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F=l : tntu not biot. vol. 1. pp J5? o 5'61'i ; ' ; ; ;" Pr6t Ltd i i ls

ninrcd in o'Ger Erirrinrt'i

INTNODUCTIONThc cariogenicPotential of Streptoco.ccusnl'ons hasbccn clearly demonstraled n animal models (Keyes.l%Z l968iand some role for this microorganism ndenral carics in man seems evidenl {Krasse et al',l96E; Lcschc et al., 1975\Scvcratattempts have been made lo study the in'traoral ecology of Slrep' nutons by the implantationin thc human mouth of Strep' mutans ltalns contaln-inr. for identificatiol purPoses'a streptomyon'resls-rait marler (Krasseet at.' 1967:Jordan et al', 1912;Edman et ol.,1975 Svanbergand Loesche'1978| butmost labellcd sltains were quickly ebminated fromrbc mourh. Edman et al. 11975) stablished abelledsuains in approximal siles on one side of the mouthin two "oiunt..tt and noted thal the implantedstrains were mainly recovered from that sidc' Theutificial fissure model (Liie. Karring and Theiladc'l9?3) can bc used to rcliably implant .slrcPromycin-,oitiun, strains n singlc occlusalsitcs (SvanbergandLocschc, 9?8).

MATENIAIS AND METHODSSubjects

The rhree subjects G, T and S) who participated:ad 38, 52 and i8 filled tooth surfaceq espectively'ro carious lesionsand no missing eelh' SubjectsGrnd S had a normal occlusion and subject T hadr class ll malocclusion.An art if iciaf issureAF) containing Strep'mutans;train which had beenmade resistanto I m8/ml of' hescnraddress: i ireborgsniversitct.dontologiskatl iniken. ack4ff i.33 Giireborg 3'Sweden

55?

0m1.9969:7E.0?0tr57t0t.00;0

INTRAORAL SPREADOF STREPTOCOCCUSUTANSIN MANMot*le L. SvrNsERctand W. J. L.oEscHrDen R sea" t""l;f .ou#:'il?; #:'|1tT'schoorfDentistrv'

Summary-Streptomycin-resislanl strainsof Streptococans urarswere reliably establisbed n3 subjecti by the use of artifical fissures AF) containing adherentgrowlh oflhe Strep.4utonsstrain.The intraoral spread of these mplantedStrep.mutans trainswas monjtored n lhesubjecrswhile the AF was in situ and for up lo 26 weeksafter the AF was removed.During$c 26 weeks. all rooth surfaoesand saliva were repeatedlysampled.The implanted Strep.mutonsspread to adjacent and anlagonistic eeth only on the side of the dentition wherethe implantations were made. Afler establishment f a streplomycin-resistanl train in onesubicct. a sterile AF was inserted multiple times in the same occlusalsurfacr. on which theimptantations had previously been madc. The srer ile AF was colonized by local spread ofSnep.murcnsstrains, as was shown by the relationshipbetween hepercenlage f streptomycin'resijgnt Snep.mutans o lotal Strep.mutans olony forming units (CFU) counts n the AFand thc salivary ffora. The streptomycin-resislanl trep.mutans epresented 0. 93.5 per cenlof rhc total Strep. mutons CFUs in the AF, but only G-1.5 per oent o[ the Strep. mutansCFUs in rhe saliva during colonizalionof the AF. The intraoral establishment nd spreadof the implanted Slrep. mutonsseemed avoured by conditions presnt n subjectswith highsljvarv concentralions of endogenousStr.ep.muransstrains and high caries experience.as.rpr.r*d by rhc high number of filled loolh surfaces.

streptomycin was placed in a molar looth in eachsubject.The AFs had been cultured in vitro and con-tained at the time of insertion l0? colon1,f6v6l1gunits (CFU) o[ Strep.mutansas adherent growth onthe AF (Svanbergand Loesche,1978).The AFs wereeach inserted hrce times in the occlusal surface ofan upper first molar in subjcct G and in a lowerfirst molar in subjectsT and S. The implanted Strep.rnutonsstrains G. T and S belonged lo serotype cand wcre re-inlroduced nto the same mouth fromwhich they originally had been solated SvanbergandLoesche.1978) .Bacterial somples

The AFs containing strains G, T and S were leftin uivo tor 7-12 days. Al 2, 4. 6, 8 and 26 weeksafler removal of the infe ctedAF. plaque sampleswerecollected iom all surfaccs f all teeth n each subject.Twenty-sir approximal toolh surfaceswere sampledwith dental floss and 56 smoolh tooth surfaccswithffattened hypodermic needles. Twenty-eight plaguesamples rom marginsof fillings and fissureswere col'lected with 26-gauge hypodermic needles. Thesampleswere laken in the morning after the subjectshad refrained from all oral hygiene on the eveninSand morning before sampling. Two to four salivarysampleswere collectedper week during this 26weekperiod.Colonizationof sterileAFs

Strain S established n the nalural tooth surfacesin subjectS for a prolongedperiod of t ime. Duringthis interval.steri leAFs were insertedseveral imesin the same toolh as that used earlier for the AFcontaining adherent gro*'th of 5trep. ntutans.Th e

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55 8r 9vqt , lsFEAt 78Table. Persisrence[ sr rot s * p.^, i ;;""', ;X,?'^:.ilci n esista sraiDr In sUt laCeSn v9lUj1gg1s.

Weeks after removal ofinfectedAF'4 6 8 t 6

Mona L. Svanbcrgand W. J. Loesche

The ,saliva and AF:: nf :';:;, t"' ,ffi'I#:::#',,'r.rll":.,f.y.rraT -erelaed in 2.5ml of RTF anJ norirrrt.,9l:1.0.0.05-rnrtisuors,;; ;p;;";;,.i i i i ,,o*were preadnmiris-salivariu,..;rrr..lnr_'iirsgf1Gotd.ordan nd an.Houre,sll) _dT;'ilN,rrosucro^segar SyedndLoebche,rzjy *iitr anj *i*r_out 0.2mgtnl srrepromycin.. ;no"uirrc1'"prrr.,wereplacedn ananacrbbi.r,.r*i *irr,ii i6 rrunof collecrionf samplesnd ncubaiedi"jz;i ro,48h in.anarmospheref Sjp.;';"i *-r, , o.r'on,H2 and 5 per cent CO,.ldentification of colonies of .Srrep.muranssuainson rhe various media was bascd o,i ,f,.ii .{ur""r"r_isric colonial morphology, supplernenredif ;.;rr"rywi hbiochemicatesrsS'trraii'aao.*",' r ili ""0y examinarion irh fluorescenl,iU"iy-Oi[.,.dagainst,he variousS rc ,-,1u2on, cr ypes Grenier,EveJandnd Loeschc,9Z3). heSr*p-'"lr_r-"r"r"were obrajncd rom MSB agar ana f-rn'-frl"frfOsucrose gar.Tlrecounts f rhisrrepromy"ir_*r;.i_,Strep.mutans ereobrainedrom de M'Mib';;"*agarwf1fi^5lyspromycin.The counrs f ir;;;.-ii,'r*,on .MM l0 sucrose garminus rh. "ounr".of '.Srr"p.mu ta n s o n MMJ 0 s u o o s c a p e r w i rh . , , - : ^ : . : :wereorsider.a,o.pi.?"Til.":3,"t'j"J"HffgenousS_rrep. utolrs.hc rotal .otony-iorr;n, 'rnucounrs CFU) wasobrainedro_ ri,. Vti'ii; ';;;r.ae.ar.

sterileAF was eft n sjrl for I and2 dayseachon]^f::i?"" tor 5 days.on r occasronsnd for 2ldayson I occasion,her'eafrert. .onr.nioi,ir. nrwas ulrured. hebacrerial.rnpr., ".i.'iri.oi.,}ytacedn reduedransponi,io nii.i'iil.a"."aLoesche, 1972)and dispersedUy sonincJtioi ;r';.(BransonmodelW lg5'D, N.y. l ----"-" 'v ' JB ac er o q ical .procedures

NESULTSlntrooral spread. f, 1 , lfs , conraining rhe srreprornycin-labelledslrarnsserved o inoculare hc orher ,oo,f, irrf ]"_whife rhe-vwere in viuo. Atter ,.ro".f 'oi',f,eJ';;*the srreprom.vcin-resisuntstrains of Sorp,.-rr_ion,couldbe derecred uring he nexl 26 ,n.Jr-Jn ; '; ; ;r-

SubjecrSubjecrSubject2 1 0 0l 0 6 7 8t 2 2 0 t 4 4

006'Artificial fissurc.t Numbcr of tooth surfaceswirh streptomycinresistanltrainsof Srrep. alans.ing numbcrof roorh 11rj:*: flable lI In subjccrG, ryo roorhsurfaces erecolonizeJ"iirrJz"*or,sampling bur rhereafs,:,,e,;,;;,,1il; :,,:Xl.:Tt::Ttaccswcre coloniacd aJrhcmpraniedffi ;-i,dtl.*f,frX':j1uiltzurfaccs6 weeks frer he.,.ro""i'll ii. ii. r"ubjccr,asmany s20-roorhuJ".., irj'I,?Lufstreptomvcin-resistantrernovarr he F. r,":'ij;l#;?:"tJTf""ffiafjlcenl nd.anragonisrjcecih""ty ;;;;; "rIJ,o.cof rhedcnririons thalon rrhich fr. aF f,t U"-inserredFi8s a-c).F1m 2 trrb.iC,'fil"r.) ,o6. subjecr , Fig tc) separare.oorh surfaesw34colonizedar some dme' by._rhe rp-r""i.i.s".pzurans,srrains.n subjecr] ffig fUiif"'rpr".-O roantagonisriceerh nclude.rtori' rhe'111.iif'_i'pr..molarswhich,because r a class-iit '""i#lr. i"r\were n occlusion lirh rhe m"noUut"i moL:ir,arconrainedheAF. All roorh urt"o, .-o-lonirJi1r,r,.implanred rrainalso "onr"jn.Ja.r;,.b1il;tl, "{ndogcnous trep.munns. n all ,rui.li'-rorn."rur.faceswhichcontajned noogenous rrep. rurarrsidnol become olonizedUy rhi implan;.i;;;:'". Tlre mplanred rrep.muranstrainG wasdercctcdin the salivary "-pl.s of suuiio"C ;;."j;r,period of rirnc rhan in plaqueL_pt,,--i...-r.,|iv"ry:1npLl wereposiriveS.weeksr,.i ,J.o".fli'rr,.AF, alrhough he baocria were ound ,n ,1. "oAsurfaces nly ar rhe 2 week ,".pfiru't i.UfJ'aTwenry-four onsecutive alvary samples olleqedfromsubjecr fiom weet9 ,.;;k ,;;;;.ffii".

( o ) S u b i . c t G 1 b ) S u b i . s rT ( c ) S u b i t c ? S

o /"U,F i r s u raAl . \\ { L )

I t | |\IYVY\J

Figs r(a--c) 'nrraoral pread I implanred:r" . .y:1^ in,subjccrs , T and s. Dorred ields. oorhurfacesolonized y impJanred trrp. r=,o^.

Fi$un

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strainslcers

Intraoral prcad f Srep.mutonsn man 559

Table2. Presence of strepfomycin-resistantStrep..-mutansn saUvasamPlesn relation to persistcnccof the' microorganisms n tooth surfaccs

t -4 Weeks fter emoval f theartificial issurc4-8 8-12 t2-t6 16-20 20-26

26 NDT29t

ND

NDI0

ND

NDt a

0ND

00

27 14t0 'l

006

No. of toothsurfacescolonizedPer cenl saliva samPlespositivei.Jo. of toothsurfacescolonizedPer cent saliva samPlesposidveNo. of toothsurfacts colonizedPer cent saliva samPlespositive

mycin-

840

624

l 92045

29l 44 t t

141ND

39ubiaweckssistantE sur-ieclS.roothrF. Inctablcaftcr:ad tore si&beenla) towcreStrep.ad toI Ptc..usion' lhatry thc:ls of: sur-rs did.cctodeg.'riv.rYf tltcloottllc 2).cdodlalivc

, I lump of sugareverywaking hour fo rI DailY f luoride nnses'l Not determlneo'for strain G. Subject G was lhcn placed on a higlt.*", t.rj-.n in which he ale a lump of suSarevery;;'kir;;;"r for ll davs' Twenty nine per cenl offti. oflt"tv samplesbecameposirive for the strepte,"dn-t.r ir,"nl sUain.When the frequent ucrosn'*iiio" *u, stoPp4 no streptomycin'rcsistantSlrep'L'r-i ,rttln. were detected n his salivary samples'H',fr. .tO of thc cxperiment' al wek 26' no toothrrtf"t-*.t. posirive or strain G' In subjectsT andi. L;fr ffuoride rinscs, 'e' 5 ml of 0'4per cent stan'ioil'6uo;a. and 5 mt of 0'02 Per cenl acidulated;;il;; ;roride (lradicvo, Janar Co.,. Grand[';"p"f,r,JtJ.| wereadministcrcd or a 4wecl periodi" IrO"t to climinatcor rcduce he implanted.strain'ia iuU5".r T, thc implantcd Strep' mutatn slraln was;;;;i in 4o pcr ccnt of thc salivary samplcs priori;ilil bur'dcscas"d to 14 Pcrccnl during rinsing.oa'*"ti", daectablcin cither thc saliva or on thclootb surfaccsat wocr z6 (Tablc 2)' ln-subjeo.S tbeil;J.-';;i"g did not climinatc thc implantcd,,;;il- .t " J,i saliva. and somc rooth samples;;J positivc durinr thc wholc crperirncntal;;-T# positivesaliv-ar.v amplcs in.all subjcas'*i. au. ro low lcvch of thc implantcd straln' r'c's td/ml.

After the establishmcnl f strain S on adjacentan danragonistic ooth surfacesn subject S, a sterilc AFwas nscrtedon multiple occasions nto the molar sitc.These AFs wcre removed at various time intcrvalsand the rario of streptomycin-resistantCFUs of Strep.murans o total Strep.mutanswas determincd for thcAF and for the saliva al the time of the removal ofrhe AF. After I day. approximately 90 pcr ccnt ofrhe snep. mvtansin thc AF were strcPtomycin rcsist'anl whereas onlv 0.1 Der cenl of the .Snea..muto sin the saliva were resislanl o slrcpromycin (Tablc 3) 'Al Z 5 and 2l days, thc proportions of strcptomy-cin-resislantstrains decreastd in thc AF. but thcseDroporlions were alwaysal least lOGfold highcr thanih.'.orr.rponding ratio of these organisms in thesaliva

DlscussloNln alt subjcos, be inttaoral spread of the impla-ntcdSfteD.murorsoccurred only on the samc sidc of thedeniition as thal on which the implanutions wcrcmade.The ertcnt of the ntraoral spread varicd widclyin rhc ttirec test Personsr hich may be due lo a var-ietv of host diet and microflora factors.The dissimi-larity of the hosts s illustratcd by thcir diflerenl carics

Tablc 3. Colonization of stcrilc artificial fissures AF) by Snep' nwto'rs'/" Strepromycin-resistanlSoep. rmnans. f total'iorp.'r*tont CFU count in AF and in saliva

Daysaficrinscrtionof AF pr cfnlA F

Salivaper ctnl

I)5

) l

n = 2 8 7 . 0 , 9 0 . 0n = 2 9 3 . 5 , 0 . ?n = 3 33.0, 0.7, 0.5n = I 1 1 . 5Range 0.5-93.5er ent

n = 6n = 1 0n = 5 )n = 1 6

ave= 0. 10.10. t0.m40.0-1.5 et ctnt

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erperience. umberof retenlion iles uchas marginsof fillings and salivary Srep. mutons onccntrations.Subject S had a high number of filled toorh surfaccsand a high salivary concntrarion of endogenousSlrep.murons trains..e. 0o CFU per ml (Svanbergand Loesche,1978). ubjecrG had a lower cariesex-- perienceand a low salivaryconcenlralion f endogenous Strep.mutans trains..e.about l0r CFU pe rml. and subjectT was nrermediaren regards o theyanous paramclcrsunder discussion Svanbcrg ndLoesche,1978) .The importancr of dietary sucrose n raising thesalivaryconenuationso[ the mplantedStrep.mutonswas demonslrated n subject G, when this subjectwenl on a dietary regimenof frequentsuoose inges.tion. The implanted strain G, which had been unde-lecled in 24 consecutive alivary samples, e-emergedin the salivary flora- showing that a person can bea carrier of Strep. mutonsat undetectable cvels inplague samplescollected rom rooth surfacesusingdental floss and .sharp needleqas well as in salivasamples. This finding confirms earlier observations(Svanberg and Loesche,1978) hat a Srep. milons-infectcd rctenlion sitc, such as an AF, often mayremain undetectedby the sampling rechniques tpresnt available.Tlie implanted Strep.nutonsslrains spread o adja-cent and antagonistic ooth surfaces. he sprcadwasdetectedonly on the side of the dentirion where theimplantations were made, confirming the observa-tions of Edman et al. (1975\ This suggests hal lhenumber of streptomycin-resistanlCFU of Srrep.muransshed rom the AF is so low rhat they becomediluted in the safiva so lhat their chance to makeconlact with teethon the orher side of rhe denrir ionis slight. We have shown (Svanberg and Loesche,1978) bat AFs containingmore rhan 103 CFU ofrhe labefledSrrep.mutans hed102-103 FU of thescorganismsper ml of saliva.The sal iva conrainsaboul106 CFU of various bacteria per ml, u,hich meansrhat in homogenized holesalivaabout one in every100.0m to 1,00,000 organisms would be of thelabelledstrain. Homogenizedwhole saUva-however,is an art ifactof our culturingprocedure.n uiuo. hesaliva lowingover the sidecontaining he AF wouldbe enriched for the streptomvcin-resislantstrains,whereas the saliva flowing over the opposite sidewould mosl l ikcly havc no slreptomycin-resistantstrains.The higber salivaryconcentrat ions f Strep.murunson the implanted sidewould favour unilateralsprcad of tbe labelled organisms.This was so *'hensrerileAFs were insefled n subject S after thc cstaLlishmentof the implanted Srrep. mutans on adjaent

and antagonistic teeth. The local spread of Snep.mulansstrains was il lustrated by the relat ionshipbetween he percentageslrepromycin-resistantSrrep.muuns ol total Srrep.mulansCFU counls in the AF,i.e. 10.5-93.5per cent and in the salivary flora- i.e.0.O-1.5per cent. The salivary concentrationsof theimplanted Step. mutanswere on all occasions owerrhan those found previously to be associaredwithStep. mutanscolonization of a sterile AF (Svanbertand Loesche, 978)and of a smooth tooth surfaa(vanHouteand Greene,1974). he shedding f Srrep.murans trains rom infectedareasprobably increasesthe local salivaryconcentrat ionso such an ertenl

MonaL. Svanbcrg nd W. J. Locsche

REFENf,NCESEdman D. C., Kecnc H. J.. Shklair l. L and HK. C. 1975.Dcnral ffoss for implanrarion andol Streptococcusmuroas from approximal suhuman reerh. Archs orol Biot.2$, ia lta8.Gold O. G., Jordan H. V. and van Houlc J. l9?3tive medium tor Sreptococcus ,nuraas. Arcls or & 1 3 5 7 -1 3 6 4 .Grenicr E. M.. Eveland W. C. and Lpeschc W.ldentification ol Streptocrccusmuutns rotypcsplaque by fluoresccnt anribody rechniquci. ABiol. l& 707-r-15.Joldar H. V.. Englander H. R., Englcr W. A. andS. 1972.Observarions on rhe implanration and tsion of Srreprococcusmutons n humans. J. d5 t . 5 t 5 - , 1 1 8 .Keyes P. H. 1962. Rcccnl advanccs in dentarescarch.Boct. lnt. dent. J. 12,443-448.Kcyes P. H. and Fiugerald R. J. 1962. Denralthe Syrian hamsrer IX. z{rc}rs ral Biol.1. Z67-Keycs P. H. 1968.Research in dental carics. J. ,4ss.?6. I 35?- I 3?3.Krassc 8.. Edwardsson S.. Svensson . and Trclllmplantation of caries inducing srreprococchuman oral cavi ly . 4rc ls oral Bio l. 1 231-21Krasse B., Jotdan H. V.. Edwardsson S.. SvenssTrell L. 1968.Ttre occurrencc of cerrain .'cariing" srreprococcin human denral plaque marerio ra l B io l .1 3 .9 l l - 9 1 8 .Lit H.. Karring T. and Thciladc E. t973. Anmethod for the srudy of the microbiology of f issures. or ies Res.?, 120-129.Loesche W. J., Rowan J., Srraffon L. H. and Lof975. Associati on of Srreprococcusmurons withdental dccay. nfecr Immun. l , 1252-1260.Shklair l. L. and Xeene H. J. l9?4. A biochcmicalfor the separation of thc five varietiesof Snepmutouts. rchsorol Bio l . t9 , 1079-1081.Svanberg M. and Loesche W. J. 1977. Salivarytrations of Streptococcusmutans and Sueptococguis and the colonization of anificial fissurcsby these organisms. Arcls oral Biol.77, 441447Svanberg M. and Loesche W. J. 1978. mplanraStreptococas murons on l ooth surfaccs in mano ro l B io l . 2 3 , 5 5 1 -5 5 6 .Syed S. A. and Loesche W. J. t972. Survival of dental plague flora in various rransport mediMicrobio l. U.638-#4.

compared o whole saliva that colonizationcentandantagonistic eeth s favoured. t is,halso possible thal con_tactbetween, eethenhanceshe spread of Strep.murans trainsln all subjecrs,all rooth surfaccs that hathe implanted Strep. mutottsstrains were aonized by srreptomycin-sensitivestrains omutons. he streptomycin-scnsitivc trep.murpresumablyof endogenous9rign, as lhe strcin-resisranlmarker is stable jn uiuo (SvanLocsche,1978).This indicarcs hat srrains GS cocxistcd or a rime witb thc endogcnouof Strap. mutons.Acknowledgemenrs-Thisork was supportcd. Public Hcalrh Granrs Nos. DE-02?3t. DE-03DE{,3423 rom rheNarional nsrirurc f DcnralRDr. Thorkild Karring gencrously rovidcdsomAFs.

560