Longitudinal survey of the distribution of various serotypes of Streptococcus mutans in infants

0095-1137/79/10-0497/06$02.00/0

Longitudinal Survey of the Distribution of

Various Serotypes

of Streptococcus

mutans

in Infants

NORIO MASUDA,' NOBUO TSUTSUMI,1 SHIZUO SOBUE,' ANDSHIGEYUKI HAMADA2*

Department ofPedodontics' andDepartment of OralMicrobiology,2 Osaka UniversityDentalSchool,

Nakanoshima, Kita-ku,Osaka,530Japan

Received forpublication16July1979

Theestablishment ofvarious serotypesof Streptococcusmutans wasstudied

serologically in plaque samples collected from labial surfaces ofupperprimary

incisors of 22 infants (startingage, 5 to 13months) over aperiod of30months.

Clinicalexaminationswerealsoperformed. No clear-cut association between the

initiation of dental caries and previous detection of S.mutanswasnoted. However,

all 12oftheinfants with caries had S. mutans isolatedatsome time during the

courseof thisstudy. Themostcommonserotypeisolatedattheinitial establish-mentofS.mutansonthetooth surfaceswasserotypec, whereastypesd,e,and

g became established in a few cases. During the test period, changes in the distribution ofserotypesof S.mutanswereobserved insome cases.Theinitiation

of carious lesions could be found inafew cases evenwhenS.mutanscomprised about 1%orless of the totalstreptococcalcountof the specimen from thetooth surfaces. Serotype

dlg

strains tended to develop carious lesions on smooth

surfaces, although serotype c was isolated from almost all individuals who

developed caries.

Streptococcusmutanshasbeenconsidered to

be the mostimportant etiologicalfactor ofdental

caries induction in experimental animals and

men (11, 12, 17). Bratthall (2) classified strains

of S. mutans into at least five distinct types

designated a, b, c, d, and e according to their

serological specificities. Perch et al. (15)

dem-onstratedtwoadditionalserotypesfand g.

Sev-eral investigators showed a geographic

differ-enceinthe distributionofthe various serotypes

orbiotypes of S.mutans (13,16,20). We reported

that serotype c strains were predominant and serotypesd andewerealsousually isolated,but serotypes a and b were notfound in Japanese children (10).

Whetheror notserotypesofprevalent S.

mu-tansstrains vary insubjects overtime remains

tobeelucidated.Therefore,alongitudinalstudy

was undertaken of the distribution of various

serotypes ofS.mutans onthetoothsurfaces of

infants. Identification of theserotype ofS.

mu-tansatinitial

establishment, changes

in

predom-inant serotypeswith

time,

and incidenceof car-ies weremainly noted.

MATERIALS AND METHODS Studypopulation. The initialstudy population consistedof22 subjects (13boys and 9girls) whose

agesrangedfrom 5to13months,witha meanageof 10monthsatthestartof thestudy.Allsubjectswere under care in a day nursery. They had no visually

detectable cariesatthe initiation of the survey. Clini-cal examination andplaquesamplingwereperformed

atotal of nine timesat 3-to4-monthintervalsover30

months. At the final examination, 30 months later, population mobility reduced the study populationto 17infants, and7of themstill remained caries free.

Sample collection and bacteriological proce-dure. Plaque samples were obtained using a sterile

excavatorfrom thelabial surfaces of the upper central incisors. When the upper incisorswere notavailable, the lower incisors or gingival ridge were used for sampling. Immediatelyafter the collection ofplaque samples, they were homogenized in 2 ml of sterile salinecontaining0.1% yeastextractbyultrasonication for 30 s, and 10-fold dilutions were made. A0.1-ml volume of the appropriate dilution wascultured on

mitis salivarius agar (MS agar; Difco)

plates

and on

MS agarsupplementedwith 15%sucroseand0.2Uof bacitracin per ml(MSBagar) (8)at37°Cinan

atmo-sphere of 95% nitrogenand 5% carbon dioxide for24 h. MostS. mutansgrownonMSorMSB agarplates,

or both, could be recognized on the basis of their

colonial morphology. To confirm the identity of S. mutans, thesynthesisofadherentextracellular

poly-saccharide fromsucroseandthefermentation of

man-nitoland sorbitolweretestedonpure culture isolates (7,9).

Colony-forming units ofS. mutans were enumer-ated and expressedasa percentage of total strepto-cocci (total colony-forming units on MS agar plate

culture). Classification was made by the following criterion: -, 0; +,<0to1; +,>1 to10; ++,>10to50; +++,>50 to 100%of totalstreptococcalcountwasS.

mutans. When the numberofS.mutansonMSagar

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498 MASUDA ET AL.

plate culture was very low orzero, the MSB plate

agar,which ishighlyselective medium for S. mutans,

wasused.

Serological procedure. Serotype-specific anti-sera wereprepared bythe techniques described pre-viously (10).Heat-killed cells of S.mutansE49 (sero-type a), BHT (b), Ingbritt (c), B13 (d), MT703 (e), SEll (f), andOMZ65 (g)wereusedasvaccines.Ten colonies resembling S. mutans on MS or MSBagar plates from eachsubjectwererandomly selected, sub-cultured on Trypticase soy agar(BBL Microbiology Systems), andchecked for purity. Crudeantigen ex-tractsfromthe isolated strainswerepreparedas pre-viouslydescribed (10). The antigenextracts of

refer-ence strains of S. mutansE49, BHT, Ingbritt, B13, MT703, SE11,andOMZ65werealsoprepared. Using adiffusioninagargeltechnique, antigenextractsof S.

mutansstrains weretested andserotyped by the

re-action ofidentitywith the referenceantigenextracts (10).

Dental examination. The incidence of caries in theuppercentralprimary incisorswasexamined

clin-ically byasingle examinerusinga mouthmirror and

probe. Noradiographic examinationwasmade. After

cleaninganddrying,the cariousdegreeofthesurface was classified into three categories accordingto the depthofcariouspenetration:"sound,"nopenetration;

"grade 1," enamel surfaces showed a white opaque lesionor there was a break in the continuity ofthe enamelperceptible withaprobe; "grade 2," the surface

showedashallow dentin caries.

RESULTS

Changes in the prevalence of S. mutans

with time. Changes in the number of S. mu-tansdetected in theplaquesamples during the

test period were tabulated in Table 1. During

thecourseof thestudy, S. mutanswasdetected

in 20 of 22subjects. At 0-, 4-, 7-, and 11-month

samplings, S. mutans strainswere isolated less

frequently. Once S. mutans wasestablishedon

the tooth surface, the number of S. mutans increased with time. But this tendencywasnot necessarily observed in all subjects; in some cases,especially those with low levels of S.

mu-tans, wide fluctuations were noted. Nine

chil-dren, i.e., 40% of the subjects, were found to

harbor S. mutans by theage of 2years. Atthe 30-monthexamination, 17 subjects were

exam-ined, and 4 of them were found to harbor S. mutans atlevels ofmorethan 50% of their total

streptococci. Themeanpercentage ofS.mutans

of totalcultivablestreptococciin the 17subjects

atthis timewas 14%(range, 0-93%).

Distribution of various serotypes of S. mutans. Table 2 shows the serotype distribu-tion of S.mutansstrains isolated from thesame

subjects listed in Table 1. SerotypecS. mutans wasisolated from 18to20 subjects(90%)known

TABLE 1. Prevalenceof S. mutans in plaque samples PrevalenceofS. mutans" Codeof Startingage Sex

subjects (mo) 0' 4 7 11 14 19 23 26 30

A 5 F' - - - + + - +

B 6 F - - + - - + ± + +

C 6 M - - - + - ND'

D 6 M - - -

-E 7 M - - + ++ +++ ++ ++ +++ +++

F 8 M ND - - - ND ± ND + +

G 8 F - - - - + ++ ++ + +++

H 8 M - - - ND + + +

I 11 F - - - ND ND + ND - +

J 11 M - - - + - - +

K 11 M - - + - ND ++ ++ + +++

L 11 F - - - + + -

-M 11 M ND ++ ++ + ++ ++ ++ ND +

N 11 M ND - - - + - + + ND

0 12 F - - - - + ++ ND ND ND

P 12 F - - - + + +

Q 12 M - - - - + +++ ND ND ND

R 12 M - - + - ND - + ND +

S 13 M - - ± + + ++ ++ ++ +++

T 13 F - - + - ND ND - -

-U 13 M ND - - - ND - - - ND

V 13 F ND - - - ND - ND - +

Percentageoftotalstreptococcalcount on MS agar platewhich were S.mutans:-,notdetected + 0 to1%;

+, 1to10%; ++, 10to50%; +++,>50%.

'Experimentalperiod (months).

'Sex: M,male;F,female.

"ND, Notdetermined.

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DISTRIBUTION OF S. MUTANS SEROTYPES IN INFANTS 499

TABLE 2. Distribution of S.mutans serotype inplaquesamples

Code of Distribution of S. mutansserotype'

subjects 4h 7 11 14 19 23 26 30

A d d d >e

B e e>c e>c c> e c

C c>g ND'

D

E c c c d c c c

F ND c ND c c

G d d d d d

H ND c e> c c

I ND ND c ND c

J c e>c

K c ND c>d,e d> e, c d e> c, d

L c c

M c c>e c>e c c>e c ND c

N c c c ND

0 c c ND ND ND

p c c c>d

Q c d>g ND ND ND

R c ND c>d ND e>c

S c c c c c c c

T c ND ND

U ND ND

V ND ND c

"Serotype distribution: forexample, d> e means serotype d strains were detected predominantly, and serotype estrainswerealso isolated.

'Experimentalperiod (months).

'ND, Notdetermined.

toharbor S. mutans. Serotype d,e, or g strains

were isolated from six, four, and two subjects,

respectively.

Serotype a or b strains were not

detectedatall.

Atthe initialestablishmentofS.mutans,only

serotype c strains were found in 17 out of 20

subjects, serotype d was detected twice, and

serotype e wasdetected once. Insubject C, se-rotypecandgstrainsweredetected

simultane-ouslyinwhichserotypecwasdominant.

With theincreaseof the numberofS.mutans,

the

prevalence

of isolation of

plural

serotypesof S. mutans tendedto increase. Pluralserotypes

of S. mutans were simultaneously detected in

half of the20

subjects harboring

S.mutans. In

thesecases,thedominant serotype was

generally

thatonewhich had been detectedatthe initial

establishment of S. mutans.

However,

the

pre-dominantserotypewasfoundtovary with time

(e.g.,

subject

K). In other cases, the serotype

distribution of S. mutans could change over

time. In subject

E,

for

example,

serotype c strains were isolated at 7-, 11-, and 14-month

examinations,but serotype d strainswere

tran-siently detectedat19months. After23

months,

onlyserotype cstrainswere detected

again.

In

subjectQ,

only

serotypecstrainsweredetected

at 14

months,

whereas

only

serotypes d and g wereisolated5monthslater(at 19

months).

Relation between bacteriological

find-ings and dental caries

development.

The

incidence of caries

in

the

upper

primary incisors

where bacteriological samples were taken is

shown inTable 3.During the experimental

pe-riod, halfof the subjectsdevelopeddental caries

in these samplingsites.Labialsmooth and

me-dial

approximal

surfaces ofthe teethshowed the

greatest caries activity. Only subject H devel-opedacariouslesiononthe

palatal

surface.No

carious lesion of distal

approximal

surface was

detectedin anysubject.

It is apparent from Tables 1 and 3 that no

carious lesion wasfound before S. mutans was

detected. Furthermore, a

high

prevalence

ofS.

mutans in relation tototal

streptococci

(++

-+++; Table 1) always resulted inthe

develop-ment of carious lesions. When a carious lesion

was

first

detected on a toothsurface, the per-centage of S. mutans showed wide

variability.

However, carieswasinitiated beforethe

estab-lishment of++ countsin subjects E,G, andS,

and subjectsK, M, and

Q developed

caries at

the same site of examination at which

high

countshad beenobtained.

Therelationshipbetween vanousserotypes of

S.mutansand

patterns

of dental carieswasalso studied

(Tables

2 and3).

Although

serotypes d andgwere

preferably

affiliated with thecarious VOL. 10,1979

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TABLE 3. Incidence of dental caries on the upper primaryincisors

Code of Incidence of dentalcaries"

subjects 4h 7 11 14 19 23 26 30

A~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

A - - - _ _ _

B - - - A1 A-1 A.1

C - - - ND'

D - - - _ _ _

E - L.1 L.1 LA.2 LA.2 LA.2 LA.2 LA-2

F - - - ND A.1 ND A.1 A.2

G - - - L.1 L.i L.1 LA.2 LA.2

H - - - - ND LX1 LP*1 LPA* 2

I - - ND ND - ND -

-J - - - _ _ _

K - - - ND L.1 L.2 L.2 LA.2

L - - - A-1

M A-1 A-1 LA.1 LA.1 LA.2 LA.2 ND LA.2

N - - - L.1 L.1 ND

0 - - - ND ND ND

P - - - _ _ _

Q - - - - L.2 ND ND ND

R - - - ND - - ND

-S - - - A-2 A-2 A.2 ND A-2

T - - - ND ND - -

-U - - - ND - - - ND

V - - - ND - ND - A.1

"Abbreviations: tooth surface: L,smoothlabial; A,medianapproximal; P,smoothpalatal; caries degree: -,

sound;1,enamel cariesincludingwhitespot;2,shallow dentin caries.

'Experimentalperiod(months).

'ND,Notdetermined.

processof the smooth surface (e.g., subjects G,

K, and Q), no significant association between

serotypeandpattern ofdentalcaries was noted.

DISCUSSION

Many cross-sectional studies have shown a

relation between theprevalenceofS.mutansin

plaque andthedevelopment of dental caries(14,

17).However, fewinvestigatorshaveattempted

longitudinal

studies

relating

S. mutans to the

development of dental caries on specific sites

(12, 16).It isimportantforboth clinical practice

and research toknow the time ofinitial

estab-lishment ofS.mutansand the minimumlevelof

this organism for the development of carious

lesions. Further, it is questioned whether a spe-cific serotype may be relatedto aspecific type of dental caries.

During the test period, samples were collected

from specific sites, i.e., labial surfaces of upper

primary incisors, since the prevalence of S.

mu-tans seems influenced markedly by the sites examined (10, 12).

Inthe present study, using MSB agar, a highly selective medium (8), S. mutans was not isolated from predentate infants, confirming previous

findings(4) thatS. mutans could be established

in the oral cavity after tooth eruptions. S. mu-tans wasisolated from 9 to 22 infants (41%) who

were between 13 and 24 months of age. The

earliestdetection of S.mutansamongthe infants

was 13monthsafterbirth in the case of subject

B (Table 1). However, samples at subsequent

sampling timeswerenegative.There is the

pos-sibility offalse negatives for technical reasons,

but it remains to be elucidated whether the

infantwasactually infectedortransiently

colo-nized. Similar findings were also observed in

subjectJ. Relatedtothisproblem, Carlsson (3)

reported thatS. mutansbecame established in

halfthe number ofthe infants whenthey had

reached5yearsold.Further,Berkowitz etal. (1)

found that S. mutans was detected in nine

plaquesamples collected from40 infants (23%)

withonly theirprimaryincisors.

The present investigation also revealed that, once S. mutans was established, this organism

appeared tobe isolated thereafter, although an

occasional decrease in relative prevalence of S. mutans (+/+ --) was observed in some sub-jectsduringthecourseoftheexaminations. The percentage of total streptococcal counts which wereS. mutans increased graduallyover time. The initiation ofacariouslesion seemed to occur even with a low number of S. mutans in the

plaque. Swenson et al. (19) investigated

longi-tudinallythe association between the recoveries

ofS.mutansinplaquecollected from noncarious

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surfaces of firstpermanent molars and the de-velopment of dental caries. They observed that

aninitialcariouslesion could develop when the

infective level ofS.mutans was greater than 1%

of total streptococcal counts. In the present

study, however, half ofthe children with caries

developed theinitial caries even when the

infec-tive level of S. mutans wasless than 1%. This

findingsuggests thepossibility that the low level

ofS.mutans in a verysusceptible subject may

result in caries development. As indicated by

Swensonetal. (19), the establishment of a

mi-crobialrisk profilewould be necessary to predict and hence to avoid the development of initial

caries in infants.

Ourserologicalstudy(Table 2) demonstrated

that serotype c strains were most prevalent in

thepopulation sampled. Types d,e, and g were

also detected, but their occurrence was low or rare.Typesa, b, and

f

were notfoundin any of

the samples examined. These results are in

agreement withpreviousfindings (10, 16, 20). It isofinterest that the relative population of serotypes from isolates of a single subject was

found to change with time. This observation

suggests that the microflora on the tooth surface in an infant is unstable,

especially

in terms of

the ecology ofS. mutans. It has beenreported

that

implantation

of S. mutans into the oral

cavitywasverydifficultinadult humans (5). In

addition,theinterdentaland intraoral

transmis-sionofthisorganism doesnoteasilyoccur(18).

It isimportanttodetermineif specific serotypes may be particularly related tospecifictypes of

dental caries. Unfortunately, however, there is

no clear-cut relationshipbetween serotype and

type of carious lesions, whereas serotype

dig

strains appear to be related to smooth labial surface caries (Tables 2 and 3). However, the

predominant presence ofserotype c strains in

almost allsubjectswhodeveloped caries made

itdifficulttoknow the relation ofspecific

sero-typesof S. mutans tocariousstatus.

Qureshi

et al.(16) could notincriminateaspecificserotype

inthedecayof aparticulartooth surface. In this

connection,wehave foundusingananimal

car-iessystem that serotype dS.mutans

preferen-tially

induced smooth surface

caries,

whereas types

c/e/f

strains

preferably

inducedcaries

pri-marily

originating

from

pits

andfissures

(11).

Surveys of the prevalence ofserotypes ofS. mutans in the oral cavities of infants will con-tribute needed information for effective vacci-nation againstdental caries. Avaccine

eliciting

specific salivary

immunoglobulin

A antibodies

shouldplay an importantrole in the control of dental caries(6).

The mode of transmission ofS.mutansisnot

clearly defined. Berkowitz et al. (1) suggested

thepossibility ofmaternal transferof S.mutans.

In preliminary survey (unpublished data), we

havefound achild harboringserotype

f

S.

mu-tanspredominantly. Subsequently, her mother

andsister werealso found toharborserotypef

strains, although prevalence of this serotype is very rare in the world. Thepatternofbacteriocin productionalso suggests the possible

transmis-sion of S. mutans among the children in the same nursery (unpublished data). However,

more extensive epidemiological surveys should

beperformedtoclarifythe sourceand

transmis-sionroute of thisorganism.

ACKNOWLEDGMENT

We thank RosemaryLinzerfor assistance with theEnglish version ofthe manuscript.

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