Department of Biotechnology, University of Turku, Tykistökatu 6A, FIN Turku, Finland

Transcription

1 953 Journal of Food Protection, Vol. 63, No. 7, 2000, Pages Copyright, International Association for Food Protection Qualitative Detection of Tetracycline Residues in Milk with a Luminescence-Based Microbial Method: The Effect of Milk Composition and Assay Performance in Relation to an Immunoassay and a Microbial Inhibition Assay JUSSI KURITTU,* STEFAN LÖNNBERG, MARKO VIRTA, AND MATTI KARP Department of Biotechnology, University of Turku, Tykistökatu 6A, FIN Turku, Finland MS : Received 4 October 1999/Accepted 22 December 1999 ABSTRACT Performance of Tet-Lux, a newly developed microbiological test for the detection of tetracycline residues in raw milk, based on tetracycline-controlled luminescence activation of the test bacteria, was evaluated in bovine milks with variable amounts of somatic cells, bacteria, fat, protein, and natural inhibitory compounds. The sensitivity of Tet-Lux was also compared to a commercially available tetracycline immunoassay (Snap, Idexx Laboratories Inc.) and to a microbial inhibition test (Delvotest SP, Gist-Brogades). There were slight differences in the luminescence signals between different milk samples, but no single factor could be pointed out to be responsible for them. There appeared to be a modest inverse relationship between luminescence and increasing fat and protein content. The amount of somatic cells, bacteria, and the natural inhibitors lysozyme and lactoferrin did not affect the luminescence response. The test fulfilled the sensitivity requirement specified by the European Union (maximum residue limit 100 ng/ml for tetracyclines). The Tet-Lux test was clearly more sensitive to all tetracyclines tested (oxytetracycline, tetracycline, chlortetracycline, doxycycline, demeclocycline, methacycline, minocycline) than Delvotest SP, and for five tetracyclines out of seven more sensitive than Snap. The test provides a fast, simple, and robust microbial method for the qualitative detection of tetracycline residues in milk. Detection of antibiotic residues in milk is indispensable, because some residues may cause technological problems in dairy fermentation processes and some antibiotics may result in allergic reactions in humans (5, 9). In addition, trace amounts of antimicrobial compounds in milk favor the development of antibiotic-resistant bacterial populations (15). The control of antibiotic residues is commonly accomplished with three types of methods: microbial inhibition tests, group- or substance-specific tests, and quantitative confirmatory methods. Screening of milk samples for antibiotic residues is done with microbial inhibition tests and group- or substance-specific tests that are mainly qualitative or semiquantitative (11). Microbial inhibition tests are based on inhibition of growth and metabolism of test bacteria by antibiotic residues, and they usually detect a broad spectrum of antimicrobials in a nonspecific manner. Groupor substance-specific tests include enzymatic tests as well as receptor assays and immunoassays. Unlike microbial inhibition tests, group- or substance-specific tests outline the possible antibiotics in the sample for instance into a certain group, thereby facilitating the performance of more laborious confirmatory methods. They can also be employed to detect antibiotics that are frequently overlooked by screening tests. The final identification and quantification of antibiotic residues is performed with sophisticated confirma- * Author for correspondence. Tel: ; Fax: ; juskur@utu.fi. tory methods, such as mass spectrometry and liquid chromatography (4, 6, 12, 17). Several microbial inhibition tests have been developed for the detection of antibiotic residues in milk. Most of them detect very sensitively widely used -lactam antibiotics but are usually not able to detect tetracyclines with required sensitivity, i.e., below the European Union maximum residue limits (MRL; 100 ng/ml for tetracyclines) (5, 10, 14). Most microbial inhibition tests are also rather timeconsuming, and they do not provide any specificity for a certain antibiotic or group of antibiotics. Group-specific tests developed for the detection of tetracycline residues, such as immunoassays and receptor-based assays, are usually quite fast and sensitive. On the other hand, they are more expensive than microbiological tests, and some of them utilize radioactive labels (11). Furthermore, even they do not always achieve the required sensitivity (13, 18). There is an obvious need for new, simple, inexpensive, nonhazardous, and sensitive tests for the detection of tetracycline residues. We have previously developed a luminescence-based microbiological assay for the specific detection of tetracyclines (7). It uses Escherichia coli bacteria carrying a sensor plasmid, in which a tetracycline-specific control unit regulates the expression of bacterial luciferase genes. The presence of tetracycline residues in a sample causes an increase in the light emission of the test bacteria. The sensitivity and functionality of this assay has been preliminar-

2 954 KURITTU ET AL. J. Food Prot., Vol. 63, No. 7 ily tested in milk matrix (our unpublished results). The assay is able to detect from 4 to 35 ng/ml of tetracycline, oxytetracycline, chlortetracycline, doxycycline, demeclocycline, methacycline, and minocycline. However, milk matrix is a complex and rather heterogeneous mixture of various components, some of which are known to disturb existing residue assays (21). These interfering factors may also affect the performance of an assay that is using luminescent bacteria as a test organism. Furthermore, the variation in milk composition between different cows and dairy farms can be considerable. Thus, one objective of this study was to evaluate the performance of our tetracycline-specific microbiological test in raw bovine milks with alternating amounts of fat and protein, as well as somatic cells, bacteria, and natural antimicrobial compounds. Another purpose of this study was to compare the sensitivity of our assay to a commercially available tetracycline immunoassay and to a widely used screening assay. MATERIALS AND METHODS Chemicals. Tetracycline, oxytetracycline, chlortetracycline, doxycycline, demeclocycline, methacycline, and minocycline hydrochlorides, trans-1,2-diaminocyclohexane-n,n,n,n -tetraacetic acid, 2-(N-morpholino)ethanesulfonic acid, and lysozyme (chicken egg white) were obtained from Sigma (St. Louis, Mo.). Bovine lactoferrin was obtained from ICN Biomedicals (Aurora, Ohio). Tryptone and yeast extract were from Difco (Detroit, Mich.). Milk samples. Raw bovine milk samples were obtained from Maito-Aura dairy (Turku, Finland) and from different farms in Denmark. Fat, protein, and somatic cell contents of the milks were determined with Milkoscan 4000 (Foss Electric A/S, Hillerød, Denmark). Bacterial content of the milks was determined with Bactoscan FC (Foss Electric A/S). Tetracycline-spiked milk samples were prepared as follows: Tetracycline stock solutions were prepared in 99% ethanol, 0.01 M or 0.1 M HCl, or water. Further dilutions were made in water if needed, after which raw milk samples were spiked with proper concentrations of tetracyclines. (The maximum amount of antibiotic diluent in milk samples was always less than 0.5%.) Tetracycline-spiked milk samples were heat-treated (82 C 10 min; water bath) prior to use in the assays. When the effects of lysozyme and lactoferrin were studied, these compounds were added into milk samples prior to heat treatment like tetracyclines. All the samples were analyzed as duplicates or triplicates. Test bacteria. The Tet-Lux test is based on specific, tetracycline-controlled expression of bacterial luciferase genes that code for enzymes responsible for light emission (7). When a milk sample contains tetracyclines, genetically engineered E. coli K12(pTetLux1) cells used as a test organism emit visible light in a dose-dependent manner. Detailed mechanisms for light emission and luminescent properties of the test bacteria used in Tet-Lux assay have been described elsewhere (7). The Tet-Lux test utilizes freeze-dried E. coli test bacteria. Freeze-drying of the bacteria has been described elsewhere (8). Tet-Lux test procedure. Lyophilized sensor bacteria were rehydrated with buffered (100 mm 2-[N-morpholino]ethanesulfonic acid; ph 6.0) Luria Bertani medium (16) (20 ml Luria Bertani medium per cell ampoule), and the cells were allowed to recover at room temperature (22 C) for 120 min. First, 25 l of 250 mm trans- 1,2-diaminocyclohexane-N,N,N,N -tetraacetic acid solution was pipetted into the wells of white microtitration plates (Labsystems, Helsinki, Finland), after which 100 l of heat-treated milk sample was added. Finally, 100 l of recovered sensor bacteria suspension was added into the wells. The plates were incubated at 37 C for 120 min after which the luminescence was measured with Victor 1420 multilabel counter (EG&G Wallac, Turku, Finland). Reference methods. Commercial tests, Snap Tetracycline Test (Idexx Laboratories, Westbrook, Maine) and Delvotest SP (Gist-Brocades, Delft, The Netherlands), were performed according to manufacturers instructions. Snap Tetracycline Test is an enzyme-linked immunoassay for the qualitative detection of tetracycline, chlortetracycline, and oxytetracycline residues in raw commingled bovine milk. The interpretation of the results of the Snap Tetracycline Test was performed by visually comparing the color intensity of sample and control spots according to the instructions. Delvotest SP is a microbial inhibition assay for the screening of raw milk for a wide range of antimicrobial compounds. It uses Bacillus stearothermophilus as a test organism, and it is based on inhibition of the germination and growth of B. stearothermophilus spores by antibiotics. The Delvotest SP was visually observed twice: at control time, e.g., when the negative control sample turns from purple to yellow, and after 3 h incubation. The results of Delvotest SP were considered negative ( ) when the test agar turned yellow and positive ( ) when the agar remained purple. Results were considered doubtful ( ) when only a faint color change was observed. Sensitivity comparison of different tests. The sensitivity comparison of the Tet-Lux test, Snap, and Delvotest SP was performed on average raw bovine milk from an 80,000-liter milk silo. The tests were performed with identical milk samples, except that for the Tet-Lux test, the samples were heat-treated before testing. RESULTS AND DISCUSSION Typical dose response curves of the Tet-Lux test for three tetracyclines is presented in Figure 1. As can be seen, a high concentration of tetracyclines causes a decrease in the luminescence response obtained with the test because of the toxic effect of tetracyclines on the test organism. However, the occurrence of false-negative results due to the high concentration of tetracyclines can be avoided by diluting the sample or by adjustment of test conditions (7). The effect of variation between different milk samples on the performance of the Tet-Lux test was evaluated by analyzing 105 bovine milk samples collected from dairy farms. Each milk was a sample from a pool of all milk from one farm. Milk samples contained variable amounts of fat, protein, somatic cells, and bacteria (ranges in different milk samples: fat content, 2.9 to 6.3%; protein content, 2.9 to 4.3%; somatic cells, to /ml; bacteria, to /ml). All samples were tested without tetracycline spiking (control level) and spiked with 100 ng/ ml (European Union MRL) of oxytetracycline, tetracycline, and chlortetracycline. The milk samples were tested in six panels, each containing 15 to 19 milks. Results of one panel are presented in Figure 2. Even in the case of oxytetracycline, which is clearly the weakest activator of luminescence of the test bacteria, there is still a clear difference in the luminescence signal between the highest control signal and the lowest signal obtained with oxytetracycline-spiked milk. The results obtained with all six milk panels were

3 J. Food Prot., Vol. 63, No. 7 DETECTION OF TETRACYCLINE RESIDUES IN MILK 955 FIGURE 1. Typical dose response curves of the Tet-Lux test for oxytetracycline hydrochloride ( ), tetracycline hydrochloride ( ), and chlortetracycline hydrochloride ( ) in bovine milk. Dashed line shows the European Union maximum residue limit (100 ng/ml) for tetracyclines in milk. highly similar: there was always at least a six- to ninefold difference between the highest signal obtained with nonspiked milk and the lowest signal obtained with milk spiked with 100 ng/ml of oxytetracycline. For other tested tetracyclines, tetracycline and chlortetracycline, that are more effective activators of luminescence, this difference was approximately 40- to 50-fold on the MRL level (Fig. 2). The variation (relative standard deviation) in luminescence signal between different samples was 15 to 25% within each milk panel. Therefore, the difference in luminescence signal between negative samples and samples containing an MRL level of tetracyclines appears to be adequate in order to avoid the occurrence of false-negative samples. Although the Tet-Lux test functioned well in all the milk samples tested, there were slight variations in luminescence signals between the samples. However, somatic cell and bacterial content of milk was not found to affect the luminescence signal levels obtained with the Tet-Lux test (Table 1). This was obvious, because heat treatment of the milk samples would be expected to have inactivated or killed both somatic and bacterial cells. Fat and protein content of milk appeared to have a modest inhibitory effect on FIGURE 2. Performance of the Tet-Lux test in 19 farm milks without spiking (control) and spiked with 100 ng/ml (EU MRL) of oxytetracycline hydrochloride (Otc), tetracycline hydrochloride (Tc), and chlortetracycline hydrochloride (Ctc). Square ( ) shows the average luminescence signal (RLU) of all 19 milks; bars ( ) show the maximum and minimum luminescence signals.

4 956 KURITTU ET AL. J. Food Prot., Vol. 63, No. 7 TABLE 1. Performance of Tet-Lux test in milks with different composition Milk sample Fat (%) Protein (%) Somatic cells/ml Bacteria/ml Negative control a OTC (100 ng/ml) b , , , , , , , , , ,407 a Tet-Lux assay without spiking (average luminescence signal [RLU] of two replicas). b Tet-Lux assay spiked with 100 ng/ml oxytetracycline (average luminescence signal [RLU] of two replicas). the luminescence response obtained with oxytetracyclinespiked samples (Table 1). There was no clear correlation, but the luminescence signals obtained with oxytetracyclinespiked samples were somewhat lower in milks containing high amounts of fat and protein ( 5% and 4%, respectively). This may be partly explained by the binding of tetracyclines into protein molecules that decreases the availability of tetracyclines (1, 19). However, no single factor studied could be pointed out to be responsible for the between-sample variations. Most importantly, the variations were so slight that they do not significantly affect the performance of the Tet-Lux test. During mastitis and colostrum, the amount of the natural inhibitors lysozyme and lactoferrin in milk is often abnormally high, e.g., mastitic milk may even contain 3 g/ml of lysozyme and 8,000 g/ml of lactoferrin (2). These compounds are known to be responsible for falsepositive results in microbiological residue assays both alone and in combination (2). In order to evaluate the effect of these inhibitors on the performance of the Tet-Lux test, we spiked raw milk with lysozyme (up to 3 g/ml), lactoferrin (up to 5,000 g/ml), and their combination (up to lysozyme 3 g/ml lactoferrin 2,500 g/ml). These inhibitors had TABLE 2. Comparison of the sensitivity of the Tet-Lux test, Snap Tetracycline test, and Delvotest SP a Antibiotic Tet-Lux Snap Delvotest SP Tetracycline Oxytetracycline Chlortetracycline b Doxycycline Demeclocycline b Methacycline Minocycline b a This table shows the concentrations of tetracyclines (ng/ml) that gave a clear positive result with the test. For the Tet-Lux test the value is the first concentration tested above the detection limit (average 3 SD; 99% confidence). b The highest concentration tested was 500 ng/ml. At this concentration chlortetracycline, demeclocycline, and minocycline gave borderline ( ) results. A concentration of 200 ng/ml of each tetracycline gave negative results with Delvotest SP. no effect on the Tet-Lux test: the test was able to detect tetracyclines with equal sensitivity in the absence and presence of lysozyme, lactoferrin, or their combination (data not shown). This was probably due to the heating step that at least partly inactivates them. Heating of the samples is widely used in other microbiological residue assays to eliminate the influence of these inhibitory substances (3, 13, 20). The sensitivity and performance of the Tet-Lux test was compared to Snap, an enzyme-linked immunosorbent assay for tetracyclines, and to Delvotest SP, a widely used screening assay for antibiotic residues in milk. The Tet-Lux test was found to be clearly more sensitive to all tetracyclines than Delvotest SP and for five tetracyclines out of seven more sensitive than Snap immunoassay (Table 2). The Tet-Lux test was as sensitive to tetracycline as Snap and slightly less sensitive to oxytetracycline. We noticed that Snap was able to detect all tetracyclines with rather good sensitivity, although according to the manufacturers instructions it is intended to detect oxytetracycline, tetracycline, and chlortetracycline. Sensitivity comparison was performed in silo milk (from an 80,000-liter milk silo) that represents average raw milk, because we found that Snap test did not function at all in milks containing high amounts of fat and protein ( 6% and 4%, respectively). The performance of Delvotest SP was not affected by the fat and protein content of the milk. The Tet-Lux test proved to fulfill the sensitivity requirements specified by the European Union in all kinds of milk matrices tested, and it was found to be very competitive with or even superior to widely used commercial reference tests. The Tet-Lux test is very simple to perform, because it requires only the incubation of a heat-treated milk sample with the test bacteria and the measurement of luminescence. It is also easily adjustable for different assay formats with different volumes and capacity depending on the needs, and there are several luminometers on the market, from single-tube inexpensive ones up to high-capacity luminometers using, e.g., 384-microwell format. Furthermore, production of the test is very inexpensive compared to, for instance, immunoassays, because the test organism itself provides all the enzymes and other proteins, sub-

5 J. Food Prot., Vol. 63, No. 7 DETECTION OF TETRACYCLINE RESIDUES IN MILK 957 strates, etc. needed in the assay. The Tet-Lux test is also a very fast microbiological test. Because the performance of the test does not require replication of the test organism, which is necessary for the performance of most available microbial inhibition assays, results are obtained in as soon as 2 h. We have also demonstrated that antibiotics other than tetracyclines, possibly present in the sample at concentrations that could be encountered as residues in milk, do not markedly interfere with the performance of the test (our unpublished results). Therefore, the Tet-Lux test may provide a new qualitative method for the specific detection of tetracycline residues in milk. Still, it must be noted that this study was performed only with tetracycline-spiked milk samples. The confirmation of the performance of the Tet- Lux test in relation to currently available screening tests and confirmatory methods still requires extensive analysis of samples from cows receiving tetracycline therapy. ACKNOWLEDGMENTS We express our gratitude to Foss Electric A/S (Hillerød, Denmark) for providing and analyzing raw bovine milk samples and to Maito-Aura dairy (Turku, Finland) for providing silo milk. REFERENCES 1. Aureli, P., A. M. Ferrini, and V. Mannoni Study of the effect of some proteolytic enzymes to improve the sensitivity of the microbial method for the detection of antibiotics and sulfonamide residues in milk, p In Proceedings of the Symposium on Residues of Antimicrobial Drugs and Other Inhibitors in Milk. International Dairy Federation, Brussels, Belgium. 2. Beukers, R Some special aspects of Delvotest. Bull. Int. Dairy Fed. 283: D Haese, E., H. J. Nelis, W. Reybroeck, and H. 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