F=

!
0m1.9969:7E.0?0t 4r57t0t.00;0

I'1

l:tntu not biot. vol. !1. pp J5? lo 5'61' Pr6t Ltd iils ninrcd in o'Ger Erirrin ;';;;" i !r t' i

:l

MUTANS I N T R A OR A L S P R E A DOF STREPTOCOCCUS IN MAN

Mot*le L. SvrNsERct and W. J. L.oEscHr

r" t""l;f .ou#:'il?;! esea uI R Den

#:'|1tT'

ofDentistrv' schoor

murars were reliably establisbedin Summary-Streptomycin-resislanl strains of Streptococans (AF) containing adherentgrowlh oflhe Strep.4utons 3 subjecti by the use of artifical fissures strain. The intraoral spread of theseimplanted Strep.mutansstrainswas monjtored in lhe subjecrswhile 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 teeth only on the side of the dentition where of a streplomycin-resistanl strain in one the implantations were made. Afler establishment subicct. a sterile AF was inserted multiple times in the same occlusal surfacr. on which the imptantations had previously been madc. The srerile AF was colonized by local spread of of streptomycin' Snep.murcnsstrains, as was shown by the relationshipbetweenrhe percenlage (CFU) mutans lotal Strep. colony forming units counts in the AF to mutans Snep. resijgnt Strep. mutansrepresented 10.!93.5 per cenl and thc salivary ffora. The streptomycin-resislanl of rhc total Strep. mutons CFUs in the AF, but only G-1.5 per oent o[ the Strep. mutans and spread CFUs in rhe saliva during colonizalionof the AF. The intraoral establishment of the implanted Slrep. mutons seemedfavoured by conditions presnt in subjectswith high murans strains and high caries experience.as sljvarv concentralions of endogenousStr.ep. .rpr.r*d by rhc high number of filled loolh surfaces.

streptomycin was placed in a molar looth in each subject.The AFs had been cultured in vitro and conznl'ons has tained at the time of insertion l0? colon1,f6v6l1g Thc cariogenic Potential of Streptoco.ccus (Keyes. units (CFU) o[ Strep.mutansas adherent growth on models animal in demonstraled bccn clearly l%Z l968iand some role for this microorganism in the AF (Svanbergand Loesche,1978).The AFs were denral carics in man seems evidenl {Krasse et al', each inserted lhrce times in the occlusal surface of an upper first molar in subjcct G and in a lower l96E; Lcschc et al., 1975\ first molar in subjectsT and S. The implanted Strep. Scvcrat attempts have been made lo study the in' traoral ecology of Slrep' nutons by the implantation rnutonsstrains G. T and S belonged lo serotype c in thc human mouth of Strep' mutanssltalns contaln- and wcre re-inlroducedinto the same mouth from inr. for identificatiol purPoses'a streptomyon'resls- which they originally had beenisolated(Svanbergand rait marler (Krasse et at.' 1967: Jordan et al', 1912; L o e s c h e . 1 9 7 8 ) . Edman et ol.,1975 Svanbergand Loesche'1978| but Bacterial somples most labellcd sltains were quickly ebminated from established labelled The AFs containing strains G, T and S were left rbc mourh. Edman et al. 11975) in uivo tor 7-12 days. Al 2, 4. 6, 8 and 26 weeks suains in approximal siles on one side of the mouth and noted thal the implanted afler removal of the infectedAF. plaque sampleswere in two "oiunt..tt collectedfiom all surfaccsof all teeth in each subject. strains were mainly recovered from that sidc' The Twenty-sir approximal toolh surfaceswere sampled utificial fissure model (Liie. Karring and Theiladc' l9?3) can bc used to rcliably implant .slrcPromycin- with dental floss and 56 smoolh tooth surfaccs with (Svanbergand ffattened hypodermic needles. Twenty-eight plague ,oitiun, strains in singlc occlusalsitcs samplesfrom margins of fillings and fissureswere col' Locschc, l9?8). lected with 26-gauge hypodermic needles. The sampleswere laken in the morning after the subjects MATENIAIS AND METHODS had refrained from all oral hygiene on the eveninS Subjects and morning before sampling. Two to four salivary who participated The rhree subjects(G, T and S) sampleswere collected per week during this 26week :ad 38, 52 and i8 filled tooth surfaceq respectively' period. G leelh' Subjects ro carious lesions and no missing Colonizationof sterile AFs rnd S had a normal occlusion and subject T had r classlll malocclusion. on the nalural tooth surfaces Strain S established a Strep'mutans (AF) containing An artificiaffissure subject S for a prolonged period of time. During in lo I m8/ml of ;train which had been made resistant this interval.sterile AFs were insertedseveral times in the same toolh as that used earlier for the AF Odontologiska ' hescnraddress: universitct. Giireborgs containing adherent gro*'th of 5trep. ntutans. The Sweden 33' tliniken.Fack4ffi. 33 Giireborg INTNODUCTION 55?

- 6?

tt 78. 9vqt,lsFEA
558
Mona L. Svanbcrgand W. J. Loesche

Loesche, 1972)and dispersed Uy sonincJtioi ----"-" 'v' ;r';. J (Branson model W lg5'D, N.y.l B acterio lq ical .pr ocedures The ,saliva and AF

sterileAF wasleft jn sjrl for I and 2 dayseachon tor 5 days.on r occasrons and for 2l ]^f::i?"" days on I occasion, wher'eafrer rt. .onr.nioi,ir. nr wasculrured. The bacrerial ,.rnpr., ptaced in redued rranspon ni,io".i.'iri.oi.,}y

Table l. Persisrence o[ srr n-resista nt sr raiDs ot st* p. ^, i i;;""', ;X,?'^:.ilci rIn sUtlaCeS in v9lUj1gg1s.
Weeks after removal of infected AF' 4 6 8 t

fnii.i'iil.a"."a

fissurc. 0.05-rnr atisuors f,;; ;p;;";;,.iiii,,o* 9l:1.0. t Numbcr of tooth surfaceswirh were spread on miris-salivariu, streptomycinu..;rrr..ln .r_'iirsgf resistanl strainsof Srrep. Jordan 1Gotd. nalans. andvan.Houre, lsll) _dT;'ilN,rro sucro^se agar(Syed andLoebche, rrzjy *iitr anj *i*r_ ing numbcr of roorh out 0.2 mgtnl srrepromycin. flable lI In subjccr n. ;no"uirrc1'"prrr.,G, ryo roorhsurfaces 11rj:*: wereplaced were colonizeJ"iirrJz"*or, in an anacrbbi. .r,.r*i *irr,ii i6 rrun sampling bur rhereaf of collecrion of samples and incubaied ,i"jz;i ro, 48h in.anarmosphere .f Sjp.;';"i *-r, , o.r'on, taccs wcre coloniacd aJ H2 and 5 per cent CO,.
ldentification of colonies of .Srrep. murans suains on rhe various media was bascd o,i ,f,.ii .{ur""r"r_ isric colonial morphology, supplernenred if ;.;rr"ry

s.y.rraT w-ere plaed in 2.5ml of RTF anJ

:'I#:::f#',,'r.rll":.,f r,ffi ::-nf :';:;, t"' 'Artificial norirrrt.,

Subjecr G Subjecr T Subject S

2 1 0 l0 6 t 2 2

0 7 0 t

0 4 8 4

0 0 6

s,:,,e,;,;;,,1il;!:,,:Xl.:Tt::TjtJi;i

S, Fig tc) separare. loorh surfaes w34 colonizedar some dme' by._rhe irp-r""i.i.s".p zurans,srrains. ln subjecr ffig f Uiif"'rpr".-O ro ] antagonisric reerhinclude.rt bori' rhe'111.i iif'_i'pr.. molarswhich,because or a class-iit'""i#lr.i"r\ were in occlusionrlirh rhe m"noUut"i were corsider.a,o r.pi.?"Til.":3,"t'j"J"Hff moL:ir,ar 'rnu conrained rheAF. All roorh.urt"o, .-o-lonirJi1r,r,. genous S_rrep. mutolrs. ihc rotal .otony-iorr;n, implanred srrain also counrs{CFU) wasobrained fro_ ri,. Vti'ii;';;;r. "onr"jn.Ja.r;,.b1il;tl, Strep. gndogcnous munns. ae.ar. ln all ,rui.li'-rorn."rur. "{ faces which contajnedenoogenous Srrep.rrurarrs did nol become colonizedUy rhi implan;.i;;;:'" NESULTS Srrep. murans strainG wasdercctcd . Tlre implanred lntrooral spread in the salivary,"-pl.s of suuiio"C ;;."j;r, f,!1 ,lfs conraining rhe srreprornycin-labelled period of rirnc rhan in plaque L_pt,,--i...-r.,|iv"ry . , slrarnsserved to inocularerhc orher ,oo,f, :1npLl were posirive S.weeks,r,.i ,J.o".fli'rr,. 'oi',f,eJ';;* irrf]"_ AF, alrhoughrhe baocria were found whife rhe-vwere in viuo. Atter ,.ro".f ,n ,1. l"oA surfaces only ar rhe 2 week ,".pfiru'ti.UfJ'a the srreprom.vcin-resisuntstrains of Sorp,. rr_ion, Twenry-fourconsecutive could be derecred salvary samplescolleqed during rhe nexl 26 ,n.Jr-Jn ;';;;rfrom subjecr G fiom weet 9 ,.;;k ,;;;;.ffii".
(o) Subi.ctG 1 b ) S u b i . s rT
(c) Subitc? S

by examinarion wirh fluorescenl ""0 _,iU"iy-Oi[.,.d against, the variousStrcp, scr o types (Grenier, -,1u2on, EveJand and Loeschc, I9Z3). The Sr*p-'"lr_r-"r"r" were obrajncd from MSB agar ana f-rn'-frl"frffO sucrose agar.Tlrecounts of rhisrrepromy"ir_*r;.i_, mutans Strep. wereobrained from de M'Mib';;"* agarwf1fi^5lyspromycin. The counrs.f ir;;;.-ii,'r*, '.Srr"p. on MM l0 sucrose agar minus rh. . m u t a n s o n M M J 0 s u o o s c a p e r w i "ounr".of rh .,,-:^:.::

wirh biochemicat resrs r raii'aao lS'tr i.*",'

riili

streptomvcin-resistant

zurfaccs 26 weeks afrerrhe.,.ro""i'll ii. ii. r" subjccr S,asmany as20-roorh ,uJ".., irj'I,?Luf

rhc impranied ;ffi ;-i,dtil.*f,frX':j1uilt

"rIJ,o.c afjlcenland.anragonisrjc recih ;;;;; of rhedcnririon ""tyrfr. aF as thal on rrhich f,t U"inserred (Fi8s ta-c). F1m 2 trrb.iC,'fil"r.) ,o 26.(subjecr

rernovar orrhe AF. rr,":'ij;l#;?:"tJTf""ffi

o
A
l . \

Firsura

/"U,
Fi$un

\{
I t |

L)
|

\IYVY\J

Figs r(a--c)' Inrraoral spreadoI

implanred t:r"!..y:1^ in,subjccrs G, T and s. Dorredfields. roorh surfaces colonized by impJanred Strrp. ^r=,o^.

63
A

in man Intraoralsprcadof Srep.mutons strains lcers

559

in relation to persistcncc of the in saUvasamPles Strep..-mutans of strepfomycin-resistant Table2. Presence ' on tooth surfaccs microorganisms

t-4

of the artificialfissurc Weeks afterremoval 16-20 20-26 t2-t6 8-12 4-8

26 0 0 6
mycin-

rubia
wecks sistant

No. of tooth surfaces colonized Per cenl saliva samPles positive i.Jo. of tooth surfacescolonized Per cent saliva samPles posidve No. of tooth surfacts colonized Per cent saliva samPles positive , I lump of sugar every waking hour for I DailY fluoride nnses' l Not determlneo'

NDT
27 t0 14 'l 8 40
29t ND

NDI 0 ND

0 ND 0 0

l9 20 45

29
l4 4tt

141 ND

ND
ta

24

39

E suriecl S.
rooth rF. In ctablc aftcr :ad to re si& been la) to wcre Strep. ad to I Ptc.. usion ' lhat ry thc :ls of : surrs did .cctod eg.' riv.rY f tltc loottl lc 2). cdod lalivc

was lhcn placed on a higlt for strain G. Subject G he ale a lump of suSar every which in .*", t.rj-.n Twenty nine per cenl of davs' ll for ;;'kir;;;"r posirive for the strepte became samples fti. oflt"tv ln' When the frequentsucros sUain. ,"dn-t.rir,"nl Slrep' streptomycin'rcsistant no stoPp4 *u, *iiio" samples' ,rttln. were detected in his salivary L'r-i wek 26' no tooth H',fr. .tO of thc cxperiment' al posirive for strain G' In subjectsT and rrtf"t-*.t. per cent stan' i. L;fr ffuoride rinscs,i'e' 5 ml of 0'4 acidulated cenl of 0'02 mt 5 and Per ioil'6uo;a. (lradicvo, Janar Co.,. Grand ;roride ;;il;; were administcrcd for a 4wecl period [';"p"f,r,JtJ.| the implanted.strain' i" IrO"t to climinatc or rcduce Strep' mutatn slraln was implantcd thc T, ia iuU5".r samplcs prior ;;;;i in 4o pcr ccnt of thc salivary rinsing during ccnl to 14 Pcr bur'dcscas"d i;ilil thc saliva or on thc cither daectablcin .oa'*"ti", (Tablc 2)' ln-subjeo.S tbe lootb surfaccsat wocr z6 climinatc thc implantcd not did il;J.-';;i"g " somc rooth samples and saliva. ,,;;il- .t J,i thc wholc crperirncntal durinr positivc ;;J samplcs in.all subjcas positive saliv-ar.v ;;-T# '*i. straln' r'c' au. ro low lcvch of thc implantcd s td/ml.

of strain S on adjacent and After the establishmcnl anragonisticlooth surfacesin subject S, a sterilc AF was inscrtedon multiple occasionsinto the molar sitc. These AFs wcre removed at various time intcrvals CFUs of Strep. and the rario of streptomycin-resistant muransto total Strep.mutans was determincd for thc AF and for the saliva al the time of the removal of rhe AF. After I day. approximately 90 pcr ccnt of rhe snep. mvtansin thc AF were strcPtomycin rcsist' anl whereas onlv 0.1 Der cenl of the .Snea..muto s in the saliva were resislanl lo slrcpromycin (Tablc 3)' Al Z 5 and 2l days, thc proportions of strcptomycin-resislant strains decreastd in thc AF. but thcse Droporlions were always al least lOGfold highcr than ih.'.orr.rponding ratio of these organisms in the saliva DlscussloN ln alt subjcos, lbe inttaoral spread of the impla-ntcd SfteD.murors occurred only on the samc sidc of the deniition as thal on which the implanutions wcrc made.The ertcnt of the intraoral spread varicd widcly which may be due lo a varin rhc ttirec test Personsr ietv of host diet and microflora factors. The dissimilarity of the hosts is illustratcd by thcir diflerenl carics

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

Days aficr inscrtion of AF ) 5

) l

AF
pr cfnl

Saliva per ctnl

87.0,90.0 n=2 9 3 . 51 , 0.? n=2 , 0.5 , 0 . 71 n = 3 3 3 . 01 11.5 n= I Range10.5-93.5 Percent

n = 6 n=10
n = 5 ) n = 1 6

ave = 0.1 0.1 0.t

0.m4

pet ctnt 0.0-1.5

b4

560

Mona L. Svanbcrg and W. J. Locsche

erperience. number of retenlionsilessuchas margins comparedro whole saliva that colonization of adia, of fillings and salivary Srep. mutonsconccntrations. cent and antagonisticteeth is favoured. lt is, howevcr, Subject S had a high number of filled toorh surfaccs also possible thal con_tact between, teeth grcatly and a high salivary concntrarion of endogenous enhances the spread of Strep. muransstrains. Slrep.murons strains. i.e. l0o CFU per ml (Svanberg ln all subjecrs,all rooth surfaccs that harboured and Loesche,1978). SubjecrG had a lower cariesex- the implanted Strep. mutottsstrains were also col. - perienceand a low salivaryconcenlralion of endo onized by srreptomycin-sensitivestrains of Slrep. genous Strep. mutans strains. i.e. about l0r CFU per mutons. The streptomycin-scnsitivc Strep. murals were ml. and subjectT was inrermediare in regards ro the presumablyof endogenous 9rign, as lhe strcptomy. yanous paramclcrsunder discussion (Svanbcrg and cin-resisranlmarker is stable jn uiuo (Svanbcig and Loesche,1978). Locsche,1978).This indicarcs that srrains G, i anO The importancr of dietary sucrose in raising the S cocxistcd for a rime witb thc endogcnous strains salivaryconenuations o[ the implantedStrep. mutons of Strap. mutons. was demonslrated in subject G, when this subject wenl on a dietary regimen of frequent suoose inges. Acknowledgemenrs-This work was supportcd by U.S. tion. The implanted strain G, which had been unde- . Public Hcalrh Granrs Nos. DE-02?3t. DE-030i1 and lecled in 24 consecutive salivary samples,re-emerged DE{,3423from rhe Narional Insrirurc of Dcnral Rcscarch. salivary the florashowing that a person can be Dr. Thorkild Karring gencrously in providcd somc of thc AFs. a carrier of Strep. mutonsat undetectablelcvels in plague samples collected from rooth surfacesusing dental floss and .sharp needleq as well as in saliva samples. This finding confirms earlier observations (Svanberg and Loesche, 1978) rhat a Srep. milonsREFENf,NCES infectcd rctenlion sitc, such as an AF, often may Edman D. C., Kecnc H. J.. Shklair l. L and Hocrmal remain undetectedby the sampling rechniquesat K. C. 1975. Dcnral ffoss for implanrarion and satnplin3 presnt available. ol Streptococcusmuroas from approximal surfaccs of Tlie implanted Strep.nutons slrains spreadro adjahuman reerh.Archsorol Biot.2$, ialta8. The sprcad was Gold O. G., Jordan H. V. and van Houlc J. l9?3. A scteccent and antagonistic looth surfaces. tive medium tor Sreptococcus ,nuraas. Arcls orol Biol. detected only on the side of the dentirion where the r& 1357-1364. implantations were made, confirming the observations of Edman et al. (1975\ This suggeststhal lhe Grenicr E. M.. Eveland W. C. and Lpeschc W. J. l9?3. ldentification ol Streptocrccus muutnssrotypcs in denul number of streptomycin-resistanl CFU of Srrep. plaque by fluoresccnt anribody rechniquci. Archs ool muransshed from the AF is so low rhat they become Biol. l& 707-r-15. diluted in the safiva so lhat their chance to make Joldar H. V.. Englander H. R., Englcr W. A. and Kulcyzt conlact with teeth on the orher side of rhe denririon S. 1972.Observarions on rhe implanration and transmrs. We (Svanberg have shown and Loesche, is slight. sion of Srreprococcus mutons in humans. J. denr. Res. 5t.5t5-,118. 1978) tbat AFs containing more rhan 103 CFU of mutans shed 102-103 rhe labefledSrrep. CFU of thesc Keyes P. H. 1962. Rcccnl advanccs in dental caries rescarch. Boct. lnt. dent. J. 12,443-448. organismsper ml of saliva.The saliva conrains aboul 106 CFU of various bacteria per ml, u,hich means Kcyes P. H. and Fiugerald R. J. 1962. Denral caries in the Syrian hamsrer IX. z{rc}rsoral Biol.1. Z67-27E. whole salivaabout one in every rhat in homogenized Keycs P. H. 1968. Research in dental carics. J. AnL aeil. 100.0m to 1,00,000 organisms would be of the ,4ss.?6. I 35?- I 3?3. labelled strain. Homogenized whole saUva-however, Krassc 8.. Edwardsson S.. Svensson l. and Trcll L. 196?. ln uiuo.the is an artifact of our culturing procedure. lmplantation of caries inducing srreprococci in the flowing over the containing would side the AF saliva human oral cavily. 4rcls oral Biol. l1 231-216. be enriched for the streptomvcin-resislantstrains, Krasse B., Jotdan H. V.. Edwardsson S.. Svensson I. and Trell L. 1968. Ttre occurrencc of cerrain .'caries-induc. whereas the saliva flowing over the opposite side would mosl likcly havc no slreptomycin-resistant ing" srreprococciin human denral plaque marerial. lrcis o r a l B i o l .1 3 . 9 l l - 9 1 8 . of Strep. strains.The higber salivary concentrations Lit H.. Karring T. and Thciladc E. t973. An in rirp murunson the implanted side would favour unilateral method for the srudy of the microbiology of occlusal sprcad of tbe labelled organisms.This was so *'hen f i s s u r e sC . o r i e s R e s .? , 1 2 0 - 1 2 9 . srerileAFs were insefled in subject S after thc cstaL Loesche W. J., Rowan J., Srraffon L. H. and Loos p. J. lishment of the implanted Srrep. mutans on adjaent f 975. Association of Srreprococcusmurons with human and antagonistic teeth. The local spread of Snep. d e n t a l d c c a y . I n f e c r I m m u n .l l , 1 2 5 2 - 1 2 6 0 . mulans strains was illustrated by the relationship Shklair l. L. and Xeene H. J. l9?4. A biochcmical schemc for the separation of thc five varieties of Sneprococors between lhe percentageslrepromycin-resistantSrrep. m u t o u t sA . r c h so r o l B i o l . t 9 , 1 0 7 9 - 1 0 8 1 . muuns ol total Srrep.mulans CFU counls in the AF, i.e. 10.5-93.5per cent and in the salivary flora- i.e. Svanberg M. and Loesche W. J. 1977. Salivary concentrations of Streptococcusmutans and Sueptococas sn. 0.O-1.5per cent. The salivary concentrations of the guis and the colonization of anificial fissurcs in man Step. were mutans on all occasions implanted lower by these organisms. Arcls oral Biol.77, 441447. rhan those found previously to be associared with Svanberg M. and Loesche W. J. 1978. lmplanrarion of Step. mutanscolonization of a sterile AF (Svanbert Streptococas murons on looth surfaccs in man. ,4rclrr and Loesche,1978)and of a smooth tooth surfaa orol Biol.23, 551-556. (vanHoute and Greene,1974). The sheddingof Srrep. S y e d S . A . a n d L o e s c h e W . J . t 9 7 2 . S u r v i v a l o f h u m a n dental plague flora in various rransport media_ AppL strains from infected areas probably increases murans Microbiol. U.638-#4. the local salivary concentrationslo such an ertenl