of mucoid colonies on sucrose agar under aerobic conditions by 3 strains of group K streptococci,

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1 THE EFFECT OF CARBON DIOXIDE ON POLYSACCHARIDE PRODUCTION BY STREPTOCOCCUS BOVIS JOEL A. DAIN, A. L. NEAL,' AND H. W. SEELEY Department of Biochemistry and Nutrition, and Laboratory of Bacteriology, College of Agriculture Cornell University, Ithaca, N. Y. Polysaccharide production by streptococci from sucrose or raffinose has generally been examined by one of two methods-streak or pour plates containing carbohydrate and incubated under air or broth cultures containing the carbohydrate. In the former method polysaccharide production is accompanied by the development of large, confluent moist colonies, usually slimy in appearance. With the latter method, polysaccharide formation is accompanied by an increased viscosity of the broth, sometimes to the point of solidification. Niven et al. (1941a) and Sherman et al. (1943) showed that all cultures of Streptococcus salivarius in their collection produced a polysaccharide on agar and in broth media. Of a considerable nuimber of other streptococci representative of Lancefield's Groups A through H, and members of Sherman's lactic, enterococcus and viridans divisions, only 1 culture of a collection of 54 strains of Streptococcwu bovis showed typical slime production on sucrose agar. Niven et al. (1941b) identified this material as a dextran. In a later publication, Niven et al. (1946) showed polysaccharide production both on agar and in broth by 7 additional strains of S. bovis, and to a lesser extent by Streptococcus s.b.e. (Streptococcus sanguis), and rarely, and in broth only, by Streptococcus mitis. Polysaccharide production in sucrose broth has been reported for the group H streptococci (Neil et al., 1941; Porterfield, 1950) and for Streptococcus "DS" (probably Streptococcus sanguis) (Hehre, 1948; Hehre and Neill, 1946). In studies on streptococci from endocarditis, Hehre and Neill (1946) and Porterfield (1950) demonstrated that some cultures that failed to form slime under aerobic conditions, formed slime in broth culture (i. e., relatively anaerobic conditions) and on plates incubated anaerobically. Farmer (1953) reported the production Received for publication January 9, Supported in part by a grant from the Moorman Manufacturing Company, Quincy, fllinois. 209 of mucoid colonies on sucrose agar under aerobic conditions by 3 strains of group K streptococci, but no gel formation in sucrose broth or on sucrose agar under anaerobic conditions. The polysaccharide produced by streptococci has been identified as dextran in the majority of instances in which chemical identification has been undertaken. S. salivarius, however, has been shown to produce some dextran along with larger amounts of levan. The work reported in the present paper concerns observations on slime formation by 87 strains of Streptococcus bovis. Of this number, 71 strains formed the large, confluent moist colonies typical of the polysaccharide producers when small amounts of CO2 were included in the atmosphere. An occasional strain produced slime on sucrose gelatin agar under air. Slime production appears to be dependent on the presence of CO2 and not on other conditions obtained by aerobic or anaerobic incubation. We have found, however, that "tween 80" can substitute for CO2 in enhancing slime production by S. bovis under anaerobic conditions. Among various strains a relationship has been observed between slime production, mannitol fermentation, and action on blood that may be of value in classification problems in the species. MATERIALS AND METHODS Table 1 shows the source and number of strains from each source used in this study. Sucrose gelatin agar plates (Niven et al., 1941a) were streaked from 18-hr thioglycolate broth (Brewers modified, BBL) cultures and incubated for 18 hr at 37 C in desiccators containing the desired atmosphere. In addition to giving heavier growth, thioglycolate broth proved to be superior to beef infusion broth in maintainmg cultures of this species. The polysaccharide was obtained for identification experiments by scraping slime from sucrose

2 210 DAIN, NEAL, AND SEELEY [vol. 72 TABLE 1 Sources of strains Isolated by of Strainsm Sample Type Animals (Number of Animals) Dain and Seeley* 46 Feces Cows (6), Calves (5), Goat (1), others (5) 18 Rumen Cows (3), Sheep (3) 7 Raw milk 3 Samples Cornell collection 3 2 from subacute bacterial en- Human (2) docarditis Bryant 5 Rumen Calves (2), Cows (2) Hungate 2 Perry 1 Rumen Sharpe 1 Feces Infant Shattock 4 Feces Cows (4) * Isolation procedures will be described in a future publication. gelatin agar plates that had been incubated Effect of different atmospheres upon 8lim4 production. The 2 strains of Streptococcus bovis used in under an atmosphere of CO2 for 24 hr at 37 C, and was purified by the method of Niven et al. the following experiments were Hi, isolated as (1946). A 2 per cent suspension of the purified polysaccharide was hydrolyzed in 1 N HCI for 4 hr at 100 C. The hydrolyzate was spotted on Whatman No. 1 filter paper, and the ascending technique was employed to develop the paper chromatograms using the two different solvents, 1-butanol-pyridine-water (0.3, 0.2, 0.15 by volume) (Hamerman et al., 1955) and 1-butanolacetic acid-water (0.4, 0.1, 0.5 by volume) (Block et al., 1955). After developing the chromatograms for 18 hr at 20 C, they were dried and sprayed with a 0.2 M m-phenylenediamine solution in 76 per cent ethanol (Block et al., 1955). The sprayed chromatograms were heated in an oven at 105 C for 15 min after which they were examined under ultraviolet light. EXPERIMENTAL RESULTS Table 2 gives a summary of reactions that proved to be valuable in grouping the 87 strains of Streptococcus bovis. Using slime production under CO2, mannitol fermentation, and blood reaction as pertinent tests, three rather distinct varieties emerge. The first and predominant variety (71 strains) consistently produced slime, never fermented mannitol, and in most cases produced greening (a) on horse-blood agar. The second variety (12 strains) produced no slime, always fermented mannitol, and appeared to have no effect (y) upon horse-blood agar. The third variety (4 strains) produced no slime, failed to ferment mannitol, and produced a greening on horse-blood agar. a predominant organism in a sheep suffering from experimentally produced overfeeding sickness and 470, isolated as one of the predominant organisms in a bloated heifer which had apparently been fed considerable grain. Table 3 lists the various atmospheres and their effect upon slime production. The data of table 3 may be interpreted as follows: Experiments 1 to 5, inclusive, show that CO2 is apparently involved in the formation of slime. By comparing experiments 1, 2,3, and 4, it is seen that in lower concentrations, a decrease in the CO2 content of the atmosphere is accompanied by a decrease in slime production, and that the amount of CO2 required for maximum slime production varies with the strain. Experiments 5, 6, and 9 indicate that the CO2 requirement is independent of the oxygen in the atmosphere. The ability of tween 80 to substitute for CO2 under anaerobic conditions, but not under aerobic conditions, is indicated by experiments 5, 7, and 8. Slime production as related to various carbohydrates. A 5 per cent carbohydrate gelatin agar was prepared with the following carbohydrates and combinations of carbohydrates: arabinose, ribose, glucose, fructose, lactose, maltose, sucrose, raffinose, melizitose, inulin, starch, glucose + fructose, fructose + maltose, glucose + inulin, fructose + starch, starch + inulin, esculin, a-methylglucoside, and a-methylmannoside. The results were identical with the 5 strains tested. All strains produced a heavy

3 1lo] C62 EFFECT ON S. BOVIS POLYSACCHARIDE PRODUCTION 211 TABLE 2 Number of strains producing various reactions after 24 hours at 87 C Sucrose Gelatin Agar Horse Blood Agar Number of Strains 5% CO,, 95% Air Air Fernnenttion Heavy slime Weak slime Weak slime producers producers producers * * All these strains produced a heavy slime in the presence of C02. In addition, some strains listed here as producing no slime in air after 24 hr showed a tendency to produce slime on prolonged incubation, i.e., 48 hr or more. TABLE 3 Production of slime by Streptococcus bovis under various atmospheres* after 24-hours incubation at 87 C Experimnent Atmosphere Strain HI Air S 2 0.5% C02 HS HS 3 0.3% C02 HS S 4 0.2% C02 HS 5 100% N % N % CO2 or HS HS 100% C % Tween 80t, air S 8 0.1% Tween 80100% N2 HS HS 9 0.5% C0, + 40% 02 HS HS * Unless otherwise indicated, the difference in atmosphere was air. t Tween 80 was added to the agar. HS = Heavy slime production; S = Weak slime production; = Growth, but no slime production. slime from sucrose medium in the presence of 5 per cent C02 in the atmosphere. No slime was produced in the air. Furthermore, no slime was produced on any of the other substrates tested either in the presence of 5 per cent C02 in the atmosphere or in air. On all substrates there was more luxuriant growth under 5 per cent C02. Identification of the CO2-dependent slime from strain Hi as a glucose-containing polysaccharide. The slime produced from strain Hi was identified as a glucose-containing polysacebaride as follows. The solubility properties in water and ethanol for the polysaccharide from strain Hi appeared to be the same as those for the dextran described TABLE 4 Comparison of color* and Rf values of chromatographed materials after 18 hours at 20 C Rf Values 1 2 Color of Spott Hydrolyzate Violet Glucose Violet Fructose Pale yellow * 1 = 1-Butanol-pyridine-water (0.3, 0.5, 0.15 by volume) (Hamerman, Bartz, and Reife, 1955). * 2 = 1-Butanol-acetic acid-water (0.4, 0.1, 0.5 by volume) (Block, Durrum, and Zweig, 1955). t Observed under ultraviolet light after treating chromatograms with m-phenylenediamine. by Niven et al. (1946), and Hehre and Neill (1946). A violet spot was observed on the developed chromatograms of the hydrolyzed purified slime when they were examined under ultraviolet light. The Rf values in the 2 solvents used and the color of the spot corresponded to those for glucose (table 4). Chromatographing a mixture of glucose and the hydrolyzate under similar conditions gave a single spot, whereas a mixture of fructose and the hydrolyzate gave 2 spots that were identical in Rf values to those of a mixture of glucose and fructose. It is therefore evident that the hydrolyzate contains only glucose and that the slime from Hi is a glucose polysaccharide. DISCUSSION Although S. bovis has been studied extensively over the years, its role as a producer of slime from sucrose has not been fully realized.

4 212 DAIN, NEAL, AND SEELEY [VOL. 72 An influence of carbon dioxide on the metabolism of amylolytic streptococci of sheep rumen origin (probably S. bovis) was reported by Macpherson (1953) who noted stimulation of growth by an atmosphere of 10 per cent CO2 and 90 per cent N2. The growth of two strains of S. bovis examined by Prescott et al. (1955) and Prescott and Stutts (1955) was found to be greatly stimulated by including CO2 in a synthetic medium in which arginine was the only source of amino nitrogen. It may be of interest that these 2 strains, at least one of which (P10) was of endocarditis origin, appear in our collection in the mannitol-fermenting, non-slimeproducing group. A noteworthy effect of CO2 on the growth habit of the "minute" streptococci of Lancefield's groups F and G was reported by Liu (1954) and Deibel and Niven (1955). Not only were colony size and numbers increased significantly, but the latter investigators reported enhanced hemolytic action on pour plates under C02. Deibel and Niven also showed that tween 80 would substitute for C02 as a growth stimulating substance, an observation that led us to try to replace CO2 by tween 80 as a stimulant of dextran production. With the strains we have tested, tween 80 would substitute for CO2 in the stimulation of polysaccharide production only upon anaerobic incubation. All the strains we employed (87), including those that failed to form slime under C02 tension, grew in 5 per cent sucrose broth producing a dense turbidity. The cells tended to remain in suspension even after several days and viscosity of the medium was increased, although in no case did complete solidification occur. Mann et al. (1954) and Mann and Oxford (1955) pointed out a correlation among reactions in a group of amylolytic streptococci from the calf rumen that corresponds to our observations, i. e., the strains producing greening on blood agar failed to ferment mannitol, whereas the strains indifferent on blood agar fermented mantol. It is a well established fact that Streptococcus bovie is widespread among ruminants and occurs in considerable numbers (Briggs, 1955). In the light of the work of Hungate et al. (1955), which pointed out that slime production may be asociated in an important way in bloat of ruminants, the problem of possible slime production by S. bovis in the rumen might merit some attention. ACKNOWLEDGMENTS We express our appreciation to Dr. M. P. Bryant, Dr. R. E. Hungate, and Dr. M. E. Sharpe, from whom we secured some of the cultures used in this study; and to Dr. R. W. Dougherty who made some experimental materials available. SUMMARY A requirement of C02 for slime formation on sucrose gelatin agar was demonstrated in the majority of strains of Streptococcus bovis tested. In most cases there appears to be a correlation between slime formation on sucrose gelatin plates incubated in the presence of C02, inability to ferment mannitol, and greening on horse-blood agar. The C02 requirement for slime production from sucrose gelatin agar plates can be replaced, in the 2 strains tested, by the inclusion of 0.1 per cent tween 80 in the sucrose gelatin agar medium and by incubating these plates anaerobically. The COrdependent slime from one strain was purified and identified as a glucosecontaining polysaccharide by paper chromatography of the hydrolyzate. REFERENCES BLOCK, R. J., DURRUM, E. L., AND ZWEIG, G.1955 A manual of paper chromatography and paper electrophoresis. Academic Press Inc., New York. BRIGGS, C. A. E Viable bacteria in the rumen. Dairy Sci. Abstr., 17, DEIBEL, R. H. AND NIVEN, C. F., JR The "minute" streptococci: further studies on their nutritional requirements and growth characteristics on blood agar. J. Bacteriol., 70, FARMER, E. D Streptococci of the mouth and their relationship to subacute bacterial endocarditis. Proc. Roy. Soc. (London), 48, HAmERMAN, O., BARTZ, K. W., AND REIFE, A Separation of a mixture of nine monosaccharides by two-dimensional ascending paper chromatography. Anal. Chem., 27, HEHrE, E. J Dextran-forming streptococci from the blood in subacute endocarditis and from the throats of healthy persons Bull. N. Y. Acad. Med., 24, HEm, E. J. AND NEILL, J. M Formation of serologically reactive dextrans by streptococci from subacute bacterial endocarditis. J. Exptl. Med., 83,

5 1956] C10EFFECT ON S. BOVIS POLYSACCHARIDE PRODUCTION 213 HUNGATE, R. E., FLETCHER, D. W., DOuGHERTY, R. W., AND BARRENTINE, B. F Microbial activity in the bovine rumen: Its measurement and relation to bloat. Appl. Microbiol., 3, Liu, P Carbon dioxide requirement of group F and minute colony G hemolytic streptococci. J. Bacteriol., 68, MACPHERSON, M. J Isolation and identification of amylolytic streptococci from the rumen of the sheep. J. Pathol. Bacteriol., 66, MANN, S. O., MASSON, F. M., AND OxFoRD, A. E Facultative anaerobic bacteria from the sheep's rumen. J. Gen. Microbiol., 10, MANN, S. 0. AND OXFORD, A. E Relationships between viable saccharolytic bacteria in rumen and abomasum of young calf and kid. J. Gen. Microbiol., 12, NEILL, J. M., SUGG, J. Y., HEHRE, E. J., AND JAFFE, E Influence of sucrose upon production of serologically reactive material by certain streptococci. Proc. Soc. Exptl. Biol. Med., 47, NIVEN, C. F., JR., KIZIUTA, Z., AND WMTE, J. C Synthesis of a polysaccharide from sucrose by Streptococcus s.b.e. J. Bacteriol., 51, NIVEN, C. F., JR., SMLEY, K. L., AND SHERMAN, J. M. 1941a The production of large amounts of a polysaccharide by Streptococcus 8alivarius. J. Bacteriol., 41, NIVEN, C. F., JR., SMaLEY, K. L. AND SHERMAN, J. M. 1941b The polysaccharides synthesized by Streptococcus salivariuw and Streptococcus bovis. J. Biol. Chem., 140, PORTERFIELD, J. S Classification of the streptococci of subacute bacterial endocarditis. J. Gen. Microbiol., 4, PRESCOTT, J. M., STUTTS, A. L., AND LYMAN, C. M Role of C02 in growth of Streptococcus bovis. Federation Proc., 14, 264. PRESCOTT, J. M. AND STuTTs, A. L Effects of carbon dioxide on the growth and amino acid metabolism of Streptococcus bovis. J. Bacteriol., 70, SHERMAN, J. M., NIVEN, C. F., JR., AND SMILEY, K. L Streptococcus salivarius and other non-hemolytic streptococci of the human throat. J. Bacteriol., 45, Downloaded from on August 15, 2018 by guest