Microbiological Research 163 (2008) 168172

tStreptococcus mutans in Turkish children

E. Esin Hames--KocabAtac- Uzelb, Ali Rza A

aEge University, Science TechnologbBasic and Industrial Microbiology35100 Bornova, Izmir, TurkeycDepartment of Pedodontics, Facu

Accepted 27 March 2006

AP PCR;to determine the possibility of intra-familial transmission in a group of Turkishchildren and their parents. A total of 56 children participated in the study together

significant positive correlation (po0:001) was found between the infected children

in which mutans streptococci play a major role (Alaluusua, 1991; Alaluusua and Renkonen, 1983;Thibodeau and OSullivan, 1999). The major routeof early acquisition of mutans streptococci indi-cated the transmission from mother to child

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Corresponding author.E-mail address: esin.kocabas@ege.edu.tr(Caufield and Walker, 1989; Hardie, 1986; Li

0944-5013/$ - see front matter & 2006 Elsevier GmbH. All rights reserved.doi:10.1016/j.micres.2006.03.016

(E.E. Hames--Kocabas-).and their parents with high S. mutans counts.& 2006 Elsevier GmbH. All rights reserved.

Introduction

Dental caries is a transmissible infectious disease

(Mattos-Graner et al., 2001). Early acquisition ofthese bacteria contributes to increased cariesprevalence in the primary and permanent dentitionTransmission with their parents (20 fathers and 49 mothers). Saliva samples were collected fromthe individuals and cultivated on S. mutans selective TYCSB agar. The typical isolatesof S. mutans were identified by using classical microbiological methods, as well asmolecular typing of S. mutans clones which was performed by using AP PCR withOPA5 primer for the detection of transmission. The vertical transmission of salivaryS. mutans was detected among 14 motherfatherchild, 35 motherchild (one twins)and 6 fatherchild combinations. The homologies of strain types were recorded as24% and 16.6% for motherchild and fatherchild combinations, respectively. AKEYWORDSStreptococcusmutans;as-a,, Fusun Uc-arb, Nazan Kocatas- Ersinc,lpozc

y Center, 35100 Bornova, Izmir, TurkeySection, Department of Biology, Ege University Faculty of Science,

lty of Dentistry, Ege University, 35100 Bornova, Izmir, Turkey

SummaryThe aim of the study was to establish the colonization of Streptococcus mutans andColonization and vertical ransmission of

www.elsevier.de/micres

(such as bacteriocin typing), serotypic and geno-typic characterizations, including plasmid DNA

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Colonization and vertical transmission of S. mutans in Turkish children 169profiling, restriction endonuclease analysis, arbi-trarily primed polymerase chain reaction (AP PCR)and ribotyping. Although each method has its owndrawbacks, the classification based upon genotypictraits appears more reliable than the phenotypictraits (Caufield and Walker, 1989). Especially APPCR is relatively easy and fast to perform comparedto the other genotypic methods; hence, it appearsto be an attractive alternative method. The majoradvantage of the method is that no prior knowledgeof the DNA sequence of the target bacterial speciesis required. The results also highlight that AP PCRhas good discriminative ability in differentiatingamong the mutans streptococci clones (Saarelaet al., 1996).It is not clear whether there are varieties in

different populations and countries, as to intra-familial distribution (Li et al., 2000). The aim ofthis study was to establish the colonization ofS. mutans in a group of Turkish children and theirparents and to determine the possible intra-familial transmission of S. mutans by using AP PCR.

Material and methods

The study group was comprised of 49 mothers, 20fathers and 56 children with a mean age of33.8711.7 months. The subjects were selectedfrom a group of children attending to a private daycare center located in an urban area of Izmir.Experimental procedures were approved by theEthical Committee of Ege University, Izmir, Turkey.All subjects had a similar high socio economicstatus. None of the subjects participated in to thisstudy had chronic diseases, daily medicine usage orantibiotic treatment within the last 1 month priorto the assessment. Children and parents were orallyet al., 2000; Redmo Emanuelsson and Wang, 1998).However, other possible transmission routes, suchas extra-familial acquisition of mutans streptococciin children, intra-familial transmission betweenfather and child, and the transmission amongspouses have also been reported. In most of thesereports, the individuals with high levels of mutansstreptococci in their mouths and who are infrequent contact with the children were suggestedto be the principal source of transmission (Alaluu-sua, 1991; Berkowitz and Jones, 1989; Kozai et al.,1999; Redmo Emanuelsson et al., 1998; Saarelaet al., 1996).Methods for examining mutans streptococci

transmission include phenotypic characterizations,examined with a mirror under daylight. Cariesprevalence in the children and the parents wererecorded in accordance with the WHO criteria(1997).

Microbial sampling, isolation and cultivationof S. mutans

Parents and children were instructed not to brushtheir teeth 2 h prior to the assessment. Isolates ofS. mutans were obtained between 10 and 12 AMfrom unstimulated saliva (0.31.0ml) of the sub-jects. Samples were dispersed in a vortex mixer toobtain a homogeneous suspension of saliva andwere cultivated on selective tryptoneyeastcys-teine (TYCSB) agar supplemented with 0.2 U/mlbacitracin (Sigma Chemical Co., St. Louis, MO, USA)and sucrose 15% (Hardie and Whiley, 1992; VanPalenstein Helderman et al., 1983). The plateswere incubated in 5% CO2 at 37 1C for 48 h. One tothree isolates of S. mutans were recovered fromeach of the individuals. The isolates were examinedunder a light microscope (cell morphology and gramstaining reaction) and identified by their distinctivecolony and confirmed by sugar fermentation tests(acid production from mannitol, sorbitol, melli-biose, raffinose, starch, inulin and dextrin), hydro-lysis of arginine and esculin, VogesProskauer andresistant to bacitracin. S. mutans NCTC 10449 wasused as a reference strain (Hardie, 1986; Hardieand Whiley, 1992; Smibert and Krieg, 1994). Thenumbers of S. mutans isolates were countedand interpreted according to criterion describedby Roeters et al. (1995). Low level o104 cfuof S. mutans/ml of saliva, moderate level 104po106 cfu of S. mutans/ml of saliva and highlevel X106 cfu of S. mutans /ml of saliva. Theisolates were preserved in 50% sterile glycerol andstored at 70 1C for further DNA extraction.

Extraction of DNA

The method used to isolate chromosomal DNAfrom bacteria was briefly as follows (Saarela et al.,1996). S. mutans isolates were grown in 5ml ofToddHewitt broth incubated in 5% CO2 at 37 1C for48 h. Thereafter, bacterial cells were harvested bycentrifugation (1500g for 10min) and cells werewashed with 1ml of TE buffer (10mM Tris base,1mM EDTA, pH 8.0). Cells were resuspended in100 ml of TE, 50 ml of 10% sodium dodecyl sulfatewas added and cells were incubated for 30min at65 1C. The suspension was centrifuged (2000g for5min) and the supernatant was removed. TheEppendorf tubes containing the cells were placed

MannWhitney and Spearman rank correlation testswere used for comparisons and correlations amongeach group. The statistical level of significance wasset at po0:05.

Results

In our study, S. mutans was isolated from 33 ofthe 56 children, 43 of the 49 mothers and 18 of 20fathers. The characteristic specifications of chil-dren with the colonization of salivary S. mutans arepresented in Table 1. The prevalence of S. mutans

children are given in Table 2. No significant

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Gender Girls 9 11

E.E. Hames--Kocabas- et al.170in a microwave oven (490W) and heated for 2min30 s. The pellets were dissolved in 250 ml of TE andthe Eppendorf tubes were frozen at 20 1C. BeforeAP PCR assay, the suspension was melted, centri-fuged and the supernatant was used in AP PCR.

AP PCR assay

AP PCR was performed by using primer50AGGGGTCTTG-30 (OPA5) as previously described(Saarela et al., 1996). PCRs were carried out in a25 ml reaction mixture containing the DNA template(1 ml DNA (2050 ng)), 0.2mM of each deoxynucleo-side triphosphate, 0.4 mmol primer (ThermoBioScience GmbH, Ulm, Germany), 4.0mM MgCl2and 2.5 U Taq DNA polymerase (MBI Fermentas,Vilnius, Lithuania) according to manufacturersrecommendations. DNA amplification was per-formed in a thermalcycler (Crocodile III, Appligene,Oncor, Illkirch, France) with an initial denaturationat 94 1C for 5min, followed by 35 cycles ofdenaturation (94 1C, 1min), annealing (36 1C,2min) and extension (72 1C, 2min). The finalextension step was at 72 1C for 5min. A blankreagent, which contained all the components of thereaction mixture with the exception of templateDNA (which was substituted with sterile distilledwater), was included in every PCR assay. Amplifica-tion reactions were performed twice to check theconsistency and reproducibility of the method(Saarela et al., 1996).PCR products were analyzed electrophoretically

with 1% agarose gel containing 0.5 mg/ml ethidiumbromide and visualized with transilluminator. GeneRuler DNA ladder mix (1 kb, 10010000 bp) (MBI,Fermentas, Vilnius, Lithuania) was run as a mole-cular-size marker in one of the lanes, for sizecomparison.

Analysis of AP PCR banding patterns

The AP PCR fingerprints were analyzed by side-by-side visual comparison. Fingerprints were con-sidered identical when all major bands were thesame. Any repeatable difference regarding thestrong bands was considered discriminatory. Inaddition, the size of the base pairs was measuredby BioDoc Analyze (Biometra Ti5, Goethingen,Germany).

Statistical analysis

Data was analyzed with SPSS 10.0 version soft-ware (SPSS Inc., Chicago, IL, USA). w2 test was usedfor the analysis of the categorical variables.Boys 24 12

Caries Active 9 Free 24 23correlation was found between the parents andtheir children in regards to the S. mutans level.The study covered a total of 43 mothers and 18

fathers but the transmission was evaluated only for25 mothers and 12 fathers due to the undetectableS. mutans. The results of AP PCR revealed that 6 of25 motherchild pairs (24%) and 2 of the 12fatherchild pairs (16.6%) showed identical geno-types, respectively (Fig. 1). None of the individualsin the parental pairs showed identical S. mutansgenotypes.

Discussion

Studies in using bacteriocin profiles, serotypingand genotyping suggest that the mothers areprincipal source of mutans streptococci to theirchildren because of the fact that the mothers are infrequent and intimate contact with their infants in

Table 1. Characteristic specification of children withcolonization of S. mutans.

S. mutansdetected (n)

S. mutans notdetected (n)

Age o26 7 12X26 26 11was higher for the group of children who were 26months or older, compared with younger agegroups. The colonization of S. mutans was foundto be related to both age and the caries of thechildren (Spearman correlation test, po0:05). Nosignificant correlation was found between thecolonization of S. mutans and the gender. The levelof salivary S. mutans of mothers, fathers and their

(1974) also reported that successful transmission ofbacteria among the human hosts was complex anddepended on a variety of interrelated factors,including bacterial affinity to potential colonizationsites and number of infecting bacterial cellsavailable for attachment.The results of the present study showed that six

of 25 children (24%) and 2 of 12 children (16.6%)harbored genotypes of S. mutans identical to thoseof their mothers and fathers, respectively. Therewere five spouses in the study population and noneof the individuals in the parental pairs showedidentical S. mutans genotypes. It could be ex-plained by the persistence of oral microbiota inadults. These results were in accordance with thereports of Redmo Emanuelsson et al. (1998) whofound no transmission of S. mutans among thespouses. The authors suggested that all of thechildren were attending to private day care centerfrom where they could have acquired S. mutansfrom the individuals that they were in frequentcontact; this has also been reported by Mattos-Graner et al. (2001), Redmo Emanuelsson et al.,

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Colonization and vertical transmission of S. mutans in Turkish children 171Table 2. Prevalence of salivary S. mutans in mothers,fathers and their children.

n S. mutans score

0 1 2 3

Children 56 23 19 13 1Mothers 49 6 7 29 7Fathers 20 2 5 10 3

0 not detected.1 Low; o104 cfu of S. mutans/ml of saliva.2 Moderate; 104po106 cfu of S. mutans /ml of saliva.3 High; X106 cfu of S. mutans/ml of saliva.

R M C M C F C M C M C M C F C M C N S

3000

10000

2000

1031

bpthe first 2 yr of life (Gronroos et al., 1998; RedmoEmanuelsson and Wang, 1998). However, it hasbeen suggested that there were examples offathers who may have been the source and thepossibility of extra-familial transmission (Kozaiet al., 1999; Tedjosasongko and Kozai, 2002).The frequency of intra-familial transmission

varied among the populations due to differentand cultural practices which could have an influ-ence on the degree of contact between childrenand their parents and other individuals. Forexample, matching mutans streptococci genotypeswere observed in 71% of the motherchild pairs inthe American population (Li and Caufield, 1995);however this frequency was lower in the Swedishfamilies as only 24% of 3-yr-old children showed thesame genotype with their mothers and none ofthem showed their fathers genotypes (RedmoEmanuelsson et al., 1998). In Japan, 31.4% of thegenotypes harbored by 011-yrold childrenmatched genotypes detected in their fathers (Kozaiet al., 1999). In addition, Van Houte and Green

1998 and Kozai et al. (1999) as well.In the present study it was also demonstrated

500

100

Figure 1. Identical AP PCR patterns of S. mutans isolateswith OPA5. M mother, F father, C child, N negativecontrol, R reference strain (S. mutans NCTC 10449),and S size marker.that high levels of S. mutans in parents was asignificant factor for S. mutans transmission tochildren (MannWhitney, po0:005) (Fig. 2). Theauthors have suggested that these transmissionpatterns could reflect an underestimation of thetrue level of the homology because of the limited

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5

0

5

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15

20

25

30

35

40

45

0 1 2 3

num

ber o

f mot

hers

and

fath

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transmission detected

transmission not detected

Figure 2. Colonization levels of S. mutans in parents andthe distribution of the parents who infected theirchildren in black. 0; not detected, 1; low o104 cfu ofS. mutans/ml of saliva, 2; moderate 104po106 cfu ofS. mutans/ml of saliva, 3; high X106 cfu of S. mutans/ml of saliva.

of transmission to their children. It could be

mutans establishment and dental caries experience

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E.E. Hames--Kocabas- et al.172in children 24 years old. Scand. J. Dent. Res. 91,453457.

Caufield, P.W., Walker, T.M., 1989. Genetic diversitywithin Streptococcus mutans evident from chromoso-mal DNA restriction fragment polymorphisms. J. Clin.Microbiol. 27, 274278.

Gronroos, L., Saarela, M., Matto, J., Tannner-Salo, U.,Vuorela, A., Alaluusua, S., 1998. Mutacin productionby Streptococcus mutans may promote transmission ofbacteria from mother to child. Infect. Immun. 66,25952600.

Hardie, J.M., 1986. Genus Streptococcus. In: Sneath,P.H.A., et al. (Eds.), Bergeys Manual of SystematicBacteriology, vol. II. Williams &Wilkins, Baltimore,USA, pp. 10431071.This study was supported by the Scientific andTechnical Research Council of Turkey (TUBITAK,TBAG-AY/299-I02T225) and Ege University ScienceTechnology Center (E04/15/02), Turkey.

References

Alaluusua, S., 1991. Transmission of mutans streptococci.Proc. Finn. Dent. Soc. 87, 443447.

Alaluusua, S., Renkonen, O.-V., 1983. Streptococcusrecommended that several preventive programmescould be beneficial to prevent the intra-familialtransmission by directing antibacterial measures athighly colonised mothers and fathers.

Acknowledgmentsmutans in their saliva. In our study, 55% of theparents who had a high salivary S. mutans counts intheir mouths had been the source of transmission totheir children. However, the children and theirparents participated to the study were selectedfrom an urban area that had high socio-economicalbackground. The majority of the participants werecaries free and the parents were aware of theimportance of oral hygiene and they had good oralhygiene habits. For these reasons, this study couldbe considered as a population-specific study and itcould be suggested that the frequency of transmis-sion found in the study could not be extrapolated tothe whole population in Turkey.

Conclusion

It could be concluded that parents who had highlevels of S. mutans in their saliva were the sourcecus Oral. In: Balows, A., et al. (Eds.), TheProkaryotes. second ed., vol. II. Springer, New York,pp. 14211449.

Kozai, K., Nakayama, R., Tedjosasongko, U., 1999.Intrafamilial distribution of mutans streptococci inJapanese families and possibility of father-to-childtransmission. Oral Microbiol. Immunol. 43, 99106.

Li, Y., Caufield, P.W., 1995. The fidelity of initialacquisition of mutans streptococci by infants fromtheir mothers. J. Dent. Res. 74, 681685.

Li, Y., Wang, W., Caufield, P.W., 2000. The fidelity ofmutans streptococci transmission and caries statuscorrelate with breast-feeding experience amongChinese families. Caries Res. 34, 123132.

Mattos-Graner, R.O., Li, Y., Caufield, P.W., Duncan, M.,Smith, D.J., 2001. Genotypic diversity of mutansstreptococci in Brazilian nursery children suggestshorizontal transmission. J. Clin. Microbiol. 39,23132316.

Redmo Emanuelsson, I., Wang, W., 1998. Demonstrationof identical strains of mutans streptococci withinChinese families by genotyping. Eur. J. Oral Sci. 106,788794.

Redmo Emanuelsson, I., Li, Y., Bratthall, D., 1998.Genotyping shows different strains of mutans strepto-cocci between father and child and within parentalpairs in Swedish families. Oral Microbiol. Immunol. 13,271277.

Roeters, F.J.M., van de Hoeven, J.S., Burgersdijk,R.C.W., Schaedenn, M.J.M., 1995. Lactobacilli, mu-tans streptococci and dental caries: a longitudinalstudy in 2-year-old children up to the age of 5 years.Caries Res. 29, 272279.

Saarela, M., Hannula, J., Matto, J., Asikainen, S.,Alaluusua, S., 1996. Typing of mutans streptococciby arbitrarily primed polymerase chain reaction. Arch.Oral Biol. 41, 821826.

Smibert, R.M., Krieg, N.R., 1994. Phenotypic character-ization. In: Gerhardt, P., et al. (Eds.), Methods forGeneral and Molecular Bacteriology. American Societyfor Microbiology, Washington, DC, pp. 603655.

Tedjosasongko, U., Kozai, K., 2002. Initial acquisition andtransmission of mutans streptococci in children at daynursery. ASDC J. Dent. Child. 69, 284288.

Thibodeau, E.A., OSullivan, D.M., 1999. Salivary mutansstreptococci and caries development in the primaryand mixed dentitions of children. Community Dent.Oral Epidemiol. 27, 406412.

Van Houte, J., Green, D.B., 1974. Relationship betweenthe concentration of bacteria in saliva and thecolonization of teeth in humans. Infect. Immun. 9,624630.

Van Palenstein Helderman, W.H., Ijsseldijk, M., Huis intVeld, J.H.J., 1983. A selective medium for the twomajor subgroups of the bacterium Streptococcusmutans isolated from human dental plaque and saliva.Arch. Oral Biol. 7, 599603.

WHO, 1997. Oral Health Surveys: Basic Method, fourthed. World Health Organization, Geneva.number of the parents who had high levels of S. Hardie, J.M., Whiley, R.A., 1992. The genus Streptococ-

Colonization and vertical transmission of Streptococcus mutans in Turkish childrenIntroductionMaterial and methodsMicrobial sampling, isolation and cultivation of S. mutansExtraction of DNAAP PCR assayAnalysis of AP PCR banding patternsStatistical analysis

ResultsDiscussionConclusionAcknowledgmentsReferences