COMPARISON OF SIX PHYSIOLOGIC CHARACTERISTICS OF STAPHYLOCOCCI FROM LABORATORY SPECIMENS

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1 THE AMERICAN JOURNAL OF CLINICAL PATHOLOGY Vol., No., pp. -8 August, 9 Copyright 9 by The Williams & Wilkins Co. Printed in U.S.A. COMPARISON OF SIX PHYSIOLOGIC CHARACTERISTICS OF STAPHYLOCOCCI FROM LABORATORY SPECIMENS CHARLES D. JEFFRIES, PH.D. WITH THE TECHNICAL ASSISTANCE OF BABBARA FLOOD WAILES Department of Microbiology, College of Medicine, Wayne State University, Detroit 7, Michigan The isolation of staphylococci from specimens submitted to the laboratory for bacteriologic examination poses problems of interpretation for both the bacteriologist and physician. The increase in antibioticresistant strains is a further complication and intensifies the need to detect rapidly those strains which are potentially pathogenic. Laboratory procedures based on the phsyiologic characteristics of Staphylococcus aureus have been devised to aid in the detection of pathogenic staphylococci. Among these characteristics have been hemolytic activity, pigment production, coagulase activity, mannite fermentation, and resistance to sodium chloride. In addition to the above reactions, it has been reported that the ability to hydrolyze deoxyribonucleic acid can be correlated with the coagulase reaction. ' ' i 9 Burns and Holtman have described a series of tests, including the above, with which they graded the pathogenicity of the organism. In the study described in this paper, the author attempted to correlate physiologic characteristics of 9 staphylococci isolated from routine specimens in the laboratory of Detroit Receiving Hospital. These strains were tested for their responses in the coagulase test, hydrolysis of deoxyribonucleic acid (DNA), mannite fermentation, and pigment production. Their ability to reduce triphenyltetrazolium and to grow on tellurite glycine agar was also studied. Received, December, 9; accepted for publication May, 9. Dr. Jeffries is Assistant Professor of Microbiology. This work was supported in part by the Receiving Hospital Research Corporation, and by a grant (E-9), from the National Institutes of Health, Public Health Service, Department of Health, Education, and Welfare. METHODS The cultures, obtained from the laboratory in the tubes in which the coagulase activity had been determined, were streaked on tryptose agar (Difco). Isolated colonies were transferred to tryptose agar slants which, after incubation, served as the source of inoculums for the test mediums. The staphylococci isolated from lesions included specimens from pus, wounds, abscesses, and ulcers; under respiratory specimens were sputum, throat swabs, and bronchial aspirations; urine specimens included those voided and catheterized; the miscellaneous category included those that had no source indicated, as well as those from any other source not included above or which were from blood samples. Pigment production was determined after 8 to hr. growth on tryptose agar, supplemented with per cent skimmed milk. Any trace of golden pigment in the growth was regarded as being a positive reaction. The coagulase activity was determined by transfer of a loopful of growth from the milk agar plate to a tube of. ml. of blood bank plasma, diluted with an equal volume of tryptose broth (Difco) and incubated at 7 C. for hr. Mannite fermentation was determined by growth in phenol red mannite broth (Difco) at 7 C. for hr. The deoxyribonuclease () activity was determined by means of growing the bacteria on tryptose agar supplemented with. per cent DNA for 8 to hr. at 7 C, and the plate was then flooded with per cent hydrochloric acid in order to precipitate undigested DNA. 7 The ability to reduce triphenyltetrazolium (TPTZ) to the colored formazan was determined on tryptose agar that contained mg. of TPTZ per liter. The tellurite glycine Downloaded from on February 8

2 Aug. 9 PHYSIOLOGIC CHARACTERISTICS OF STAPHYLOCOCCI TABLE THE RESPONSES OF 9 CULTURES OF STAPHYLOCOCCI TO PHYSIOLOGIC TESTS, RELATED TO THE SOURCE OF THE SPECIMEN* Mannite Pigment TGt TPTZJ /8 / / /9 9/7 / / 78/ / 8/88 8/8 7/ /8 7/7 / /7 8/ / / 7/ / 9/87 8/ /9 / 7/ / /7 88/ 7/ / /7 8/7 9/77 /8 /9 * The numbers in the various columns, e.g., /8, indicate positive reaction/negative reaction. t Tellurite glycine agar. % Triphenyltetrazolium reduction. agar (Difco) permits *S. aureus to grow as a black colony on the surface of agar plates and inhibits other organisms. RESULTS The responses of the 9 strains of staphylococci to the physiologic tests are listed in Table, according to the source of the specimen. The organisms were observed to be approximately evenly divided into positive and negative types in the coagulase reaction, activity, mannite fermentation, pigment production, and the ability to grow readily on tellurite glycine agar. The ability to reduce TPTZ was possessed by approximately % of the strains. Table lists the distribution of types according to the combined responses in the coagulase reaction, activity, pigment production, and mannite fermentation for the various specimen sources. Two hundred and one (.9 per cent) of the cultures were positive for all responses, 8 (7.7 per cent) were negative for all tests, 9 (9.9 per cent) were positive for of the tests, and 9 (.9 per cent) yielded or positive reactions. When of these tests were positive, activity was among them, i.e., the absence of activity was accompanied by the absence of at least of the other reactions. It should be noted also that only of the possible reaction patterns were obtained with these strains, and that 78. per cent of the strains occurred in the groups in which the TABLE Reactions* DCMP DCM DCP DMP DM DP D C M P None THE COMBINED RESPONSES OF DEOXYRIBO- NUCLEASE ACTIVITY, COAGULASE ACTIVITY, MANNITE FERMENTATION, AND PIGMENT PRO DUCTION ACCORDING TO THE SOURCE OF THE SPECIMEN * D indicates deoxyribonuclease activity; C indicates coagulase activity; M indicates mannite fermentation; P indicates pigment production. reactions were either all positive or all negative. Among the coagulase-positive strains, 9. per cent of those from lesions, 8.9 per cent from the respiratory tract, 7.9 per cent from blood, 8. per cent from urine, and 8.7 per cent from miscellaneous sources hydrolyzed DNA, produced pigment, and fermented mannite. In only instances (. per cent) was the coagulase Downloaded from on February 8

3 JEFFRIES Vol. TABLE THE RELATION BETWEEN THE SELECTIVITY OF THE TELLURITE GLYCINE AGAK AND THE COAGULASE AND DEOXYRIBONUCLEASE ACTIVITIES OF STAPHYLOCOCCI BY THE SOURCE OF THE SPECIMEN Tellurite Glycine Agar Tellurite Glycine Agar test alone positive; of these were isolated from urine and the other from a throat swab. On the other hand, cultures produced but failed to clot plasma, of which were negative in either mannite fermentation or pigment production, or both. Mannite was negative in 9 instances among the coagulase-positive strains, allowing detection of 9.8 per cent of the potentially pathogenic strains, and strains failed to produce pigment; thus, 9. per cent of the coagulase-positive strains would have been detected. The correlation between the reaction on the tellurite glycine plates and the coagulase test (8 per cent) was not as good as that encountered between coagulase activity and mannite fermentation, pigment production, or the reaction. Growth on the tellurite glycine agar was observed to correlate with activity (87 per cent) better than with coagulase activity (Table ). DISCUSSION The correlation between the production of the enzymes, coagulase, and, is moderately good, with 9. per cent of the cultures tested possessing like abilities. It is to be emphasized that most of the disagreement resulted from production of without coagulase; indeed, only cultures possessed coagulase activity while failing to produce, ferment mannite, or produce pigment. The results described here fail to support those obtained by DiSalvo, who reported complete correlation between coagulase and activities in all of the cultures studied, but seem to be similar to the results obtained by Weckman and Catlin. 9 This difference may be owing to the use of a commercial preparation of rabbit plasma by DiSalvo, which is reported to be superior to human plasma for determination of coagulase activity. Only coagulase-positive strains would have gone undetected if activity were used as a screening method; therefore, activity could achieve a prominence in clinical bacteriology as a screening procedure. DiSalvo reported a slight discrepancy between production of pigment and the elaboration of and coagulase. There were approximately times as many discrepancies observed in the 9 strains reported here as among the group described by DiSalvo. In general, pigment production no longer is regarded as an important characteristic of pathogenic staphylococci, and Macintosh 8 has recently commented on this characteristic in unfavorable terms. Further, it was not considered warrantable to separate the pathogenic staphylococci on the basis of pigment in the 7th edition of "Bergey's Manual of Determinative Bacteriology." It would seem, however, from the results reported here and by DiSalvo Downloaded from on February 8

4 Aug. 9 PHYSIOLOGIC CHARACTERISTICS OF STAPHYLOCOCCI 7 that in the primary identification of staphylococci, pigment may serve a function. Mannite has been incorporated into a number of selective mediums in order to aid in the detection of pathogenic staphylococci. In this study, the correlation between coagulase production and mannite fermentation was approximately the same as that between coagulase and deoxyribonuclease, although one would detect fewer coagulasepositive strains by means of mannite fermentation than with the test. Zebovitz and associates observed that coagulase-positive strains of staphylococci grew well on the tellurite glycine agar, and reported per cent detection of coagulasepositive cultures. Some coagulase-negative cultures, however, were observed to grow on tellurite glycine agar with the characteristics of coagulase-positive strains. Of the 9 strains of staphylococci studied, 8 (. per cent) of the coagulase-positive strains failed to grow on the tellurite glycine agar. Approximately times as many of the cultures were coagulase-negative, but grew well with black colonies on tellurite glycine agar. The correlation was slightly better between activity and the tellurite glycine agar reaction. The ability of the staphylococci to accomplish the series of reactions described here may have a direct relation to pathogenicity, and in fact Burns and Holtman believed that strains that were positive in these reactions, as well as certain others, were primarily pathogenic. A gradation from pathogenic to saprophytic types was arranged as the tests in the battery used by those authors were observed to be negative. The demonstration of the virulenceenhancing effect of coagulase for strains of staphylococci that do not produce that enzyme serves to reaffirm the importance of coagulase in determining pathogenic potential. Pigment production and mannite fermentation permitted detection of not less than 9 per cent of the coagulasepositive strains studied; on the other hand, the determination of activity permitted detection of 98.7 per cent of the coagulase-positive cultures. It would seem from this study that activity, mannite fermentation, or pigment production could be used as a preliminary test of potential pathogenicity among the staphylococci, and that activity would be superior to the others. The tellurite glycine agar was less suitable than the above mediums as a screening method, and the reduction of TPTZ seemed to be of very little value. SUMMARY Six physiologic tests have been applied to 9 cultures of staphylococci isolated in a hospital laboratory. The coagulase activity, deoxyribonuclease activity, mannite fermentation, and pigment production were observed to be the same for 8 (78. per cent) of the strains. At least 9 per cent of the coagulase-positive strains could have been detected by mannite fermentation, and 98.7 per cent by deoxyribonuclease. Tellurite glycine agar was inferior to deoxyribonuclease, pigment production, or mannite fermentation. The ability to reduce triphenyltetrazolium was the least reliable test for coagulase-positive staphylococci. SUMMARIO IN INTERLINGUA Sex tests physiologic esseva applicate a 9 culturas de staphylococcos isolate in un laboratorio de hospital. Esseva trovate que le activitate de coagulase, le activitate de disoxyribonuclease, le fermentation de mannitol, e le production de pigmento esseva le mesmes pro 8 del racias {i.e., 78. pro cento). Al minus 9 pro Mento del racias positive pro coagulase haberea potite esser detegite per le fermentation de mannitol e 98.7 pro cento per le activitate de disoxyribonuclease. Le test de agar a tellusiteglycina esseva inferior a illos de disoxyribonuclease, production de pigmento, o fermentation de mannitol. Le capacitate de reducer triphenyltetrazolium esseva le criterio le minusfidel in le recognition de staphylococcos a positivitate pro coagulase. REFERENCES. BREED, R. S., MURRAY, E. G. D., AND SMITH, \ N. R.: Bergey's Manual of Determinative Bacteriology, Ed. 7. Baltimore: The Williams & Wilkins Company, 97, pp. -.. BURNS, J., AND HALTMAN, D. F.: Biochemical properties of virulent and avirulent staphylococci. Ann. New York Acad. Sci., 88: -, 9. Downloaded from on February 8

5 8 JEFFRIES Vol.. DISALVO, J. W.: Deoxyribonuclease and coagulase activity of micrococci. Med. Technicians Bull., 9:9-9, 98.. EKSTEDT, R. D., AND YOTIS, W. W.: Studies on staphylococci. II. Effect of coagulase on the virulence of coagulase negative strains. J. Bact., 8: 9-, 9.. FUSILLO, M. H., AND WEISS, D. L.: Qualitative estimation of staphylococcal deoxyribonuclease. J. Bact., 78: -, 99.. GREER, J. E.: Laboratory identification and classification of staphylococci. Am. J. M. Technol., : 9-9, JEFFRIES, C. D., HOLTMAN, D. F., AND GUSE, D. G.: Rapid method for determining the activity of microorganisms on nucleic acids. J. Bact., 7: 9-9, MACINTOSH, O. C: The ubiquitous staphylococcus does not always show its true colours. Canad. M. A. J., 8:-, WECKMAN, B. G., AND CATLIN, B. W.: Deoxyribonuclease activity of micrococci from clinical sources. J. Bact., 7: 77-7, 97.. ZEBOVITZ, E., EVANS, J. B., AND NIVEN, C. F., JR.: Tellurite glycine agar: A selective plating medium for the quantitative detection of coagulase-positive staphylococci. J. Bact., 7: 8-9, 9. Downloaded from on February 8