Bull Vet Inst Pulawy 49, 419-422, 2005
DETECTION OF STAPHYLOCOCCUS AUREUS IN MILK BY TUBE COAGULASE TEST HULYA TURUTOGLU1, FULYA TASCI2 AND SENAY ERCELIK1 1
Departments of 1Microbiology, and 2Food Hygiene and Technology, Faculty of Veterinary Medicine, Akdeniz University, 15100 Burdur, Turkey e-mail:
[email protected] Received for publication August 02, 2005.
Abstract The milk-tube coagulase test (mTCT) was utilized for direct detection of coagulase-positive staphylococci in 287quarter milk samples from cows with subclinical mastitis. The test was performed in sterile tubes by adding 0.5 ml of milk sample to 0.5 ml of reconstituted rabbit plasma. Coagulase activity was detected only in the samples from which Staphylococcus aureus had been isolated. When compared with culture results, sensitivity and specificity of the mTCT were 91% and 100%, respectively. The number of S. aureus colonies grown on Baird Parker agar from samples considered as positive for coagulase activity was 102 colony forming units/ml and higher. Although the positive reactions in mTCT occurred as the number of S. aureus increased in the quarter milk samples, a statistically significant correlation was not observed between the number of S. aureus cultured from the milk sample and the reaction time to yield a positive mTCT. It is concluded that the assay appears to be useful only in detecting coagulase-positive S. aureus from subclinical mastitic milk.
Key words: milk, milk-tube coagulase test, Staphylococcus aureus, mastitis. Subclinical mastitis is a disease of major economic importance to dairy industry causing reduced milk quality and leading to a loss in its production (19). In spite of increasing frequency of isolation of coagulase-negative staphylococci (CNS) from the bovine mammary glands, coagulase-positive Staphylococcus aureus is recognized worldwide as a major pathogen causing subclinical intramammary infection in dairy cows (12, 14, 18). S. aureus produces coagulase, an extracellular enzyme that binds to protein to form a complex with thrombin-like activity which converts fibrinogen to fibrin (9, 10). Although this enzyme is also produced by Staphylococcus intermedius and some Staphylococcus hyicus strains (7), coagulase production is one of the most reliable criteria for the identification of S. aureus (9, 15). Yazdankhah and Olsen (20) described the tube coagulase test for direct detection of S. aureus in milk samples of cows with subclinical mastitis. We aimed to adjust the method more useful in practice for the
detection of S. aureus and other coagulase-positive staphylococci in milk from cows with subclinical mastitis.
Material and Methods Samples. Randomly selected lactating cows from 91 dairy farms in Burdur province of Turkey were examined for subclinical mastitis by California Mastitis Test (CMT). In total CMT-positive 287 quarter milk samples were aseptically collected and transported icecooled to the laboratory within 2 h from the collection. Milk tube coagulase test (mTCT). The mTCT was performed in sterile glass tubes (13 mm diameter) by adding 0.5 ml of milk sample to 0.5 ml of reconstituted rabbit plasma (Bactident Coagulase EDTA, Merck KGaA, Darmstadt, Germany). After mixing by gentle rotation, the tubes were incubated at 37°C. Clotting was evaluated at 30 min intervals for the first 4 h of the test and then after 24 h incubation. The reaction was considered positive, if any degree of clotting was visible within the tube when tilted. As a positive control, the culture of a coagulase-positive S. aureus strain grown in tryptone soya broth (Oxoid Ltd, Hampshire, England), containing 108 bacteria/ml, was added to milk from mastitis-free cow at 1:5 dilution. Milk from mastitis-free cow according to CMT and culture results was used as a negative control. Isolates of Staphylococcus aureus. From each milk sample, 0.1 ml was plated on 7% sheep blood agar (Oxoid) plate and incubated at 37°C for 48 h. After presumptive identification based on colony morphology and microscopic morphology, biochemical and growth characteristics of the isolates were determined (9). To classify the presumptively identified staphylococci, the tube coagulase test with rabbit plasma was applied to each of them. In addition, coagulase positive isolates were identified based on haemolytic activity, acetoin production (Voges-Proskauer test) and anaerobic fermentation of mannitol (9, 11). Enumeration of S. aureus colonies. From each milk sample, 10 ml was added aseptically to 90 ml of 0.1% sterile peptone water. After a 10-fold serial
420 dilution to 10-8, each dilution was spread onto plate containing Baird Parker (B-P) agar (Oxoid) with 20% egg yolk telluride emulsion (Merck) by drop plating technique. Plates were incubated aerobically at 37°C for 24 - 48 h. Typical S. aureus colonies as circular, smooth, convex, moist, 1-3 mm in diameter, black (telluride reaction), and surrounded by an opaque zone with an outer clear zone (lecitinase reaction) were enumerated using a manual method. Atypical S. aureus colonies were taken for confirmatory testing, e.g. catalase, coagulase, and mannitol fermentation. This experiment was repeated three times, and the means of the number of colony forming units (cfu) were calculated (4, 21). Statistical analysis. The data were evaluated by Spearman correlation analysis using SPSS (version 11.0. SPSS Inc., Chicago, Illinois).
Results The aerobic bacteria were demonstrated in 260 (90.6%) from CMT-positive milk samples; no pathogens were found in 27 samples. From most of the samples, S. aureus (88 isolates), CNS (108 isolates), Streptococcus sp. (18 isolates), Escherichia coli (19 isolates), and Arcanobacterium pyogenes (2 isolates) were isolated
alone. Two organisms were isolated from 25 samples (Table 1). Positive mTCT results were recorded in 90 of 99 milk samples containing S. aureus (Table 1). All of the mTCT positive samples were positive for S. aureus in culture of milk on blood agar. No positive reactions were recorded in quarter milk samples containing other bacteria, e.g. CNS, Streptococcus sp., E. coli, and A. pyogenes (Table 1). In mTCT, some milk samples contaminated with E. coli became in appearance as if curdled and since typical coagulation was not formed these samples were considered negative. When compared with culture results, sensitivity and specificity of the mTCT were 91% and 100%, respectively (Table 2). Each milk sample positive for S. aureus resulted in typical S. aureus colonies (black, circular, smooth, moist, and surrounded by an opaque zone with an outer clear zone) on B-P agar. The number of S. aureus colonies in samples determined as positive for coagulase activity was 102 cfu/ml and higher (Table 3). Although the positive reactions in mTCT occurred as the number of S. aureus increased in the samples, a statistically significant correlation was not observed between the number of S. aureus cultured from the sample and the reaction time to yield a positive mTCT (r = - 0.464; P> 0.01).
Table 1 Bacterial species and mTCT results in quarter milk samples from cows with subclinical mastitis (n = 287) mTCT Bacterial species (number of samples) Positive Negative S. aureus (88) 79 9 CNS (108) 0 108 Streptococcus sp. (18) 0 18 E. coli (19) 0 19 A. pyogenes (2) 0 2 Streptococcus sp. and E. coli (3) 0 3 CNS and E. coli (11) 0 11 S. aureus and CNS (10) 10 0 S. aureus and E. coli (1) 1 0 No growth 0 27 Total 90 197
Table 2 Comparison of the mTCT results with culture results of S. aureus isolated from quarter milk samples of cows with subclinical mastitis (n = 260), sensitivity = 91%, specificity = 100% S. aureus culture mTCT Positive Negative Total
Positive 90 9
Negative 0 161
Total 90 170
99
161
260
421 Table 3 Number of S. aureus colonies identified from 99 S. aureus positive milk samples and reaction time to yield a positive mTCT Number of milk samples
Number of S. aureus colonies (cfu/ml)
mTCT Reaction time of positive results
6 11 44 32
107 bacteria/ml). In this study, S. hyicus and S. intermedius were not isolated from samples and coagulase activity was only detected in milk samples from which S. aureus was isolated. The clinical mastitis can develop anytime in subclinically infected mammary glands. In clinical mastitis cases, the treatment should be started immediately (5). By performing mTCT as preliminary test to CMT-positive milk samples, it can be determined whether the agent is S. aureus or not. The specific treatment against S. aureus can be quickly initiated instead of blind treatment before the laboratory results based on culture and antibiotic susceptibility tests are obtained. S. aureus still remains one of the most significant organisms associated with bovine mastitis and the current mastitis control protocols are comprehensive. The present study showed that mTCT for the detection of S. aureus in milk of cow affected with mastitis without apparent clinical sign is a very cheap and simply applicable method in practice by the
422 veterinarians, especially at repeated monitoring of health status of the mammary gland prior to and following a therapy. Nevertheless, the use of culture and biochemical classification is still the golden standard for the definitive diagnosing of the etiological agent responsible for subclinical mastitis.
Acknowledgments: The authors thank the Scientific Research Council of Akdeniz University for financial support.
9.
10. 11.
12.
References 1.
2.
3. 4. 5. 6.
7.
8.
Aarestrup F.M., Dangler C.A., Sordillo L.M.: Prevalence of coagulase gene polymorphism in Staphylococcus aureus isolates causing bovine mastitis. Can J Vet Res 1995, 59, 124-128. Cifrian E., Guidry A.J., Marquardt W.W.: Role of the milk fractions, serum, and divalent cations in protection of mammary epithelial cells of cows against damage by Staphylococcus aureus toxins. Am J Vet Res 1996, 57, 308-312. Collen D., Lijnen H. R.: Basic and clinical aspects of fibrinolysis and thrombolysis. Blood 1991, 12, 31143124. FDA Bacteriological Analytical Manual: Staphylococcus aureus, In: AOAC International, 8th rev. ed., Gaithersburg, MD, 1998, pp. 12.01-12.05 Francis P.G.: Update on mastitis. III. Mastitis therapy. Br Vet J 1989, 145, 302-311. Fox L.K., Besser T.E., Jackson S.M.: Evaluation of a coagulase-negative variant of Staphylococcus aureus as a cause of intramammary infections in a herd of dairy cattle. J Am Vet Med Assoc 1996, 209, 1143-1146. Holt J.G., Krieg N.R., Sneath P.H.A., Staley J.T., Williams S.T.: Gram-positive cocci. In: Bergey’s Manual of Determinative Bacteriology, Williams and Wilkins, Baltimore, 1994, pp. 527-558. Kawano J., Shimizu A., Kimura S.: Coagulase typing of Staphylococcus aureus isolated from animals. Zentralbl Bacteriol Microbiol Hyg 1986, 261, 407-410.
13.
14. 15. 16. 17. 18.
19. 20. 21.
Koneman E.W, Allen S.D., Janda W.M., Schreckenberger P.C., Winn W.C.: Gram-positive cocci part I: Staphylococci and related organisms. In: Color Atlas and Textbook of Diagnostic Microbiology, JP Lippincott Company, Philadelphia, 1992, pp. 405-429. McDevitt D., Vaudaux P., Foster T.J.: Genetic evidence that bound coagulase of Staphylococcus aureus is not clumping factor. Infect Immun 1992, 60, 1514-1523. Roberson J.R., Fox L.K., Hancock D.D., Beser T.E.: Evaluation of methods for differentiation of coagulasepositive staphylococci. J Clin Microbiol 1992, 30, 32173219. Roberson J.R., Fox L.K., Hancock D.D., Gay J.M., Beser T.E.: Prevalence of coagulase-positive staphylococci, other than Staphylococcus aureus, in bovine mastitis. Am J Vet Res 1996, 57, 54-58. Schlegelova J., Dendis M., Benedik J., Babak V., Rysanek D.: Staphylococcus aureus isolates from dairy cows and humans on a farm differ in coagulase genotype. Vet Microbiol 2003, 92, 327-334. Smith R.E., Hagstad H.V.: Infection of the bovine udder with coagulase-negative staphylococci. Prev Vet Med 1986, 4, 35-43. Sperber W., Tatini S.R.: Interpretation of the tube coagulase test for identification of Staphylococcus aureus. J Appl Microbiol 1975, 29, 502-505. Sprong R.C., Hulstein M.F.E., Van der Meer R.: Bactericidal activities of milk lipids. Antimicrob Agents Chemother 2001, 45, 1298-1301. Tizard I.R.: Veterinary Immunology: An Introduction. W.B. Saunders Company, Philadelphia, 2000 Turutoglu H., Mudul S., Pehlivanoglu F.: Antibiotic susceptibility and β-lactamase prevalence for staphylococci isolated from bovine mastitic milk samples. Acta Vet (Beograd) 2002, 52, 337-344. Yalcin, C.: Cost of mastitis in Scottish dairy herds with low and high subclinical mastitis problems. Turkish J Vet Anim Sci 2000, 24, 465-472. Yazdankhah S.P., Olsen E.: Simple and direct detection of Staphylococcus aureus in milk by a tube coagulase test. Lett Appl Microbiol 1998, 27, 111-115. Yousef A.E., Carlstrom C.: Staphylococcus aureus. In: Food Microbiology: A Laboratory Manual, A WileyInterscience Publication, USA, 2003, pp. 121-127.
