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Presence of mutans streptococci and Candida spp. in dental plaque/dentine of carious teeth and early childhood caries

Presence of mutans streptococci and Candida spp. in dental plaque/dentine of carious teeth and early childhood caries
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  Presence of mutans streptococci and  Candida spp. in dental plaque/dentine of carious teethand early childhood caries Fabı´olaGalbiattideCarvalho a, *,DaniloSouzaSilva b,1 ,JosimeriHebling a,2 ,Luis Carlos Spolidorio b,1 , Denise Madalena Palomari Spolidorio b,1 a Department of Orthodontics and Pediatric Dentistry, Araraquara Dental School,Sa˜o Paulo State University, UNESP, Brazil b Department of Physiology and Pathology, Araraquara Dental School, Sa˜o Paulo State University,UNESP, Brazil Accepted 5 June 2006 Archives of Oral Biology (2006)  51 , 1024— KEYWORDS Early childhood caries,Mutans streptococci, Candida albicans  Summary  This study determined the presence of mutans streptococci and  Candida spp. in supragingival dental plaque and infected dentine of caries-free children, withearly childhood caries and caries. Pooled samples of dental plaque and infecteddentine were collected from 56 children aged 1—5 years, which were divided into 3groups: early childhood caries (ECC); caries and caries-free. Infected dentine wascollected in ECC and caries groups to compare the frequency of these microorganismsin the collected sites. The samples were inoculated in SB20 and SA medium, formutans streptococci and  Candida  spp., respectively, and incubated at 37  8 C for 48 h.Colony growth was verified and the identification was performed by biochemical testsand CHROMagar Candida. Fisher’s test or chi-square ( x 2 ) were applied (  p  = 0.05). Themoreprevalentspecieswere S.mutans  and Candidaalbicans  inECC(85.4%and60.4%,respectively), independently of the sample site.  S. mutans   only was significantlyassociated with carious teeth, whether in early childhood caries or not. However, thefrequency of   C. albicans   in ECC was higher when compared to caries and caries-freegroups. There is a significant association between the presence of   C. albicans   andearly childhood caries. # 2006 Elsevier Ltd. All rights reserved. * Corresponding author at: Av: Dr. Paulo de Moraes, 1671 apto 11, 13400-853 Piracicaba, Sa˜o Paulo, Brazil. Tel.: +55 19 3412 5345;fax: +55 19 3412 5218. E-mail addresses: (F.G. de Carvalho), (D.S. Silva), (J. Hebling), (L.C. Spolidorio), (D.M.P. Spolidorio). 1 Present address: R. Humaita´, 1680, 14801-903 Araraquara, Sa˜o Paulo, Brazil. Tel.: +55 16 33016480; fax: +55 16 33016488. 2 Present address: R. Humaita´, 1680, 14801-903 Araraquara, Sa˜o Paulo, Brazil. Tel.: +55 16 33016328; fax: +55 16 33016488.0003–9969/$ — see front matter # 2006 Elsevier Ltd. All rights reserved.doi:10.1016/j.archoralbio.2006.06.001  Introduction Early childhood caries (ECC) is defined as the pre-sence of one or more decayed (noncavitated orcavitated lesions), missing (due to caries), or filledtooth surfaces in any primary tooth 1 and is asso-ciated with the consumption of foods or fluids swee-tened with fermentable carbohydrates, usuallyfrom the bottle, over an extended period. 2 ECCrepresents a serious public health problem in dis-advantaged communities in both developing andindustrialized countries, dietary habits and earlymutans streptococci infection have been identifiedas the important predisposing factors. 3 Studies onthe microflora of ECC began in the 1980s 4 and thepredominant microorganisms identified weremutans streptococci, although others have sincebeen isolated, e.g.  Candida  spp.,  Lactobacillus  spp.,  Actinomyces   spp. and  Veillonella  spp. 5,6 Cross-sectional studies focused on mutans strepto-cocci and lactobacilli, because they were beinglooked at as markers, and there were selectivemedia available to isolate them. However, the roleof other microorganisms in infecting the oral florabecome important, due to their presence in the oralsamples of infants with ECC 2,6 and the possibilitythat they will be associated with the disease pro-cess. Candidaalbicans  haveahighacidogenicpotentialand biofilm formation may have been affected bydietary sugars. 7 Moreover, there is evidence to sug-gest that  C. albicans   may have a cariogenic poten-tial, 8 although its role in caries aetiology has notbeen established.Most reports investigate the microflora of earlychildhood caries in the dental plaque of affectedchildrencomparedwiththat of caries-free children;however infected dentine is not frequently col-lected. Furthermore, there is no comparison avail-able in the literature, between the microorganismsof early childhood caries and those of caries. Due toinsufficient information regarding the species of mutans streptococci and  Candida  spp. related tothese types of caries and infected dentine, such acomparison is important for a better understandingof the colonization processes and induction of thisdisease. Material and methods Subjects After the approval by the Araraquara Dental SchoolResearch and Ethics Committee, Sa˜o Paulo StateUniversity, 56 infants ranging from 2 to 5 years of age were recruited for this study. Written informedconsent was obtained from at least one of theirparentsorguardiansbeforethestudy,inaccordancewith the Ethical Guidelines of the Declaration of Helsinki (1975). These infants were selected on thebasis of feeding habits and clinical characteristics of caries type. They had only primary teeth, were ingood general health and had not made use of anti-biotics for at least 1 month before the investigation.Three groups were formed:   earlychildhoodcaries(ECC)group( n  = 24):atleastone decayed upper incisor with a specific clinicalpattern of early childhood caries (lesions at thecervical third of the clinical crown) 1 and history of prolonged, at libidum breast-feeding and/or babybottle with fermentable carbohydrates.   caries group ( n  = 11): at least 1 upper incisor withstrictly approximal caries in enamel and dentine(the cervical margin was sound), and childrenreceived bottle or breast feeding only while theywere in the nursing period, and from then on theywere no longer bottle or breast-fed.   caries-free group ( n  = 21): children received bot-tle or breast feeding only while they were in thenursing period, and from then on they were nolonger bottle or breast-fed. Sample collection and biochemical tests Supragingival dental plaque samples were collectedfrom the upper incisor buccal surface most affectedby caries, using a sterilized wooden toothpick. Sam-ples were collected from enamel in clinically soundgingival areas for the caries-free group, and aroundthe affected enamel for the ECC and caries groups.Immediately after sample collection, each woodentoothpick was placed in a sterilized tube containing1 mL saline solution. The infected dentine sampleswere collected at the center lesion of the sameupper incisor with a sterilized excavator no. 11.5(Duflex-SS.WHITE), taking care not to allow theexcavator to touch the adjacent and cervicalenamel to prevent sample contamination. Dentinesamples were placed in sterilized tubes containing5 mL BHI broth and incubated at 37  8 C for 24 h incandle jars. In a pilot study, both samples of dentineand dental plaque were placed in a sterile salinesolution, but only dental plaque grew. The authorssuggested that in dentine the microorganisms werein involved; they were enveloped by dentine andneeded a broth to grow in. Otherwise, in dentalplaque the microorganisms were more dispersed,and grew more easily than in dentine. Thus, dentalplaque samples were placed in saline solution anddentine in BHI broth.Presence of mutans streptococci and Candida spp. In dental plaque/dentine 1025  All samples were dispersed by vortexing withsterile 3.5—4.5 mm diameter glass beads for 30 sto disperse bacterial and yeast segregates and werediluted in decimal series from 10  1 to 10  4 in 0.15 Msalinesolution.Aliquotsofeachdilutionwereinocu-lated in Bacitracin Sucrose Agar/SB-20 9 and Sabour-aud Agar medium 10 for mutans streptococci and Candida  spp., respectively, and then incubated at37  8 C for 48 h. Mutans streptococci were identifiedto the species level on the basis of biochemical tests(fermentation of mannitol, sorbitol, melibiose andraffinose, hydrolysis of arginine, resistant to baci-tracin and H 2 O 2  produced) and physiological char-acteristics in accordance with Bergey’s Manual. 11 Candida  spp. were identified on CHROMagar Can-dida(Probac/Brazil),andthosenotidentifiedbythismedium were confirmed by biochemical identifica-tion tests (germ-tube tests, chlamydospore forma-tion, the assimilation and fermentation of sugars). 10 To differentiate  C. albicans   from  C. dubliniensis  , allsamples of   C. albicans   identified by CHROMagarCandida were tested for growth at 42 and 45  8 C. 10 Statistical analysis To evaluate the independence between species andcarious type (ECC, caries and caries-free), Fisher’stest or chi-square ( x 2 ) were applied (SPSS, Release6.0 software, SPSS Inc., Chicago, IL, USA). All sta-tistical tests were considered at the level of sig-nificance of 0.05%. Results Independence was observed between sampling siteand all species of mutans streptococci and  Candida ,suggestingthatthefrequenciesofpositivegrowthof microorganisms were similar for dental plaque andinfected dentine. In light of this, dental plaque andinfected dentine were considered jointly to deter-mine the association between the presence of microorganisms and the carious type; with theexception of caries-free group, in which just dentalplaque was evaluated (Table 1). For mutans strep-tococci, the prevalent species were  S. mutans  ,followed by  S. sobrinus  , both being present at ahigher frequency in ECC (85.4—12.5%) (Table 1).When the frequency of   S. mutans   was comparedbetween ECC and caries groups in dental plaque +dentine there was no significant difference (85.5—77.2%) (  p > 0.05) (Table 1).When the caries-free group was compared withthe early childhood caries and caries group, therewas only significant association between the pre-senceof   S.mutans  (  p < 0.0001).Thus,forthe other1026 F.G. de Carvalho et al. Table 1  Frequency of mutans streptococci according to carious type and sampling siteGroupsEarly childhood caries ( n  = 24) Caries ( n  = 11) Caries-free( n  = 21)Plaque Dentine Plaque + dentine Plaque Dentine Plaque + dentine Plaque S. mutans   87.5 (21) 83.3 (20) 85.4 (41) n.s. 72.7 (8) 81.8 (9) 77.2 (17) n.s. 19 (4) a,* S. sobrinus   12.5 (3) 12.5 (3) 12.5 (6) 9.0 (1) 9.0 (1) 9.0 (2) 9.5 (2)Other species 12.5 (3) 19.1 (8) 20.7 (10) 18.1 (2) 18.1 (2) 18.1 (4) 4.7 (1) n.s.: values did not differ statistically (Fisher’s test,  p > 0.05). For  S. sobrinus   and other species the values did not differ statistically(Fisher’s test,  p > 0.05). a Percentage of isolates (number of samples positive). * Values differed statistically (Fisher’s test,  p > 0.05). Table 2  Frequency of   Candida  spp. according to carious type and sampling siteGroupsEarly childhood caries ( n  = 24) Caries ( n  = 11) Caries-free( n  = 21)Plaque Dentine Plaque + dentine Plaque Dentine Plaque + dentine Plaque Candida albicans   50 (12) 70.8 (17) 60.4 (29) * 18.2 (2) 36.4 (4) 12.5 (6) n.s. 14.3 (3) n.s. a C. tropicalis   16.7 (4) 4.2 (1) 10.4 (5) 9.1 (1) ND 4.5 (1) ND C. krusei  4.2 (1) 8.3 (2) 6.2 (3) ND ND ND ND n.s.: values did not differ statistically (Fisher’s test,  p > 0.05). For  C. tropicalis   and  C. krusei  the values did not differ statistically(Fisher’s test,  p > 0.05). ND: not detected. a Percentage of isolates (number of samples positive). * Values differed statistically (Fisher’s test,  p > 0.05).  species, similar frequencies were detected for car-ies-free children and those with caries (Table 1).For the  Candida  species, the most prevalent were C. albicans  , followed by  C. tropicalis   and  C. krusei ,which were present at a higher frequency in ECC(60.4%, 10.4%, 6.2%, respectively) (Table 2). All  C.albicans  isolatesexhibitedgrowthat42  8 C/45  8 Candno presence of   C. dubliniensis   was observed. Thefrequency of   C. albicans   in ECC (60.4%) was signifi-cantly greater than for the caries-free (14.3%) andcaries(12.5%)groups(Table2).Thus,itmaysuggestasignificant association between the presence of thisyeast and early childhood caries (  p < 0.05). Discussion These data confirm previous reports about the highfrequency of   S. mutans   in early childhood carieslesionscomparedwiththefrequencyfoundindentalplaque on sound enamel surfaces of pre-school chil-dren. 3,4,12 In the present study the lowest preva-lenceof  S.mutans  wasfoundincaries-freechildren.According to Nyvad and Kilian, 13 in these children,or on sound enamel surfaces, the more prevalentspecies were  S. sanguinis   and  S. mitis  , since theseare the primary colonizers and play an antagonisticrole with  S. mutans,  which usually constitutes lessthan 1% of the dental plaque flora in children withnegligible caries activity. 14 Similar frequency of   S. mutans   was found in twocarious types (ECC and caries) in dental plaque +dentine. This may be because 72.7% of the childrenin the caries group were on a highly cariogenic diet;although they did not have the night breast-feedinghabit, sucrose was another diet source, favoring anincrease of   S. mutans   in the oral environment. 15 S. mutans   was more prevalent than  S. sobrinus  ,which is in agreement with Alaluusua et al. 16 andSpolidorio et al. 17 These two species are clearlyimplicated in the caries process. The majority of epidemiological studies exploring the associationbetween streptococci and caries have determinedthe combined numbers of the two species. 15 The differential media used in this investigationwas CHROMagar Candida (Probac/Brazil), an easyand reliable method for the presumptive identifica-tion of   C. albicans  ,  C. tropicalis   and  C. krusei , 18—20 and widely used in clinical microbiology labora- tories. 20 Yu¨cesoy and Marol 20 found values at99.4% sensitivity and 100% specificity for  C. albicans  strains identified by CHROMagar Candida.The prevalence of   Candida  spp., mainly  C. albi-cans  , was higher in ECC when compared with chil-dren of  the caries-free group, corroborating otherreports. 2,21 Furthermore,  C. albicans   is the mostcommonly found species in the oral cavity followedby  C. tropicalis  . 2 The results demonstrated a significant associa-tion between the presence of   C. albicans   and earlychildhood caries (Fisher’s test,  p < 0.05, Table 2).Unfortunately, there are no reports in the literatureevaluating  C. albicans   and early childhood caries.There are evidences that  C. albicans   may have acariogenic potential  in vitro 8 and carious dentinehas a high concentration of   Candida  spp., providinga significant ecologic niche for the dissemination of these yeasts. 22 The role of   C. albicans   in dentalcaries has not been clearly established, but someauthors 21,23,24 have evidenced a relationshipbetween the presence of this yeast and caries,indicating that poor oral hygiene, dentinal cariouslesionsandcariogenicdietmaybeimportantfactorsfor the presence of   C. albicans   in oral cavity.Furthermore, the effect of dietary sugars on Candida  biofilm formation has also been studiedfrom an oral point of view, since dietary carbohy-drates modulate microbial colonization of the latterniche.Twomonosaccharides,glucoseandgalactose,have been extensively investigated for their effecton candidal adhesion. 7 Several studies have demon-strated that culture media rich in carbohydrates,such as glucose, sucrose, and especially galactose,increase  C. albicans   adherence to epithelial cellsand to acrylic dental surfaces, 25 due to the addi-tional production of the fibrillar—floccular surfacelayer, which makes the yeast more resistant. 26 There are no reports in the literature evaluatingthe formation of the fibrillar—floccular layer in thebiofilm and carious dentine of children with earlychildhood caries, although these children fre-quently have the habit of night bottle-feeding. 3 Milkcontains lactose, which will be degraded to galac-tose and glucose following the addition of sucrose inbaby bottles, suggesting that in these children,there could be higher adhesion of   C. albicans  , dueto the presence of these sugars. This study foundthat there is an association between  C. albicans   andECC, thus it was suggested that  C. albicans   may playa role in the caries process. The higher numberscould, however, be no more than a consequence of the higher sugar levels in the diet of children withECC. Further investigations are necessary to verifywhether the growth of   C. albicans   is favored byfactors involved in the caries process or to this yeasthaving a role in caries aetiology. Conclusion Higher frequency of   S. mutans   was found in cariesand ECC groups, followed by  S. sobrinus  .  C. albicans  Presence of mutans streptococci and Candida spp. In dental plaque/dentine 1027  was the most prevalent  Candida  species and theirfrequency in ECC was significantly higher than in thecaries-free and caries groups, suggesting an associa-tion between the presence of this yeast and earlychildhood caries. Acknowledgements The authors are grateful to the laboratory techni-cians Carina BentoLuizandAndre´iaCristina Cellifortheir technical support. References 1. Drury TF, Horowitz AM, Ismail IA, Maertens MP, Rozier GR,Selwitz RH. Diagnosing and reporting early childhood cariesfor research purposes.  J Public Health Dent  1999; 59 :192—7.2. Marchant S, Brasilsford SR, Twomey AC, Roberts GJ, BeightonD. The predominant microflora of nursing caries lesions. Caries Res  2001; 35 :397—402.3. Ramos-Gomes FJ, Weintraub JA, Gansky SA, Hoover CI,Featherstone JDB. Bacterial, behavior and environmentalfactors associated with early childhood caries.  J Clin Pediatr Dent  2002; 26 :165—73.4. van Houte J, Gibbs G, Butera C. Oral flora of children with‘‘nursing bottle caries’’.  J Dent Res  1982; 62 :382—5.5. Matee MIN, Mikx FHM, Maselle SYM, Van Palenstein Helder-man WH. Mutans streptococci and Lactobacilli in breast-fedchildren with rampant caries.  Caries Res  1992; 26 :183—7.6. Starr JR, White TC, Leroux B, Luis HS, Bernardo M, Leita˜o J, et al.  Persistence of oral  Candida albicans   carriage in healthyPortuguese schoolchildren followed for 3 years.  Oral Micro-biol Immunol  2002; 17 :304—10.7. Jin Y, Samaranayake LP, Samaranayake Y, Yip HK. Biofilmformation of   Candida albicans   is variably affected by salivaand dietary sugars.  Arch Oral Biol  2004; 49 :789—98.8. Nikawa H, Yamashiro H, Makihira S, Nishimura M, Egusa H,Furukawa M,  et al.  In vitro cariogenic potencial of   Candidaalbicans  .  Mycoses  2003; 46 :471—8.9. Davey AL, Rogers AH. Multiple types of the bacterium  Strep-tococcus mutans   in the human mouth and their intra-familytransmission.  Arch Oral Biol  1984; 29 :453—60.10. Sullivan D, Coleman D.  Candida dubliniensis:  characteristicsand identification.  J Clin Microbiol  1998; 36 :329—34.11. Hardie JM, Oral streptococci.Sneath PHA, Mair NS, SharpeME, Holt JC, editors.  Bergey’s manual of systematic bacter-iology  , vol. 2. Baltimore: Willians and Wilkins; 1984—1986.  p.1054—63. 12. Milnes AR, Bowden GHW. The microflora associated withdeveloping lesions of nursing caries.  Caries Res  1985; 19 :289—97.13. Nyvad B, Kilian M. Comparison of the initial streptococcalmicrofloraondentalenamelincaries-activeecaries-inactiveindividuals.  Caries Res  1990; 24 :267—72.14. Berkowitz R. Etiology of nursing caries: a microbiologicperspective.  J Public Health Dent  1996; 56 :51—4.15. Colby SM, Russell RRB. Sugar metabolism by mutans strepto-cocci.  J Appl Microbiol Sym Suppl  1997; 83 :80S—8S.16. Alaluusua S, Ma¨tto¨ J, Gro¨nrooos L, Innila¨ S, Torkko H, Asi- kainen S,  et al.  Oral colonization by more than one clonaltype of mutans streptococcus in children with nursing-botlledental caries.  Arch Oral Biol  1996; 41 :167—73.17. Spolidorio DMP, Hofling JF, Rosa EAR, Pereira CV, Moreira D,Gonc ¸ alvesRB.Salivarybiotypesofmutansstreptococcilevelsin schoolchildren aging 6—8-year old having a socioeconomicbase.  Braz J Oral Sci  2004; 3 :390—4.18. Odds FC, Bernaerts R. CHROMagar Candida, a new differen-tial isolation medium for presumptive identification of clini-callyimportantCandidaspecies.  JClinMicrobiol 1994; 32 (8):1923—9.19. Beighton D, Ludford R, Clark DT, Brailsford SR, Pankhurst CL,Tinsley GH,  et al.  Use of CHROMagar Candida medium forisolation of yeasts from dental samples.  J Clin Microbiol 1995; 33 (11):3025—7.20. Yu¨cesoy M, Marol S. Performance of CHROMagar Candida andBIGGY agar for identification of yeasts species.  Ann ClinMicrobiol Antimicrob  2003; 2 (8):1—7.21. Radford JR, Ballatyne HM, Nugent ZJ, Beighton D, RobertsonM, Longbottom C,  et al.  Caries — associated microorganismsin infants from different socio-economic backgrounds inScothland.  J Dent  2000; 28 :307—12.22. Hossain H, Ansari F, Schulz-Weidner N, Wetzel WE, Chakra-borty T, Domann E. Clonal identity of   Candida albicans   in theoral cavity and the gastrointestinal tract of pre-school chil-dren.  Oral Microbiol Immunol  2003; 18 (5):302—8.23. Moalic E, Gsetalin A, Quinio D, Gest PE, Zerilli A, Le FlohicAM. The extent of oral fungal flora in 353 students andpossible relationships with dental caries.  Caries Res  2001; 35 :149—55.24. Akdeniz BG, Koparal E, Sen BH, Ates M, Denizci AA. Preva-lence of   Candida albicans   in oral cavities and root canals of children.  J Dent Child   2002; 69 :189—292.25. Pires MFC, Correa B, Gambale W, Paula CR. Experimentalmodel of   Candida albicans   (serotypes A and B) adherence invitro.  Braz J Microbiol  2001; 32 :163—9.26. McCourtie J, Douglas J. Relationship between cell surfacecomposition, adherence and virulence of   Candida albicans  . Infect Immun  1984; 45 :6—12. 1028 F.G. de Carvalho et al.
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