STREPTOCOCCUS SALIVARIUS C. E. SAFFORD, J. M. SHERMAN AND H. M. HODGE Laboratory of Bacteriology, College of Agriculture, Cornell University, Ithaca, New York Received for publication August 17, 1936 During the past twenty years, with the application of improved methods and more extensive tests, a number of the natural groups or species of the streptococci have become fairly clearly defined. Among the more adequately described groups are those typified by Streptococcus pyogenes, the "animal pyogenes," Streptococcus equi, Streptococcus mastitidis, Streptococcus lactis, Streptococcus fecalis, Streptococcus liquefaciens, Streptococcus zymogenes, Streptococcus bovis and Streptococcus thermophilus. On the other hand, some of the streptococci which have been well known since the relatively early days of bacteriology have not been extensively studied by the more modern methods, and the differentiation of these forms from some of the better established groups is not entirely clear. In such a state are the characteristic non-hemolytic streptococci of the human throat, commonly designated as the "viridans group," Streptococcus mitior, Streptococcus salivarius, Streptococcus mitis, etc. The present study was limited to the predominating streptococci in the throats of healthy and presumably normal persons. The isolations were made by the application of quantitative dilution methods to freshly taken swab samples, so as to eliminate not only organisms of fortuitous occurrence, but also even streptococci native to the throat which may occur in small numbers. Under these quantitative restrictions true beta-hemolytic streptococci were not obtained among the 322 cultures which were isolated at various times from 20 healthy individuals. 263
264 C. E. SAFFORD, J. M. SHERMAN AND H. M. HODGE METHODS The material was collected from the throats (not the mouths) of supposedly normal individuals by means of sterile cotton swabs. Serial dilutions of the material obtained were made in broth, from which poured agar plates were prepared immediately. The agar medium used contained the following nutrients: lactose, 1 per cent; glucose, 0.1 per cent; beef extract, 0.3 per cent; and 0.5 per cent each of yeast extract and peptone. Only well-isolated colonies were subcultured from the plates and all cultures were thoroughly checked for purity before adding to the collection. Action on blood in poured agar plates was determined on subsurface colonies, the methods recommended by Brown (1919) being faithfully followed. The temperature limits of growth were determined in litmus milk, the cultures being adjusted to incubation temperature in a water bath immediately after inoculation and incubation carried out in sealed tubes in accurately controlled incubators. Thermal resistance tests were made in milk. Ten cubic centimeters of sterile skimmed milk were added to 1 cc. of culture and the heavily seeded mixture heated in a sealed tube in a water bath. Tests for ammonia production were made on cultures incubated for one week at 370C. in 4 per cent peptone solution. Action on starch was determined by plating in proper dilution (not streaking) in starch agar, the plates being flooded with iodine solution after incubation for three days at 370C. Substances for the fermentation tests were sterilized separately in 10 per cent solutions and added to sterile yeast-extract peptone broth so as to give a 1 per cent concentration of the test substance. Maltose, inulin and salicin were sterilized by filtration, while the other test substances were autoclaved. Certain other tests which have distinct value in the differentiation of particular groups of streptococci-such as the pentose sugars and sorbitol (Orla-Jensen, 1919), trehalose (Edwards, 1932), esculin (Meyer and Schdnfeld, 1926), inhibition by methylene blue (Sherman and Albus, 1918), and salt tolerance (Sherman and Stark, 1934)-were not used in the present study
STREPTOCOCCUS SALIVARIUS because of lack of presumptive relevance. Since the group under study was found to be neatly differentiated from the better established species on the basis of the methods used, it was not necessary to utilize those and other tests which might have been called upon. THE CHARACTERISTICS OF STREPTOCOCCUS SALIVARIUS Of the 322 cultures studied, 290 (group I) represented a remarkably homogeneous group. The remaining 32 cultures (groups II, III, IV, and V) showed some variation from the type, but hardly sufficient, we believe, to warrant designation as distinct species at the present time. Group I (290 cultures) The typical throat streptococcus, represented by 290 cultures, was found to have the following characteristics. Morphology. The characteristic grouping is in short chains, the length of chains being somewhat longer, as a rule, in broth than in milk cultures. The size of the individual cells in milk cultures is distinctly larger than those of most streptococci; they are plump and give the impression of being very thrifty. The larger size of the cells is less marked and may be scarcely noticeable in broth cultures. Blood agar. In horse-blood agar the reaction varies from gamma to alpha, "weak alpha" being characteristic. Very few cultures were encountered which could be classified as truly gamma types, while relatively few gave what might be termed a strong or typical alpha reaction. Temperature limits of growth. No growth takes place at 10 C. nor at 470C. The maximum temperature for growth is about 450C., a minority of the cultures being able to grow at this temperature. The maximum growth temperature of this group appears to be, therefore, somewhat higher than for those groups of streptococci represented by Streptococcus pyogenes, S. mastitidis and S. lactis, but definitely lower than for S. bovis, S. thermophilus, S. fecalis, S. liquefaciens, S. zymogenes, etc. (Sherman and Albus, 1918; Orla-Jensen, 1919; Sherman and Stark, 1931, 1934; Sherman and Wing, 1935; Hansen, 1935.) 265
266 C. E. SAFFORD, J. M. SHERMAN AND H. M. HODGE Thermal resistance. There is no survival in milk when heated for 30 minutes at 600C. Litmus milk. Litmus milk is promptly acidulated and curdled without previous reduction, but with marked though not quite complete reduction after curdling. There is no apparent digestion of casein. The end result of the action of this streptococcus on litmus milk is much like that given by S. mastitidis, but the mouth streptococcus is on the average somewhat faster in bringing about coagulation. Final ph. In glucose broth a final ph of 4.4 to 4.0 is attained. (These limits were not. exceeded by the 290 cultures which made up this "typical" group; the relatively few varieties or aberrant strains which gave atypical results are noted below.) Sodium hippurate. Sodium hippurate is not hydrolyzed. Ammonia production. Ammonia is not produced from peptone. Starch. Starch is not hydrolyzed. Gelatin. There is no liquefaction of gelatin. Fermentation reactions. Glucose, maltose, lactose and sucrose are fermented. Glycerol and mannitol are not fermented. Raffinose (93 per cent positive), inulin (80 per cent positive), and salicin (95 per cent positive) are usually fermented. Pathogenicity. There is no apparent pathogenicity for rabbits by intravenous injection nor for white mice injected intraperitoneally. Not all cultures were tested for virulence, but random selections from this group, in addition to representatives from groups II to V, were used. One cubic centimeter amounts of 0.1 per cent glucose broth cultures were employed. Viability. One of the noticeable characteristics of this organism is its lack of viability in artificial culture media, notwithstanding its rather vigorous growth in such substrates. It is readily lost with infrequent transfers, being, in our experience, rather more delicate in this respect than the pathogenic streptococci, and decidedly more so than the other non-hemolytic types with the exceptions of freshly isolated cultures of S. thermophilus and Streptococcus equinus. Habitat. Human mouth and throat.
STREPTOCOCCUS SALIVARIUS Group II (19 cultures) The cultures of this group differ from those in group I in having limiting ph values of 5.2 to 4.9, with a correlated tendency (by no means rigid) toward a stronger or more typical alpha reaction on blood agar. These cultures do not ferment salicin and only a minority of them ferment raffinose or inulin. Obviously these weak acid-producing strains do not act so vigorously in milk, some failing to cause coagulation. With a number of correlated divergencies from the typical, these cultures have some right to a group or variety status. However, the differences from the type are all "majority" ones, with the exception of the high limiting ph. The final hydrogenion concentration, introduced by Ayers, Johnson and Davis (1918), has proved of great value as a group characteristic in the streptococci. On the other hand, individual strains which are weak acid producers are found in all species. Group III (4 cultures) This small group is distinguished by the production of an alpha prime reaction on blood agar (Brown, 1919). Again associated with a stronger action on blood is a tendency toward weak acidproducing power. Three of the cultures gave final ph values of 5.4 to 5.2, but the correlation was spoiled by the fourth culture which produced a final acidity of ph 4.2. One of the four cultures ferments raffinose and inulin, while none ferments salicin. Group IV (8 cultures) This group is composed of eight cultures, all of which produce ammonia from peptone and give final ph values in glucose broth between 5.3 and 4.9. On blood, and on fermentation tests, they agree perfectly with the members of group I. In some respects this group appears to have rather good claims for recognition as a definite variety, but such a contention in the present state of our knowledge would probably be a mistake. The production of ammonia from peptone was introduced into the study of streptococci by Ayers, Rupp and Mudge (1921) 267
268 C. E. SAFFORD, J. M. SHERMAN AND H. M. HODGE and constitutes a valuable test which deserves wider usage. Except for the contributions of Ayers and his associates and a number of papers from this laboratory, little has been reported on the production of ammonia by streptococci. Until the method comes into more general use it is probably wise not to emphasize too strongly the possible taxonomic significance of such observations as are here reported. Group V (I culture) The one culture which makes up this "group" does not ferment lactose, and therefore has no visible action on milk. When litmus milk is supplemented by the addition of 2 per cent glucose, however, it causes rapid curdling followed by marked and almost complete reduction of the litmus in a manner entirely characteristic of the "milk reaction" of the cultures in group I. Aside from its inability to ferment lactose, it agrees in all characteristics studied with the members of group I, including the fermentation of raffinose, inulin and salicin. It would be easy to follow custom and call this organism Streptococcus equinus, but from a careful study of it in comparison with the characteristic lactose-non-fermenting streptococci of horse feces, we feel that it is probably an aberrant strain of the S. 8alivarius group, and should not be confused with the true S. equinus of Andrewes and Horder (1906). In this connection it should be mentioned that streptococci which fail to ferment lactose have been obtained frequently, and apparently in substantial numbers, from the human throat (Arnold, 1920). THE STREPTOCOCCUS MITIS OF ANDREWES AND HORDER The question naturally arises as to whether we should consider as one species only the rather homogeneous collection of cultures contained in group I, or include also the small groups (II to V) as representing variants from the type. Further work may show that more than one true species may be involved, but we feel that in the light of our present knowledge of the streptococci it would be unfortunate to encumber the literature with more names based on rather shaky foundations.
STREPTOCOCCUS SALIVARIUS Those who would divide the organisms here studied into more than one species could combine groups II and III to form a type which is usually marked by a higher limiting ph and a stronger or more definitely alpha type of action on blood, and which usually does not ferment raffinose, inulin or salicin. If such a species were established the appropriate name would appear to be Streptococcus mitis. Andrewes and Horder (1906) described S. mitis as usually not clotting milk nor fermenting raffinose, as opposed to S. salivarius which usually did. They did not claim that S. mitis never ferments raffinose, nor that S. salivarius always does. In fact, some 15 per cent of the cultures assigned by them to the species S. mitis fermented either raffinose or inulin. The foregoing paragraph has been criticized by a competent reviewer of this manuscript on the ground that Andrewes and Horder's "Type Form" of S. salivarius did not ferment salicin, and that some bacteriologists have more recently differentiated S. salivarius from S. mitis on the basis of the salicin reaction, those types fermenting this substance being regarded as S. mitis. From this point of view it would appear that we are reversing the definitions of Andrewes and Horder; we think, however, that our own descriptions agree with those of Andrewes and Horder, and that the reversal of their distinctions is the work of intervening bacteriologists. This point demands some detailed consideration of the data and statements of Andrewes and Horder. It appears that the emphasis on the salicin reaction in the differentiation of S. salivarius from S. mitis probably originated in the work of Holman (1916). This investigator classified the streptococci on the basis of their respective reactions on blood, lactose, mannitol, and salicin; he did not use some of the other tests which, we think, Andrewes and Horder considered of greater importance than salicin. These remarks are in no sense a criticism of the admirable work of Holman whose classification is a model of ingenuity, simplicity, and workability. With reference to S. salivarius (pp. 712-713) Andrewes and Horder state: "It clots milk almost always and in its typical form reduces neutral red, though variants occur which fail to do 269
270 C. E. SAFFORD, J. M. SHERMAN AND H. M. HODGE this. The characteristic fermentation reactions are saccharose, lactose, and raffinose, the last named less constantly than the first two. The glucoside reactions may be added, and rarely inulin." Again (p. 776) they state: "The common positive chemical reactions are clotting of milk, reduction of neutral red, and acid formation with saccharose, lactose, and often raffinose, but not with mannite. Reactions with the glucosides are often added...." In connection with S. mitis (p. 712) Andrewes and Horder remark: "It is a short-chained form... and it gives a marked acid reaction in milk, though no clotting.... Its typical positive reactions on Gordon's tests are saccharose and lactose with or without the glucosides.... Such forms may with fair propriety be regarded as variants of Type D (S. salivarius) in which the power of clotting milk has been suppressed." It would appear from these quotations that Andrewes and Horder considered the clotting of milk, reducing action on neutral red, and the fermentation of raffinose as the primary distinguishing characteristics between S. salivarius and S. mitis, and that they regarded salicin as of subsidiary importance. Of more importance than what Andrewes and Horder say, however, is what their data show. In table 3 (p. 776) are listed 14 varieties of S. salivarius, and 12 of them fermented salicin. However, they designate as the "Type Form" one of the two which did not ferment salicin, but which did ferment raffinose, clot milk, and reduce neutral red. "This form is regarded as the type in virtue of its frequency of occurrence in normal saliva, not in disease. We have met with it once only as a pathogenic agent in a case of malignant endocarditis." Still more to the point perhaps are the "Reaction Curves" (p. 853) on streptococci. These present graphically the distribution of fermentation reactions for large numbers of cultures. These curves represent: all streptococci, saprophytic and parasitic; parasitic streptococci; streptococci producing suppuration; streptococci from cases of malignant endocarditis; and human saprophytic streptococci, based on salivary and fecal specimens. In each group, salicin was the most frequently fermented test substance with the exceptions of sucrose and lactose. The test substances
STREPTOCOCCUS SALIVARIUS 6 271 included sucrose, lactose, raffinose, inulin, mannitol, salicin, and coniferin. Salicin was fermented by 70 per cent of all the streptococci studied, and by 67 per cent of the "human saprophytic" group from saliva and feces. It would appear that, "with or without the glucosides," our group I on the bases of strong acid production (low ph and coagulation of milk), rather strong reducing action, and, in general, the fermentation of raffinose, corresponds to Andrewes and Horder's S. salivarius; and that our groups II and III correspond to their S. mitis, which "forms may with fair propriety be regarded as variants (of S. salivarius) in which the power of clotting milk has been suppressed." The confusion, the hazy borderline, and the fact that Andrewes and Horder themselves considered their S. mitis type as probably only a variant of S. salivarius, add weight to our previously expressed view that an additional species should not be recognized in this general group in the present state of our knowledge. If a species of S. mitis were accepted, the eight cultures which make up our group IV would perhaps have equal claims to recognition. These cultures, while typical of S. salivarius on blood and the fermentation tests, produce ammonia from peptone and have high limiting ph values, the two aberrant characteristics being perfectly correlated in the small group. It is needless to add that we do not at present recommend the recognition of this group, either as a species or as a definite variety. THE RELATION OF STREPTOCOCCUS SALIVARIUS TO OTHER SPECIES The important point, which we wish to emphasize especially, is that whether the species is restricted to the typical cultures of group I, or expanded to include the variant types, the group is sufficiently homogeneous and clearly defined so that it can be readily differentiated from any of the adequately described species of the streptococci. It is unnecessary to point out the numerous points which distinguish this group of streptococci from the hemolytic pathogens, the enterococcii" (S. fecalis and its relatives), or the
272 C. E. SAFFORD, J. M. SHERMAN AND H. M. HODGE "lactic" group (S. kactis and its varieties). From S. mastitidis it is markedly differentiated on the bases of sodium hippurate and blood reactions; in the majority of cases also by the fermentation tests; and, by one familiar with the two groups, slight but significant differences are to be noted in morphology and maximum growth temperatures. S. thermophilus, which conceivably might be confused with this organism on superficial study, is widely separated by its much higher maximum temperature of growth, a much higher thermal death point, and its inability, as a rule, to ferment maltose. S. bovis has a higher thermal death point, a somewhat higher maximum growth temperature, hydrolyzes starch, and ferments arabinose. The organism which could be most readily confused with the typical human throat streptococcus is that representative of the "bovis group" which occurs characteristically in the bovine mouth, designated by Ayers and Mudge (1923) simply as S. bovis variety B, but given the specific name of Streptococcus inulinaceus by Orla-Jensen (1919). This organism differs from the typical S. bovis in not fermenting arabinose and not hydrolyzing starch, or attacking it very feebly. It practically invariably ferments raffinose and inulin. Even here the differences from the human throat type appear fairly clearly defined in that S. inulinaceus has a higher thermal death point and a slightly higher maximum temperature of growth, usually ferments mannitol, and is usually less vigorous in its action on litmus milk from the standpoint of rate of acid production and extent of reduction of the litmus (Sherman and Stark, 1931). It is of interest and significance to note in this connection that in their valuable paper on the streptococci of the bovine mouth, Ayers and Mudge (1923) stated that on the basis of their unfinished studies of the S. salivarius group of the human mouth it appeared that these organisms are distinct. There is practically no doubt that the non-hemolytic streptococcus which Andrewes and Horder (1906) named Streptococcus salivarius is the same as the organism here described. Since it is proposed, for the present at least, not to recognize more than one species in this group, it might well be argued that the older
STREPTOCOCCUS SALIVARIUS name, Streptococcus mitior (Schottmiiller, 1903), be used. This, in fact, is the view taken by us in a preliminary report on this group of organisms (Safford and Sherman, 1936). However, from a careful review of Schottmiller's paper it must be admitted that the types of non-hemolytic streptococci with which he worked could not be now identified. Although Schottmiiller worked with throat forms, among others, he apparently used the name S. mitior in much the same broad and non-specific sense as "S. viridans" is used today. The limited description given of his organisms would apply to several of the now clearly defined species of the streptococci. For additional authority on this point we would quote the Winslows (1908). These authors deplored the fact that Andrewes and Horder did not make use of the older names in the descriptions of some of their species, but stated: "One of their short-chained forms... might well have been identified with Str. mitior of Schottmiiller. Andrewes and Horder, however, preferred to give new names to their types; and since they have done so their names must stand, as they, for the first time, have described streptococcal types with sufficient clearness and detail to make them definitely recognizable." There would appear to be little doubt that Streptococcus salivarius is the name which should be retained for this species. SUMMARY 273 A study was made of 322 cultures of non-hemolytic streptococci isolated from human throats. The isolations were made by the application of quantitative methods so as to limit the collection to the predominating types. Streptococcus salivarius was found to be the prevailing type and 290 of the cultures studied are believed to be typical of the species. A full description of the organism is given. The remaining 32 cultures varied somewhat from the type but scarcely enough to be considered separate species in the light of present knowledge of the streptococci. Streptococcus salivarius, as herein described, may be readily differentiated from any of the clearly established species of the streptococci.
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