Genetic diversity within Streptococcus mutans evident from chromosomal DNA restriction fragment polymorphisms

Copyright © 1989, American Society forMicrobiology

Genetic

Diversity

within

Streptococcus

mutans

Evident

from

Chromosomal

DNA

Restriction

Fragment

Polymorphisms

P. W. CAUFIELD*ANDT. M. WALKER

Instituteof Dental Research, University of Alabama, Birmingham, Alabama 35394 Received 26July 1988/Accepted 25 October 1988

Attempts tostudy the acquisition, transmission, and otheraspects ofthe naturalhistoryofStreptococcus

mutans infections in humans have been hampered by limitations and inconsistencies in methods by which phenotypic characteristics of individual isolates areexamined. Because most mutansstreptococci associated withhuman dental caries fall within the biotypeI (serotypescandf) grouping, designated S. mutans, these typing methodsareof littlevalue indistinguishingindividual isolates. Hereweshow thatstrainsof S.mutans obtained from over 30 individuals demonstrate unique "fingerprints" of chromosomal DNA digested with restriction endonucleaseHaeIII. To demonstratethat thispolymorphisminrestrictionfragmentscanbeused to study the acquisition and transmission of this organism, we examined isolates of S. mutans from three mother-infant pairs obtainedatthetimethe infantfirstbecame colonized bythis organism. Results indicate that strains of S. mutans found in infants exhibit restriction fragment profiles identical to those of their mothers, stronglysupportingthe notion that motherstransmitthis organismtotheirinfants. Also, weshow that strains of S. mutans with the same restriction fragment profile were stably maintained over a 3-year interval in theonemother-infant pair studied. Moreover, we foundthat mothers and their infants harbored onlyafewindividualstrains, suggestingthattransmission of thisorganismisprobablyconfined withindiscrete family cohorts. Collectively, these findings demonstrate the potential utility of genomic fingerprinting in studyingthenatural historyof S. mutans infections in humans.

The phenotypically similargroupofbacteria knownasthe

mutansstreptococci canbe divided into atleastsix distinct species on the basis ofDNA hybridization, moles percent

guanine plus cytosine,and biochemical andserological char-acteristics (6, 15, 16). The subgroup of the mutans strepto-cocci most commonly found in humans, and therefore of major interest in the study of dental caries, fallswithin the genetically defined species designated Streptococcus

mu-tans, which includes the c, e, and fserotypes. The c and f

serotypesexhibit similarbiochemical profilesandhave been designated biotype1(16). Although methodsareavailableto

distinguish between the biotypes or serotypes of mutans

streptococci, they have notbeen very useful for differenti-ating individual strains because most humans harbor the serotype c (biotype I) subgroup. The ability to further characterize individual strains of S. mutans would be of tremendous value in studies onthe natural history of infec-tion with this organism, particularly as the natural history pertainstothetransmission andacquisition ofthisbacterium in human populations.

Thequestionthenariseswhether sufficientdiversityexists within the serotype c subgroup of S. mutans via some phenotypicor,preferably, genotypic trait which would allow

characterization of individual strains. Previous work with whole-cell extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis failed to show

differ-ences in protein profilesamongserotypec strains, although

serotypescould bedifferentiated (14). Otherinvestigations, however, suggestdiversity within theserotype c group.For example, serotypecorbiotypeIstrainscanbedistinguished onthe basisofbacteriocinproductionandimmunity profiles (1, 7, 13). Previously, we differentiated a subgroup of

bio-type I strains on the basis of plasmid DNA profiles (2-5). More recent data from Gilmore and co-workers (8) show

* Corresponding author.

evidence ofgenetic diversity by demonstratingdistinct elec-trophoretictypesfor several of 16enzymesisolatedfrom six serotype cstrains.

Here we report the use of restriction enzyme digests of chromosomal DNAtoobservepolymorphismin restriction fragment lengthsamongstrains of S. mutansobtained from different individuals. DNA fingerprinting was then used to

study thetransmission andacquisition of S. mutanswithin familycohortsandtoexaminetheconservation of strainsas afunction of time within afamily.

(Portions of this research were reported at the Interna-tional Association of Dental Research Annual Meeting [P. W.Caufield,T.Walker,and D.Perkins,J.Dent. Res.66:43, abstr. no. 66, 1988].)

MATERIALS ANDMETHODS

Sourceand confirmation of isolatesof S. mutans. Strains of S. mutans wereobtained from three different sources. The prototypestrains ofserotypecstrains 10449 andGS-5were obtained from theAmericanTypeCultureCollection (Rock-ville, Md.) and D. B. Clewell (Institute of Dental Research, AnnArbor,Mich.), respectively. Twenty-eight isolatesof S.

mutansfrom unrelated individuals wereselected for charac-terization at random by using a random-number generation

program for a patient population ofover 400. S. mutans

isolates from these individualswereoriginallycultured from mitis-salivarius-bacitracin (MSB) medium (9). S. mutans

wasalso obtainedfrom threemother-infant pairs whowere participants in an epidemiological trial studying the trans-mission ofS. mutans within family cohorts. Samples were obtained from the saliva of each mother and from plaque from her infant whenS.mutanswasfirst detected inplaque of the infant (averageage, 14.7 months). Ten isolates of S.

mutans perindividualwereselectedatrandom fromprimary

isolation plates of MSB medium. If differences in colony morphologywereevident,effortsweremadetoinclude each 274

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ofthedifferent colony types. In mother-infant pair 1,

sam-plesof S. mutans were obtained from both members at initial

detection of colonization in the infant (11.5 months) and 3 years later. This was done to examine the stability of different types within a family. Isolates were obtained in a similar manner from mother-infant pairs 4and 13 at initial ages of acquisition in the infants of 21 and 11.6 months, respectively.All isolates, including those from mother-infant pairs, were confirmed as S. mutansbiotype I by biochemical means (16) and were maintained at -70°C until needed.

Chromosomal DNA isolation. The method used to isolate chromosomal DNA from S. mutans was as follows. Cultures of S. mutans were initiated from frozen stocks, pure streaked for single colonies, andgrownovernight in 5mlof Todd-Hewitt broth (THB; Difco Laboratories, Detroit, Mich.) in an atmosphere of 85% N2, 5%C02, and 10%H2.

Three milliliters of this culture was inoculated into 100-ml volumes of THB and allowed togrowfor 4 to 6h at 37°C to late log phase (opticaldensity at660nm, 0.35).Glycine(final concentration, 5%) was then added to disrupt cell wall synthesis; the culture was gentlyagitatedfor 45 min at 37°C (12). Cells were pelleted by centrifugation, rinsed once in TES buffer (0.03 MTrishydrochloride, 0.005 MEDTA,0.05 M NaCI [pH 8.0]), and suspended in 2.5 ml of TES buffer with 25% sucrose.

Cells

were then exposed to lysozyme

(chicken egg white lysozyme L-6876; Sigma Chemical Co., St. Louis, Mo.), for a final concentration of 1.0 mg/ml in TES, for 2 h at 37°C under gentle agitation to disrupt the integrity ofthe cell wall. EDTA was added (0.1 mlof a 5 M aqueous EDTA stock solution), and the solution was

al-lowed to stand for 15 min at room temperature before

addition of proteinase K (0.5 ml of a 5-mg/ml solution in TES, predigested for 30 min at 37°C) (P-0390; Sigma).

Proteinase Kdigestionproceeded for 30 min at 37°C.

Result-ingprotoplasts were thenlysed in a 1%(final concentration)

solution ofSarkosyl (L-5125; Sigma) at 37°C for 1 h. The lysate was cleared of cell debris and unlysed cells by centrifugation at 4,400 x g for 10 min.DNA from the cleared lysate was then partitioned in a CsCl-ethidium bromide

gradient by ultracentrifugation performed twice.

Chromo-somalDNA was removedfrom the gradient, dialyzed over-night in 4 liters of0.1x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodiumcitrate) (12) and stored at 4°C until digested

with restriction endonucleases. The concentration of chro-mosomal DNAwasdetermined spectrophotometrically (260 nm) by standardprocedures (10).

Endonuclease digests. By an empirical approach, we ini-tially selected a group of six-base recognition enzymes,

which included BamHI,

HindIII,

EcoRI, SalI, XBAI, and the four-base cutter HaeIII. Restriction endonuclease and

corresponding 10x buffers were obtained from Bethesda

Research Laboratories, Inc. (Gaithersburg, Md.). In our hands and under theconditions described here,HindIII and

HaeIII gave the best resolution of bands for the 6- to

20-kilobase fragments. Digests containing 2 to 3 ,ug of

chromosomal DNA and 5 to 10 U ofrestriction enzymewere

performed in30-,ul volumes at37°C for 2 h. Each digest was repeated on at least three different occasions to ensurethat gels represented complete, notpartial, digests.

Gel electrophoresis and photography. We obtained good

resolution offragments using a 0.55%agarose gel(IBI, New Haven, Conn.) run in Tris-borate-EDTA buffer (10) and

electrophoresed at 40 V for 16 h. Photographs were taken with Polaroid 667 film and aFotodyne MP-4 camera under UV illumination (Fotodyne UV 300) after the bands were

Ch

C a me

Co

Covc :> «: CoCouCDnv-NLun CO

Kbp

23.1 - h

9.4

-15.5

6.6

-4.4

-2.3-

-Wui

FIG. 1. Randomly selected biotype I strains of S. mutans chro-mosomal DNA digested with HaeIII. Lane X, Lambda DNA cut with HindIII used as the restriction fragment size standard; lanes 10449 and GS-5, chromosomal DNA from the serotype c, biotype I prototypes; remaining lanes, chromosomal DNA patterns from 10 randomly selected biotype I strains of S. mutans obtained from different individuals. The arrow on the right indicates the migration distance of the 15.5-kbp restriction fragment.

made visible by staining the gel in ethidium bromide (1

,ug/ml)for 1 h and rinsing briefly in water. RESULTS

DNA isolation procedure. The DNA isolation procedure we used yielded 200 to 500,ug of DNA per 100-ml culture. The DNA/protein ratio averaged 1.8, as judged from UV spectrophotometry at 260 and 280 nm, respectively.

Restriction fragment patterns of biotype I strains. Figure 1 shows chromosomal DNA obtained from 10 strains of S. mutans and digested with HaeIII. Lanes 10449 and GS-5 represent chromosomal DNA from serotype c prototype strains 10449 and GS-5, respectively. The remaining lanes represent chromosomal DNA obtained from randomly se-lected biotype I isolates. Strains of S. mutans display a marked degree ofpolymorphism, particularly evident in the larger fragments (9 to 20 kilobase pairs [kbp]) (Fig. 1). An additional 18 strains of randomly selected biotype I S. mutans strains were also digested with HaeIII; each dem-onstrated a unique fragment pattern (data not shown). In-cluded in lane A of this and the othergels was a molecular size standard of bacteriophage lambda DNA cut with HindIII. The distinctive restriction endonuclease patterns exhibited by the different isolates convinced us that this method would be useful in distinguishing isolates from mother-infant pairs to study thetransmission and acquisition ofS. mutans in this population.

Figure 1 also shows a restriction fragment of approxi-mately 15.5 kbp that is common to chromosomal DNA patterns in 8 of the 10 strains illustrated. Afragment of this size also appeared in 14 (78%) of 18 randomly selected

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A

Kbp 23.1

-

9.4-

6.6-4.4

-À

.MW

t>.,:

.'e,

'l' :1.

l'.

B

À

C H

g

h

3-16-85

4-2-88

A

F H

c

O

R

n

Kbp

23.1-

9.46.6 -

4.4-FIG. 2. Restriction fragment patterns for mother-infant pair 1.

(A) Lanes A, F, H, andc, HaelII digests of chromosomal DNA from strains of S. mutans obtained from mother 1 and her infant when S. mutanswasfirst detected in the infantat11.5months. (B) Lanes O, R, and n, Haelll-cut chromosomal DNA from strains obtained from mother-infant pair i 3 years later. Capital letters denote strains from the mother, and small letters denote infant strains. LaneX, Lambda size standard.

biotype

I strains (datanot shown) andin 7of7strainsfrom the mother-infant

pairs

shown in

Fig.

2 to4.

Mother-infant

pairs.

Restriction fragment patterns from the HaeIIIdigests shown in

Fig.

2 represent three distinct strains of S. mutansfound in the saliva ofmother i and a

single

strainfoundin

plaque

from her infant when the infant was firstcolonized with S. mutantsat11.5 months.The three strainsfrom mother i

(Fig.

2A,lanesA, F, andH)

represent

unique

strains from 10

original isolates,

with the

pattern

in laneH

being

themostcommon

(6

of10) andthe patterns in lanes A and F less common (2 of 10

each).

Ail 10 isolates

from the infant

(Fig.

2, lane c) were identical

(data

not

shown)

and matched the

pattern

of the mother in lane H.

Figure

2B shows the distinct restriction fragment patterns

from isolates

obtained

from the same

mother-infant

pair

3 yearslater. Patterns from strains O and Rrepresentthetwo

distinct strainsfound in mother 1 and match the

original

patterns

represented

in lanes F and H,

respectively.

The

strainwith

pattern

Rremainedthepredominant straininthe

mother(6of10). Thepattern in lane Awasnotfoundamong

the 10 isolates obtained 3 years later. The patterns of 10

infant isolates obtained 3 years later

(Fig.

2, lane n) were

identical and matched the

original

pattern shown in lane c

(infant),

lane H

(mother,

first

isolation),

and lane

R

(mother,

second

isolation). Hence,

formother-infant pair1, the child

apparently acquired

from the mother a

single

strain

(and

apparently

the

predominant

strain) which was conserved over the

3-year

interval. Mother 1, on the other hand,

originally

demonstrated three distinct

strains,

two ofwhich

were conserved

for

the

3-year

interval.

Our second

isolation

apparently

failed

to

detect strain A from the mother. Thisp

was not

unexpected,

because

we selected

isolates for

study

FIG. 3. Restriction fragmentpatterns for mother-infant pair 4. Lanes C and H, HaeIII digests of chromosomal DNA from two strains ofS. mutans foundin mother4; lanesgandh, profiles for twostrainsfound in infant4.Isolateswereobtainedfromtheinfant atinitialcolonizationat21monthsofage.The DNAprofiles forthe infantappearidenticaltothosefor the mother. Lane X, Lambda size standard.

randomly and strain A was less prevalent among theoriginal isolates.

Figure 3 illustrates restriction fragment patterns from HaeIII digests for another mother-infant pair (no. 4). Iso-lates were obtained from both members at the time of initial acquisition of S. mutansby the infant at21 months of age. Two distinct strains emerged from 10 original isolates from each individual (the patterns in lanes C andHi are for the mother, and those in lanes g and harefor the infant). The infantapparently acquired both genotypes from the mother, because the restriction fragment patterns appear identical.

Therestrictionfragment patterns for mother-infant pair 13

(Fig. 4) illustrate the use of several different enzymes to discern differences among strains. The first three lanes show patterns for strains A and E from the mother and strain a from the infant. As with mother-infant pair 1, the infant harbored only one strain type. Isolate E from the mother appeared identical to isolate a from the infant. Because of themarkedsimilarities infragmentsevidentwith the HindIII digest of isolates A and E from the mother and isolateafrom theinfant,weused HaeIII and EcoRItofurther characterize these strains. All threedigests showed polymorphism,and strain E from the mother and strain a from the infant

appeared identical(Fig. 4).

DISCUSSION

The abilitytodistinguish individual isolatesof S. mutans by restriction enzyme digests of chromosomal DNA repre-sents apowerfultool forexaminingthenaturalhistoryof this infectious disease. More specifically, we believe that this

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AE

a

A E

a

AE

a À

Kbp

23.1

9.4

6.6

4.4

2.3 2.0

HindIII

Haelil EcoRI

FIG. 4. Restrictionfragmentpatternsfor mother-infant pair13. Multiple enzyme digests were included to discern differences

be-tweenstrains A and E from mother 13. HindIIIdigestsof DNA from strains A and E from the mother show similar but distinctprofiles. Additional digests with HaeIII and EcoRI demonstrate additional polymorphism between these strains. DNA profiles for strain a

fromthe infant match those for strain E from the mother. Samples from the infant were obtained at 11.6 months of age. Lane X,

Lambda size standard.

method will allow us to examine the transmission and acquisition of S. mutans within human populations and possiblyreveala strain-specific relation tothedevelopment of dental caries. Inaddition, DNAfingerprinting may prove

useful in answering several questions pertaining to the ontogeny of S. mutans and other members of the biota indigenous in humans. For example, how stable are theS. mutans populations within an individual? Do S. mutans populations changewith time ordisease status? Howmany

different strains of S.mutansdoesanindividual harbor? Are

some strains associated with pathogenesis while others are not? Our preliminary investigation suggests that adult

hu-mans harbor multiple genotypes, and in at least in one example, S. mutansisrelativelystableovera3-year period. Long-term studies andmoreextensive samplingareneeded

to confirm theseobservations, however.

Thatgenetic diversity existswithin thebiotypeI strains of

S.mutans,asevidencedby restriction-fragment-length poly-morphisms of chromosomal DNA, isnottotally unexpected. Previous results fromourlaboratory showed diversitywithin biotype I strains based on the presence or absence of plasmid DNA and even genetic and phenotypic diversity within plasmid-containing strains (2-5). Serological typing hasalso revealed that humans often harbor multiple strains ofS. mutans (11). Otherphenotypic traits, such as bacteri-ocin production and immunity, employed previously to

distinguish strains, indicate diversity within the biotype I, serotype c strains of S. mutans, but withconflicting results probablyduetodifferencesin methodology. Forexample,a study by Berkowitz and Jordan (1) demonstrated that

be-tween6and 17differentbacteriocintypescouldbe found in

a

single

individual.

Rogers (13),

ontheother

hand,

reported

thatanindividual exhibited

only

a

single

bacteriocintypeof

S. muitans.

Later, Davey

and

Rogers

(7),

using

a modifica-tion oftheir

previous method,

found thatmostindividuals of families studied harboredas manyas fourdifferent

bacteri-ocin types.

Despite

the

conflicting

results, differences in

phenotypic

expression

of bacteriocin

production

and

immu-nity

support

genetic diversity

within

biotype

Istrains.

The three mother-infant

pairs

discussed here

point

to several

interesting findings. First,

in all three cases, the

infant

acquired

a genotype identical to one found in the mother. This

study

shows forthe first that the strains ofS. mutans

initially colonizing plaque

ininfants areidentical to

thoseofthe

mother, strongly

suggesting

that thesebacteria

are transmitted

vertically

from mother to child.

Second,

mothers harbor a more

heterogeneous

population

of S. mutans than do infants at initial

acquisition, although

the

repertoire

appearstobe limitedto

only

two orthree strains. If children

acquire

S. mutans

primarily

fromtheir

mothers,

wewouldexpectthe numberof strains tobe limitedto

only

those few harbored

by

themother. So

far,

thisappearstobe

thecase. We cannotbe sure that saliva reflectsall

possible

genotypes

withinan

individual; however,

we

believe,

asdo

others,

thatsalivaconstitutesthe

major

vehicle of transfer of this

organism

between individuals.

Lastly,

wedemonstrated

that inatleastonemother-child

pair, genotypes

ofS.mutans

remained

relatively stable,

because twoof three

genotypes

from the mother and one from the infant were recovered

after a

3-year

interval. This

finding

supports the results of

studies in which bacteriocin types

appeared

stable over a

6-month interval

(7, 13).

Interestingly,

we noticedthat in 29

(83%)

of35 individual strains whichwe

fingerprinted

with

HaeIII,

adiscrete

frag-ment of

approximately

15.5

kbp

was present. Whether this

common

fragment

represents a

highly

conserved

15.5-kbp

sequence in the

biotype

IS. mutans genomeawaits

hybrid-ization studies. Further characterization of this common

fragment

may be warranted to obtain a

possible genomic

probe

useful for

detecting biotype

I strains ofS. mutans.

In

conclusion,

we found

genomic

fingerprinting

of S. mutans isolates a useful tool for

distinguishing

individual

strains. Further

application

of the

technique

may prove valuable in

studying

the

acquisition

and transmission of

other members of the

indigenous

biota

colonizing

the oral

cavity.

One

question

we are

currently

addressing

is whether

childrencan

acquire

S.mutansfromsourcesother than their

mothers. Atleastin the threemother-infant

pairs

examined

thus

far,

wefound

complete parity

betweenstrains harbored

by

infants and those harbored

by

their mothers. Had the infant

acquired

a strain not harbored

by

the

mother,

we

would suspect

acquisition

from othersources, such asfrom

the father or someothercontact. This was notthecase. In

fact,

we have obtained isolates of S. mutans from two

families

including fathers;

the strains from the fathers show restriction patterns

clearly

distinct from those for motheror infant

(P.

W.

Caufield,

and T. M.

Hagan,

manuscript

in

preparation).

This suggests to usthat inthese two

families,

fathers were

probably

not a source of S. mutans for their children.

ACKNOWLEDGMENTS

Wethank Don B. Clewell forhis valuedobservationsand for the

use of his laboratory during the development of this

technique.

Susan Hollingshead contributed to the editing and clarity of the

manuscript.

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This work was supported by Public Health Service grant 2P50 DE02670-18 and contract RFP5-83-3R-DE42552 from the National Instituteof Dental Research.

LITERATURE CITED

1. Berkowitz, R. J., and H. V. Jordan. 1975. Similarity of bacte-riocins ofStreptococcus mutansfrommother and infant. Arch. Oral Biol. 20:1-6.

2. Caufield, P. W., N. K. Childers, D. N.Allen, and J. B. Hansen. 1985.Distinct bacteriocin groups correlate withdifferentgroups ofStreptococcus mutansplasmids. Infect. Immun. 48:51-56. 3. Caufield, P. W., N. K. Childers, D. N. Allen, J. B. Hansen, K.

Ratanapridakul, D. M. Crabb, and G.Cutter. 1986. Plasmids in Streptococcus mutans: usefulness as epidemiological markers and association with mutacins, p. 217-223. In S. Hamada, S. Michalek, H. Kiyono, L. Menaker, and J. McGhee (ed.), Molecular microbiology andimmunobiology ofStreptococcus mutans. Elsevier Science Publishing,Inc., New York. 4. Caufield, P. W., K. Ratanapridakul, D. N. Allen, and G. R.

Cutter. 1988. Plasmid-containing strains ofStreptococcus mu-tans cluster within family and racial cohorts: implications for naturaltransmission. Infect. Immun.56:3216-3220.

5. Caufield, P. W., Y. M. Wannemuehler, and J. B. Hansen. 1982. Familial clustering of theStreptococcus mutanscryptic plasmid strain ina dental clinic population. Infect. Immun.38:785-787. 6. Coykendall, A., and K. B. Gustafson. 1986. Taxonomy of

Streptococcusmutans, p. 21-28. InS.Hamada,S.Michalek,H. Kiyono, L.Menaker,and J. McGhee (ed.),Molecular microbi-ology and immunobimicrobi-ology ofStreptococcus mutans. Elsevier Science Publishing,Inc., NewYork.

7. Davey, A. L., and A. H. Rogers. 1984. Multiple types of the bacterium Streptococcus mutansin the human mouth and their

intra-family transmission. Arch. Oral Biol. 29:453-460. 8. Gilmore,M.N.,T. S.Whittam, M. Kilian, and R. K. Selander.

1987. Genetic relationship among the oral streptococci. J. Bacteriol. 169:5247-5257.

9. Gold,O.G.,H. V.Jordan,andJ. van Houte. 1973. A selective mediumfor Streptococcus mutans. Arch. Oral Biol. 18:1357-1364.

10. Maniatis, T.,E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning:a laboratory manual, p. 446-468. Cold SpringHarbor Laboratory, ColdSpring Harbor, N.Y.

11. Masuda, N., N. Tsutsumi, S. Sobue, and S. Hamada. 1979. Longtitudinalsurveyofthedistributionof variousserotypesof Streptococcusmutansin infants.J. Clin. Microbiol. 10:497-502. 12. Reader, J. L., and F. L.Macrina.1976. PlasmidDNAisolation in Streptococcus mutans: glycine-enhanced cell lysis, p. 725-736. In H. M. Stiles,W. J. Loesche, andT. C. O'Brien(ed.), Proceedings: microbialaspectsof dental caries(aspecial sup-plement to Microbiology Abstracts), vol. 3. Information Re-trieval, lnc.,Washington, D.C.

13. Rogers, A. H. 1977.Evidence forthetransmissibility ofhuman dentalcaries. Aust. Dent. J. 22:53-56.

14. Russell, R. R. B.1976. Classification of Streptococcusmutans strains by SDSgel electrophoresis. Microbios Lett. 2:55-59. 15. Schlekfer, K. H., R. Kilpper-Basz, J. Kraus, and F. Gehring.

1984. Relatedness and classification of Streptococcus mutans and "mutans-like" streptococci. J. Dent. Res. 63:1047-1050. 16. Shklair, I. L.,andH.J. Keene. 1976. Biochemical

characteri-zation and distributionof Streptococcusmutansin threediverse populations,p. 201-210. InH. M. Stiles, W.J. Loesche, and T. C. O'Brien (ed.), Proceedings: microbialaspects of dental caries(aspecialsupplementtoMicrobiology Abstracts), vol.1. InformationRetrieval, Inc., Washington, D.C.

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