purchased as a 0.01 M, ph 7, solution. It was devoid of free phenolphthalein and

GLUCURONIDASE PRODUCTION BY STREPTOCOCCUS PYOGENES' JOHN J. ROBINSON, CHARLES W. BLINN, AND PAUL F. FRANK Naval Medical Research Unit 4, Great Lakes, Illinois Received for publication April 25, 1952 Glucuronidase, identified by the liberation of phenolphthalein from phenolphthalein glucuronidate substrate, is produced by numerous bacteria that are essentially nonpathogenic for man (Odell et al., 1950; Karunairatnam and Levvy, 1951). Jacox (1951) reported a much higher incidence of glucuronidase production by streptococci isolated from rheumatic fever patients than by strains from other sources. Since distinguishing features of streptococci presumably involved in rheumatic fever have not been established, further data on the production of glucuronidase by Streptococcus pyogenes seemed essential to investigation of the interesting possibility mentioned by Jacox (1951) that rheumatic fever. strains might uniquely possess this enzyme. This report describes glucuronidase assay methods and the results of testing 2,491 strains of streptococci giving typical beta hemolysis on sheep blood agar. MATERIALS AND ME:THODS Substrate. Phenolphthalein glucuronidate (Sigma Chemical Company) was purchased as a 0.01 M, ph 7, solution. It was devoid of free phenolphthalein and remained stable during several months storage at 4 C. Dilutions were made in 0.01 M, ph 6, acetate buffer. Quantitative test for glucuronidase. Three ml volumes of 0.3 per cent neopeptone in brain-heart infusion broth were inoculated with the test organism. After 16 to 20 hours' incubation at 37 C, the culture was centrifuged and the streptococcal residue (approximately 0.2 ml) was suspended in 0.20 ml each of 0.01 M, ph 6, acetate buffer, and 0.0001 M phenolphthalein glucuronidate. The suspension was held at 37 C one hour, 1.40 ml of 0.15 M Na2COS solution were added, and then centrifuged clear. The red supernatant resulting from the liberated, basic phenolphthalein was measured for optical density at a wavelength of 540 mju. Next, 0.50 ml of 1 N HCI was added and the cleared supernatant again similarly measured for optical density. This optical density was multiplied by 1.2, because of the 20 per cent dilution factor in adding the HCl, and subtracted from the initial optical density to give the net optical density free from nonphenolphthalein opacities. Data relating optical density with phenolphthalein were linear when plotted arithmetically. The amount of phenolphthalein was a function of k X optical density, with k a constant of 37.5 when phenolphthalein concentrations were expressed in micrograms. I The opinions herein expressed are those of the authors and cannot be construed as reflecting the views of the Navy Department or of the Naval Service at large. Mention of a commercially available material in this report does not mean an endorsement of that product. 719

720 J. J. ROBINSON, C. W. BLINN, AND P. F. FRANK [VOL. 64 Micro test for glucuronidase. A flamed loop, 1 mm in diameter, made from 28 gauge platinum wire, was filled approximately half full of S. pyogenes grown on sheep blood agar. These organisms were mixed in a small aluminum foil depression with 0.008 ml of 0.001 M phenolphthalein glucuronidate solution. About 10 depressions were prepared at one time by placing a 2 by 6 cm sheet of thin wrapping foil over snoothed modeling clay and gently pressing down with a 3 mm diameter rounded clean glass rod. Substrate, 0.001 M solution, was dispensed with a 1 ml syringe and 26 gauge needle, one small drop averaging 0.008 ml, just prior to mixing so that evaporation would be minimized. A few seconds of stirring gave a turbid suspension which was taken up in a 1 mm by 8 cm glass tube through capillary action. The tube was held about 30 degrees to the horizontal during filling, momentarily held upright to allow the suspension to fall a cm or so below the end, and then placed in modeling clay at a numbered location. The nonwetting and disposable features of the aluminum foil mixing hollows were convenient and nearly all of the suspension entered the capillary tube. Capillary tubes containing streptococci with substrate were prepared 10 at a time and placed in an air 37 C incubator for 60 to 90 minutes. White spot plates were prepared with 0.03 ml of 0.15 M Na2CO3 in numbered hollows. The capillary tubes were removed, the ends embedded in the clay were broken off, and their contents expelled into designated spots of the alkaline solution. Allowing the suspension to gravitate below the free end of a tube prevented drying which frequently occluded the opening. For expelling tube contents, a medicine dropper bulb with a small hole in the top for the tube was used. Such an open bulb permitted application to the tube without expulsion of contents until desired, when a finger would be placed over the open base of the bulb. The prompt appearance of a distinct red color in the Na2CO3 solution indicated a culture positive for glucuronidase. Cultures. Most cultures tested were taken during the course of epidemiologic studies at Great Lakes. Beta hemolytic streptococci were isolated on sheep blood agar and subjected to Swift (1947) and Lancefield's serologic classification after growing 18 to 24 hours at 37 C in brain-heart infusion broth with 0.3 per cent added dextrose and neopeptone. The following specific test sera were used: groups A, C, G; types 1, 2, 3, 4, 5, 6, 10/12, 11, 13, 14, 15, 17, 18, 19, 22, 23, 24, 25, 26, 28, 29, 30, 31, 32, 33, 36, 37, 38, 39, 40, 41, 42, 43, 44, 46, and 47. Cultures were stored at 4 C on sealed sheep blood agar slants, the majority being 1 to 2 months old when tested for glucuronidase by the micro method, with a storage range from 1 to 120 days. Cultures were selected at random for glucuronidase determinations without previous knowledge of group, type, or clinical source data. The clinical conditions most frequently represented were epidemic acute pharyngitis and rhinitis. The following laboratory stock cultures of S. pyogenes were repeatedly tested for glucuronidase production in the development of testing methods: strains NY-5(A-10/12), Richards(A-3), 98(A-not typable, Rammelkamp), Fontaine (A-14, rheumatic fever source), T28(A-28), 039127(A-5), Blackmore(A-11), and C(group C).

1952] GLUCURONIDASE PRODUCTION BY S. PYOGENES 721 RESULTS Supernatants of streptococcal broth cultures did not yield detectable amounts of glucuronidase. Grinding with sand and ph 6 buffer in a mortar released the TABLE 1 Glucuronidase production by Blackmore (A-li) and group C (C) streptococci grown 16 hour.s at 37 C in brain-heart infusion (BHI) and tryptose phosphate (TP) broths pg PHENOLPHTHALErN PRODUCEID IN: INGREDIENTS ADDED 0.3 PFR CENT TO BROTH: BHI broth by streptococcus TP broth by streptococcus group group A-1l C A-li C None.2.8 4.0 2.0 3.4 Dextrose.1.2 2.0 0.5 0.8 Neopeptone.7.0 7.4 3.6 4.6 Proteose peptone no. 3.5.0 6.0 2.8 4.2 Dextrose and neopeptone.1.01.8 0.4 1.0 TABLE 2 Glucuronidase production by cultures of Streptococcus pyogenes isolated during epidemiologic studies of acute respiratory diseases GLUCURONIDASE GROUP TYP NO. _ Negative Positive % Positive A 1 13 12 1 7.8 A 3 535 530 5 0.9 A 5 228 224 4 1.7 A 6 9 9 0 0 A 10/12 35 33 2 5.7 A 14 189 189 0 0 A 18 193 191 2 1.0 A 19 4 4 0 A 24 7 7 0 A 28 3 3 0 A 33 1 1 0 A 40 2 2 0 A 41 2 2 0 A 44 3 3 0 A 47 1 0 1 Subtotal 1,225 1,210 15 1.2 A not typable 347 299 48 13.8 C 241 147 94 39.0 G 114 54 60 52.6 Not groupable 393 274 119 30.3 Data absent... 163 136 27 16.5 Total... 2,483 2,120 363 14.6 enzyme in positive streptococci, while other procedures such as repeated freezing and thawing, washing with buffers, saline, and water did not. Increased concentrations of glucose appeared to hinder glucuronidase formation, while neopeptone and proteose peptone no. 3 (Difco) enhanced its produc-

722 J. J. ROBINSON, C. W. BLINN, AND P. F. FRANK [VOL. 64 tion. Table 1 summarizes data on the production of glucuronidase by the Blackmore and group C stock strains grown in 10 different broths. These two strains were positive for glucuronidase from 2 hours' to at least 40 days' incubation in broth at 37 C, and for several months on blood agar cultures stored at 4 C. The following stock strains were consistently negative for glucuronidase production under variable conditions of media, incubation, and storage: NY-5, Richards, Fontaine, 98, T28, and 039127. Four strains of S. pyogenes isolated from patients with acute pharyngitis followed shortly by the onset of rheumatic fever were studied, and all were negative for the production of glucuronidase. They were of the following group A types: 5, 14, 10/12, and 18. Sixty to 90 minutes' incubation at 37 C was adequate to demonstrate glucuronidase production under conditions of the previously mentioned test methods. About 100 duplicate quantitative and micro tests were run with complete agreement as to positive or negative results. End points with both methods were distinct and free of troublesome doubtful readings. The quantitative test was optimum for finer differentiation as in media comparisons, while the micro test was convenient and efficient in screening cultures. Production of glucuronidase among 2,483 separately isolated cultures of S. pyogenes is given in table 2. These cultures were tested by the micro method and were obtained from epidemiologic studies of acute respiratory infections. The 1,225 group A, typed strains had a total incidence of 1.2 per cent for glucuronidase production, while other strains of S. pyogenes had a much higher incidence of the enzyme. DISCUSSION The amounts of substrate employed were adequate to yield a measurable red color upon the formation of free phenolphthalein by glucuronidase from the conjugated substrate and subsequent alkalinization. Such red colors were stable over several days. The 0.0001 M concentration when used in 0.20 ml volume (2 X 10-8 moles) provided between 7 and 8 ug of phenolphthalein on complete break down. Since data characterizing enzymes, such as velocity constant determinations, require an excess of substrate to enzyme and short (30 minutes or less) incubation times, this small amount of substrate would not yield valid characterization data. However, the high cost of the substrate made minimum workable test quantities desirable. In the routine micro test, 8 X 10-9 moles or 0.008 ml of 0.001 M phenolphthalein glucuronidate solution was used as substrate, enabling nearly a thousand tests to be run per 1 ml of the 0.01 M substrate solution purchased. A tenfold increase in this substrate concentration did not offer superior end points, while a tenfold dilution gave faint but still readable results. The micro technique was also efficient because it required only a few mg of streptococci scraped from slants held in storage, and the disposable aluminum foil mixing dishes and capillary tubing eliminated the effort and materials incident to additional cultural procedures.

1952] GLUCURONIDASE PRODUCTION BY S. PYOGENES 723 The ph of streptococci-substrate mixtures approximated 6, which was close to the optimum reported in other studies (Karunairatnam and Levvy, 1951) on bacterial glucuronidase action. Significant numbers of streptococci associated with rheumatic fever were not studied. However, since these generally are typable group A strains, it seems possible that they also might have the low glucuronidase incidence of 1.2 per cent observed in this study among typed group A streptococci. The four socalled rheumatic fever strains of S. pyogenes studied did not produce glucuronidase, and none of the 15 patients yielding the glucuronidase-positive, typable group A streptococci of table 2 had either scarlet fever or rheumatic fever. These data do not suggest a unique production of glucuronidase by streptococci associated with rheumatic fever. The high production of glucuronidase by numerous saprophytes as well as nongroup A streptococci makes it unlikely that bacterial glucuronidase in itself plays a significant part in hman disease. ACKNOWLEDGMENTS The staff of Naval Medical Research Unit 4, under the direction of CDR J. R. Seal, MC, USN, kindly supplied many cultures studied and aided this investigation in various ways. SUMMARY Methods for testing streptococci for glucuronidase production are presented. A micro technique has proved efficient, accurate, and economical. Of 2,491 strains of Streptococcus pyogenes studied for glucuronidase production, 1.2 per cent of group A typable strains produced the enzyme. Group A nontypable strains, nongroupable strains, and groups C and G had a much higher incidence of the enzyme. Group A, typed strains isolated from four patients during an acute pharyngitis followed by rheumatic fever did not produce glucuronidase. Rheumatic fever was not associated with any of the glucuronidase-positive, typable group A streptococci encountered. REFERENCES JACOX, R. F. 1951 Beta glucuronidase production by beta hemolytic streptococci. J. Clin. Invest., 30, 652. KARUNAIRATNAM, M. C., AND LEvvy, G. A. 1951 A glucuronide-decomposing enzyme from rumen micro-organisms. I. Preparation and assay. Biochem. J., 49, 210-215. ODELL, L. D., PRDDLE, H. D., AND BURT, J. C. 1950 Activity of beta glucuronidase in human female genital tissues and in vaginal secretions. Am. J. Clin. Path., 20, 133-140. SWIFT, H. F. 1947 The relationship of streptococcal infections to rheumatic fever. Appendix: The technic of grouping and typing streptococci. Am. J. Med., 2, 168-189.