) Potravin. V dy 12, (9) Hozová, B., Hudecová, D., & Kratmüllerová, M. (1998) Czech. J. Food Sci. 16, (10) EEC (1992) Off. J. Eur. Com. 73, 8 14 (11) Luitz, M., Suhren, G., & Heeschen, W. (1996) Milchwissenschaft 51, (12) Hozová, B., & Kratmüllerová, M. (2001) Czech. J. Food Sci. 19, (13) IDF (1991) Detection and Confirmation of Inhibitors in Milk and Milk Products, pp 1 99 (14) Slinker, B.K. (1998) J. Mol. Cell. Cardiol. 30, (15) Suhren, G., Reichmuth, J., & Walte, H.G. (1996) Milchwissenschaft 51, (16) Charm, S.E., & Zomer, E. (1995) in Residues of Antimicrobial Drugs and Other Inhibitors in Milk, August 28 31, Kiel, Germany, International Dairy Federation (17) Suhren, G., & Reichmuth, J. (1998) Lebensmittelindustrie und Milchwirtschaft 119, (18) Nouws, J., Egmond, H., Smulders, I., Loeffen, G., Schouten, J., & Stegeman, H. (1999) Int. Dairy J. 9, 85 90