Anaerobe 35 (2015) 54e59
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Anaerobe journal homepage: www.elsevier.com/locate/anaerobe
Pathogenesis and toxins
Penetration of Streptococcus sobrinus and Streptococcus sanguinis into dental enamel Susanne Kneist a, *, Sandor Nietzsche b, Harald Küpper a, Gerhard Raser a, Brita Willershausen c, Angelika Callaway c a b c
Research Laboratory for Dental Materials, Center of Dentistry, University Hospital, Jena, Germany Center of Electron Microscopy, University Hospital, Jena, Germany Department of Operative Dentistry, University Medical Center of the Johannes Gutenberg University, Mainz, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 August 2014 Received in revised form 21 January 2015 Accepted 30 January 2015 Available online 21 March 2015
The aim of this pilot study was to assess the difference in virulence of acidogenic and aciduric oral streptococci in an in vitro caries model using their penetration depths into dental enamel. 30 caries-free extracted molars from 11- to 16-year-olds were cleaned ultrasonically for 1 min with deionized water and, after air-drying, embedded in epoxy resin. After 8-h of setting at room temperature, the specimens were ground on the buccal side with SiC-paper 1200 (particle size 13e16 mm). Enamel was removed in circular areas sized 3 mm in diameter; the mean depth of removed enamel was 230 ± 60 mm. 15 specimens each were incubated anaerobically under standardized conditions with 24 h-cultures of Streptococcus sanguinis 9S or Streptococcus sobrinus OMZ 176 in Balmelli broth at 37 ± 2 C; the pH-values of the broths were measured at the beginning and end of each incubation cycle. After 2, 4, 6, 8, and 10 weeks 3 teeth each were fixed in 2.5% glutaraldehyde in cacodylate buffer for 24 h, washed 3 and dehydrated 30e60 min by sequential washes through a series of 30e100% graded ethanol. The teeth were cut in half longitudinally; afterward, two slits were made to obtain fracture surfaces in the infected area. After critical-point-drying the fragments were gold-sputtered and viewed in a scanning electron microscope at magnifications of 20-20,000. After 10 weeks of incubation, penetration of S. sanguinis of 11.13 ± 24.04 mm below the break edges into the enamel was observed. The invasion of S. sobrinus reached depths of 87.53 ± 76.34 mm. The difference was statistically significant (paired t test: p ¼ 0.033). The experimental penetration depths emphasize the importance of S. sanguinis versus S. sobrinus in the context of the extended ecological plaque hypothesis. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Plaque hypotheses Caries model experiment Human enamel Cariogenic bacteria Penetration depths
1. Introduction Various species of facultative anaerobic streptococci are capable of establishing themselves on different sites in the human oral cavity, and are considered as part of the normal indigenous microflora, co-existing during health with the host in a harmonious relationship [1,2]. Streptococcus sanguinis belongs to the pioneer microorganisms, which are the first to colonize and adhere to pellicle-covered sound enamel surfaces. There, dental biofilm represents a community of microorganisms in a matrix of polymers derived from the host or bacteria; endogenous substrates allow
* Corresponding author. Hütergasse 2, D-99084 Erfurt, Germany. E-mail address: [email protected] (S. Kneist). http://dx.doi.org/10.1016/j.anaerobe.2015.01.013 1075-9964/© 2015 Elsevier Ltd. All rights reserved.
only relatively slow cell division and growth, and only moderate amounts of acids are formed. In contrast, mutans streptococci (Streptococcus mutans and Streptococcus sobrinus), clearly implicated in caries initiation, are normally only present as a small proportion of the total plaque flora [3]. Caries is the result of an imbalance in the dental plaque flora. In the case of an increase in frequency and amount of intake of fermentable carbohydrates, and in particular of sucrose, longer intervals of low pH ranges persist [4]. Such conditions favor survival of aciduric and strongly acidogenic microorganisms, for example mutans streptococci and lactobacilli, shifting the balance between de- and remineralization towards loss of minerals, and invasion of cariogenic bacteria, in particular mutans streptococci, into dental enamel can occur [5e10]. Later, it was found that also non-mutans streptococci can adapt under severe and prolonged
S. Kneist et al. / Anaerobe 35 (2015) 54e59
acidic conditions from initially moderate acid producers into aggressive, cariogenic strains [11e20]. Accordingly, the ecological [1] and later the extended ecological [19] plaque hypothesis were formulated, which can better than earlier plaque hypotheses take into account the clinical course of the disease and preventive approaches. The aim of the study was to model biofilm formation and bacterial penetration into dental enamel, using two different cariogenic species, the acidogenic S. sanguinis and the acidogenic and aciduric S. sobrinus. 2. Material and methods 2.1. Preparation of specimens To exclude biological variabilities like differences in the degree of sclerotisation of the dental hard tissues, only caries-free permanent molars from 11- to 16-year-olds, extracted for orthodontic reasons, were included in the present study. The teeth were cleaned ultrasonically for 1 min with de-ionized water and, after air-drying, embedded in epoxy resin (Epofix, Struers, Germany). After 8 h of setting at room temperature, the specimens were ground on the buccal side with SiC-paper 1200 (particle size 13e16 mm) (ExaktMikroschleifsystem 400CS, Exakt Apparatebau Norderstedt, Germany) to remove the resin. Then, enamel was removed in circular areas sized 3 mm in diameter; the depth of removed enamel should be 200e250 mm (Fig. 1A, B).
55
2.3. Scanning electron microscopy of infected tooth areas After 2, 4, 6, 8, and 10 weeks 3 teeth each were first cut longitudinally (Exakt-Trennschleifsystem 300CP, Exakt Apparatebau Norderstedt, Germany) into halves; afterward, two slits were made to obtain fracture surfaces in the grinding buccal area (Fig. 2A, B), broken in the infected area of the window, resulting in a maximum of 12 fragments for each time period and streptococcal strain. The specimens were fixed in 2.5% glutaraldehyde in cacodylate buffer for 24 h, washed 3x in cacodylate buffer, and dehydrated for 30e60 min by sequential washes through a series of 30e100% graded ethanol. After critical-point drying, the fragments were sputtered with gold and viewed in a scanning electron microscope (LEO 1450VP, Zeiss, Oberkochen, Germany) at magnifications of x20-20,000. The minimal and maximal enamel thickness in the tooth fragments, and the largest penetration depth of the species were measured in a standardized way as shown in Fig. 3, using parallel lines orthogonal to the axis of the tooth. 2.4. Statistical analyses The data were analyzed with the statistics program SPSS (Version 20.0 Chicago, Ill. USA). Descriptive analyses were made for enamel thickness, penetration depths and pH values; data are expressed as means ± SD (standard deviations). The paired t test was used to test differences between the penetration depths of the two bacterial strains and in the changes in pH after 8 days. The significance level was set at p < 0.05 for all tests.
2.2. Microbiological procedures 3. Results Of the 30 specimens embedded in epoxy resin, 15 each were incubated anaerobically under standardized conditions at 37 ± 2 C (95% N2, 5% CO2; VT 5042 EK, Heraeus, Hanau, Germany) in 20 ml Balmelli broth (10 g trypticase, 5 g yeast extract, 5 g K2HPO4, 3 g meat extract, 50 g sucrose, ad 1000 ml aqua dest.; pH 7.2), inoculated with 1 ml of 24 h-cultures of the same broth of S. sanguinis 9S or S. sobrinus OMZ 176 (Heraeus Lamin Air, HBB 2448, Hanau, Germany) to simulate the caries initiation process in an in vitro model under highly standardized conditions with mono-species biofilm formation on and subsequent penetration into dental enamel. S. sobrinus OMZ 176 was chosen as a representative strain of mutans streptococci and S. sanguinis 9S as an example for low-pH non-mutans streptococci [21e23]. After 8 days each, the teeth were transferred into 20 ml of fresh Balmelli broth, inoculated again with 1 ml of the respective 24 h-cultures in Balmelli broth, and further incubated. The pH-values of the broths were measured at the beginning and end of each incubation cycle for five specimens each.
Enamel was removed in circular areas sized 3 mm in diameter; the mean depth of removed enamel was 230 ± 60 mm. The minimal enamel thickness was 619.55 ± 222.02 mm and the maximal enamel thickness was 1224.42 ± 222.73 mm. The analysis of the depth of penetration showed that after two and four weeks of incubation with the streptococcal strains a bacterial invasion could not yet be detected. After six weeks of incubation S. sobrinus was detected in 8 fragments and S. sanguinis in 4 fragments, but the number of samples was not sufficient for a statistical analysis. After 8 und 10 weeks in 16 fragments with regions inoculated with S. sobrinus and in 22 inoculated with S. sanguinis bacterial invasion could be detected and measured in a scanning electron microscope (Table 1). After 10 weeks of incubation, S. sanguinis reached depths of 11.13 ± 24.09 mm below the fracture edges into the enamel (Fig. 4AeD). S. sobrinus reached depths of 87.53 ± 76.34 mm
Fig. 1. (A) Embedding of specimens in epoxy resin (Epofix, Struers, Germany); (B) grinding of specimens on the buccal side with SiC-paper 1200 (particle size 13e16 mm) (ExaktMikroschleifsystem 400CS, Exakt Apparatebau, Norderstedt, Germany) with circular areas sized 3 mm in diameter.
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S. Kneist et al. / Anaerobe 35 (2015) 54e59
Fig. 2. (A) Cutting of specimens in half longitudinally (Exakt-Trennschleifsystem 300CP, Exakt Apparatebau, Norderstedt, Germany); (B) halves with two slits to obtain fracture surfaces in the infected area inoculated with S. sobrinus or S. sanguinis.
4. Discussion
Fig. 3. Exemplary electron micrograph of a tooth fracture surface for the screening of penetrated bacteria. E enamel, D e dentine, G e grinding surface, R e resin, P e deepest penetration of bacteria (x), min e minimum enamel thickness of the specimen, max e maximum enamel thickness.
(Fig. 5AeD). The difference was statistically significant (paired t test: p ¼ 0.033) (Fig. 6). Both bacteria were embedded in exopolysaccharides (EPS), of which the formation took place prior to the penetration and could also be shown in broth (Fig. 4A) after 24 h of incubation. The pH-values of the broths were measured at the beginning and end of each incubation cycle for five specimens each, over a period of ten weeks. After 24 h of growth, a pH drop occurred from the original 7.33 ± 0.06 in fresh broth to 4.21 ± 0.08 for S. sobrinus and to 4.54 ± 0.13 for S. sanguinis. On the 8th day of growth, a slightly lower pH of 4.02 ± 0.05 for S. sobrinus and of 4.37 ± 0.04 for S. sanguinis was measured. For both strains the pH differences after 24 h and on day 8 as well as the differences between the two strains were statistically significant (paired t test p < 0.001).
In the present study, mono-species biofilm formation on and subsequent penetration into dental enamel of extracted human teeth was investigated to simulate the caries initiation process in an in vitro model under highly standardized conditions. The thickness of the enamel was determined, and it was shown that a sufficient enamel depth was present to measure a comparable penetration for both strains. Due to their highly acidogenic and aciduric properties, mutans streptococci can continue to metabolize carbohydrates in acidic environments. Both species possess mechanisms to deal with acid stress, some of which have been characterized [24e26]. However, only for cells of S. sobrinus, but not of S. mutans, grown at pH 5.0 an increase in the glucose phosphoenolpyruvate:sugar phosphotransferase system was observed [24]. Moreover, it was shown in a longitudinal study with children aged 8e12 years [23] that the presence of S. sobrinus in carious lesions led to a significant increase in tooth decay (decayed, missing, filled teeth ¼ DMFT index). Furthermore, Torlakovic et al. [27] could show a significant association of S. mutans with the development of white spot lesions in their in vivo model with human teeth. This model included 16 premolars, destined to be extracted for orthodontic reasons, which received orthodontic bands to study plaque accumulation; after 7 weeks, 75% of the teeth had developed early carious lesions. Bacteria could be detected in the enamel for the first time after 6e8 weeks of incubation, with EPS preceding the bacterial front; however, a statistical interpretation based on the number of samples was only meaningful after a period of 8 weeks. In view of the large amount of EPS produced by both strains, which was seen in the electron micrographs, it could be confirmed in vitro that in the absence of external substrate the EPS could be degraded and serve as energy source for the bacteria. The clinical picture of hidden caries might reflect this process [28], because early caries lesions can remineralize and invaded bacteria can survive by metabolizing EPS and further progress towards the pulp. The clinical picture of this carious progress underneath the remineralized enamel surface
Table 1 Penetration depth (mm) of S. sobrinus and S. sanguinis below the fracture edges into the human enamel in vitro (n: number of specimens positive for bacteria/specimens analyzed). Incubation (week)
n
S. sobrinus mean ± SD
n
S. sanguinis mean ± SD
2 4 6 8 10
0/12 0/12 8/12 11/12 8/12
0 0 18.05 ± 14.46 57.66 ± 81.73 8753 ± 76.34
0/12 0/12 4/12 10/12 12/12
0 0 15.65 ± 10.09 6.20 ± 6.41 7.08 ± 14.90
S. Kneist et al. / Anaerobe 35 (2015) 54e59
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Fig. 4. (A) Extracellular polysaccharides produced by Streptococcus sanguinis in Balmelli broth; (B) biofilm of S. sanguinis on human enamel surface after 10 weeks of incubation; (C) extracellular polysaccharides in the enamel; and (D) invasion of S. sanguinis into the human enamel.
Fig. 5. (A) Biofilm of S. sobrinus with extracellular polysaccharides on human enamel surface after 10 weeks of incubation; (B) S. sobrinus detail from A; (C) invasion of S. sobrinus into the human enamel; and (D) detail from C.
escapes the diagnosis by the dentist. In the past many studies investigating bacterial invasion into dental enamel have been performed using rats as experimental animal [29e32] or analyzing enamel caries lesions in humans in vivo or in situ [27,33e36]. From a study with animals [31] it was concluded that invasion by different bacteria is difficult to compare, because the number of entry sites into the enamel (initial micro-defects) can vary. In the
present study, such micro-defects were avoided by creating as entry site for S. sobrinus and S. sanguinis windows of comparable size by means of removing defined amounts of enamel. Furthermore, in contrast to Schwendicke et al. [37], who determined the cariogenic potential of the tested strains by measuring the mineral loss in dental tissues by means of transverse micro-radiography, the focus here was instead to measure the penetration depth at a given period of time.
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S. Kneist et al. / Anaerobe 35 (2015) 54e59
Fig. 6. Penetration depth (mm) of S. sobrinus and S. sanguinis below the fracture edges into the human enamel in vitro.
After 10 weeks of incubation, a deeper penetration depth into the enamel of S. sobrinus (mean: 87.5 mm) versus S. sanguinis (mean: 11.1 mm) could be confirmed. The viability of the two bacteria e especially of the less aciduric S. sanguinis e was maintained by discontinuous cultivation for periods of 8 days each by supplying fresh culture medium, and in addition, a sufficiently high amount of sucrose as substrate was available. For both strains the mean pH differences in the broth after 24 h (S. sobrinus 4.21, S. sanguinis 4.54) and on day 8 (S. sobrinus 4.02, S. sanguinis 4.37) as well as the differences between the two strains were statistically significant. It was previously shown that a pH range of 5.2e5.5 induces the demineralization of the enamel [38]. Without doubt, however, the final pH values reached by the two bacteria in the nutrient broths were sufficient for penetration into the dental enamel. The difference between the two strains observed in the present study could almost be predicted by the in vitro studies concerning the adaptive acid tolerance response of S. sobrinus [24], the acid induced tolerance and acidogenicity of non-mutans streptococci [6] and the transient acid-impairment of growth ability of S. sanguinis [25]. It remains undisputed that mutans streptococci continue to play, in the context of the extended ecological plaque hypothesis, an important role in the etiopathogenesis of caries, and this fact was confirmed here as well as previously by numerous authors [1,2,10,17,19,27,38e41] and by clinical-experimental own studies [42,43]. In summary, this pilot study could serve as model for future studies to determine bacterial penetration as measure for virulence of potentially cariogenic bacteria. Furthermore, this way the success of certain caries protective measures could be also monitored. Conflict of interest and funding There is no conflict of interest in the present study for any of the authors. Funding was provided by the Medical Faculty, University Hospital, Jena, Germany. References [1] P.D. Marsh, Microbial ecology of dental plaque and its significance in health and disease, Adv. Dent. Res. 8 (1994) 263e271. [2] P.D. Marsh, Dental plaque as a microbial biofilm, Caries Res. 39 (2004)
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Margolis, The association of mutans streptococci and non-mutans streptococci capable of acidogenesis at a low pH with dental caries on enamel and root surfaces, J. Dent. Res. 72 (1993) 508e516. [15] S. Alam, S.R. Brailsford, S. Adams, C. Allison, E. Sheehy, Zoitopoulos, et al., Genotypic heterogeneity of Streptococcus oralis and distinct aciduric subpopulations in human dental plaque, Appl. Environ. Microbiol. 66 (2000) 3330e3336. [16] J.J. De Soet, B. Nyvad, M. Kilian, Strain-related acid production by oral streptococci, Caries Res. 34 (2000) 486e490. [17] I. Kleinberg, A mixed-bacteria ecological approach to understanding the role of the oral bacteria in dental caries causation: an alternative to Streptococcus mutans and the specific-plaque hypothesis, Crit. Rev. Oral Biol. Med. 13 (2002) 108e205. [18] J.A. Aas, A.L. Griffen, S.R. Dardis, A.M. Lee, I. Olsen, F.E. Dewhirst, E.J. Leys, B.J. 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Dent. 7 (2006) 19e24. [24] M.M. Nascimento, J.A. Lemos, J. Abranches, R.B. Gonçalves, R.A. Burne, Adaptive acid tolerance response of Streptococcus sobrinus, J. Bacteriol. 186 (2004) 6383e6390. [25] M. Horiuchi, J. Washio, H. Mayanagi, N. Takahashi, Transient acid-impairment of growth ability of oral Streptococcus, Actinomyces, and Lactobacillus: a possible ecological determinant in dental plaque, Oral Microbiol. Immunol. 24 (2009) 319e324. [26] A.R. Martinez, J. Abranches, J.K. Kajfasz, J.A. Lemos, Characterization of Streptococcus sobrinus acid-stress response by interspecies microarrays and proteomics, Mol. Oral Microbiol. 25 (2010) 331e342. [27] L. Torlakovic, V. Klepac-Ceraj, B. Øgaard, S.L. Cotton, B.J. Paster, I. Olsen, Microbial community succession on developing lesions on human enamel, J. Oral Microbiol. (2012), http://dx.doi.org/10.3402/jom.v4i0.16125. [28] K.L. Weerheijm, J.J. de Soet, J. de Graaf, W.E. van Amerongen, Occlusal hidden caries: a bacteriological profile, ASDC J. Dent. Child. 57 (6) (1990) 428e432. [29] A.R. Luoma, H. Luoma, A. Pelltari, Microbial invasion and subsurface colonization of rat enamel in early fissure caries observed by scanning electron microscopy, Scand. J. Dent. Res. 92 (1984) 120e126. €, S. Spets-Happonen, M. Tulla, K. Pelkonen, Early penetra[30] H. Luoma, L. Seppa tion of Streptococcus mutans into fissure enamel of gnotobiotic rats, Oral Microbiol. Immunol. 2 (1987) 30e34. €, H. Luoma, H. Forss, S. Spets-Happonen, S. Markkanen, K. Pelkonen, [31] L. Seppa Invasion of Streptococcus mutans and Lactobacillus salivarius in early caries lesions of gnotobiotic rats, Caries Res. 23 (1989) 371e374. [32] Y. Kobayashi, M. Ozeki, A. Ogawa, S. Matsumoto, M. Sanjo, T. Moriyama, Invasion of Streptococcus mutans, Streptococcus intermedius and Propionibacterium acnes into the teeth of gnotobiotic rats, Caries Res. 26 (1992) 132e138.
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[39] D. Beighton, The complex oral microflora of high risk individuals and groups and its role in the caries process, Community Dent. Oral Epidemiol. 33 (2005) 248e255. [40] J.M. Tanzer, J. Livingston, A.M. Thompson, The microbiology of primary dental caries in humans, J. Dent. Educ. 65 (2001) 1028e1037. [41] A.C.R. Tanner, J.M.J. Mathney, R.L. Kent Jr., N.I. Chalmers, C.V. Hughes, C.Y. Loo, N. Pradhan, E. Kanasi, J. Hwang, M.A. Dahlan, E. Papadopolou, F.E. Dewhirst, Cultivable anaerobic microbiota of severe early childhood caries, J. Clin. Microbiol. 49 (2011) 1464e1474. €ßer, Acid production by human strains [42] S. Kneist, T. Bergholz, T. Fischer, L. Sto of mutans streptococci, Caries Res. 33 (1999) 290. [43] S. Kneist, H. Kubieziel, B. Willershausen, H. Küpper, A. Callaway, Modeling of S. mutans and A. naeslundii acid production in vitro with caries incidence of lowand high-risk children, Quint. Int. 43 (2010) 413e420.
Contents lists available at ScienceDirect
Anaerobe journal homepage: www.elsevier.com/locate/anaerobe
Pathogenesis and toxins
Penetration of Streptococcus sobrinus and Streptococcus sanguinis into dental enamel Susanne Kneist a, *, Sandor Nietzsche b, Harald Küpper a, Gerhard Raser a, Brita Willershausen c, Angelika Callaway c a b c
Research Laboratory for Dental Materials, Center of Dentistry, University Hospital, Jena, Germany Center of Electron Microscopy, University Hospital, Jena, Germany Department of Operative Dentistry, University Medical Center of the Johannes Gutenberg University, Mainz, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 August 2014 Received in revised form 21 January 2015 Accepted 30 January 2015 Available online 21 March 2015
The aim of this pilot study was to assess the difference in virulence of acidogenic and aciduric oral streptococci in an in vitro caries model using their penetration depths into dental enamel. 30 caries-free extracted molars from 11- to 16-year-olds were cleaned ultrasonically for 1 min with deionized water and, after air-drying, embedded in epoxy resin. After 8-h of setting at room temperature, the specimens were ground on the buccal side with SiC-paper 1200 (particle size 13e16 mm). Enamel was removed in circular areas sized 3 mm in diameter; the mean depth of removed enamel was 230 ± 60 mm. 15 specimens each were incubated anaerobically under standardized conditions with 24 h-cultures of Streptococcus sanguinis 9S or Streptococcus sobrinus OMZ 176 in Balmelli broth at 37 ± 2 C; the pH-values of the broths were measured at the beginning and end of each incubation cycle. After 2, 4, 6, 8, and 10 weeks 3 teeth each were fixed in 2.5% glutaraldehyde in cacodylate buffer for 24 h, washed 3 and dehydrated 30e60 min by sequential washes through a series of 30e100% graded ethanol. The teeth were cut in half longitudinally; afterward, two slits were made to obtain fracture surfaces in the infected area. After critical-point-drying the fragments were gold-sputtered and viewed in a scanning electron microscope at magnifications of 20-20,000. After 10 weeks of incubation, penetration of S. sanguinis of 11.13 ± 24.04 mm below the break edges into the enamel was observed. The invasion of S. sobrinus reached depths of 87.53 ± 76.34 mm. The difference was statistically significant (paired t test: p ¼ 0.033). The experimental penetration depths emphasize the importance of S. sanguinis versus S. sobrinus in the context of the extended ecological plaque hypothesis. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Plaque hypotheses Caries model experiment Human enamel Cariogenic bacteria Penetration depths
1. Introduction Various species of facultative anaerobic streptococci are capable of establishing themselves on different sites in the human oral cavity, and are considered as part of the normal indigenous microflora, co-existing during health with the host in a harmonious relationship [1,2]. Streptococcus sanguinis belongs to the pioneer microorganisms, which are the first to colonize and adhere to pellicle-covered sound enamel surfaces. There, dental biofilm represents a community of microorganisms in a matrix of polymers derived from the host or bacteria; endogenous substrates allow
* Corresponding author. Hütergasse 2, D-99084 Erfurt, Germany. E-mail address: [email protected] (S. Kneist). http://dx.doi.org/10.1016/j.anaerobe.2015.01.013 1075-9964/© 2015 Elsevier Ltd. All rights reserved.
only relatively slow cell division and growth, and only moderate amounts of acids are formed. In contrast, mutans streptococci (Streptococcus mutans and Streptococcus sobrinus), clearly implicated in caries initiation, are normally only present as a small proportion of the total plaque flora [3]. Caries is the result of an imbalance in the dental plaque flora. In the case of an increase in frequency and amount of intake of fermentable carbohydrates, and in particular of sucrose, longer intervals of low pH ranges persist [4]. Such conditions favor survival of aciduric and strongly acidogenic microorganisms, for example mutans streptococci and lactobacilli, shifting the balance between de- and remineralization towards loss of minerals, and invasion of cariogenic bacteria, in particular mutans streptococci, into dental enamel can occur [5e10]. Later, it was found that also non-mutans streptococci can adapt under severe and prolonged
S. Kneist et al. / Anaerobe 35 (2015) 54e59
acidic conditions from initially moderate acid producers into aggressive, cariogenic strains [11e20]. Accordingly, the ecological [1] and later the extended ecological [19] plaque hypothesis were formulated, which can better than earlier plaque hypotheses take into account the clinical course of the disease and preventive approaches. The aim of the study was to model biofilm formation and bacterial penetration into dental enamel, using two different cariogenic species, the acidogenic S. sanguinis and the acidogenic and aciduric S. sobrinus. 2. Material and methods 2.1. Preparation of specimens To exclude biological variabilities like differences in the degree of sclerotisation of the dental hard tissues, only caries-free permanent molars from 11- to 16-year-olds, extracted for orthodontic reasons, were included in the present study. The teeth were cleaned ultrasonically for 1 min with de-ionized water and, after air-drying, embedded in epoxy resin (Epofix, Struers, Germany). After 8 h of setting at room temperature, the specimens were ground on the buccal side with SiC-paper 1200 (particle size 13e16 mm) (ExaktMikroschleifsystem 400CS, Exakt Apparatebau Norderstedt, Germany) to remove the resin. Then, enamel was removed in circular areas sized 3 mm in diameter; the depth of removed enamel should be 200e250 mm (Fig. 1A, B).
55
2.3. Scanning electron microscopy of infected tooth areas After 2, 4, 6, 8, and 10 weeks 3 teeth each were first cut longitudinally (Exakt-Trennschleifsystem 300CP, Exakt Apparatebau Norderstedt, Germany) into halves; afterward, two slits were made to obtain fracture surfaces in the grinding buccal area (Fig. 2A, B), broken in the infected area of the window, resulting in a maximum of 12 fragments for each time period and streptococcal strain. The specimens were fixed in 2.5% glutaraldehyde in cacodylate buffer for 24 h, washed 3x in cacodylate buffer, and dehydrated for 30e60 min by sequential washes through a series of 30e100% graded ethanol. After critical-point drying, the fragments were sputtered with gold and viewed in a scanning electron microscope (LEO 1450VP, Zeiss, Oberkochen, Germany) at magnifications of x20-20,000. The minimal and maximal enamel thickness in the tooth fragments, and the largest penetration depth of the species were measured in a standardized way as shown in Fig. 3, using parallel lines orthogonal to the axis of the tooth. 2.4. Statistical analyses The data were analyzed with the statistics program SPSS (Version 20.0 Chicago, Ill. USA). Descriptive analyses were made for enamel thickness, penetration depths and pH values; data are expressed as means ± SD (standard deviations). The paired t test was used to test differences between the penetration depths of the two bacterial strains and in the changes in pH after 8 days. The significance level was set at p < 0.05 for all tests.
2.2. Microbiological procedures 3. Results Of the 30 specimens embedded in epoxy resin, 15 each were incubated anaerobically under standardized conditions at 37 ± 2 C (95% N2, 5% CO2; VT 5042 EK, Heraeus, Hanau, Germany) in 20 ml Balmelli broth (10 g trypticase, 5 g yeast extract, 5 g K2HPO4, 3 g meat extract, 50 g sucrose, ad 1000 ml aqua dest.; pH 7.2), inoculated with 1 ml of 24 h-cultures of the same broth of S. sanguinis 9S or S. sobrinus OMZ 176 (Heraeus Lamin Air, HBB 2448, Hanau, Germany) to simulate the caries initiation process in an in vitro model under highly standardized conditions with mono-species biofilm formation on and subsequent penetration into dental enamel. S. sobrinus OMZ 176 was chosen as a representative strain of mutans streptococci and S. sanguinis 9S as an example for low-pH non-mutans streptococci [21e23]. After 8 days each, the teeth were transferred into 20 ml of fresh Balmelli broth, inoculated again with 1 ml of the respective 24 h-cultures in Balmelli broth, and further incubated. The pH-values of the broths were measured at the beginning and end of each incubation cycle for five specimens each.
Enamel was removed in circular areas sized 3 mm in diameter; the mean depth of removed enamel was 230 ± 60 mm. The minimal enamel thickness was 619.55 ± 222.02 mm and the maximal enamel thickness was 1224.42 ± 222.73 mm. The analysis of the depth of penetration showed that after two and four weeks of incubation with the streptococcal strains a bacterial invasion could not yet be detected. After six weeks of incubation S. sobrinus was detected in 8 fragments and S. sanguinis in 4 fragments, but the number of samples was not sufficient for a statistical analysis. After 8 und 10 weeks in 16 fragments with regions inoculated with S. sobrinus and in 22 inoculated with S. sanguinis bacterial invasion could be detected and measured in a scanning electron microscope (Table 1). After 10 weeks of incubation, S. sanguinis reached depths of 11.13 ± 24.09 mm below the fracture edges into the enamel (Fig. 4AeD). S. sobrinus reached depths of 87.53 ± 76.34 mm
Fig. 1. (A) Embedding of specimens in epoxy resin (Epofix, Struers, Germany); (B) grinding of specimens on the buccal side with SiC-paper 1200 (particle size 13e16 mm) (ExaktMikroschleifsystem 400CS, Exakt Apparatebau, Norderstedt, Germany) with circular areas sized 3 mm in diameter.
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Fig. 2. (A) Cutting of specimens in half longitudinally (Exakt-Trennschleifsystem 300CP, Exakt Apparatebau, Norderstedt, Germany); (B) halves with two slits to obtain fracture surfaces in the infected area inoculated with S. sobrinus or S. sanguinis.
4. Discussion
Fig. 3. Exemplary electron micrograph of a tooth fracture surface for the screening of penetrated bacteria. E enamel, D e dentine, G e grinding surface, R e resin, P e deepest penetration of bacteria (x), min e minimum enamel thickness of the specimen, max e maximum enamel thickness.
(Fig. 5AeD). The difference was statistically significant (paired t test: p ¼ 0.033) (Fig. 6). Both bacteria were embedded in exopolysaccharides (EPS), of which the formation took place prior to the penetration and could also be shown in broth (Fig. 4A) after 24 h of incubation. The pH-values of the broths were measured at the beginning and end of each incubation cycle for five specimens each, over a period of ten weeks. After 24 h of growth, a pH drop occurred from the original 7.33 ± 0.06 in fresh broth to 4.21 ± 0.08 for S. sobrinus and to 4.54 ± 0.13 for S. sanguinis. On the 8th day of growth, a slightly lower pH of 4.02 ± 0.05 for S. sobrinus and of 4.37 ± 0.04 for S. sanguinis was measured. For both strains the pH differences after 24 h and on day 8 as well as the differences between the two strains were statistically significant (paired t test p < 0.001).
In the present study, mono-species biofilm formation on and subsequent penetration into dental enamel of extracted human teeth was investigated to simulate the caries initiation process in an in vitro model under highly standardized conditions. The thickness of the enamel was determined, and it was shown that a sufficient enamel depth was present to measure a comparable penetration for both strains. Due to their highly acidogenic and aciduric properties, mutans streptococci can continue to metabolize carbohydrates in acidic environments. Both species possess mechanisms to deal with acid stress, some of which have been characterized [24e26]. However, only for cells of S. sobrinus, but not of S. mutans, grown at pH 5.0 an increase in the glucose phosphoenolpyruvate:sugar phosphotransferase system was observed [24]. Moreover, it was shown in a longitudinal study with children aged 8e12 years [23] that the presence of S. sobrinus in carious lesions led to a significant increase in tooth decay (decayed, missing, filled teeth ¼ DMFT index). Furthermore, Torlakovic et al. [27] could show a significant association of S. mutans with the development of white spot lesions in their in vivo model with human teeth. This model included 16 premolars, destined to be extracted for orthodontic reasons, which received orthodontic bands to study plaque accumulation; after 7 weeks, 75% of the teeth had developed early carious lesions. Bacteria could be detected in the enamel for the first time after 6e8 weeks of incubation, with EPS preceding the bacterial front; however, a statistical interpretation based on the number of samples was only meaningful after a period of 8 weeks. In view of the large amount of EPS produced by both strains, which was seen in the electron micrographs, it could be confirmed in vitro that in the absence of external substrate the EPS could be degraded and serve as energy source for the bacteria. The clinical picture of hidden caries might reflect this process [28], because early caries lesions can remineralize and invaded bacteria can survive by metabolizing EPS and further progress towards the pulp. The clinical picture of this carious progress underneath the remineralized enamel surface
Table 1 Penetration depth (mm) of S. sobrinus and S. sanguinis below the fracture edges into the human enamel in vitro (n: number of specimens positive for bacteria/specimens analyzed). Incubation (week)
n
S. sobrinus mean ± SD
n
S. sanguinis mean ± SD
2 4 6 8 10
0/12 0/12 8/12 11/12 8/12
0 0 18.05 ± 14.46 57.66 ± 81.73 8753 ± 76.34
0/12 0/12 4/12 10/12 12/12
0 0 15.65 ± 10.09 6.20 ± 6.41 7.08 ± 14.90
S. Kneist et al. / Anaerobe 35 (2015) 54e59
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Fig. 4. (A) Extracellular polysaccharides produced by Streptococcus sanguinis in Balmelli broth; (B) biofilm of S. sanguinis on human enamel surface after 10 weeks of incubation; (C) extracellular polysaccharides in the enamel; and (D) invasion of S. sanguinis into the human enamel.
Fig. 5. (A) Biofilm of S. sobrinus with extracellular polysaccharides on human enamel surface after 10 weeks of incubation; (B) S. sobrinus detail from A; (C) invasion of S. sobrinus into the human enamel; and (D) detail from C.
escapes the diagnosis by the dentist. In the past many studies investigating bacterial invasion into dental enamel have been performed using rats as experimental animal [29e32] or analyzing enamel caries lesions in humans in vivo or in situ [27,33e36]. From a study with animals [31] it was concluded that invasion by different bacteria is difficult to compare, because the number of entry sites into the enamel (initial micro-defects) can vary. In the
present study, such micro-defects were avoided by creating as entry site for S. sobrinus and S. sanguinis windows of comparable size by means of removing defined amounts of enamel. Furthermore, in contrast to Schwendicke et al. [37], who determined the cariogenic potential of the tested strains by measuring the mineral loss in dental tissues by means of transverse micro-radiography, the focus here was instead to measure the penetration depth at a given period of time.
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Fig. 6. Penetration depth (mm) of S. sobrinus and S. sanguinis below the fracture edges into the human enamel in vitro.
After 10 weeks of incubation, a deeper penetration depth into the enamel of S. sobrinus (mean: 87.5 mm) versus S. sanguinis (mean: 11.1 mm) could be confirmed. The viability of the two bacteria e especially of the less aciduric S. sanguinis e was maintained by discontinuous cultivation for periods of 8 days each by supplying fresh culture medium, and in addition, a sufficiently high amount of sucrose as substrate was available. For both strains the mean pH differences in the broth after 24 h (S. sobrinus 4.21, S. sanguinis 4.54) and on day 8 (S. sobrinus 4.02, S. sanguinis 4.37) as well as the differences between the two strains were statistically significant. It was previously shown that a pH range of 5.2e5.5 induces the demineralization of the enamel [38]. Without doubt, however, the final pH values reached by the two bacteria in the nutrient broths were sufficient for penetration into the dental enamel. The difference between the two strains observed in the present study could almost be predicted by the in vitro studies concerning the adaptive acid tolerance response of S. sobrinus [24], the acid induced tolerance and acidogenicity of non-mutans streptococci [6] and the transient acid-impairment of growth ability of S. sanguinis [25]. It remains undisputed that mutans streptococci continue to play, in the context of the extended ecological plaque hypothesis, an important role in the etiopathogenesis of caries, and this fact was confirmed here as well as previously by numerous authors [1,2,10,17,19,27,38e41] and by clinical-experimental own studies [42,43]. In summary, this pilot study could serve as model for future studies to determine bacterial penetration as measure for virulence of potentially cariogenic bacteria. Furthermore, this way the success of certain caries protective measures could be also monitored. Conflict of interest and funding There is no conflict of interest in the present study for any of the authors. Funding was provided by the Medical Faculty, University Hospital, Jena, Germany. References [1] P.D. Marsh, Microbial ecology of dental plaque and its significance in health and disease, Adv. Dent. Res. 8 (1994) 263e271. [2] P.D. Marsh, Dental plaque as a microbial biofilm, Caries Res. 39 (2004)
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