Coagulase Activity: Plasma Suitability and Anticoagulants Tech Com: Microbiology

by Cheryl V. Maloney, MT(ASCP) Downloaded from https://academic.oup.com/labmed/article/10/6/358/2640021 by guest on 25 September 2021

Pathogenic Staphylococcus aureus pro anticoagulant tubes were pooled respec duces coagulases that accelerate the tively and each mixed thoroughly. To clotting of plasmas from a number of obtain sterile guinea pig plasma, citrated animal sources. Although coagulase ac guinea pig whole blood (CIBCO Diag tivity correlates with pathogenicity, the nostics, Madison, Wl) was aseptically enzyme itself has a little known role transferred to sterile vials and cen- in the pathogenesis of staphylococcal trifuged. The sterile plasma was then infections. transferred to new sterile vials. Human serum was prepared by collecting blood Several animal plasmas including hu in glass tubes, letting the blood clot man plasma will serve as a satisfactory and pooling several samples. Rabbit substrate for coagulase testing.1 We in plasma (BBL, Cockeysville, MD) was vestigated the nature and action of coagu prepared according to the manufac lase activity by ten isolates of Staphy turer's instructions. lococcus aureus and tested the suitability of four human plasma pools and rabbit To test the sterility of each plasma plasma as substrates. Since guinea pig pool, aliquots were cultured in brain plasma is not suitable for coagulase heart infusion broth (BBL) and observed 2 testing, we studied guinea pig plasma for growth after 24 hours of incubation. to determine if there is a deficiency of coagulase reacting factor (CRF) or if an The tube method of Lenette, Spauld- 3 inhibitor is present. The results of these ing, and Truant served as the reference studies are of practical importance. method for coagulase testing. In this method, 0.5 ml of sterile plasma must be aseptically added to Kahn tubes Materials and Methods containing four drops of sterile trypti case soy broth. In our study, the tubes The isolates of Staphylococcus aureus were inoculated with each of the ten were obtained from wound and swab isolated cultures of Staphylococcus specimens submitted to the Western aureus (previously identified by a positive Pennsylvania Hospital Bacteriology Labo tube coagulase test using rabbit plasma ratory. Primary cultures were subcul- as a substrate) along with a positive Cheryl V. Maloney, tured once to trypticase soy agar and control (ATCC reference strain of S. MT(ASCP), is with maintained at 37°C. the Clinical aureus) and a negative control (S. epi- Chemistry To prepare each human plasma pool, dermidis). The plasmas tested were: Laboratory at the University Health blood specimens were collected in each (1) rabbit coagulase plasma with EDTA; Center of of four Vacutainer tubes (Becton-Dickin- (2) guinea pig plasma with EDTA; (3) Pittsburgh in son, Rutherford, IL) containing different human plasma with heparin; (4) human Pittsburgh, Pennsylvania. anticoagulants: EDTA, heparin, oxalate plasma with EDTA; (5) human plasma and citrate. After centrifugation, the with oxalate; (6) human plasma with plasmas derived from each of the four citrate.

358 0007-5027/79/0600/358 $00.70 © American Society of Clinical Pathologists Table I—Tube Test Reactions After 24 Hours at 37°C.

Positive Negative % Growth Growth Cultures of Staphylococcus Aureus Sensitivity Control Control 1 2 3 4 5 6 7 8 9 10 Obtained* Rabbit Coagulase Plasma + - + + + + + + + + + + 100

Guinea Pig Plasma + - + - + - + - + + - - 55

Human Plasma-EDTA + - + + + + + + + + + + 91

Human Plasma-Oxalate + - + + + + + + + + + 100 + Downloaded from https://academic.oup.com/labmed/article/10/6/358/2640021 by guest on 25 September 2021

Human Plasma-Heparin + - + + + + + + + + + +• 100

Human Plasma-Citrate + - + - - + + - - + - - 45

Human Serum + - + 0

+ Positive Reaction - Negative Reaction

* The % sensitivity was calculated by dividing the number of positive cultures by the total number of Staphylococcus aureus cultures for each type of plasma. Results for the negative control are not included in the calculations.

Human serum served as an additional negative Results control. Following the addition of plasma and bac The results of the coagulase tube test are shown teria, the tubes were incubated in a water bath at in Table I. The results of the slide test are shown 37°C and observed at four hours for clot formation. in Table II. Any degree of coagulation, ranging from a sus pended clot to a firm clot, was considered a positive The tube tests were easier for us to interpret than reaction. Negative tubes were reincubated and the slide tests because the clots were so distinct. observed again for clot formation at 24 hours. The slide test gave results that were hard to interpret in some cases, especially when using citrated human The rapid slide test, as described by Bailey and 1 plasma. The positive slide test reactions (heparinized Scott, was a second method used. A small portion human plasma) detected only 73% of the coagulase- of an isolated colony was emulsified in a drop of positive strains, while the best tube tests (oxalated distilled water on a microscope slide. One loop of or heparinized human plasma) detected 100% of plasma was added and the emulsion was thoroughly coagulase-positive strains. mixed for five seconds. Coagulase-positive reactions were observed macroscopically as the formation of The dilution tubes using guinea pig and rabbit white clumps. plasmas as substrate were positive for coagulase activity. Therefore, there was no evidence for an In order to obtain the correct dilutions in testing inhibitor of coagulase reactivity or the lack of CRF for the presence of an inhibitor or lack of coagulase in guinea pig plasma. However, the tube test reacting factor in guinea pig plasma, the following utilizing guinea pig plasma detected only 55% of the dilutions were made and inoculated with the positive coagulase-positive strains. control culture: Tube #1—0.2 ml guinea pig plasma and 0.8 ml rabbit coagulase plasma; Tubes #2 through #7—Serial twofold dilutions of the guinea Discussion pig plasma were made using rabbit plasma as the Pathogenic Staphylococcus aureus produces a diluent; Tubes #8 through #14—The above pro coagulase that accelerates the clotting of a number cedure was repeated, but the proportions of guinea of different animal plasmas. Since the formation of pig and rabbit plasma were reversed. such clots is one of the characteristics of pathogenic

LABORATORY MEDICINE • VOL. 10, NO. 6, JUNE 1979 359 Table II—Slide Test Reactions After 5 Seconds at Room Temperature.

Positive Negative % Growth Growth Cultures of Staphylococcus Aureus Sensitivity 41 Control Control 1 2 3 4 5 6 7 8 9 10 Obtained Rabbit Coagulase Plasma + - + - + + - + - - + + 64

Guinea Pig Plasma + - 9

Human Plasma -EOT A + - - 4. + - - - - + + + 55

Human Plasma-Oxalate + - - - - + + + - - + + 55 Downloaded from https://academic.oup.com/labmed/article/10/6/358/2640021 by guest on 25 September 2021

Human Plasma-Heparin + + - - + + + + — + + 73

Human Plasma-Citrate - - + - - + + - - - - - 27

Human Serum ------0

+ Positive Reaction - Negative Reaction

Staphylococcus aureus in man and in animals, it is fibrinolysis and false-negative reactions. Our stud natural to relate coagulase activity and pathogenicity; ies would indicate that either pooled human plasma however, coagulase has no known role in the patho or rabbit plasma are adequate as substrates for genesis of infection. coagulase testing.

According to the classification of Rammelkamp,4 Guinea pig plasma, according to Smith and Hale,7 there are three types of coagulase factors: Type I, does not clot following reaction with coagulase. the cell-bound enzyme, is liberated by autolysis of These investigations postulated that susceptibility bacteria, acts directly on susceptible fibrinogen, and to coagulase depended on an activator in plasma, is neutralized by antibody; Type II, the free factor, and that the activator was deficient in guinea pig reacts only in the presence of the activator sub plasma. The activator resembles prothrombin in stance and is not neutralized by antibody;2 Type many respects, although its chemical identity is III is thought to be related to both Types I and II. still unknown.8 Our dilution studies would indicate The tube method measures all three types of coagu that guinea pig plasma can be clotted by Staphy lase while the slide method measures only Type I. lococcus aureus, but that the reaction is less re liable than with human or rabbit plasmas. To be acceptable for coagulase testing, a plasma must have an adequate amount of coagulase re The type of anticoagulant used for the plasma leasing factor and of fibrinogen, be relatively free substrate is critical. Other investigators have noted of fibrinolytic activity due to activation of the that citrate is an unreliable anticoagulant for use in plasminogen-plasmin system, and be free of signif coagulase testing.9 Citrate may cause false-positive icant amounts of inhibitors. Plasma suitability for reactions through the action of contaminating micro coagulase testing varies among animals of different organisms that utilize the citrate in the plasma, species and among different animals of the same species. For example, plasmin activity is greater in both rabbit and human plasmas than in swine * The Mueller factor is a heat-stable staphylococcal product plasmas.5 The Mueller factor* produced by staphy responsible for activating a proteolytic enzyme precursor in mammalian sera. The Mueller phenomenon is a proteolytic lococci activates the plasminogen-plasmin system of reaction resulting from activation of plasminogen by staphylo- swine plasma, and the plasmin formed causes kinase.6

360 LABORATORY MEDICINE • VOL. 10, NO. 6, JUNE 1979 causing clotting. In our studies, citrated plasma staphylococci. We demonstrated that human plasma gave a high percentage of false-negative reactions. gives results comparable to those obtained with The best anticoagulants for preparation of human commercial rabbit plasma; however, positive and plasma appear to be oxalate and heparin, both of negative controls must be included in all coagulase which gave results comparable to commercial rabbit procedures. Citrate is unacceptable as the anti plasma. coagulant, but oxalate and heparin give good results. When using human plasma for coagulase testing, Guinea pig plasma is not suitable for use in antibody production and circulating antibiotics must coagulase testing since it detected only 53% of also be considered, since both can give false- coagulase-positive staphylococci in our study. negative reactions.10 These problems sometimes The tube test proved more reliable in our hands arise when pooled or outdated blood bank plasma is than the slide test, detecting 35% more positive used for coagulase testing. Since most blood bank reactions. plasma contains citrate, our study would indicate Downloaded from https://academic.oup.com/labmed/article/10/6/358/2640021 by guest on 25 September 2021 that pooled human plasma obtained from other References sources would be acceptable as long as positive and negative controls are included. 1. Bailey, R.W., and Scott, E.G., 1973. Diagnostic Microbiology. 4th Edition. St. Louis, Mosby, pp. 112-114. 2. Wilson, G.S., and Miles, A.A., 1957. Topley and Wilson's Principles A final consideration in coagulase testing is the of Bacteriology and Immunity. 4th Edition. Baltimore, Williams and choice of tube or slide tests. Slide tests have the WilkinsCo., p. 1152. advantage of speed and convenience. However, 3. Lenette, E.H., Spaulding, E.H., and Truant, J.F., 1974. Manual of in our hands they are harder to interpret and yield Clinical Microbiology. Washington, D.C., American Society for Micro biology, p. 93. a high percentage of false-negative results. Tube 4. Dubos, R.J.. 1958. Bacterial and Mycotic Infections in Man. 3rd tests are more time consuming, but also more Edition. Philadelphia, J.B. Lippincott, p. 316. reliable. A practical solution might be to screen all 5. Parisi, J.T., Baldwin, J.N., and Sottile, M., 1973. Pour plate method for the detection of coagulase production by Staphylococcus aureus. organisms with a slide test and consider those that Appl. Microbiol. 25:558. give positive reactions as Staphylococcus aureus. 6. Cohen, JO., 1972. The Staphylococci. New York, Wiley Interscience, Negative reactions should then be retested by the pp. 222-223. tube method. 7. Smith, W., and Hale, J.H., 1944. Br. J. Exp. Pathol. 25:101. 8. Smith, D.T., and Conant, N.F., 1960. Zinsser Microbiology. 12th Edition. New York, Appleton-Century-Crofts, Inc., p. 196. Conclusion 9. BBL insert for preparation and use of coagulase plasma. 1972. Cockeysville, Maryland, Division of Becton-Dickinson and Co. Coagulase testing is the most widely used in vitro 10. Morton, H.E., and Cohn, J., 1972. Coagulase and deoxyribonuclease activities of staphylococci isolated from clinical sources. Appl. test to determine the potential pathogenicity of Microbiol. 23:732. •

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LABORATORY MEDICINE • VOL 10, NO 6, JUNE 1979 361