salitarlus had this property while similar cultures were easily SALIVARIUS certain non-hemolytic streptococci produce a polysaccharid,

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1 THE PRODUCTION OF LARGE AMXIOtUNTS OF A POLYSACCHARID BY STREPTOCOCCUS SALIVARIUS CHARLES F. NIVEN, JR., KARL L. SMILEY AND J. M. SHERNIAN Laboratory of Bacteriology, College of Agriculture, Cornell University, Ithece, New York Received for publication August 7, 1940 Oerskov (1930; Oerskov and Poulsen, 1931) has reported that certain non-hemolytic streptococci produce a polysaccharid, resulting in relatively large mucoid colonies, when grown on 2 per cent sucrose agar and to a lesser extent on raffinose agar. As Oerskov did not report tests for the production of carbon dioxide or volatile acids by his organisms, it is prol)able that many bacteriologists have thought, as did we, that lhe dealt with members of the genus Leuconostoc or Betacoccus, which organisms are well known to produce heavy mucoid growth under such conditions. In a footnote, Oerskov and Poulsen stated that this suggestion had been made by Dr. P. Arne Hansen but was not investigated. Through the kindness of Dr. Rebecca C. Lancefield we obtained two cultures of "indifferent streptococci" which produced mucoid colonies on sucrose agar. Upon detailed study these cultures were found not, to be betacocci, lout proved to be very typical strains of Streptococcus salivarius. We then found that a considerable proportion of our own stock cultures of Streptococcus salitarlus had this property while similar cultures were easily isolated from the human throat. When tested in 2 per cent concentration, the production of the mucoid material is greater on sucrose than on raffinose, but is apparently just as abundant on raffinose if used in a 50 per cent higher concentration in order to give equivalent amounts of the same constituent monosaccharids. The polysaccharid is also 479

2 sbr c. F. NIVEN, JR., K. L. SMILEY AND J. M. SHERMAN abundantly produced in well-buffered liquid media, crude tests indicating that as much as 40 per cent of the sucrose may be accounted for in the production of this material. The amount of mucoid material, and resulting size of colony on agar, may be increased by using 5 per cent sucrose, further increased if buffered with phosphate or calcium carbonate, and apparently still further increased if 5 per cent gelatin is added to the agar medium. The accompanying photographs show the comparative size of colonies of a typical culture of Streptococcus salivarius when grown on 5 per cent glucose and sucrose agars, the basic medium _ F...:...O _ ' 1 2 M F.::W '"~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~. ''...';....: extract,~~~~~~~~~~~~~~~~ 0. pe cent K2HO45l pe cen geatn an 1. per ce tagar.. (Rte strkin resut maobtained... be by"'sub-! qt.... stituting 0~~~~~~~.5 perlcetccffr ifh h hsht urs FIGa. nt SURFACE COLONIES OF STREPTOCOCCUS SALIVARIUS ON a PER CENT GLUCOSE (1 ) AND SUCROSE (2) AGARS in each case containing: 1 per cent tryptone, 0.5 per cent yeast extract, 0.3 per cent K2HPO4, 5 per cent gelatin, and 1.5 per cent agar. (Rather striking results may be obtained by sub- the sucrose stituting 0.5 per cent CaCO3 for the phosphate in agar. In this case, equally large colonies develop which are in addition surrounded by a clear halo in the opaque medium.) On a well-buffered 5 per cent sucrose agar the surface colonies of strains of Streptococcus salivarius having this property attain about the size of the colonies of coliform bacteria and yeasts. A more apt comparison is with the large, clear, soft colonies which are produced by most varieties of the genus Rhizobium.

3 PRODUCTION OF A POLYSACCHARID BY S. SALIVARIUS 481 In the study of a fairly large collection of "salivarius group" streptococci an interesting correlation appears: all of the cultures of Streptococcus salivarius which produce the mucoid colonies on sucrose agar ferment inulin, and vice versa. One may naturally be skeptical concerning whether this correlation will hold rigidly as larger numbers of cultures are studied. So far as our experience goes, all strains of Streptococcus salivarius which ferment inulin also ferment raffinose, but cultures of the salivarius group which ferment raffinose, but not inulin, do not produce mucoid colonies on sucrose agar. As will be noted later, inulin-fermenting streptococci belonging to other species, with a few possible exceptions, do not produce large mucoid colonies on sucrose agar. Streptococcus salivarius cultures which produce the mucoid material from sucrose and raffinose do not produce it from the following carbohydrates and combinations, though all of them are fermented: glucose; fructose; galactose; maltose; lactose; inulin; glucose + fructose; glucose + fructose + galactose; fructose + maltose; fructose + lactose; glucose + inulin. Five representative strains were used in these tests. As sucrose and raffinose have fructose as a constituent monosaccharid, and since chemical tests indicate that fructose is the chief hydrolytic product of the polysaccharid formed, it is of interest that the mucoid material is not produced from the fructosan inulin. The fructosecontaining trisaccharid melizitose was not fermented and the polysaccharid was not produced from this substance. The following cultures of hemolytic streptococci did not produce the large mucoid colonies on sucrose agar: group A (5), group B (8), group C (5), group D (12), group E (3), group F (5), group G (5), group H (2), and an inulin-fermenting hemolytic streptococcus (3) which does not belong to any of the recognized Lancefield serological groups. The figures in parentheses indicate the numbers of cultures of the various groups which were tested. Among the cultures of groups B, C, D, and G were strains of both human and animal origin. Negative results were also obtained with the following non-hemolytic organisms: Streptococcus fecalis (5), Streptococcus liquefaciers (5), Streptococcus lactis (5), Streptococcus cremoris (5), Streptococcus

4 482 C. F. NIVEN, JR., K. L. SMILEY AND J. M. SHERMAN thermophilus (5), "Bargen streptococcus" (2), Streptococcus uberis (2; inulin-fermenting), Streptococcus equinus (46; 20 inulin-fermenting) and pneumococci (15 cultures representing 13 types) of which 13 fermented inulin. Streptococcus bovis represents a special case. Tests were made on sucrose agar of 54 strains of this organism, 33 of which fermented inulin. Some cultures of this species at times produce somewhat larger and more luxuriant colonies on sucrose than on glucose agar, but the colonies are rather dry and tough, not resembling the large, moist, soft, mucoid colonies which are produced by Streptococcus salivarius. However, one culture of Streptococcus bovis consistently produced large mucoid colonies on sucrose agar which appeared to the eye to be identical with those formed by Streptococcus salivarius. This particular culture was isolated from the throat of a calf and its other characteristics indicate that it is a strain of Streptococcus bovis, rather than belonging to the salivarius group. It survived heating at 60TC. for 30 minutes in skimmed milk, which is quite out of line for Streptococcus salivarius; actively hydrolyzed starch, which is very characteristic of Streptococcus bovis but relatively rare among organisms belonging to the salivarius group; and fermented mannitol and sorbitol, which characteristics are not rare among strains of Streptococcus bovts but have never been reported for a culture of Streptococcus salivarius, so far as we are aware, by any investigator. However, the mucoid material which was produced by this culture of Streptococcus bovis, although not subjected to chemical study, appeared on the basis of solubility and some other characteristics to differ from that produced by Streptococcus salivarius. Oerskov and Poulsen (1931) studied 25 cultures which had the property of producing mucoid colonies on sucrose agar, 24 of which were obtained from the human throat. Although they did not use inulin, all of these 24 cultures fermented raffinose and failed to ferment mannitol, making it highly probable, in view of their source, that Oerskov and Poulsen also dealt with Streptococcus salivarius. The other culture studied by them was obtained from milk; it did not ferment raffinose and differed in

5 PRODUCTION OF A POLYSACCHARID BY S. BALIVARIUS 483 some additional characteristics from their other cultures, and the polysaccharid produced appeared to be different. In the absence of other critical tests, it is entirely possible that this organism belonged to the genus Leuconostoc, members of which are of common occurrence in milk. We have subjected to detailed study 95 cultures of Streptococcus salivarius which have the property of producing mucoid colonies on sucrose agar. These cultures represent a very homogeneous group, considerably more homogeneous than the "salivarius group" as a whole. All of these cultures have characteristics which would place them within the group which Safford, Sherman, and Hodge (1937) considered the "typical" Streptococcus salivarius, 80 per cent of which fermented inulin. If it should prove permissible to consider the production of the mucoid material as species-specific for Streptococcus salivarius, the resulting group would be as clearly marked and as homogeneous as any "species" in the genus Streptococcus. The other and less homogeneous members of the "salivarius group" could be considered as something else, most logically the Streptococcus mitis of Andrewes and Horder (1906). However attractive and convenient such an arbitrary procedure might be, the distinction does not appear justified on the basis of present knowledge. It is true that most cultures of the salivarius group which do not have the property of producing mucoid material from sucrose are "atypical" in other respects, but some strains appear otherwise identical with the mucoid-forming cultures with the exception of their inability to ferment inulin. Also, some non-mucoid-forming strains appear to belong to the same serological types as do some which produce the mucoid material. Although we have been entirely unsuccessful in our attempts to produce group-specific sera for Streptococcus salivarius, it is of interest to note that about 50 per cent of the mucoid-forming cultures belong to one serological type; but a small percentage of the non-mucoid-forming strains also react with this type serum. However, there can be no doubt that the production of mucoid colonies on sucrose agar and the fermentation of inulin would prove to be simple and helpful methods for the identification of "typical" strains of Streptococcus salivarius

6 484 C. F. NIVEN, JR., K. L. SMILEY AND J. M. SHERMAN among the many cultures of "indifferent streptococci" and "Streptococcus viridans" with which all bacteriological laboratories have to deal. SUMMARY Many cultures of Streptococcus salivarius produce a large amount of a polysaccharid from sucrose, resulting, when grown on solid media, in the formation of large mucoid colonies. The material is also produced in liquid media. The mucoid material is likewise produced from raffinose, but not from the other carbohydrates which have been tested. In the salivarius group, the ability to produce the polysaccharid from sucrose appears to be correlated with the ability to ferment inulin, though the mucoid material is not produced from inulin. Large mucoid colonies were not produced on sucrose agar by cultures of the other groups and species of hemolytic and nonhemolytic streptococci, including many inulin-fermenting strains. However, a somewhat similar material may rarely be produced from sucrose by strains of Streptococcus bovis. REFERENCES ANDRZWEs, F. W., AND HORDzR, T. J A study of the streptococci pathogenic for man. Lancet, 2, , , OERSKOV, J Untersuchungen uber einen exzessiv Polysaccharid-bildenden Streptokokkus. Zentr. Bakt. Parasitenk., Orig., 119, OUBSKOV, J., AND POULSEN, K. A Das hliufige Vorkommen von Streptokokken im menschlichen Rachen, die bei Wachstum auf der Oberftche fester, Saccharose (oder Raffinose) enthaltender Substrate exzessiv Polysaccharid produzieren. Zentr. Bakt. Parasitenk., Orig., 120, SAFFORD, C. E., SHERMAN, J. M., AND HODGE, H. M Streptococcus salivarius. J. Bact., 33,