Journal of Investigative and Clinical Dentistry (2011), 2, 197–200
ORIGINAL ARTICLE Oral Microbiology
Differential adhesion of Streptococcus mutans to metallic brackets induced by saliva from caries-free and caries-active individuals Ademir Franco do Rosa´rio Junior1, Lue´gya Amorin Henriques Knop1, Fernanda Brasil Baboni1, Alinne Ulbrich Mores Rymovicz1, Orlando Motohiro Tanaka1 & Edvaldo Antonio Ribeiro Rosa2 1 Department of Orthodontics, The Pontifical Catholic University of Parana´, Curitiba, Brazil 2 Xenobiotics Research Unit, The Pontifical Catholic University of Parana´, Curitiba, Brazil
Keywords adhesion, caries experience, metallic bracket, saliva, Streptococcus mutans. Correspondence Prof. Edvaldo Antonio Ribeiro Rosa, Xenobiotics Research Unit, Biological and Health Sciences Centre, The Pontifical Catholic University of Parana, 1155 Imaculada Conceicao Street, Curitiba, Parana 80215 901, Brazil. Tel: +55-41-3271-1497 Fax: +55-41-3271-1405 Email: [email protected] Received 29 January 2011; accepted 1 February 2011. doi: 10.1111/j.2041-1626.2011.00058.x
Abstract Aim: The purpose of this study was to evaluate the influence of saliva obtained from caries-free and caries-active individuals on the adhesion rates of Streptococcus mutans to metallic brackets. Methods: The unstimulated whole saliva of four caries-free (decayed, missing, or filled surfaces = 0) volunteers and four caries-active (decayed, missing, or filled surfaces >12) patients were collected. The saliva samples from each group were mixed and clarified. Acquired pellicles were formed onto 30 metallic edgewise brackets for premolars for each saliva group. The brackets were put in contact with planktonic cells of Streptococcus mutans ATCC 25175. Adhesion rates were assessed by crystal violet retention technique. Results: A higher streptococcal adhesion pattern (P < 0.00001) was observed on acquired pellicles formed by saliva from caries-active donors. Conclusions: The results showed that saliva from caries-active patients tends to increase the mutans adhesion to surfaces, which is a point of concern for orthodontists.
Introduction One undesirable side-effect of orthodontic therapy is the increment in caries risk due to biofilms that entrap acidogenic organisms.1–4 Such biofilms can be formed onto exposed dental or abiotic adjacent surfaces. The initial process of bacterial adhesion to these surfaces is influenced by acquired pellicle.5 Saliva with different compositions might imply a qualitative variability of acquired pellicle that will interfere in primary bacterial adhesion. It has been demonstrated that hydroxyapatite covered with glucosyltransferase-rich, acquired pellicles also presents glucans, which enhance streptococcal adhesion.6 Thus, it is reasonable to suppose that saliva from individuals with different caries ª 2011 Blackwell Publishing Asia Pty Ltd
experiences might determine the microbial colonization performance. It is important to note that brackets could play a significant impact in caries events because they remain attached to dentition throughout the entire period of orthodontic treatment, and their complex design provides a unique environment that impedes proper access to tooth surfaces for cleaning.7 Also, brackets seems to corroborate with the increase of caries casuistic.8 The cariogenic bacterium Streptococcus mutans has been found on in vivo orthodontic brackets.9 The understanding of how dental caries begins and develop in orthodontic patients is an indispensable necessity. Based on this premise, the aim of this study was to evaluate if saliva from caries-free and caries-active 197
A.F. do Rosa´rio Junior et al.
Streptococcus mutans adhesion and saliva
Methods Ten-milliliter aliquots of unstimulated whole saliva were obtained from four volunteers with a decayed, missing, or filled surface (DMFS) index of 0 (22.5 ± 1.3 years; two men and two women), and from four patients with active caries and an DMFS index higher than 12 (25.4 ± 2.7 years; two men and two women). The World Health Organization10 caries diagnostic criteria for DMFS were used to evaluate dental caries status by an expert dental surgeon (LAHK). Clinical examinations were carried out under artificial light, using a sharp sickle explorer, flat-surface mouth mirror, gauze, and compressed air. Three bite-wing radiographs were taken. Saliva donors carefully brushed their teeth under supervision of a clinician, and after this, avoided eating for at least 2 h before saliva collection. All saliva collections were carried out simultaneously at 9 h. Donors were instructed to swallow saliva for 1 min, after which, harvestings were carried out for 5 min. Collections were performed using sterile polypropylene straws and glassware, avoiding contact with the vermilion borders. Saliva aliquots from each group were immediately mixed in order to increase the number of receptors.11 Immediately after collection, saliva mixtures were cooled in saltcrushed ice for 30 min and centrifuged (16 000 · g, 4C, 5 min) to remove microorganisms and food debris. Gram stainings confirmed the lack of clumped and free-living bacteria/fungi in supernatants of saliva mixtures. The saliva from two groups (caries free and caries active) was used immediately after processing. Streptococcus mutans ATCC 25175 was grown in brain heart infusion 1:20 plus 100 mm glucose under 10% pCO2, 100 rpm at 37C. After 24 h incubation, cells were harvested at 3000 · g and washed twice with sterile 145 mm NaCl. Washed cells were resuspended in sterile 145 mm NaCl to an optical density (OD)660 nm of 1.0 (2 · 09 CFU/mL).12 Two set of 30 metallic 0.022-inch slot edgewise brackets for premolars (Dental Morelli, Sorocaba, Brazil) were immersed in clarified saliva samples from caries-free and caries-active donors for 2 h and washed twice with sterile 145 mm NaCl. Saliva-conditioned brackets were immersed in a suspension of Streptococcus mutans ATCC 25175 (2 · 109 CFU/mL) for 2 h at 37C.12 The brackets were gently washed in a water bath, and the cells that remained attached were further fixed with 99% methanol for 15 min. After fixing, the brackets covered with bacterial cells were air dried and immersed in 0.5% crystal violet for 20 min. Excess crystal violet was removed by 198
gently washing with water. Finally, the entrapped crystal violet was released by adding 250 lL of 33% acetic acid. Aliquots of 100 lL were transferred to flat-bottom polystyrene microtitration plates (TPP, Trasadingen, Switzerland), and OD560 nm was determined using a TP reader (ThermoPlate, Sa˜o Paulo, Brazil).12 Thirty OD560 nm values for each group were plotted in MS Excel spreadsheets (Microsoft, Redmond, WA, USA) and transferred to the interface of BioEstat 5.0 statistical package (IDS Mamiraua´, Tefe´, Brazil). Statistical differences between the results of both groups were assessed by Student’s t-test, with a P-threshold of 0.05. The execution of this study was approved by the institution’s Committee of Ethics in Research with Humans. Results Overall, the obtained data revealed that streptococcal adhesion rates were higher in pellicles formed with saliva from caries-active individuals than those from caries-free individuals (P < 0.00001) (Figure 1). Discussion To our knowledge, the results obtained have never been presented by others and might contribute to the current
0.7 0.5% CV retention by adhered cells (OD560 nm)
patients interferes in the adhesion patterns of Streptococcus mutans to metallic brackets.
P < 0.00001 (Student’s t-test) 0.6
0.5
0.4
0.3
0.2
0.1
0.0
Saliva from patients with DMFS >12
Saliva from patients with DMFS = 0
Figure 1. Differential adhesion of Streptococcus mutans ATCC 25175 on acquired pellicles formed with saliva from individuals with different caries experiences. Error bars indicate the standard error of mean (SEM). CV, crystal violet retention; DMFS, decayed, missing, or filled surfaces; OD, optical density.
ª 2011 Blackwell Publishing Asia Pty Ltd
A.F. do Rosa´rio Junior et al.
Streptococcus mutans adhesion and saliva
understanding of cariogenic bacteria attachment to teeth and of orthodontic-related caries dynamics. Patients with a previous history of high-activity caries tend to be more densely colonized by mutans streptococci than the remaining population. Among the virulence attributes of such bacterium, secreted glucosyltransferases (Gtf) (EC 2.4.1.5) play a considerable role, indirectly favoring cellular adhesion to biotic and abiotic intraoral hard surfaces.13 There are substantial data supporting the idea that significant amounts of streptococcal Gtf activity can be localized in vivo.14,15 Gtf is present in whole human saliva and is incorporated into initial salivary pellicle, which forms on teeth,6,16–18 increasing throughout initial stages of pellicle formation.19 In addition, the activity of Gtf is enhanced when GtfB, GtfC, and GtfD enzyme isoforms are adsorbed to the surface when compared with its activity in solution.20–23 These enzymes act in extracellular environments, converting transitory sugars, such as sucrose, into extracellular glucan that mediate mutans streptococci attachment to acquired pellicle-related glucan by glucan-binding protein B.24 Results from other studies have shown that glucan synthesis catalyzed by streptococcal Gtfs can enhance the pathogenic potential of dental plaque by promoting the accumulation of large numbers of cariogenic streptococci on the teeth of humans and experimental animals,25 as well as on hydroxyapatite beads.7,25 Using the isoenzymatic variant GtfB as a predictive caries marker once in caries-active patients, it was recently proposed that higher salivary amounts of this enzyme strongly correlate with higher DMFS values and with elevated mutans counts.26 Based on such a premise and our results, we consider that increments in streptococcal adhesion seen here might involve mostly Gtf glucan-binding mechanisms than any other. Hypothetically, other possible pathways involving mutans surface adhesin AgI/II could be considered, as various salivary components, such as sIgA, b2-microglobulin, histidine-rich polypeptide, lysozyme, lactoferrin, and
References 1 Noble J, Cassolato S, Karaikos N, Wiltshire WA. Point of care. Preventive and interceptive measures for improving and maintaining good oral hygiene and cariogenic control in orthodontic patients. J Can Dent Assoc 2009; 75: 441–3. 2 Opsahl Vital S, Haignere-Rubinstein C, Lasfargues JJ, Chaussain C. Caries risk and orthodontic treatment. Int Orthod 2010; 8: 28–45.
ª 2011 Blackwell Publishing Asia Pty Ltd
high molar mass glycoproteins might act as receptors for this adhesin.27 However, it was demonstrated that AgI/II analog adhesins fail to bind selectively to salivary pellicles formed with saliva samples from caries-free and cariesactive individuals.28 In addition, other host-related salivary molecules seem to not vary between patients from these two cohorts, as demonstrated by parotidean–saliva protein profiles.29,30 This study was conduced with brackets and with no other materials because brackets present intricate architecture that causes brush cleaning to be effective.8 Also, they serve as obstacles for the salivary clearance of microbes interfering saliva rheology and favoring bacterial retention.31,32 The clinical importance of the results here presented resides on the premise that patients with active caries or with recent caries history might have salivary conditions that favor mutans streptococci adhesion to orthodontic devices, even if these bacteria are minor in number among wider-diversity microbiota. Orthodontists must be aware of this, and adopt strategies that protect patients from further caries. Based on the data generated here, we propose that the caries experience exerts a direct influence on the colonization pattern of mutans streptococci onto metallic brackets. It is important to bear in mind that this increase in streptococcal colonization might determine caries development, and that preventive attitudes must be implemented or improved. Acknowledgments This study was conducted using intramural funds from The Pontifical Catholic University of Parana´. The study was subject to a grant from the National Council for Scientific and Technological Development (CNPq protocol 80621-08/9) as part of the Scientific Initiation Program of Ademir Franco do Rosa´rio Junior.
3 Sanpei S, Endo T, Shimooka S. Caries risk factors in children under treatment with sectional brackets. Angle Orthod 2010; 80: 509–14. 4 Chaussain C, Opsahl Vital S, Viallon V et al. Interest in a new test for caries risk in adolescents undergoing orthodontic treatment. Clin Oral Investig 2010; 14: 177–85. 5 Teixeira EH, Napimoga MH, Carneiro VA et al. In vitro inhibition of streptococci binding to enamel
acquired pellicle by plant lectins. J Appl Microbiol 2006; 101: 111–6. 6 Schilling KM, Bowen WH. Glucans synthesized in situ in experimental salivary pellicle function as specific binding sites for Streptococcus mutans. Infect Immun 1992; 60: 284–95. 7 Ahn SJ, Lee SJ, Lim BS, Nahm DS. Quantitative determination of adhesion patterns of cariogenic streptococci to various orthodontic brackets. Am J Orthod Dentofacial Orthop 2007; 132: 815–21.
199
A.F. do Rosa´rio Junior et al.
Streptococcus mutans adhesion and saliva
8 Mitchell L. Decalcification during orthodontic treatment with fixed appliances: an overview. Br J Orthod 1992; 19: 199–205. 9 Anhoury P, Nathonson D, Hughes CV, Socransky S, Feres M, Chou LL. Microbial profile on metallic and ceramic brackets. Angle Orthod 2002; 72: 338–43. 10 World Health Organization. Dentition status and treatment needs. Oral Health Surveys: Basic Methods, 4th edn. Geneva: World Health Organization, 1997: 40–7. 11 Teixeira EH, Napimoga MH, Carneiro VA et al. In vitro inhibition of oral streptococci binding to the acquired pellicle by algal lectins. J Appl Microbiol 2007; 103: 1001–6. 12 Baboni FB, Guariza-Filho O, Moreno AN, Rosa EAR. Influence of cigarette smoke condensate on cariogenic and candidal biofilm formation upon orthodontic materials. Am J Orthod Dentofacial Orthop 2010; 138: 427–34. 13 Vacca-Smith AM, Bowen WH. Binding properties of streptococcal glucosyltransferases for hydroxyapatite, saliva-coated hydroxyapatite, and bacterial surfaces. Arch Oral Biol 1998; 43: 103–10. 14 Ro¨lla G, Ciardi JE, Schultz SA. Adsorption of glucosyltransferase to saliva coated hydroxyapatite. Possible mechanism for sucrose dependent bacterial colonization of teeth. Scand J Dent Res 1983; 91: 112–7. 15 Scheie AA, Eggen KH, Ro¨lla G. Glucosyltransferase activity in human in vivo formed enamel pellicle and in whole saliva. Scand J Dent Res 1987; 95: 212–5.
200
16 Kopec LK, Vacca-Smith AM, Bowen WH. Structural aspects of glucans formed in solution and on the surface of hydroxyapatite. Glycobiology 1997; 7: 929–34. 17 Steinberg D, Kopec LK, Bowen WH. Adhesion of Actinomyces isolates to experimental pellicles. J Dent Res 1993; 72: 1015–20. 18 Vacca-Smith AM, Venkitaraman AR, Quivey RG, Bowen WH. Interactions of streptococcal glucosyltransferases with a-amylase and starch on the surface of salivacoated hydroxyapatite. Arch Oral Biol 1996; 41: 291–8. 19 Vacca-Smith AM, Bowen WH. In situ studies of pellicle formation on hydroxyapatite discs. Arch Oral Biol 2000; 45: 277–91. 20 Schilling KM, Bowen WH. The activity of glucosyltransferase adsorbed onto saliva-coated hydroxyapatite. J Dent Res 1988; 67: 2–8. 21 Hannig C, Hannig M, Attin T. Enzymes in the acquired enamel pellicle. Eur J Oral Sci 2005; 113: 2–13. 22 Vacca-Smith AM, Venkitaraman AR, Schilling KM, Bowen WH. Characterization of glucosyltransferase of human saliva adsorbed onto hydroxyapatite surfaces. Caries Res 1996; 30: 354–60. 23 Hannig C, Ruggeri A, Al-Khayer B et al. Electron microscopic detection and activity of glucosyltransferase B, C, and D in the in situ formed pellicle. Arch Oral Biol 2008; 53: 1003–10. 24 Hajishengallis G, Michalek SM. Current status of a mucosal vaccine
25
26
27
28
29
30
31
32
against dental caries. Oral Microbiol Immunol 1999; 14: 1–20. Tanzer JM, Freedman ML, Fitzgerald RJ. Virulence of mutants defective in glucosyltransferase, dextran mediated aggregation, or dextranase activity. In: Mergenhagen SA, Rosan B, ed. Molecular Basis of Oral Microbial Adhesion. Washington, DC: American Society for Microbiology, 1985: 204–11. Vacca-Smith AM, Scott-Anne KM, Whelehan MT, Berkowitz RJ, Feng C, Bowen WH. Salivary glucosyltransferase B as a possible marker for caries activity. Caries Res 2007; 41: 445–50. Koga T, Oho T, Shimazaki Y, Nakano Y. Immunization against dental caries. Vaccine 2002; 20: 2027–44. Tovar JA, Dura´n C, Rodrı´guez A, Jaramillo L. Adhesion of salivary components to Streptococcus mutans peptides. Acta Odontol Latinoam 2006; 19: 53–8. Anderson LC, Lamberts BL, Bruton WF. Salivary protein polymorphisms in caries-free and caries-active adults. J Dent Res 1982; 61: 393–6. Dodds MW, Johnson DA, Mobley CC, Hattaway KM. Parotid saliva protein profiles in caries-free and cariesactive adults. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1997; 83: 244–51. Demling A, Elter C, Heidenblut T et al. Reduction of biofilm on orthodontic brackets with the use of a polytetrafluoroethylene coating. Eur J Orthod 2010; 32: 414–8. Steinberg D, Eyal S. Initial biofilm formation of Streptococcus sobrinus on various orthodontics appliances. J Oral Rehabil 2004; 31: 1041–5.
ª 2011 Blackwell Publishing Asia Pty Ltd
ORIGINAL ARTICLE Oral Microbiology
Differential adhesion of Streptococcus mutans to metallic brackets induced by saliva from caries-free and caries-active individuals Ademir Franco do Rosa´rio Junior1, Lue´gya Amorin Henriques Knop1, Fernanda Brasil Baboni1, Alinne Ulbrich Mores Rymovicz1, Orlando Motohiro Tanaka1 & Edvaldo Antonio Ribeiro Rosa2 1 Department of Orthodontics, The Pontifical Catholic University of Parana´, Curitiba, Brazil 2 Xenobiotics Research Unit, The Pontifical Catholic University of Parana´, Curitiba, Brazil
Keywords adhesion, caries experience, metallic bracket, saliva, Streptococcus mutans. Correspondence Prof. Edvaldo Antonio Ribeiro Rosa, Xenobiotics Research Unit, Biological and Health Sciences Centre, The Pontifical Catholic University of Parana, 1155 Imaculada Conceicao Street, Curitiba, Parana 80215 901, Brazil. Tel: +55-41-3271-1497 Fax: +55-41-3271-1405 Email: [email protected] Received 29 January 2011; accepted 1 February 2011. doi: 10.1111/j.2041-1626.2011.00058.x
Abstract Aim: The purpose of this study was to evaluate the influence of saliva obtained from caries-free and caries-active individuals on the adhesion rates of Streptococcus mutans to metallic brackets. Methods: The unstimulated whole saliva of four caries-free (decayed, missing, or filled surfaces = 0) volunteers and four caries-active (decayed, missing, or filled surfaces >12) patients were collected. The saliva samples from each group were mixed and clarified. Acquired pellicles were formed onto 30 metallic edgewise brackets for premolars for each saliva group. The brackets were put in contact with planktonic cells of Streptococcus mutans ATCC 25175. Adhesion rates were assessed by crystal violet retention technique. Results: A higher streptococcal adhesion pattern (P < 0.00001) was observed on acquired pellicles formed by saliva from caries-active donors. Conclusions: The results showed that saliva from caries-active patients tends to increase the mutans adhesion to surfaces, which is a point of concern for orthodontists.
Introduction One undesirable side-effect of orthodontic therapy is the increment in caries risk due to biofilms that entrap acidogenic organisms.1–4 Such biofilms can be formed onto exposed dental or abiotic adjacent surfaces. The initial process of bacterial adhesion to these surfaces is influenced by acquired pellicle.5 Saliva with different compositions might imply a qualitative variability of acquired pellicle that will interfere in primary bacterial adhesion. It has been demonstrated that hydroxyapatite covered with glucosyltransferase-rich, acquired pellicles also presents glucans, which enhance streptococcal adhesion.6 Thus, it is reasonable to suppose that saliva from individuals with different caries ª 2011 Blackwell Publishing Asia Pty Ltd
experiences might determine the microbial colonization performance. It is important to note that brackets could play a significant impact in caries events because they remain attached to dentition throughout the entire period of orthodontic treatment, and their complex design provides a unique environment that impedes proper access to tooth surfaces for cleaning.7 Also, brackets seems to corroborate with the increase of caries casuistic.8 The cariogenic bacterium Streptococcus mutans has been found on in vivo orthodontic brackets.9 The understanding of how dental caries begins and develop in orthodontic patients is an indispensable necessity. Based on this premise, the aim of this study was to evaluate if saliva from caries-free and caries-active 197
A.F. do Rosa´rio Junior et al.
Streptococcus mutans adhesion and saliva
Methods Ten-milliliter aliquots of unstimulated whole saliva were obtained from four volunteers with a decayed, missing, or filled surface (DMFS) index of 0 (22.5 ± 1.3 years; two men and two women), and from four patients with active caries and an DMFS index higher than 12 (25.4 ± 2.7 years; two men and two women). The World Health Organization10 caries diagnostic criteria for DMFS were used to evaluate dental caries status by an expert dental surgeon (LAHK). Clinical examinations were carried out under artificial light, using a sharp sickle explorer, flat-surface mouth mirror, gauze, and compressed air. Three bite-wing radiographs were taken. Saliva donors carefully brushed their teeth under supervision of a clinician, and after this, avoided eating for at least 2 h before saliva collection. All saliva collections were carried out simultaneously at 9 h. Donors were instructed to swallow saliva for 1 min, after which, harvestings were carried out for 5 min. Collections were performed using sterile polypropylene straws and glassware, avoiding contact with the vermilion borders. Saliva aliquots from each group were immediately mixed in order to increase the number of receptors.11 Immediately after collection, saliva mixtures were cooled in saltcrushed ice for 30 min and centrifuged (16 000 · g, 4C, 5 min) to remove microorganisms and food debris. Gram stainings confirmed the lack of clumped and free-living bacteria/fungi in supernatants of saliva mixtures. The saliva from two groups (caries free and caries active) was used immediately after processing. Streptococcus mutans ATCC 25175 was grown in brain heart infusion 1:20 plus 100 mm glucose under 10% pCO2, 100 rpm at 37C. After 24 h incubation, cells were harvested at 3000 · g and washed twice with sterile 145 mm NaCl. Washed cells were resuspended in sterile 145 mm NaCl to an optical density (OD)660 nm of 1.0 (2 · 09 CFU/mL).12 Two set of 30 metallic 0.022-inch slot edgewise brackets for premolars (Dental Morelli, Sorocaba, Brazil) were immersed in clarified saliva samples from caries-free and caries-active donors for 2 h and washed twice with sterile 145 mm NaCl. Saliva-conditioned brackets were immersed in a suspension of Streptococcus mutans ATCC 25175 (2 · 109 CFU/mL) for 2 h at 37C.12 The brackets were gently washed in a water bath, and the cells that remained attached were further fixed with 99% methanol for 15 min. After fixing, the brackets covered with bacterial cells were air dried and immersed in 0.5% crystal violet for 20 min. Excess crystal violet was removed by 198
gently washing with water. Finally, the entrapped crystal violet was released by adding 250 lL of 33% acetic acid. Aliquots of 100 lL were transferred to flat-bottom polystyrene microtitration plates (TPP, Trasadingen, Switzerland), and OD560 nm was determined using a TP reader (ThermoPlate, Sa˜o Paulo, Brazil).12 Thirty OD560 nm values for each group were plotted in MS Excel spreadsheets (Microsoft, Redmond, WA, USA) and transferred to the interface of BioEstat 5.0 statistical package (IDS Mamiraua´, Tefe´, Brazil). Statistical differences between the results of both groups were assessed by Student’s t-test, with a P-threshold of 0.05. The execution of this study was approved by the institution’s Committee of Ethics in Research with Humans. Results Overall, the obtained data revealed that streptococcal adhesion rates were higher in pellicles formed with saliva from caries-active individuals than those from caries-free individuals (P < 0.00001) (Figure 1). Discussion To our knowledge, the results obtained have never been presented by others and might contribute to the current
0.7 0.5% CV retention by adhered cells (OD560 nm)
patients interferes in the adhesion patterns of Streptococcus mutans to metallic brackets.
P < 0.00001 (Student’s t-test) 0.6
0.5
0.4
0.3
0.2
0.1
0.0
Saliva from patients with DMFS >12
Saliva from patients with DMFS = 0
Figure 1. Differential adhesion of Streptococcus mutans ATCC 25175 on acquired pellicles formed with saliva from individuals with different caries experiences. Error bars indicate the standard error of mean (SEM). CV, crystal violet retention; DMFS, decayed, missing, or filled surfaces; OD, optical density.
ª 2011 Blackwell Publishing Asia Pty Ltd
A.F. do Rosa´rio Junior et al.
Streptococcus mutans adhesion and saliva
understanding of cariogenic bacteria attachment to teeth and of orthodontic-related caries dynamics. Patients with a previous history of high-activity caries tend to be more densely colonized by mutans streptococci than the remaining population. Among the virulence attributes of such bacterium, secreted glucosyltransferases (Gtf) (EC 2.4.1.5) play a considerable role, indirectly favoring cellular adhesion to biotic and abiotic intraoral hard surfaces.13 There are substantial data supporting the idea that significant amounts of streptococcal Gtf activity can be localized in vivo.14,15 Gtf is present in whole human saliva and is incorporated into initial salivary pellicle, which forms on teeth,6,16–18 increasing throughout initial stages of pellicle formation.19 In addition, the activity of Gtf is enhanced when GtfB, GtfC, and GtfD enzyme isoforms are adsorbed to the surface when compared with its activity in solution.20–23 These enzymes act in extracellular environments, converting transitory sugars, such as sucrose, into extracellular glucan that mediate mutans streptococci attachment to acquired pellicle-related glucan by glucan-binding protein B.24 Results from other studies have shown that glucan synthesis catalyzed by streptococcal Gtfs can enhance the pathogenic potential of dental plaque by promoting the accumulation of large numbers of cariogenic streptococci on the teeth of humans and experimental animals,25 as well as on hydroxyapatite beads.7,25 Using the isoenzymatic variant GtfB as a predictive caries marker once in caries-active patients, it was recently proposed that higher salivary amounts of this enzyme strongly correlate with higher DMFS values and with elevated mutans counts.26 Based on such a premise and our results, we consider that increments in streptococcal adhesion seen here might involve mostly Gtf glucan-binding mechanisms than any other. Hypothetically, other possible pathways involving mutans surface adhesin AgI/II could be considered, as various salivary components, such as sIgA, b2-microglobulin, histidine-rich polypeptide, lysozyme, lactoferrin, and
References 1 Noble J, Cassolato S, Karaikos N, Wiltshire WA. Point of care. Preventive and interceptive measures for improving and maintaining good oral hygiene and cariogenic control in orthodontic patients. J Can Dent Assoc 2009; 75: 441–3. 2 Opsahl Vital S, Haignere-Rubinstein C, Lasfargues JJ, Chaussain C. Caries risk and orthodontic treatment. Int Orthod 2010; 8: 28–45.
ª 2011 Blackwell Publishing Asia Pty Ltd
high molar mass glycoproteins might act as receptors for this adhesin.27 However, it was demonstrated that AgI/II analog adhesins fail to bind selectively to salivary pellicles formed with saliva samples from caries-free and cariesactive individuals.28 In addition, other host-related salivary molecules seem to not vary between patients from these two cohorts, as demonstrated by parotidean–saliva protein profiles.29,30 This study was conduced with brackets and with no other materials because brackets present intricate architecture that causes brush cleaning to be effective.8 Also, they serve as obstacles for the salivary clearance of microbes interfering saliva rheology and favoring bacterial retention.31,32 The clinical importance of the results here presented resides on the premise that patients with active caries or with recent caries history might have salivary conditions that favor mutans streptococci adhesion to orthodontic devices, even if these bacteria are minor in number among wider-diversity microbiota. Orthodontists must be aware of this, and adopt strategies that protect patients from further caries. Based on the data generated here, we propose that the caries experience exerts a direct influence on the colonization pattern of mutans streptococci onto metallic brackets. It is important to bear in mind that this increase in streptococcal colonization might determine caries development, and that preventive attitudes must be implemented or improved. Acknowledgments This study was conducted using intramural funds from The Pontifical Catholic University of Parana´. The study was subject to a grant from the National Council for Scientific and Technological Development (CNPq protocol 80621-08/9) as part of the Scientific Initiation Program of Ademir Franco do Rosa´rio Junior.
3 Sanpei S, Endo T, Shimooka S. Caries risk factors in children under treatment with sectional brackets. Angle Orthod 2010; 80: 509–14. 4 Chaussain C, Opsahl Vital S, Viallon V et al. Interest in a new test for caries risk in adolescents undergoing orthodontic treatment. Clin Oral Investig 2010; 14: 177–85. 5 Teixeira EH, Napimoga MH, Carneiro VA et al. In vitro inhibition of streptococci binding to enamel
acquired pellicle by plant lectins. J Appl Microbiol 2006; 101: 111–6. 6 Schilling KM, Bowen WH. Glucans synthesized in situ in experimental salivary pellicle function as specific binding sites for Streptococcus mutans. Infect Immun 1992; 60: 284–95. 7 Ahn SJ, Lee SJ, Lim BS, Nahm DS. Quantitative determination of adhesion patterns of cariogenic streptococci to various orthodontic brackets. Am J Orthod Dentofacial Orthop 2007; 132: 815–21.
199
A.F. do Rosa´rio Junior et al.
Streptococcus mutans adhesion and saliva
8 Mitchell L. Decalcification during orthodontic treatment with fixed appliances: an overview. Br J Orthod 1992; 19: 199–205. 9 Anhoury P, Nathonson D, Hughes CV, Socransky S, Feres M, Chou LL. Microbial profile on metallic and ceramic brackets. Angle Orthod 2002; 72: 338–43. 10 World Health Organization. Dentition status and treatment needs. Oral Health Surveys: Basic Methods, 4th edn. Geneva: World Health Organization, 1997: 40–7. 11 Teixeira EH, Napimoga MH, Carneiro VA et al. In vitro inhibition of oral streptococci binding to the acquired pellicle by algal lectins. J Appl Microbiol 2007; 103: 1001–6. 12 Baboni FB, Guariza-Filho O, Moreno AN, Rosa EAR. Influence of cigarette smoke condensate on cariogenic and candidal biofilm formation upon orthodontic materials. Am J Orthod Dentofacial Orthop 2010; 138: 427–34. 13 Vacca-Smith AM, Bowen WH. Binding properties of streptococcal glucosyltransferases for hydroxyapatite, saliva-coated hydroxyapatite, and bacterial surfaces. Arch Oral Biol 1998; 43: 103–10. 14 Ro¨lla G, Ciardi JE, Schultz SA. Adsorption of glucosyltransferase to saliva coated hydroxyapatite. Possible mechanism for sucrose dependent bacterial colonization of teeth. Scand J Dent Res 1983; 91: 112–7. 15 Scheie AA, Eggen KH, Ro¨lla G. Glucosyltransferase activity in human in vivo formed enamel pellicle and in whole saliva. Scand J Dent Res 1987; 95: 212–5.
200
16 Kopec LK, Vacca-Smith AM, Bowen WH. Structural aspects of glucans formed in solution and on the surface of hydroxyapatite. Glycobiology 1997; 7: 929–34. 17 Steinberg D, Kopec LK, Bowen WH. Adhesion of Actinomyces isolates to experimental pellicles. J Dent Res 1993; 72: 1015–20. 18 Vacca-Smith AM, Venkitaraman AR, Quivey RG, Bowen WH. Interactions of streptococcal glucosyltransferases with a-amylase and starch on the surface of salivacoated hydroxyapatite. Arch Oral Biol 1996; 41: 291–8. 19 Vacca-Smith AM, Bowen WH. In situ studies of pellicle formation on hydroxyapatite discs. Arch Oral Biol 2000; 45: 277–91. 20 Schilling KM, Bowen WH. The activity of glucosyltransferase adsorbed onto saliva-coated hydroxyapatite. J Dent Res 1988; 67: 2–8. 21 Hannig C, Hannig M, Attin T. Enzymes in the acquired enamel pellicle. Eur J Oral Sci 2005; 113: 2–13. 22 Vacca-Smith AM, Venkitaraman AR, Schilling KM, Bowen WH. Characterization of glucosyltransferase of human saliva adsorbed onto hydroxyapatite surfaces. Caries Res 1996; 30: 354–60. 23 Hannig C, Ruggeri A, Al-Khayer B et al. Electron microscopic detection and activity of glucosyltransferase B, C, and D in the in situ formed pellicle. Arch Oral Biol 2008; 53: 1003–10. 24 Hajishengallis G, Michalek SM. Current status of a mucosal vaccine
25
26
27
28
29
30
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