STREPTOCOCCUS SALIVARIUS - Journal of …jb.asm.org/content/33/3/263.full.pdf · STREPTOCOCCUS...

STREPTOCOCCUS SALIVARIUS

C. E. SAFFORD, J. M. SHERMAN AND H. M. HODGELaboratory 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 im-proved methods and more extensive tests, a number of the naturalgroups or species of the streptococci have become fairly clearlydefined. Among the more adequately described groups are thosetypified by Streptococcus pyogenes, the "animal pyogenes,"Streptococcus equi, Streptococcus mastitidis, Streptococcus lactis,Streptococcus fecalis, Streptococcus liquefaciens, Streptococcus zy-mogenes, Streptococcus bovis and Streptococcus thermophilus. Onthe other hand, some of the streptococci which have been wellknown since the relatively early days of bacteriology have notbeen extensively studied by the more modern methods, and thedifferentiation of these forms from some of the better establishedgroups is not entirely clear. In such a state are the characteristicnon-hemolytic streptococci of the human throat, commonlydesignated as the "viridans group," Streptococcus mitior, Strepto-coccus salivarius, Streptococcus mitis, etc.The present study was limited to the predominating strepto-

cocci in the throats of healthy and presumably normal persons.The isolations were made by the application of quantitativedilution methods to freshly taken swab samples, so as to eliminatenot only organisms of fortuitous occurrence, but also even strep-tococci native to the throat which may occur in small numbers.Under these quantitative restrictions true beta-hemolytic strepto-cocci were not obtained among the 322 cultures which wereisolated at various times from 20 healthy individuals.

263

on Septem

ber 26, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

http://jb.asm.org/

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. Theagar medium used contained the following nutrients: lactose, 1per cent; glucose, 0.1 per cent; beef extract, 0.3 per cent; and 0.5per cent each of yeast extract and peptone. Only well-isolatedcolonies were subcultured from the plates and all cultures werethoroughly checked for purity before adding to the collection.Action on blood in poured agar plates was determined on sub-

surface 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 awater bath immediately after inoculation and incubation carriedout in sealed tubes in accurately controlled incubators. Thermalresistance tests were made in milk. Ten cubic centimetersof sterile skimmed milk were added to 1 cc. of culture and theheavily seeded mixture heated in a sealed tube in a water bath.Tests for ammonia production were made on cultures incubatedfor one week at 370C. in 4 per cent peptone solution. Action onstarch was determined by plating in proper dilution (not streak-ing) in starch agar, the plates being flooded with iodine solutionafter incubation for three days at 370C.

Substances for the fermentation tests were sterilized separatelyin 10 per cent solutions and added to sterile yeast-extract peptonebroth so as to give a 1 per cent concentration of the test substance.Maltose, inulin and salicin were sterilized by filtration, whilethe other test substances were autoclaved.

Certain other tests which have distinct value in the differentia-tion of particular groups of streptococci-such as the pentosesugars and sorbitol (Orla-Jensen, 1919), trehalose (Edwards,1932), esculin (Meyer and Schdnfeld, 1926), inhibition bymethylene blue (Sherman and Albus, 1918), and salt tolerance(Sherman and Stark, 1934)-were not used in the present study

on Septem


.org/D

ownloaded from

http://jb.asm.org/


because of lack of presumptive relevance. Since the groupunder study was found to be neatly differentiated from the betterestablished species on the basis of the methods used, it was notnecessary to utilize those and other tests which might have beencalled upon.

THE CHARACTERISTICS OF STREPTOCOCCUS SALIVARIUS

Of the 322 cultures studied, 290 (group I) represented a re-markably 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 dis-tinct 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 broththan in milk cultures. The size of the individual cells in milkcultures is distinctly larger than those of most streptococci; theyare plump and give the impression of being very thrifty. Thelarger size of the cells is less marked and may be scarcely no-ticeable in broth cultures.

Blood agar. In horse-blood agar the reaction varies fromgamma to alpha, "weak alpha" being characteristic. Very fewcultures were encountered which could be classified as trulygamma types, while relatively few gave what might be termed astrong or typical alpha reaction.

Temperature limits of growth. No growth takes place at 10°C.nor at 470C. The maximum temperature for growth is about450C., a minority of the cultures being able to grow at thistemperature. The maximum growth temperature of this groupappears to be, therefore, somewhat higher than for those groupsof streptococci represented by Streptococcus pyogenes, S. mastitidisand 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; Sher-man and Wing, 1935; Hansen, 1935.)

265

on Septem


.org/D

ownloaded from

http://jb.asm.org/


Thermal resistance. There is no survival in milk when heatedfor 30 minutes at 600C.Litmus milk. Litmus milk is promptly acidulated and curdled

without previous reduction, but with marked though not quitecomplete reduction after curdling. There is no apparent diges-tion of casein. The end result of the action of this streptococcuson litmus milk is much like that given by S. mastitidis, but themouth streptococcus is on the average somewhat faster in bring-ing 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 madeup this "typical" group; the relatively few varieties or aberrantstrains 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 rabbitsby intravenous injection nor for white mice injected intra-peritoneally. Not all cultures were tested for virulence, butrandom selections from this group, in addition to representativesfrom groups II to V, were used. One cubic centimeter amountsof 0.1 per cent glucose broth cultures were employed.

Viability. One of the noticeable characteristics of this or-ganism is its lack of viability in artificial culture media, notwith-standing its rather vigorous growth in such substrates. It isreadily lost with infrequent transfers, being, in our experience,rather more delicate in this respect than the pathogenic strepto-cocci, and decidedly more so than the other non-hemolytic typeswith the exceptions of freshly isolated cultures of S. thermophilusand Streptococcus equinus.

Habitat. Human mouth and throat.

on Septem


.org/D

ownloaded from

http://jb.asm.org/


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 alphareaction on blood agar. These cultures do not ferment salicinand only a minority of them ferment raffinose or inulin. Ob-viously these weak acid-producing strains do not act so vigorouslyin 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 hydrogen-ion concentration, introduced by Ayers, Johnson and Davis(1918), has proved of great value as a group characteristic in thestreptococci. On the other hand, individual strains which areweak 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 associatedwith a stronger action on blood is a tendency toward weak acid-producing power. Three of the cultures gave final pH values of5.4 to 5.2, but the correlation was spoiled by the fourth culturewhich produced a final acidity of pH 4.2. One of the four cul-tures 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 brothbetween 5.3 and 4.9. On blood, and on fermentation tests, theyagree perfectly with the members of group I. In some respectsthis group appears to have rather good claims for recognitionas a definite variety, but such a contention in the present state ofour 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

on Septem


.org/D

ownloaded from

http://jb.asm.org/


and constitutes a valuable test which deserves wider usage.Except for the contributions of Ayers and his associates and anumber of papers from this laboratory, little has been reported onthe production of ammonia by streptococci. Until the methodcomes into more general use it is probably wise not to emphasizetoo strongly the possible taxonomic significance of such observa-tions 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 litmusmilk is supplemented by the addition of 2 per cent glucose,however, it causes rapid curdling followed by marked and almostcomplete reduction of the litmus in a manner entirely character-istic of the "milk reaction" of the cultures in group I. Asidefrom its inability to ferment lactose, it agrees in all characteristicsstudied with the members of group I, including the fermentationof raffinose, inulin and salicin.

It would be easy to follow custom and call this organismStreptococcus equinus, but from a careful study of it in comparisonwith the characteristic lactose-non-fermenting streptococci ofhorse feces, we feel that it is probably an aberrant strain of theS. 8alivarius group, and should not be confused with the trueS. equinus of Andrewes and Horder (1906). In this connectionit should be mentioned that streptococci which fail to fermentlactose have been obtained frequently, and apparently in sub-stantial numbers, from the human throat (Arnold, 1920).

THE STREPTOCOCCUS MITIS OF ANDREWES AND HORDER

The question naturally arises as to whether we should consideras one species only the rather homogeneous collection of culturescontained in group I, or include also the small groups (II to V)as representing variants from the type. Further work may showthat more than one true species may be involved, but we feel that inthe light of our present knowledge of the streptococci it would beunfortunate to encumber the literature with more names basedon rather shaky foundations.

on Septem


.org/D

ownloaded from

http://jb.asm.org/


Those who would divide the organisms here studied into morethan one species could combine groups II and III to form a typewhich is usually marked by a higher limiting pH and a strongeror more definitely alpha type of action on blood, and whichusually does not ferment raffinose, inulin or salicin. If such aspecies were established the appropriate name would appear to beStreptococcus mitis. Andrewes and Horder (1906) describedS. mitis as usually not clotting milk nor fermenting raffinose, asopposed to S. salivarius which usually did. They did not claimthat S. mitis never ferments raffinose, nor that S. salivariusalways does. In fact, some 15 per cent of the cultures assignedby them to the species S. mitis fermented either raffinose orinulin.The foregoing paragraph has been criticized by a competent

reviewer of this manuscript on the ground that Andrewes andHorder's "Type Form" of S. salivarius did not ferment salicin,and that some bacteriologists have more recently differentiatedS. 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 reversingthe definitions of Andrewes and Horder; we think, however, thatour own descriptions agree with those of Andrewes and Horder,and that the reversal of their distinctions is the work of interven-ing bacteriologists. This point demands some detailed considera-tion of the data and statements of Andrewes and Horder.

It appears that the emphasis on the salicin reaction in thedifferentiation of S. salivarius from S. mitis probably originatedin the work of Holman (1916). This investigator classified thestreptococci on the basis of their respective reactions on blood,lactose, mannitol, and salicin; he did not use some of the othertests which, we think, Andrewes and Horder considered of greaterimportance than salicin. These remarks are in no sense acriticism of the admirable work of Holman whose classification isa 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 typicalform reduces neutral red, though variants occur which fail to do

269

on Septem


.org/D

ownloaded from

http://jb.asm.org/


this. The characteristic fermentation reactions are saccharose,lactose, and raffinose, the last named less constantly than thefirst two. The glucoside reactions may be added, and rarelyinulin." Again (p. 776) they state: "The common positivechemical 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 oftenadded . . . ." In connection with S. mitis (p. 712) Andrewesand Horder remark: "It is a short-chained form .. . and it givesa marked acid reaction in milk, though no clotting . . . . Itstypical positive reactions on Gordon's tests are saccharose andlactose with or without the glucosides . . . . Such forms maywith fair propriety be regarded as variants of Type D (S. sali-varius) in which the power of clotting milk has been suppressed."It would appear from these quotations that Andrewes and Horderconsidered the clotting of milk, reducing action on neutral red,and the fermentation of raffinose as the primary distinguishingcharacteristics between S. salivarius and S. mitis, and that theyregarded 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 listed14 varieties of S. salivarius, and 12 of them fermented salicin.However, they designate as the "Type Form" one of the twowhich did not ferment salicin, but which did ferment raffinose,clot milk, and reduce neutral red. "This form is regarded as thetype in virtue of its frequency of occurrence in normal saliva,not in disease. We have met with it once only as a pathogenicagent in a case of malignant endocarditis." Still more to thepoint perhaps are the "Reaction Curves" (p. 853) on strepto-cocci. These present graphically the distribution of fermenta-tion reactions for large numbers of cultures. These curvesrepresent: all streptococci, saprophytic and parasitic; parasiticstreptococci; streptococci producing suppuration; streptococcifrom cases of malignant endocarditis; and human saprophyticstreptococci, based on salivary and fecal specimens. In eachgroup, salicin was the most frequently fermented test substancewith the exceptions of sucrose and lactose. The test substances

on Septem


.org/D

ownloaded from

http://jb.asm.org/


included sucrose, lactose, raffinose, inulin, mannitol, salicin, andconiferin. Salicin was fermented by 70 per cent of all thestreptococci studied, and by 67 per cent of the "human sapro-phytic" group from saliva and feces.

It would appear that, "with or without the glucosides," ourgroup I on the bases of strong acid production (low pH andcoagulation of milk), rather strong reducing action, and, in gen-eral, the fermentation of raffinose, corresponds to Andrewes andHorder's S. salivarius; and that our groups II and III correspondto their S. mitis, which "forms may with fair propriety be regardedas variants (of S. salivarius) in which the power of clotting milkhas been suppressed." The confusion, the hazy borderline,and the fact that Andrewes and Horder themselves consideredtheir S. mitis type as probably only a variant of S. salivarius,add weight to our previously expressed view that an additionalspecies should not be recognized in this general group in thepresent state of our knowledge.

If a species of S. mitis were accepted, the eight cultures whichmake up our group IV would perhaps have equal claims to recog-nition. These cultures, while typical of S. salivarius on bloodand the fermentation tests, produce ammonia from peptone andhave high limiting pH values, the two aberrant characteristicsbeing perfectly correlated in the small group. It is needless toadd that we do not at present recommend the recognition of thisgroup, 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 ofgroup I, or expanded to include the variant types, the group issufficiently homogeneous and clearly defined so that it can bereadily differentiated from any of the adequately describedspecies of the streptococci.

It is unnecessary to point out the numerous points whichdistinguish this group of streptococci from the hemolytic path-ogens, the enterococcii" (S. fecalis and its relatives), or the

6271

on Septem


.org/D

ownloaded from

http://jb.asm.org/


"lactic" group (S. kactis and its varieties). From S. mastitidisit is markedly differentiated on the bases of sodium hippurateand blood reactions; in the majority of cases also by the fermenta-tion tests; and, by one familiar with the two groups, slight butsignificant differences are to be noted in morphology and maxi-mum growth temperatures. S. thermophilus, which conceivablymight be confused with this organism on superficial study, iswidely separated by its much higher maximum temperature ofgrowth, a much higher thermal death point, and its inability,as a rule, to ferment maltose. S. bovis has a higher thermaldeath 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 bovinemouth, designated by Ayers and Mudge (1923) simply as S. bovisvariety B, but given the specific name of Streptococcus inulinaceusby Orla-Jensen (1919). This organism differs from the typicalS. bovis in not fermenting arabinose and not hydrolyzing starch,or attacking it very feebly. It practically invariably fermentsraffinose and inulin. Even here the differences from the humanthroat type appear fairly clearly defined in that S. inulinaceushas a higher thermal death point and a slightly higher maximumtemperature of growth, usually ferments mannitol, and is usuallyless vigorous in its action on litmus milk from the standpoint ofrate of acid production and extent of reduction of the litmus(Sherman and Stark, 1931). It is of interest and significance tonote in this connection that in their valuable paper on the strep-tococci of the bovine mouth, Ayers and Mudge (1923) stated thaton the basis of their unfinished studies of the S. salivarius groupof the human mouth it appeared that these organisms aredistinct.There is practically no doubt that the non-hemolytic strepto-

coccus which Andrewes and Horder (1906) named Streptococcussalivarius is the same as the organism here described. Since itis proposed, for the present at least, not to recognize more thanone species in this group, it might well be argued that the older

on Septem


.org/D

ownloaded from

http://jb.asm.org/


name, Streptococcus mitior (Schottmiiller, 1903), be used. This,in fact, is the view taken by us in a preliminary report on thisgroup of organisms (Safford and Sherman, 1936). However,from a careful review of Schottmiller's paper it must be admittedthat the types of non-hemolytic streptococci with which heworked could not be now identified. Although Schottmiillerworked with throat forms, among others, he apparently used thename S. mitior in much the same broad and non-specific sense as"S. viridans" is used today. The limited description given of hisorganisms would apply to several of the now clearly definedspecies of the streptococci.For additional authority on this point we would quote the

Winslows (1908). These authors deplored the fact that An-drewes and Horder did not make use of the older names in thedescriptions of some of their species, but stated: "One of theirshort-chained forms . . . might well have been identified withStr. mitior of Schottmiiller. Andrewes and Horder, however,preferred to give new names to their types; and since they havedone so their names must stand, as they, for the first time, havedescribed streptococcal types with sufficient clearness and detailto make them definitely recognizable." There would appear tobe little doubt that Streptococcus salivarius is the name whichshould be retained for this species.

SUMMARY

A study was made of 322 cultures of non-hemolytic streptococciisolated from human throats. The isolations were made by theapplication of quantitative methods so as to limit the collectionto the predominating types.

Streptococcus salivarius was found to be the prevailing type and290 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 ofpresent knowledge of the streptococci.

Streptococcus salivarius, as herein described, may be readilydifferentiated from any of the clearly established species of thestreptococci.

273

on Septem


.org/D

ownloaded from

http://jb.asm.org/


REFERENCESANDREWES, F. W., AND HORDER, T. J. 1906 Lancet, 2, 708.ARNOLD, L. 1920 Jour. Lab. and Clin. Med., 5, 587.AYERS, S. H., JOHNSON, W. T., JR., AND DAVIS, B. J. 1918 Jour. Infect. Dis.,

23, 290.AYERS, S. H., Rupp, P., AND MUDGE, C. 5. 1921 Jour. Infect. Dis., 29, 285.AYERS, S. H., JOHNSON, W. T., JR., AND MUDGE, C. S. 1924 Jour. Infect. Dis.,

34, 29.AYERS, S. H., AND MUDGE, C. S. 1923 Jour. Infect. Die., 33, 155.BROWN, J. H. 1919 Mon. Rockefeller Inst. Med. Res., No. 9.EDWARDS, P. R. 1932 Jour. Bact., 23, 259.HANSEN, P. A. 1935 New York (Geneva) Agr. Exp. Sta. Tech. Bul. 232.HOLMAN, W. L. 1916 Jour. Med. Res., 34, 377.MEYER, K., AND SCHNFELD, H. 1926 Centbl. Bakt., I Abt., (Orig.), 99, 402.ORLA-JENSEN, S. 1919 The Lactic Acid Bacteria. Copenhagen.SAFFORD, C. E., AND SHERMAN, J. M. 1936 Jour. Bact., 31, 572.SCHOTTMtLLER, H. 1903 Munch. Med. Wochenschrift, 50, 849.SHERMAN, J. M., AND ALIBUS, W. R. 1918 Jour. Bact., 3, 153.SHERMAN, J. M., AND STARK, PAULINE 1931 Jour. Bact., 22, 275.SHERMAN, J. M., AND STARK, PAULINE 1934 Jour. Dairy Sci., 17, 525.SHERMAN, J. M., AND WING, HELEN U. 1935 Jour. Dairy Sci., 18, 657.WINSLOW, C.-E. A., AND WINSLOW, ANNE R. 1908 The Systematic Relation-

ships of the Coccaceae. New York.

on Septem


.org/D

ownloaded from

http://jb.asm.org/

STREPTOCOCCUS SALIVARIUS - Journal of …jb.asm.org/content/33/3/263.full.pdf · STREPTOCOCCUS...

Documents

Transcript of STREPTOCOCCUS SALIVARIUS - Journal of …jb.asm.org/content/33/3/263.full.pdf · STREPTOCOCCUS...