JMSHERMANCFNIVENJRANDKLSMILEYcocciwerewellknowntobecommonlyfoundinvegetableproductsmilkbutterandcheeseandOrlaJensen1919hadreportedthemasalsooccurrnginthealimentarytractsofherbivoraInpr ID: 333898
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STREPTOCOCCUSSALIVARIUSANDOTHERNON-HEMOLYTICSTREPTOCOCCIOFTHEHUMANTHROATJ.M.SHERMAN,C.F.NIVEN,JR.ANDK.L.SMILEYLaboratoryofBacteriology,CollegeofAgriculture,CornellUniversity,Ithaca,NewYorkReceivedforpublicationAugust17,1942Thenon-hemolyticstreptococciofthehumanthroatform,simultaneously,oneoftheoldestandleastknowngroupsofthegenus.Thesefamiliarbutill-definedorganismsareusuallyreferredtoasthe"salivariusgroup,"as"Strepto-coccusviridars"and"indifferentstreptococci"accordingtothedegreeofgreen-ingproducedinbloodagar,orsimplyasthe"mouthstreptococcus."IntheirearlyclassicalworkAndrewesandHorder(1906)appliedthenamesofStreptococcussalivariusandStreptococcusmitistotheseorganisms,Strepto-coccussalivariusbeingmarkedbyitsabilitytocurdlemilk,usuaUytoreduceneutralred,andfrequentlybythefermentationofraffinoseandinulin,whereasthesepropertieswereusuallylackedbyStreptococcusmitis.ThedistinctionwasthereforeastatisticalonewithoutasharpboundaryandtheysuggestedthatStreptococcusmitismightbeconsideredonlyasa"variantbydefect"ofStreptococcussalivarius.AlthoughAndrewesandHorderwereinclinedtocon-siderStreptococcussalivarius"initsmosttypicalform"asadistinctentity,inmanyculturesitsseparationrestedonlyonthe"tenuousmilkreaction."Safford,ShermanandHodge(1937)carriedthedescriptionofStreptococcussalivariusmuchfurther,showingthatthetypicalculturesformedaveryhomo-geneousgroupmarkedbythefermentationofraffinoseandinulin,littleornogreeninginbloodagar,vigorousacidproductionasshownbyfinalpHvaluesof4.4to4.0inglucosebrothandthepromptcoagulationofmilk,togetherwithmanyothercorrelatingcharacteristics.However,astheydidnotfeeljustifiedindrawingthelinerigidlyonthebasisofinulinfermentation,therewasagradualgradationfromwhattheyconsideredthe"typical"Streptococcussali-variusthroughtheentiregroup;thedifferentiationthereforeremainingasta-tisticalonewithoutasharpboundaryline.InsuchcompilationsasthatbySherman(1937),inwhichallofthenon-hemolyticstreptococciofthehumanthroatareconsideredasStreptococcussalivarius,theresulting"species"isapoorlydefinedandsomewhatheterogeneousgroup.Oerskov(1930)andOerskovandPoulsen(1931)reportedthatcertainnon-hemolyticstreptococci,whengrownonsucroseorraffinoseagar,produceapolysaccharidewhichresultsintheformationoflargemucoidcolonies,aprop-ertylongknowntobepossessedbytheheterofermentativestreptococcalorganismsofthegenusLeuconostocorBetacoccus.Astheydidnotestablishthehomofermentativenatureoftheirorganisms,andasisolationsweremadefrommilkandanimalsourcesaswellasthehumanthroat,itisprobablethatthedoubtconcerningthenatureoftheorganismsdealtwithpreventedaproperappreciationofOerskov'simportantdiscovery.Theheterofermentativebeta-249 J.M.SHERMAN,C.F.NIVEN,JR.ANDK.L.SMILEYcocciwerewellknowntobecommonlyfoundinvegetableproducts,milk,butterandcheese,andOrla-Jensen(1919)hadreportedthemasalsooccurrnginthealimentarytractsofherbivora.Inpreviouspapers(Niven,SmileyandSherman,1941a,1941b)ithasbeenreportedthatanumberoftypicalculturesofStreptococcussalivariussynthesizealargeamountofasolublelevanfromsucroseandraffinose,somestrainsproduc-inginadditionasmalleramountofadextran.Thelevanisproducedineitherliquidorsolidmedia,resultinginthelattercaseinlargemucoidcolonies.Whengrownonawell-bufferednutrientagarcontaining5percentsucrose,Strepto-coccussalivariusproducesalarge,clear,soft,mucoidcolonyofaboutthediam-eterofthoseproducedbycoliformbacteriaandyeasts.NootherknownspeciesofstreptococcuswasfoundtoproducelargemucoidcoloniesonsucroseagarwiththeexceptionofoccasionalstrainsofStreptococcusbovis,butthepoly-saccharideformedinthiscasewasaninsolubledextran.TABLE1Somebasicnegativecharacteri8ticsofthethroatcultureswhichprecludethepresenceofpneumococci,betacocci,staphylococci,enterococci,theso-calledlacticgroupofstreptococci,andcertainviridansstreptococciBilesolubility:Notsolubleinbile.Homofermentativenature:Carbondioxidenotproducedfromglucose.Catalase:Catalasewasnotproduced.Proteolysis:Nodigestionofcasein;gelatinnotliquefied.Biletolerance:Nogrowthin30%0bileinbloodagar.Salttolerance:Nogrowthinbrothcontaining6.5%NaCl.Methylenebluetolerance:Nogrowthin0.1%solutioninmilk.Reducingaction:Litmusinmilknotreducedbeforecurdling.Thermalresistance:Killedat600C.for30minutesinskimmedmilk.Minimumtemperature:Nogrowthat100C.inbrothormilk.CULTURESSTUDIEDThe331culturesemployedinthisworkfordetailedstudywereisolatedfromthethroatsofnormalpersons.Althoughanumberofkindsofnon-hemolyticstreptococcimayoccurinsmallernumbersinhumansaliva,ifthethroatisswabbed,andtheisolationsmadeinasemi-quantitativemanner,thepre-dominatingfloraisfoundtobearelativelysimpleone.Thatthepresentcollectiondidnotcontainheterofermentativestreptococci(thegenusBetacoccusorLeuconstoc),pneumococci,normembersoftheso-calledlacticandenterococcusgroupsofstreptococciisshownbytable1.ItmaybeaddedthatthelowthermalresistanceandlowbiletoleranceoftheseorganismswouldalsoappeartoprecludethepresenceofsuchmembersoftheviridansgroupasStreptococcusbovis,StreptococcusequinusandStreptococcusthermoph-ilus.Inanydefinitivestudyoftheso-calledviridansstreptococciitisessentialtoapplyanumberof"qualifyingtests"inordertoexcludemembersofcertainrelatedgeneraaswellasrepresentativesoftheothergroupsofnon-hemolytic250 NON-HEMOLYTICSTREPTOCOCCIOFHUMANTHROATstreptococci.Ascomparedwiththeothermajorgroupsofthegenus,thetaxonomyoftheviridansstreptococciisinahighlyunsatisfactorycondition;butthecommonimpressionthattheviridansstreptococcicomposeahope-lesslyheterogeneousconglomerationislargelyduetothegeneralfailuretodeterminethebasicnatureoftheorganismsdealtwith.Amongthemanycul-turessenttothislaboratoryunderthelabelof"Streptococcusviridans"havebeennotonlynon-hemolyticenterococci,groupBstreptococciandothers,butbeta-cocciandevenstaphylococci.STREPTOCOCCUSSALIVARIUSWhenthestrainsofthethroatstreptococciarelimitedtothosewhichsyn-thesizealevanfromsucrose,amostremarkablyhomogeneousgroupoforgan-ismsresuts.ThisgroupagreesentirelywithwhatwaspreviouslydesignatedTABLE2AdditionalcharacteristicsofStreptococcussalivariusshowingtheremarkablehomogeneityofthegroupPolysaccharidesynthesis:Largemucoidcolonieson5%sucroseagar.Temperaturelimits:Growthat450C;nogrowthat100C.(Testswereconductedinbrothandinmilk.)Bloodagar:"Indifferent"reactionbymorethan95%ofcultures;nostrainproducedstronggreening.Finalacidity:InglucosebrothallstrainsgavepHvaluesbetween4.4and4.0;milkwascurdledbyallstrainswhichfermentedlactose.Esculin:Splitbyallstrainsinbroth,withoutbile.Arginine;SodiumHippurate:Nothydrolyzedbyanystrain.Inulin;Raffinose;Sucrose;Maltose;Salicin:Fermented.Mannitol;Sorbitol;Glycerol;Arabinose;Xylose:Notfermented.Lactose;Trehalose:Ofthe184strains,16failedtofermentlactose;14didnotfermenttrehalose.asthe"typical"Streptococcussalivariusbutisfurtherdefinedbysomanyothercorrelatingandapparentlyconstantcharacteristicsthatonewouldappeartobejustifiedinconsideringita"species."Itwillbenotedfromtable2thatpracti-callynovariationswereexperiencedinthemorebasiccharactersstudied,thishomogeneityextendingeventhroughthefermentationtests,withtheexceptionsonlyoflactoseandtrehalosewhichwerenotfermentedbyafewstrains.AstrikingcharacteristicofStreptococcussalivarius,asopposedtotheStrep-tococcusmitisgroupandmostotherstreptococci,istherelativelylargesizeoftheindividualcellsproducedinliquidmedia,especiallymilk.Thisistrueinneutralbrothcultures,butmaynotbenoticeableinthosewhichreachalowfinalpH.Itisidletotrytodefinea"species"ofbacteria,orevensaywhetherornotsuchanentitycanbedefined,butStreptococcussalivariusaslimitedbythecharactersherepresentedformsasdistinct,asclearlydefined,andaseasilyidentifiedanddifferentiatedaunitasanygroupinthegenusStreptococcus.Amongtheothercorrelatingcharacters,allculturesofStreptococcussalivarius251 J.M.SHERMAN,C.F.NIVEN,JR.ANDK.L.SMILEYwhichsynthesizelevanfromsucrosefermentinulin,andviceversa,butotherinulin-fermentingspeciesofstreptococcidonotsynthesizelevan;althoughitwouldscarcelyseemlikelythatthiscorrelationinStreptococcussalivariuswillbefoundtoholdinviolably,noexceptionhasyetbeenencounteredamongseveralhundredisolationswhichhavebeenmadefromthehumanthroat.ThequestionmaylogicallybeaskedastowhetherornotStreptococcussali-varius,sodefinedanddifferentiatedfromtheothernon-hemolyticthroat.strep-tococci,actuallyconstitutesatruespeciesinthebiologicalsense.Probablynot.However,theclassificationofbacteriaisofvalueforpracticalpurposes,the"species"beingunitswhichcanbeidentifiedanddifferentiatedfromothercloselyrelatedgroupswithsomedegreeofcertaintyandease.Streptococcussalivariusmeetstheserequirementsadmirably.Thepneumococciprobablyrepresentaswellestablishedanduniversallyacceptedspeciesasanygroupofbacteria.Ingeneralpracticetheidentificationofpneumococcidependsupontheirsynthesisoftype-specificpolysaccharides,butitiswellknownthatthenon-mucoidvariantsofthepneumococcidonotproducetheirspecificcapsularpolysaccharides.(Althoughitistruethatthepneumococcicontainaspecies-specific"C"antigen,sofarasweareawarethisimportantdiscoveryofTillettandFrancis(1930)isnotmadeuseofinthepracticalproblemofidentifyingthespecies.)Itwouldthereforenotseemundulyradicaltoconsiderthepro-ductionofachemically-specificpolysaccharidebyStreptococcussalivariusasaspecies-specificcharacteristic.Inotherworkinthislaboratory(NivenandSmiley,1942)ithasbeenshownthatStreptococcussalivariuscanbegrownonachemicallydefinedmediumcon-tainingonlycertainaminoacidsandvitaminsinadditiontoglucose,areducingsubstanceandinorganicsalts.Thismediumhasbeentestedwithrepresentativeculturesofallofthemoreimportantgroupsandspeciesofhemolyticandnon-hemolyticstreptococci.Noneofthese,withtheexceptionofafewstrainsofStreptococcusbovis,willgrowonthismedium.StreptococcussalivariuswillnotgrowonthesyntheticmediumwhichhasbeenusedforenterococciandgroupBstreptococci,norwilltheseorganismsgrowintheS.salivariusmedium.Ofespecialinterestinthepresentconnectionisthefactthattheothermorehetero-geneousnon-hemolyticthroatstreptococci,theStreptococcusmitisgroup,donotgrowinthissyntheticmedium.ItthereforeappearsthatamongtheotherclaimsofStreptococcussalivariustothestatusofa"species"maybeaddeditsspecificnutritiverequirements.Althoughtediousanddetailedstudiesareessentialinordertodefinemoredefinitelythelimitsofaspecies,themainvalueofsuchworkasthisistoarriveatsomesimpletestswhichcanbeappliedinthepracticalproblemofquicklyidentifyingtheorganism.TheobvioussimplepresumptivetestforStreptococcussalivariusisfortheabilitytoproducelargemucoidcolonieson5percentsucroseagar.Basedonexperiencethusfarthisappearstobearelativelysafecriterionindealingwithculturesfromhumansources.However,inaccurateworkitisnecessarytobeconstantlyonguardtoexcludetheheterofermentativebetacocciandtheoccasionalstrainsofStreptococcusboviswhichhavetheabilitytosyn-252 NON-HEMOLYTICSTREPTOCOCCIOFHUMANTHROATthesizepolysaccharidesfromsucrose.Thebetacoccicanbeeasilydetectedbytheirabilitytoproducecarbondioxidefromglucose;inaddition,theygrowat10°C.butdonotgrowat45°C.-theoppositeofthetemperaturegrowthlimitsofStreptococcussalivarius.AscomparedwithStreptococcussalivarius,Strep-tococcusbovisdiffersmarkedlyinitsgreaterbiletolerance,higherthermaldeathpoint,theproductionofmarkedgreeninginbloodagar,andusuallybytheabilityofthemajorityofstrainstofermentarabinoseandactivelyhydrolyzestarch(Sherman,StarkandSafford,1938).ADOMINANTSEROLOGICALTYPEOFSTREPTOCOCCUSSALIVARIUSAsiswellknown,theso-calledviridansstreptococcihavenotbeenshowntocontaingrouporspecies-specificantigenscomparabletothoseoftheLancefield(1933)serologicalgroupsofhemolyticstreptococci.Althoughthereisperhapsnoreasonwhythevnridansstreptococcishouldpossesssuchantigens,thisnever-thelessseemsalittlestrangeinasmuchasgroup-specific"C"substancesarecontainednotonlybyhemolyticstreptococci,butbythepneumococcus,thenon-hemolyticenterococciandnon-hemolyticvarietiesofotherserologicalgroups,andtheso-calledlacticgroup(Streptococcuslactis)ofnon-hemolyticstreptococci.(TillettandFrancis,1930;Lancefield,1934,1941;Sherman,1938;Sherman,SmileyandNiven,1940.)However,theinvestigationsofLancefield(1925a,1925b)andHitchcock(1924b,1928)gavenoevidenceofsuchgroup-specificantigensintheviridansstreptococci,althoughtheexistenceofanantigenofbroaderthantypespecificityinthehemolyticstreptococciwasatthattimeknownthroughtheworkofHitchcock(1924a).Ontheotherhand,theexistenceofmanyserologicaltypesamongtheviridansstreptococcihasofcourselongbeenknownandtheseorganismshavebeenconsideredveryheterogeneousfromtheserologicalpointofview,someinvestigatorsfindingalmostasmanysero-logicaltypesascultureswhichtheystudied.Hitchcock(1928),workingwithinulin-fermenting"indifferentstreptococci,"principallyfromthehumanthroat,foundthatabout50percentoftheseorgan-ismsbelongedtooneserologicaltype,asshownbyprecipitinandagglutinationreactions,whichhedesignatedasTypeI.Therestofhisculturesweresero-logicallyheterogeneousandhereferredtothemlooselyas"GroupX."Birk-haug(1927)likewisereportedthat75percentoftheinulin-fermenting,non-methemoglobin-formingstreptococci,isolatedinconnectionwithhisstudiesofrheumaticfever,belongedtooneagglutinativetype;andHitchcockfoundBirkhaug'stypetobephysiologicallyidenticalwithhisownindifferentstrep-tococciandtobelongtohistypeI.Small(1927)described"Streptococcuscardioarthritidis"whichheconsideredanewspecieshavingimportanceasacausativeagentinarthritisandrheumaticfever.Hegaveabriefdescriptionofhisorganism,themostrevealingcharac-teristicsfromthestandpointofitsidentitybeingitsabilitytofermentraffinoseandinulin,itsinabilitytofermentmannitol,anditsinabilitytoproduceanychangeonbloodagar.Ofthe21strainsdescribed,allwerehomogeneousintheiractiononblood,raffinose,inulin,andmannitol,but7ofthemfailedto253 J.M.SHERMAN,C.F.NIVEN,JR.ANDK.L.SMILEYfermentlactose.ItshouldbeaddedthatalthoughsomeofSmall'scultureswereobtainedfromclinicalspecimensmostofthemwereisolatedfromthethroatsofpatients.Smalldidnotclaimthatallculturesofhis"newspecies"werestrictlyhomogeneousserologicallybutstatedthat31strainshadbeenobtainedwhichbelongedtooneserologicaltype.Althoughourowneffortstoproducegroup-specificseraforStreptococcussalivariushavebeenunsuccessful,wehaveobtainedanumberoftype-specificserawhichhaveenabledustomakeapartialsurveyoftheserologicaltypeswithinthisspecies.Ofparticularinterestisthefactthatabout40percent(83of184strains)belongtooneserologicaltypeasdeterminedbythepre-cipitintechnique.Thattheunitwithwhichwearedealingisaserologicaltyperatherthanagroupisindicatedbythefactsthatallofthestrainsappearedtobelongtooneagglutinativeunitandthatintheimmunizationofrabbitstheredidnotappeartobeanybroadeningofthespecificityoftheseraastheinjectionswerecontinuedoverprolongedperiodsofimmunization.AsthisappearstobethelargestserologicaltypeofStreptococcussalivariuswehavedesignateditas"TypeI."Itwouldbeofparticularinteresttoknowwhatrelation,ifany,ourtypeIStreptococcussalivariushastotheorganismswhichhavebeendescribedbyothers.AcultureofSmall's"Streptococcuscardioarthritidis,"obtainedfromtheAmericanTypeCultureCollection,hasbeenstudiedindetailandfoundtobenotonlyanentirelytypicalstrainofStreptococcussalivariusbutalsotobelongtoourserologicaltypeI.ThroughthekindnessofDr.RebeccaC.LancefieldwehaveobtainedtwoculturesofHitchcock'sindifferentstreptococciwhichbelongedtohis"GroupX,"hisserologicallyheterogeneousgroup.Thesecul-tureshavealsobeenfoundtobetypicalpolysaccharide-synthesizingstrainsofStreptococcussalivarius,butnottobelongtoourtypeI.ItwouldthereforeseemprobablethatHitchcock'stypeIindifferentstreptococcusandourtypeIStreptococcussalivariusarethesame,sincebothhavebeenfoundasthedomi-nantserologicaltypeamongtheindifferentstreptococciisolatedfromthehumanthroat,butwehavenotasyetbeenabletolocateanystrainsofDr.Hitchcock'stypeIorganismwithwhichtotestthispoint.OurtypeIStreptococcussalivariusserumhasgivennocrossreactionswithrepresentativeculturesofthevariousserologicalgroupsofstreptococciorotherspeciesofnon-hemolyticstreptococci,withthesingleexceptionthataveryfewstrainsoftheso-calledStreptococcusmitisgroupofviridansstreptococcifromthehumanthroatgavereactions,usuallyweak,withtheserum.Especialattentionwasgiventotherelatedspeciesofviridansstreptococci,StreptococcusbovisandStreptococcusequinus,butnotasinglecrossreactionwasexperiencedwiththeseorganismsalthoughmorethanonehundredstrainsweretested.ItshouldberecalledinconnectionwiththefewreactionswhichwereobtainedwithotherthroatstreptococcithatHitchcockalsoreportedthatafewstrainsofStreptococcusviridansreactedwithhistypeIserum,whichhewasinclinedtoconsideracrossreaction.254 NON-HEMOLYTICSTREPTOCOCCIOFHUMANTHROATWehavenotdoneenoughworkwithotherserologicaltypesofStreptococcussalivariustojustifyareportatthistime.Itmaybestated,however,thatthereisatleastoneothertypewhichencompassesafairlylargeproportionofthestrainsofthisspecies,andthatanunknownnumberofothertypesexist.Itispossible,however,thatwhenthesestudiesareextended,withthelimitationofstrainsstrictlytothephysiologicallyhomogeneous,"typical"Streptococcussalivarius,thegroupmaynotbefoundtobequitesohopelesslyheterogeneousfromthestandpointofnumbersofserologicaltypesashasheretoforebeenthought.TheinterestingworkofSolowey(1942)appearedasthispaperwasbeingpreparedforpublication.Shereportsontheserologicalstudyof205strainsofviridansstreptococciobtainedfromsubacutebacterialendocarditis,humanthroatsandextractedteeth,alargeproportionofwhichsheidentifiedasbe-longingtotheStreptococcussalivariusgroup.Oftheentirecollection,136strains,or66percent,couldbeclassifiedasbelongingwithin14serologicalunits,3oftheseunits(hergroupsI,IIandIV)containingasignificantnumberofstrains.Thedominantunit,"groupI,"contained77strains,about38percentoftheentirecollection.Althoughreferredtoas"groups"itisnotclearfromherpaperwhethertheseserologicalunitsaregroups,asthistermisappliedtothehemolyticstreptococci,orserologicaltypes.ItishopedthatthroughanexchangeofculturesitmaybelearnedwhetherornotourserologicaltypeIStreptococcussalivariuscorrespondswithoneofDr.Solowey'smajorunits.STREPTOCOCCUSSALIVARIUSASANINTESTINALORGANISMAsStreptococcussalivariusisusuallythepredominatingorganisminthehumanthroatitisobviousthatmanymillionsofcellsofthisbacteriumareswallowedeachdaybyeveryone.Becauseofsomeofitscharacteristics,suchasitsrelativelackoftoleranceforbile,thereisconsiderabledoubtastowhetherornotitisabletogrowinthehumanintestine,butitwouldseemprobablethatatleastasmallproportionoftheseorganismssurvivetheacidityofthestomachandthusoccurintheintestine.Sofarasweareaware,however,Streptococcussalivariusisnotdefinitelyknowntooccurinthehumanintestine,althoughintheolderliteraturenon-hemolyticstreptococcifromfecalsampleshavebeenrecordedasStreptococcussalivariusontheinadequatebasesthattheydidnotfermentmannitolordidfermentraffinose.Sodiumazidehasbeenusedbyanumberofinvestigatorsasaninhibitoryagentinstudyingrespiratoryenzymesandasaselectivesubstancetofacilitatetheisolationofstreptococci.SnyderandLichstein(1940)haveshownthattheadditionofsodiumazide(0.01percent)tobloodagarpreventsthespreadingofProteusandlargelyinhibitscoliformbacteriawhilepermittingthegrowthofhemolyticandnon-hemolyticstreptococci.InoureffortstoisolateStreptococcussalivariusfromhumanfeces,wehavesuccessfullycombinedsodiumazideandsucroseinordertogetamediumwhich255 256J.M.SHERMAN,C.F.NIVEN,JR.ANDK.L.SMEYhasbothselectiveanddifferentialvaluesfortheisolationofthisorganism.Themediumusedhasthefollowingcomposition:Sucrose......................................5.0Tryptone......................................1.0Yeastextract.....................................0.5K,HPO4.....................................0.3Agar.....................................1.5Sodiumazide.....................................0.02pH-7.0OnthismediumStreptococcussalivariusproduceslargemucoidcolonies,asopposedtothecharacteristicsmallcoloniesofotherstreptococci,whereascoli-formbacteriaandothermiscellaneoustypeswhichproducelargecoloniesareTABLE3Streptococcussalivariusandtotalstreptococciinhumanfecesasdeterminedwithsucrose-sodiumazideagars~~~~~~~~~~SirepwosoiurimSAiUQNW3mim(PWA°(pimGRM)(pergram)(percentoftotal)19,100,0009,000,000992540,000530,0009838,000,0006,800,0008541,000,000840,000845159,000,000129,000,0008161,780,0001,320,00074723,000,00015,400,00067833,000,0007,200,00022914,200,0002,000,00014104,700,000500,000111181,0005,00061267,0002,4004131,600,00013,0000.8144,300,0002,0000.05151,640,0001,2000.0716192,000,000173,800,000183,500,000largelyorcompletelyinhibited.ItthusbecomespossibletorecognizeStrep-tococcussalivariuscoloniesonsightwithahighdegreeofaccuracy.Aswaspreviouslynoted,occasionalstrainsofStreptococcusboviscanproducemucoidcoloniesonsucroseagarandthisorganismisknowntooccursometimesinsmallnumbersinhumanfeces,butmucoidstrainsof,thisspecieswerenotencounteredinthesamplesofhumanfecesexamined.Of18personsexamined,Streptococcu-ssalivariuswasisolatedfromthefecalsamplesof15.Inthese15samplesthenumbersofStreptococcussalivariusrangedfrom1,200to129,000,000pergram,7ofthesamplescontainingmorethan1,000,000pergram.Asmaybeseenfromtable3,insomesamplesStrep- NON-HEMOLYTICSTREPTOCOCCIOFHUMANTHROATtococcussalivariuscomprisedasurprisinglylargeproportionofthetotalstrep-tococciwhichgrewonthismedium.MucoidcolonieswhichwerecountedasStreptococcussalivariuswereverifiedasstreptococcimicroscopically,andseveralculturesfromeachsample(80strainsinall)weresubjectedtodetailedstudy.AlloftheseprovedtobetypicalstrainsofStreptococcussalivariusandtoagreeperfectlywithstrainsobtainedfromthehumanthroat.Itisperhapsofinteresttonotethatthefecalstrainsshownogreatertolerancetobilethandothosefromthethroat,allbeingin-hibitedby30percentbileinbloodagarwhilemostofthemfailtogrowinthepresenceof20percentbile.Ofthe80fecalstrainstested,36(45percent)belongedtoserologicaltypeI.TheseresultsshowthegeneraloccurrenceofStreptococcussalivariusinthehumanintestineinappreciablenumbers;however,theydonotprovethatthisorganismisabletogrowintheintestine,thoughthelargenumbersfoundinsomefecalsampleswouldappeartoindicatethatgrowthprobablydoesoccurinsomecases.THESTREPTOCOCCUSXTISGROUPWhenStreptococcussalivariusissegregated,theremainingnon-hemolyticstreptococciofthehumanthroatfallintoaratherill-definedandheterogeneousgroupwhichmaybemostappropriatelydesignatedastheStreptococcusmitisofAndrewesandHorder(1906).AlthoughclearlydifferentiablefromStrep-tococcussalivariusandseveraloftheotherspeciesofviridansstreptococci,theStreptococcusmitisgroupappearstobedecidedlyheterogeneousonthebasisofthemethodsnowavailableforthestudyofstreptococci.Itmaywellbeacomplexgroupwhichawaitsmoreincisivemethodsforthesegregationofitsconstituentunits.SomeofthereactionswhichdifferentiateStreptococcussalivariusfromStrep-tococcusmitis,andalsoshowtherelativeheterogeneityofthemitisgroup,aregivenintable4.InadditiontotheinabilityofanycultureoftheStreptococcusmitisgrouptofermentinulinortosynthesizepolysaccharidefromsucrose,mostofthemdifferfromStreptococcussalivariusinanumberofothercharacteristics.Amongthe147strainsofthemitisgroupincludedinthiswork,notonedifferedfromStreptococcussalivariusonlessthanfivecharactersandthevastmajorityofthemweredifferentonsixormorereactions.AsopposedtoStreptococcussalivarius,inadditiontotheirinabilitytoproducelevanandtoattackanumberofthetestsubstances,about90percentofthestrainsoftheStreptococcusmitisgroupproducemarkedgreeningofbloodagar,veryrarelyacompletely"indif-ferent"reaction;ontheaverage,also,themitisgroupdoesnotreachsolowapHinglucosebrothandmanyculturesfailtocurdlemilk.ThattheStreptococcusmitisgroupmaycontainmorethanonedistinctunitisfutherindicatedbythefactthatanappreciableproportionofthisgrouphydrolyzedargininewiththeproductionofammonia,averyrarepropertyamongtheviridansstreptococci(Niven,SmileyandSherman,1942).Cor-257 J.M.SHERMAN,C.F.NIVEN,JR.ANDK.L.5MILEYrelatedwiththeabilitytohydrolyzearginineweretheabilityofalargerpro-portionofthestrainstogrowat45°C.,toproducealowerpHinglucosebroth,tocurdlemilk,andtoattacksalicinandesulin.Althoughthesestatisticalcorrelationsareofnopresentpracticalvalue,theydoaddtothesuspicionthat"Streptococcusmitis"mayindeedbeacomposite.Butanattempttodividethemitisgroupatthistimewouldbeamereworkoftessellation,withouttheemergenceofaclearly-linedmosaic.InasmuchIastheheterogeneityoftheStreptococcusmitisgrouphasbeenemphasized,itshouldbepointedout,ontheotherhand,thatithassomedegreeofhomogeneity.Notoneofthestrainsofthisgrouphydrolyzedsodiumhip-TABLE4RelativelyheterogeneousnatureoftheStreptococcusmitisgroupasopposedtothehomogeneityofStreptococcussalivarius,andthedifferentiationofthetwogroupsPEMCENTGIVIORCTIONS.saliwariusS.mitis(184strains)(147strains)Polysaccharidesynthesizedfromsucrose.............1000Inulinfermented........................................1000Indifferentonblood(neverstronggreening).............10010Esculinsplit.............................................10021Raffinosefermented......................................10032Growthat450C..........................................10036Argininenothydrolyzed.................................10057Milkcurdled(bylactose-fermentingstrains).............10066Salicinfermented........................................10074Trehalosefermented.....................................9315Growthin10%bile-bloodagar...........................9113Note.Inadditiontothecharactersgivenintable1,allstrainsoftheStreptococcusmitisgroup,aswellasStreptococcussalivarius,werehomogeneousinthefollowingrespects:sodiumhippurate,mannitol,sorbitol,glycerol,arabinose,andxylosewerenotattacked;sucroseandmaltosewerefermentedbyallcultures.FinalpHvaluesintheStreptococcusmitisgrouprangedfrom5.8to4.2,averagingaboutpH4.5.puratenordidasinglestrainfermentanyofthefollowingpentosesugarsandhigheralcohols:arabinose,xylose,mannitol,sorbitolandglycerol.These,however,arenotveryhelpfulastheyarecharacteristicscommontomostspeciesoftheviridansstreptococci.Itshouldneverthelessbenotedthat,inadditiontoStreptococcussalivarius,anumberofspeciesoftheviridansstreptococciareclearlyandeasilydifferentiablefromtheStreptococcusmitisgroup:Streptococcusbovisbyitsgreaterbiletoleranceandhigherthermaldeathpoint,itsabilitytosplitesculin,andbyitsusualabilitytofermentarabinose;Streptococcusequinusbyitsgreaterbiletolerance,itsabilitytosplitesculin,anditsinabilitytofermentlactose;Streptococcusthermophilusbyitsmuchhigherthermaldeathpointandmaximumtemperatureofgrowth,anditsinabilitytofermentmal-tose.Thelittle-knownStreptococcusacidominimusofAyersandMudge(1922),258 NON-HEMOLYTICSTREPTOCOCCIOFHUMANTHROATwhichoccursinthebovinevaginaandoccasionallyintheudderandinmilk,alsodiffersmarkedlyfromthemitisgroupinthatithydrolyzessodiumhippurateandhasextremelyfeebleacidtolerance,givingfinalpHvaluesoffrom6.5to5.6inglucosebrothandproducinglittleornovisiblechangeinlitmusmilk(SmithandSherman,1939).AlthoughtheStreptococcusmitisgroupcanbedifferentiatedfromthebetterdefinedspeciesofviridansstreptococci,ithasnotyetrevealedclearlymarkedcharacteristics,uniquelyitsown,whichallowitsrapidandpositiveidentifica-tionbytheuseofonlyafewsimpletests.THELACTOSE-NON-FERMENTING,NON-HEMOLYTICSTREPTOCOCCIFROMTHETHROATANDOTHERHUMANSOURCESSinceAndrewesandHorder(1906)describedStreptococcusequinus,thelactose-non-fermentingstreptococcuswhichpredominatesintheintestineofthehorse,ithasbeenthecustomtodesignateasStreptococcusequinusthenon-hemolytic,lactose-non-fermentingstreptococciwhichareisolatedfromhumansources.FloydandWolbach(1914),Broadhurst(1915),Holman(1916),Blake(1917),Arnold(1920),Porch(1941),andmanyothers,havereportedasStreptococcusequinussuchstreptococcifromhumansources,mostfrequentlyfromthethroatbutoccasionallyfromurineandfrominfections.Largecol-lectionsofnon-hemolyticstreptococciisolatedfromthehumanthroatusuallycontainafewstrainswhichareunabletofermentlactose.TheproprietyofclassifyingsuchhumanthroatstrainsasStreptococcusequinuswasquestionedbySafford,ShermanandHodge(1937),onthebasisofonlyoneculture,becausetheirstrainappearedtoresembleStreptococcussalivariusandtodifferinanumberofrespectsfromthetypicalStreptococcusequinusfromtheintestineofthehorse(HodgeandSherman,1937).Inourinvestigationsofthenon-hemolyticstreptococciofthehumanthroatanoccasionalcultureisisolatedwhichfailstofermentlactose,and21suchstrainswerestudiedindetailinthepresentworkandidentifiedasStreptococcussalivariusandStreptococcusmitis.Thatthe16strainssoclassifiedareune-quivocallyStreptococcussalivariusisshownintable5whichpresentssomeselectedcharacteristicsoftheorganismsincomparisonwiththoseof59culturesofStreptococcusequinus.Ofthe16lactose-negativestrainsofStreptococcussalivarius,8belongtoserologicaltypeI.AlthoughtheStreptococcusmitisgroupdoesnothavemanysatisfactorydistinguishingcharacteristics,itisfairlyclearlydifferentfromStreptococcusequinusonthebasesofthegreatdisparityinthebiletoleranceofthetwoorgan-ismsandtheinabilityofthemitistypetosplitesculin.Table6showsratherdefinitelythattheremaining5lactose-negativethroatstrainsbelongtotheStreptococcusmitisgroup.Theresultsreportedwouldappeartoestablishtheidentitiesoftheselactose-non-fermentingstreptococci;anditisprobablethatmostsuchlactose-negative,non-hemolyticstreptococcifromthehumanthroatbelongtothesalivarius-mitisgroup,ratherthantothatofStreptococcusequinus.Itdoesnotfollow259 J.M.SHERMAN,C.F.NIVEN,JR.ANDK.L.SMILEYthatthiswillprovetrueimthecaseofculturesisolatedfromotherhumansources.Forexample,Porch(1941)hasreported,asStreptococcusequinus,lactose-non-fermentingstreptococcifromthehumangenito-urinarytractwhichreactedwithgroupBserum.ThroughthekindnessofMissPorch(nowMrs.Frechtling)andDr.JustinaH.H-illoftheJohnsHopkinsHospital,wewereTABLE5Identityoflactose-non-fermentingorganismsfromthehumanthroatasStreptococcussalivarius,andnotasStreptoccusequinusNUEGaRowTHoNMARKEDREACTNORoGANMsoIr30%BE-GREENNGmoRAYBInOSEmmSTJtAMSBIAAGArDIWSUCROSEFR(ETEMERMENTEDTWpIHumanthroat(lactose-percUAnegative).................16--+++50S.salivarius................170-_+++40S.equinus.....5+-(90%)-(73%)0TABLE6Apparentidentificationofcertainlactose-non-fermentingorganismsfromthehumanthroatasmembersoftheStreptococcwmitisgroup,andnotasStreptococcusequinwOl--NO.OlGWTBIOODAG"WITHORLGANISSTRAMNATTACKED30obile20%obile10%obileHumanthroat(lactose-negative).5--_.S.mitis.142---(85%)-(76%)S.equinus.59++++TABLE7Identityoflactose-non-fermentingorganismsfromthehumangenito-urinarytractasgroupBstreptococci,andnotasStreptococcusequinusREACTIONHYDROLYSISO1ORGANISMGROUPBAT450C.SERUMEHippurateArgnineEculinGenito-urinary(lactose-negative).............+++_S.mwatitidis.................................+++--S.equinw.................................++abletostudy8ofthosecultures.TheynotonlygavegoodprecipitintestswithgroupBserum,buthydrolyzedsodiumhippurateandarginine,didnotsplitesculin,andfailedtogrowat45°C.-allcharacteristicreactionsoftheStreptococcusmasitidisgroup.Streptococcusequinusdoesnothydrolyzesodiuimhippuratenorarginine,doessplitesculin,andgrowsat450C.FromtimetotimewehavetestedagoodmanyrepresentativeculturesofStreptococcusequinus,isolatedfromtheintestineofthehorse,withgroupBserawithout260 NON-HEMOLYTICSTREPTOCOCCIOFHUMANTHROATnotingareaction.Theidentityofthelactose-negativeculturesfromthehumangenito-urinarytractisshownintable7.Thelactose-negativeculturesfromthegenito-urinarytractweresubjectedtodetailedstudyandfoundtobeentirelytypicalofgroupBstreptococciinotherrespects.Theydidnotgrowat10°C.,hadweakreducingaction,fer-mentedtrehalose,sucroseandmaltose,butdidnotfermentraffinose,inulin,mannitolnorsorbitol.HemolyticgroupBstreptococciarewellknowntooccurcommonlyinthehumanurinaryandreproductivetractsanditisprobablethatnon-hemolyticstrainsarenotrareinthesehabitats.TheinabilityofsomegroupBstreptococcitofermentlactoseisalsowellknownandBrown(1939)hasshownthefrequencyofsuchstrainsamonghemolyticculturesfromhumansources.ItisalsoknownthatsomebovinestrainsofgroupBst'reptococci,non-hemolyticaswellashemolytic,failtofermentlactose(Sherman,GreisenandNiven,1941).Althoughitwouldappearthatthegenerallabelingoflactose-negativestrep-tococciofhumanoriginasStreptococcusequinusisanerror,thepossibilityofitsoccurrenceinhumansources,especiallytheintestine,isbynomeansruledout.Infact,WinslowandPalmer(1910),withthebestmethodsofidenti-ficationthenavailable,reportedStreptococcusequinusfromhumanfecesmorethanthirtyyearsago.SUlMMARYAcollectionof331culturesrepresentingthepredominatingnon-hemolyticstreptococciofthehumanthroatwasstudiedindetail;184strainswereidenti-fiedasStreptococcussalivarius,andtheremaining147asmembersoftheStrep-tococcusmitisgroup.Streptococcussalivariusappearstobearemarkablyhomogeneousandclearlydefinedspecies,especiallymarkedbyitsabilitytosynthesizelargeamountsofpolysaccharidefromsucrose,tofermentbothraffinoseandinulin,itsinabilitytoproducemarkedgreeninginbloodagar,andmanyothercorrelatingandapparentlyconstantcharacteristics.About40percentofthestrainsofStreptococcussalivariusbelongtooneserologicaltype(TypeI).Thereisatleastoneotherserologicaltypewhichincludesafairlylargeproportionofthestrainsofthisspecies,andanunknownnumberofothertypes.Theso-called"Streptococcuscardioarthritidis"wasphysiologicallyandsero-logicallyidentifiedasStreptococcussalivarius.Bytheuseofanutrientagarcontainingsucrose(5percent)andsodiumazide(0.02percent),whichhasbothselectiveanddifferentialvaluesforthispurpose,itwasshownthatStreptococcussalivariuscommonlyoccursinlargenumbersinthehumanintestine.Streptococcussalivariuswasisolatedfromthefecalsamplesof15ofthe18personsexamined,thenumbersofthisorganismrangingfrom1,200to129,000,000pergram.TheorganismsidentifiedasStreptococcusmitisformaratherheterogeneousandperhapscomplexgroup,givingdiversetestsinmanyofthereactionsstudied.261 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