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A novel technique for monitoring the development of bacterial biofilms in human periodontal pockets
FEMS Microbiology Letters 191 (2000) 95^101
www.fems-microbiology.org
A novel technique for monitoring the development of bacterial bio¢lms in human periodontal pockets Jo«rg Wecke a; *, Thomas Kersten a , Kasimierz Madela a , Annette Moter b , Ulf B. Go«bel b , Anton Friedmann c , Jean-Pierre Bernimoulin c a Robert Koch-Institut, Nordufer 20, D-13353 Berlin, Germany Institut fu«r Mikrobiologie und Hygiene, Charite¨, Humboldt Universita«t zu Berlin, DorotheenstraMe 96, 10117 Berlin, Germany Abteilung Parodontologie, Zentrum fu«r Zahnmedizin der Charite¨, Humboldt Universita«t zu Berlin, Fo«hrer StraMe 15, 13353 Berlin, Germany b
c
Received 24 May 2000; received in revised form 25 July 2000; accepted 2 August 2000
Abstract A new technique is presented for analyzing subgingival bacterial plaque. Different materials (polytetrafluoroethylene, gold, dentin) kept for several days in periodontal pockets of patients suffering from periodontitis were analyzed by electron microscopy and fluorescence in situ hybridization (FISH). Those parts of the carriers extending into the deepest zone of the pockets were predominantly colonized by spirochetes and Gram-negative bacteria whereas those segments in contact with a shallower region were colonized by streptococci. Independent of the material used, the bacterial colonization of the carriers appears to be similar. FISH using eubacteria- and species-specific oligonucleotides on semi-thin cross-sections of the carriers in combination with confocal laser scanning microscopy allowed detailed analysis of the architecture of biofilms and identification of putative periodontal pathogens with single cell resolution. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Periodontal pocket; Subgingival plaque; Bio¢lm; Ultrastructure ; Fluorescence in situ hybridization
1. Introduction Though more than 400 di¡erent bacterial species have been isolated from periodontal pockets, only a limited number has been associated with periodontitis [1]. These species are thought to participate in the formation of a bio¢lm on subgingival tooth surfaces [2]. Such bio¢lm forming bacteria are obviously able to reveal a di¡erent pattern of gene expression than planktonic cells [3]. It is known that certain bacterial species frequently appear in close relationship to each other in periodontal pockets corresponding to the pocket probing depths [4]. So far, an analysis of the subgingival microbiota relied on sampling of bacteria either by paper points or by mechanical debridement. Both sampling procedures, however, disrupt the organization of bio¢lms. Bio¢lm formation has also been extensively studied using in vitro models, like £ow chambers or chemostats. However, these studies might not necessarily re£ect the situation in a periodontal pocket and
* Corresponding author. Tel. : +49 (30) 45-47-22-19 ; Fax: +49 (30) 45-47-26-06 ; E-mail : [email protected]
clearly have limitations regarding fastidious and so far uncultured microorganisms. The only method to study subgingival plaque available so far required the extraction of teeth [5]. Since extraction of teeth is often not possible, the development of alternative methods for obtaining intact bacterial bio¢lms was desirable. While a special device for collecting supragingival plaque has recently been described [6], no procedure for collecting undisturbed subgingival plaque has yet been described. The aim of this study was to present a new method for sampling and monitoring the subgingival bacterial £ora as a bio¢lm. While electron microscopy by de¢nition gives information on di¡erent bacterial morphotypes, the information on the spatial distribution has to be interpreted with caution. Fluorescence microscopy after in situ hybridization with oligonucleotide probes (FISH), however, not only allows for the identi¢cation of bacteria [7,8] but also provides valuable information on their spatial distribution [9,10]. From a previous study, it was known that extended polytetra£uoroethylene (e-PTFE) membranes used to cover super¢cial defects after surgery were colonized by pla-
0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 3 7 6 - 1
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que bacteria [11]. Therefore a further aim of this study was to compare bacterial colonization on PTFE membranes with those on di¡erent materials to get an optimized carrier for plaque bacteria. Dentin was chosen as a control material comparable to tooth surface, while gold foil has been selected because of its suitability in scanning electron microscopy. The carriers were inserted into periodontal pockets from patients su¡ering from rapidly progressive periodontitis (RPP). Bio¢lm formation was monitored by electron microscopy and FISH after di¡erent incubation periods. 2. Materials and methods A total of 12 patients scheduled for initial periodontal therapy participated in this study. Individuals who had been periodontally treated within the last 6 months or receiving any antimicrobial therapy during this period were excluded. RPP was diagnosed according to clinical and radiological criteria [12]. Distal and mesial sites were selected because of their pronounced probing depth measurements (mean 8.07 þ 1.63 mm). 2.1. Preparation of carriers and sampling of bacterial bio¢lm Carriers consisting of gold foil or e-PTFE membranes and constructed as shown in Fig. 1 were carefully inserted to reach the bottom of the pocket. The carrier was ¢xed supragingivally to the tooth surface by using cyanoacrylic glue (Octyldent1 Closure Medical Corp., Raleigh, NC, USA). The construction was guided by the assumption that the carrier positioned in the pocket might be colonized from both the tooth and the soft tissue side. To stabilize soft £exible materials like e-PTFE or gold foil, the carriers were mounted on commercially available `plast-oprobe' sticks (Maillefer, Ballaigues, Switzerland). Alternatively, 3-mm wide strips of e-PTFE or gold foil were attached to the plastic surfaces by four dots of cyanoacrylate (Tesa0 , Beiersdorf, Hamburg, Germany). The respective material was wrapped around the tips of plast-o-probes exceeding them at one side. Additionally dentin slices, not exceeding 3 mm in width each, were mounted on strips of e-PTFE. To get a better adaptation to the curvature of the dental root, it became necessary to divide the solid dentin into several segments not exceeding 3 mm in length each. Carriers were then positioned in the pockets with the longer end of the foils facing the teeth (Fig. 1) and attached to tooth surfaces. After 3 or 6 days of exposure, carriers were removed from the periodontal pockets. Only those carriers that kept their stable position during the exposure period were used for further investigations. Altogether 52 carriers (31 e-PTFE, 12 dentin, nine gold) were analyzed. For electron microscopy, specimens were ¢xed with ei-
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Fig. 1. Positioning of the e-PTFE carrier in a periodontal pocket; gold foil was inserted accordingly.
ther 2.5% (v/v) glutaraldehyde (Sigma, Munich, Germany), diluted in 0.1 M cacodylate bu¡er (pH 7) or a bu¡ered formaldehyde^glutaraldehyde ¢xative at 4³C overnight [13]. All the other treatments for transmission or scanning electron microscopy were done as previously described [11]. For FISH, four carriers were ¢xed with 3.7% (v/v) formaldehyde in phosphate-bu¡ered saline (pH 7.4) for at least 3 h. Embedding in cold polymerizing resin Technovit 8100 (Kulzer, Wehrheim, Germany) and sectioning of the specimens were performed as described earlier for tissue biopsies [9]. 2.2. FISH Group-speci¢c probe TRE I (5P-ACGCAAGCTCATCCTCAAG-3P) has been published earlier and has been shown to speci¢cally detect group I of oral treponemes, most of which are as yet uncultured [10]. EUB338 (5P-GCTGCCTCCCGTAGGAGT-3P) speci¢c for the domain Bacteria was used to visualize the entire bacterial population in the specimens [14]. Probes were labeled with Cy3 or FITC, respectively, and obtained from Interactiva (Ulm, Germany).
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J. Wecke et al. / FEMS Microbiology Letters 191 (2000) 95^101
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Fig. 2. Scanning electron micrograph showing a part of an e-PTFE carrier with `islet-like' plaque formation after 3 days. The surface of the carrier reveals the ¢brillar structure of the e-PTFE membrane (white arrows).
Hybridization bu¡er containing 0.9 M NaCl, 20 mM Tris^HCl, pH 7.3, 0.01% (w/v) sodium dodecyl sulfate, 20% (v/v) formamide and 1 WM probe each was applied on the sections. To assess the speci¢city of the probes of ¢xed cells, the following Treponema strains served as controls: Treponema vincentii (ATCC 33580 and RITZ A), both Treponema species belonging to phylogenetic group I of oral treponemes, thus serving as positive controls. Treponema denticola (ATCC 33521), Treponema pectinovorum (ATCC 33768), Treponema socranskii subsp. buccale (ATCC 35534), Treponema socranskii subsp. socranskii (ATCC 35536), Treponema maltophilum (ATCC 51939), Treponema lecithinolyticum (ATCC 700332) and Treponema phagedenis subsp. reiteri (kindly provided by B. Wilske, Munich, Germany) serving as negative controls. Control slides with ¢xed treponeme cultures were included in every hybridization experiment (data not shown). After 3.5 h of incubation in a dark humid chamber at 46³C,
sections were rinsed with distilled water, air-dried in the dark and mounted with Citi£uor AF 1 (The Chemical Laboratory of the University of Kent, UK). A confocal laser scanning microscope (CLSM) model LSM 510 (Carl Zeiss, Oberkochen, Germany) equipped with an Ar-ion laser (488 nm) and two HeNe lasers (543 and 633 nm) was used to record optical sections. Image processing was performed with a standard software package delivered with the instrument (Zeiss LSM version 1.6). 3. Results 3.1. The ultrastructure of subgingival plaque The formation of subgingival plaque was analyzed on di¡erent carriers that were kept in periodontal pockets of RPP patients for 3^6 days. After short incubation
Fig. 3. Ultrathin cross-section of a dentin carrier exposed for 3 days (part a of Fig. 1); the dentin (D) is covered by a `pellicle-like' homogeneous layer (P) with adherent Gram-negative rods (R) and spirochetes (T).
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Fig. 4. Ultrathin section of a gold carrier (part c, Fig. 1) covered by a pellicle and colonized by Gram-positive cocci (exposure 3 days).
(3 days), islets-like colonization could be observed by scanning electron microscopy (Fig. 2). The loose colonization could also be seen in cross cut ultrathin sections (Fig. 3). Independent of the three materials used (e-PTFE, dentin, gold), a homogeneous electron dense pellicle could be demonstrated on the di¡erent carriers; also the bacterial colonization was similar. Analyzing more than 4000 elec-
tron micrographs, we can state that di¡erent Gram-negative rods and treponemes were found on those parts of the carriers corresponding to the deepest part of the pockets (Figs. 3 and 5). Gram-positive cocci could preferentially be observed on the upper segments of the carriers (Fig. 4). Early colonizing bacteria were characterized by an extracellular matrix especially shown in ultrathin sections par-
Fig. 5. Ultrathin section in parallel to the e-PTFE carrier (part a, Fig. 1) after 3 days of exposure; Gram-negative rods embedded in extracellular matrix (big arrows) are in close contact to spirochetes (T).
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Fig. 6. Electron micrograph showing an ultrathin section in parallel to the e-PTFE-carrier (part a, Fig. 1) after 6 days of exposure, demonstrating the dense colonization by di¡erent bacterial morphotypes; the central Gram-positive bacteria (GP) are surrounded by some Gram-negative rods (GN) in a `rosette-like' manner. Note the high number of treponemes.
allel to the carrier surfaces (Fig. 5). Treponemes were found in close contact to this matrix (Fig. 5). When carriers were kept in the periodontal pockets for 6 days, a massive and dense colonization of bacteria could be shown (Fig. 6). After this time, a con£uent bio¢lm resulted. Besides Gram-negative bacteria, some segments revealed a massive colonization of treponemes (Fig. 6). In such areas, the treponemes seemed to be arranged in a regular pattern. The use of carriers inserted in periodontal pockets offered the opportunity to follow the development of subgingival bacterial £ora as bio¢lm and to consider the spatial arrangement of di¡erent bacterial morphotypes. Additionally the speci¢c coaggregation of di¡erent bacterial species, which is visible in Figs. 5 and 6, could be analyzed. While electron microscopical methods demonstrated the organization of subgingival bio¢lm build up with di¡erent morphotypes such as cocci, rods or spirochetes, other methods were needed to carry out a detailed identi¢cation of bacteria.
lowed exact orientation within the sample and revealed di¡erent bacterial morphotypes in cross-sectioned subgingival bio¢lm of a RPP patient (Fig. 7). The thickness of the bio¢lm could be measured with 40^45 Wm. At higher magni¢cation, di¡erent morphotypes of e.g. cocci or rods ^ some organized as microcolonies ^ could clearly be di¡erentiated (Fig. 8). Moreover, simultaneous hybridization with probe TRE I-Cy3 revealed some large spirochetes interspersed between other bacteria. The higher magni¢cation (Fig. 8) of this bio¢lm illustrates the opportunity to obtain information about the length of the treponemes or their undulation. To our knowledge, this is the ¢rst time that the numbers and the spatial arrangement of spirochetes within the periodontal bio¢lm could be demonstrated. In this part of the bio¢lm, treponemes were rather separately localized, whereas in other areas high numbers of group I treponemes were spread in the bio¢lm as also shown by electron microscopy (Fig. 6).
3.2. Identi¢cation of bacteria by FISH
4. Discussion
Since the direct CLSM analysis of bio¢lm carriers such as gold, dentin or e-PTFE was hampered by light re£ection and auto£uorescence, semi-thin sections of Technovitembedded material were used. The EUB-FITC probe al-
Here we present a method that allows the removal of subgingival plaque as widely undisturbed bio¢lm. The advantage of this method is that neither insertion nor removal of carriers requires teeth or crown removal. The precise
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Fig. 7. CLSM micrograph of a subgingival plaque taken from a RPP patient after 6 days; showing the simultaneous hybridization using probes EUBFITC (green) and TRE I-Cy3 (red). Di¡erent green-colored morphotypes are often arranged in microcolonies. While EUB-338 stains all bacteria, the probe TRE I reveals the presence of spirochetes (red).
¢xed localization of the carriers allows the association of distinct bacterial morphotypes or species with the depth of periodontal pockets. In addition, information about the time-dependent formation of bio¢lm can be monitored. Electron microscopical analysis of di¡erent carriers revealed that the nature of the carrier material does not seem to have a great in£uence on the colonization. This is in agreement with a previous observation emphasizing that as compared to supragingival conditions, the surface characteristics were less important for subgingival plaque [15], showing almost no di¡erences in subgingival plaque composition on hydrophilic or hydrophobic polymers [16]. A possible explanation for this similar colonization pattern may be the salivary pellicle on the surface of each carrier rendering the colonization conditions alike [17^ 19]. Formation of dental plaque on epoxy resin crowns has been described earlier [20,21]. However, this epoxy resin crown model is only of limited use for studying periodontitis, as any information on deep periodontal pockets is missing. High numbers of spirochetes and the presence of Gram-negative rods and coccoid cells were observed only on those parts of the carriers localized in the deepest zone of the periodontal pockets. In another ultrastructural study, Gram-positive cocci were shown as part of the apical border plaque [5]. In our study, we found Gram-positive cocci only on the most coronal segments of the plaque carriers obviously exposed to a higher oxygen tension. Modern determination of bacterial £ora via cluster analysis examining the relationship among di¡erent taxa revealed that some species do not exist separately in the periodontal pockets but rather form complexes [4]. Elec-
FEMSLE 9598 15-9-00
tron microscopy allowed to study the complexity of human plaque bio¢lm showing structural or spatial arrangements of di¡erent bacterial morphotypes (Fig. 6), described as `corncob' or `bristle-brush' formations
Fig. 8. Higher magni¢cation of a part of Fig. 7 (white frame) with large yellow/red colored treponemes (white arrows). Some microcolonies of di¡erent morphotypes (green colored) are marked (white arrow heads).
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[5,22]. The coaggregation and coadhesion of oral bacteria was summarized recently [23], however, there is doubt whether this in vitro study re£ects the situation in vivo. In contrast, the ultrastructural bio¢lm analysis enables the characterization of di¡erent bacterial morphotypes and their spatial arrangements. Further taxonomic characterizations are only possible with additional methods such as immuno-electron microscopy and/or in situ hybridization using speci¢c oligonucleotide probes. The advantage of using 16S rRNA-directed oligonucleotide probes for the identi¢cation and visualization of spatial distribution of di¡erent bacterial species was shown convincingly for environmental bio¢lms [7,8]. While the direct analysis of carriers with CLSM was hindered by re£ection and auto£uorescence of the carriers, the use of Technovit sections overcame these problems and allowed both the localization and identi¢cation of microorganisms. Detailed information on bio¢lm structure in habitats such as subgingival plaque is still lacking. FISH using di¡erent probes simultaneously may reveal the exact composition and architecture of particular bio¢lm. Thus, this novel technique presented here may contribute to an understanding of bio¢lm formation and perhaps the role of single bacterial species in the etiopathogenesis of periodontal infections. References [1] Socransky, S.S. and Ha¡ajee, A.D. (1991) Microbial mechanisms in the pathogenesis of destructive periodontal diseases : a critical assessment. J. Periodont. Res. 26, 195^212. [2] Marsh, P.D. and Bradshaw, D.J. (1997) Physiological approaches to the control of oral bio¢lms. Adv. Dent. Res. 11 (1), 176^185. [3] Burne, R.A., Quivey Jr., R.G. and Marquis, R.E. (1999) Physiologic homeostasis and stress responses in oral bio¢lms. Methods Enzymol. 310, 441^460. [4] Socransky, S.S., Ha¡ajee, A.D., Cugini, M.A., Smith, C. and Kent Jr., R.L. (1998) Microbial complexes in subgingival plaque. J. Clin. Periodontol. 25, 134^144. [5] Vrahopoulos, T.P., Barber, P.M. and Newman, H.N. (1995) The apical border plaque in severe periodontitis. An ultrastructural study. J. Periodontol. 66, 113^124. [6] Wood, S.R., Kirkham, J., Marsh, P.D., Shore, R.C., Natress, B. and Robinson, C. (2000) Architecture of intact natural human plaque bio¢lms studied by confocal laser scanning microscopy. J. Dent. Res. 79, 21^27. [7] Manz, W., Amann, R., Szewzyk, R., Szewzyk, U., Stenstro«m, T.-A., Hutzler, P. and Schleifer, K.-H. (1995) In situ identi¢cation of Legionellaceae using 16S rRNA-targeted oligonucleotide probes and confocal laser scanning microscopy. Microbiology 141, 29^39.
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[8] Amann, R., Snaidr, J., Wagner, M., Ludwig, W. and Schleifer, K.-H. (1996) In situ visualization of high genetic diversity in a natural microbial community. J. Bacteriol. 178, 3496^3500. [9] Moter, A., Leist, G., Rudolph, R., Schrank, K., Choi, B.-K., Wagner, M. and Go«bel, U.B. (1998) Fluorescence in situ hybridization shows spatial distribution of as yet uncultured treponemes in biopsies from digital dermatitis lesions. Microbiology 144, 2459^2467. [10] Moter, A., Hoenig, C., Choi, B.-K., Riep, B. and Go«bel, U.B. (1998) Molecular epidemiology of oral treponemes associated with periodontal disease. J. Clin. Microbiol. 36, 1399^1403. [11] Wecke, J., Wolf, V., Fath, S. and Bernimoulin, J.-P. (1995) The occurence of treponemes and their spherical bodies on polytetra£uoroethylene membranes. Oral Microbiol. Immunol. 10, 278^283. [12] Page, R.C., Altman, L.C., Ebersole, J.L., Vandersteen, G.E., Dahlberg, W.-H., Williamsa, P.L. and Osterberg, S.K. (1983) Rapidly progressive periodontitis ^ A distinct clinical condition. J. Periodontol. 54, 197^209. [13] Karnovsky, M.J. (1965) A formaldehyde^glutaraldehyde ¢xative of high osmolality for use in electron microscopy. J. Cell Biol. 26, 137A. [14] Amann, R.I., Binder, B.J., Olson, R.J., Chisholm, S.W., Devereux, R. and Stahl, D.A. (1990) Combination of 16S rRNA-targeted oligonucleotide probes with £ow cytometry for analyzing mixed microbial populations. Appl. Environ. Microbiol. 56, 1919^1925. [15] Quirynen, M. and Bollen, C.M.L. (1995) The in£uence of surface roughness and surface-free energy on supra- and subgingival plaque formation in man. J. Clin. Periodontol. 22, 1^14. [16] Busscher, H.J. and Van der Mei, H.C. (1997) Physico-chemical interactions in initial microbial adhesion and relevance for bio¢lm formation. Adv. Dent. Res. 11 (1), 24^32. [17] Newman, H.N. and Barber, P.M. (1995) Dental plaque structure in vivo. In: The Life and Death of Bio¢lm. Contributions Made at the Second Meeting of the British Bio¢lms Club Powys (Wimpenny, J., Handley, P., Gilbert, P. and Lappin-Scott, H., Eds.), pp. 27^32. Bio Line. [18] Glantz, P.O., Baier, R.E. and Christersson, C.E. (1996) Biochemical and physiological considerations for modelling bio¢lms in the oral cavity: A review. Dent. Mater. 12, 208^214. [19] Ellen, R.P., Le¨pine, G. and Nghiem, P.M. (1997) In vitro models that support adhesion speci¢city in bio¢lms of oral bacteria. Adv. Dent. Res. 11 (1), 33^42. [20] Listgarten, M.A., Mayo, H.E. and Tremblay, R. (1975) Development of dental plaque on epoxy resin crowns in man. A light and electronmicroscopy study. J. Periodontol. 46, 10^26. [21] Listgarten, M.A. (1994) The structure of dental plaque. Periodontology 2000 (5), 52^65. [22] Westergaard, J., Frandsen, A. and Slots, J. (1978) Ultrastructure of the subgingival micro£ora in juvenile periodontitis. Scand. J. Dent. Res. 86, 421^429. [23] Kolenbrander, P.E., Anderson, R.N., Clemans, D.L., Whittaker, C.J. and Klier, C.M. (1999) Potential role of functionally similar coaggregation mediators in bacterial succession. In: Dental Plaque Revisited ^ Oral Bio¢lms in Health and Disease (Newman, H.N. and Wilson, M., Eds.), pp. 171^186. Eastman Dental Institute University College, London.
Cyaan Magenta Geel Zwart
www.fems-microbiology.org
A novel technique for monitoring the development of bacterial bio¢lms in human periodontal pockets Jo«rg Wecke a; *, Thomas Kersten a , Kasimierz Madela a , Annette Moter b , Ulf B. Go«bel b , Anton Friedmann c , Jean-Pierre Bernimoulin c a Robert Koch-Institut, Nordufer 20, D-13353 Berlin, Germany Institut fu«r Mikrobiologie und Hygiene, Charite¨, Humboldt Universita«t zu Berlin, DorotheenstraMe 96, 10117 Berlin, Germany Abteilung Parodontologie, Zentrum fu«r Zahnmedizin der Charite¨, Humboldt Universita«t zu Berlin, Fo«hrer StraMe 15, 13353 Berlin, Germany b
c
Received 24 May 2000; received in revised form 25 July 2000; accepted 2 August 2000
Abstract A new technique is presented for analyzing subgingival bacterial plaque. Different materials (polytetrafluoroethylene, gold, dentin) kept for several days in periodontal pockets of patients suffering from periodontitis were analyzed by electron microscopy and fluorescence in situ hybridization (FISH). Those parts of the carriers extending into the deepest zone of the pockets were predominantly colonized by spirochetes and Gram-negative bacteria whereas those segments in contact with a shallower region were colonized by streptococci. Independent of the material used, the bacterial colonization of the carriers appears to be similar. FISH using eubacteria- and species-specific oligonucleotides on semi-thin cross-sections of the carriers in combination with confocal laser scanning microscopy allowed detailed analysis of the architecture of biofilms and identification of putative periodontal pathogens with single cell resolution. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Periodontal pocket; Subgingival plaque; Bio¢lm; Ultrastructure ; Fluorescence in situ hybridization
1. Introduction Though more than 400 di¡erent bacterial species have been isolated from periodontal pockets, only a limited number has been associated with periodontitis [1]. These species are thought to participate in the formation of a bio¢lm on subgingival tooth surfaces [2]. Such bio¢lm forming bacteria are obviously able to reveal a di¡erent pattern of gene expression than planktonic cells [3]. It is known that certain bacterial species frequently appear in close relationship to each other in periodontal pockets corresponding to the pocket probing depths [4]. So far, an analysis of the subgingival microbiota relied on sampling of bacteria either by paper points or by mechanical debridement. Both sampling procedures, however, disrupt the organization of bio¢lms. Bio¢lm formation has also been extensively studied using in vitro models, like £ow chambers or chemostats. However, these studies might not necessarily re£ect the situation in a periodontal pocket and
* Corresponding author. Tel. : +49 (30) 45-47-22-19 ; Fax: +49 (30) 45-47-26-06 ; E-mail : [email protected]
clearly have limitations regarding fastidious and so far uncultured microorganisms. The only method to study subgingival plaque available so far required the extraction of teeth [5]. Since extraction of teeth is often not possible, the development of alternative methods for obtaining intact bacterial bio¢lms was desirable. While a special device for collecting supragingival plaque has recently been described [6], no procedure for collecting undisturbed subgingival plaque has yet been described. The aim of this study was to present a new method for sampling and monitoring the subgingival bacterial £ora as a bio¢lm. While electron microscopy by de¢nition gives information on di¡erent bacterial morphotypes, the information on the spatial distribution has to be interpreted with caution. Fluorescence microscopy after in situ hybridization with oligonucleotide probes (FISH), however, not only allows for the identi¢cation of bacteria [7,8] but also provides valuable information on their spatial distribution [9,10]. From a previous study, it was known that extended polytetra£uoroethylene (e-PTFE) membranes used to cover super¢cial defects after surgery were colonized by pla-
0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 3 7 6 - 1
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J. Wecke et al. / FEMS Microbiology Letters 191 (2000) 95^101
que bacteria [11]. Therefore a further aim of this study was to compare bacterial colonization on PTFE membranes with those on di¡erent materials to get an optimized carrier for plaque bacteria. Dentin was chosen as a control material comparable to tooth surface, while gold foil has been selected because of its suitability in scanning electron microscopy. The carriers were inserted into periodontal pockets from patients su¡ering from rapidly progressive periodontitis (RPP). Bio¢lm formation was monitored by electron microscopy and FISH after di¡erent incubation periods. 2. Materials and methods A total of 12 patients scheduled for initial periodontal therapy participated in this study. Individuals who had been periodontally treated within the last 6 months or receiving any antimicrobial therapy during this period were excluded. RPP was diagnosed according to clinical and radiological criteria [12]. Distal and mesial sites were selected because of their pronounced probing depth measurements (mean 8.07 þ 1.63 mm). 2.1. Preparation of carriers and sampling of bacterial bio¢lm Carriers consisting of gold foil or e-PTFE membranes and constructed as shown in Fig. 1 were carefully inserted to reach the bottom of the pocket. The carrier was ¢xed supragingivally to the tooth surface by using cyanoacrylic glue (Octyldent1 Closure Medical Corp., Raleigh, NC, USA). The construction was guided by the assumption that the carrier positioned in the pocket might be colonized from both the tooth and the soft tissue side. To stabilize soft £exible materials like e-PTFE or gold foil, the carriers were mounted on commercially available `plast-oprobe' sticks (Maillefer, Ballaigues, Switzerland). Alternatively, 3-mm wide strips of e-PTFE or gold foil were attached to the plastic surfaces by four dots of cyanoacrylate (Tesa0 , Beiersdorf, Hamburg, Germany). The respective material was wrapped around the tips of plast-o-probes exceeding them at one side. Additionally dentin slices, not exceeding 3 mm in width each, were mounted on strips of e-PTFE. To get a better adaptation to the curvature of the dental root, it became necessary to divide the solid dentin into several segments not exceeding 3 mm in length each. Carriers were then positioned in the pockets with the longer end of the foils facing the teeth (Fig. 1) and attached to tooth surfaces. After 3 or 6 days of exposure, carriers were removed from the periodontal pockets. Only those carriers that kept their stable position during the exposure period were used for further investigations. Altogether 52 carriers (31 e-PTFE, 12 dentin, nine gold) were analyzed. For electron microscopy, specimens were ¢xed with ei-
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Fig. 1. Positioning of the e-PTFE carrier in a periodontal pocket; gold foil was inserted accordingly.
ther 2.5% (v/v) glutaraldehyde (Sigma, Munich, Germany), diluted in 0.1 M cacodylate bu¡er (pH 7) or a bu¡ered formaldehyde^glutaraldehyde ¢xative at 4³C overnight [13]. All the other treatments for transmission or scanning electron microscopy were done as previously described [11]. For FISH, four carriers were ¢xed with 3.7% (v/v) formaldehyde in phosphate-bu¡ered saline (pH 7.4) for at least 3 h. Embedding in cold polymerizing resin Technovit 8100 (Kulzer, Wehrheim, Germany) and sectioning of the specimens were performed as described earlier for tissue biopsies [9]. 2.2. FISH Group-speci¢c probe TRE I (5P-ACGCAAGCTCATCCTCAAG-3P) has been published earlier and has been shown to speci¢cally detect group I of oral treponemes, most of which are as yet uncultured [10]. EUB338 (5P-GCTGCCTCCCGTAGGAGT-3P) speci¢c for the domain Bacteria was used to visualize the entire bacterial population in the specimens [14]. Probes were labeled with Cy3 or FITC, respectively, and obtained from Interactiva (Ulm, Germany).
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Fig. 2. Scanning electron micrograph showing a part of an e-PTFE carrier with `islet-like' plaque formation after 3 days. The surface of the carrier reveals the ¢brillar structure of the e-PTFE membrane (white arrows).
Hybridization bu¡er containing 0.9 M NaCl, 20 mM Tris^HCl, pH 7.3, 0.01% (w/v) sodium dodecyl sulfate, 20% (v/v) formamide and 1 WM probe each was applied on the sections. To assess the speci¢city of the probes of ¢xed cells, the following Treponema strains served as controls: Treponema vincentii (ATCC 33580 and RITZ A), both Treponema species belonging to phylogenetic group I of oral treponemes, thus serving as positive controls. Treponema denticola (ATCC 33521), Treponema pectinovorum (ATCC 33768), Treponema socranskii subsp. buccale (ATCC 35534), Treponema socranskii subsp. socranskii (ATCC 35536), Treponema maltophilum (ATCC 51939), Treponema lecithinolyticum (ATCC 700332) and Treponema phagedenis subsp. reiteri (kindly provided by B. Wilske, Munich, Germany) serving as negative controls. Control slides with ¢xed treponeme cultures were included in every hybridization experiment (data not shown). After 3.5 h of incubation in a dark humid chamber at 46³C,
sections were rinsed with distilled water, air-dried in the dark and mounted with Citi£uor AF 1 (The Chemical Laboratory of the University of Kent, UK). A confocal laser scanning microscope (CLSM) model LSM 510 (Carl Zeiss, Oberkochen, Germany) equipped with an Ar-ion laser (488 nm) and two HeNe lasers (543 and 633 nm) was used to record optical sections. Image processing was performed with a standard software package delivered with the instrument (Zeiss LSM version 1.6). 3. Results 3.1. The ultrastructure of subgingival plaque The formation of subgingival plaque was analyzed on di¡erent carriers that were kept in periodontal pockets of RPP patients for 3^6 days. After short incubation
Fig. 3. Ultrathin cross-section of a dentin carrier exposed for 3 days (part a of Fig. 1); the dentin (D) is covered by a `pellicle-like' homogeneous layer (P) with adherent Gram-negative rods (R) and spirochetes (T).
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Fig. 4. Ultrathin section of a gold carrier (part c, Fig. 1) covered by a pellicle and colonized by Gram-positive cocci (exposure 3 days).
(3 days), islets-like colonization could be observed by scanning electron microscopy (Fig. 2). The loose colonization could also be seen in cross cut ultrathin sections (Fig. 3). Independent of the three materials used (e-PTFE, dentin, gold), a homogeneous electron dense pellicle could be demonstrated on the di¡erent carriers; also the bacterial colonization was similar. Analyzing more than 4000 elec-
tron micrographs, we can state that di¡erent Gram-negative rods and treponemes were found on those parts of the carriers corresponding to the deepest part of the pockets (Figs. 3 and 5). Gram-positive cocci could preferentially be observed on the upper segments of the carriers (Fig. 4). Early colonizing bacteria were characterized by an extracellular matrix especially shown in ultrathin sections par-
Fig. 5. Ultrathin section in parallel to the e-PTFE carrier (part a, Fig. 1) after 3 days of exposure; Gram-negative rods embedded in extracellular matrix (big arrows) are in close contact to spirochetes (T).
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Fig. 6. Electron micrograph showing an ultrathin section in parallel to the e-PTFE-carrier (part a, Fig. 1) after 6 days of exposure, demonstrating the dense colonization by di¡erent bacterial morphotypes; the central Gram-positive bacteria (GP) are surrounded by some Gram-negative rods (GN) in a `rosette-like' manner. Note the high number of treponemes.
allel to the carrier surfaces (Fig. 5). Treponemes were found in close contact to this matrix (Fig. 5). When carriers were kept in the periodontal pockets for 6 days, a massive and dense colonization of bacteria could be shown (Fig. 6). After this time, a con£uent bio¢lm resulted. Besides Gram-negative bacteria, some segments revealed a massive colonization of treponemes (Fig. 6). In such areas, the treponemes seemed to be arranged in a regular pattern. The use of carriers inserted in periodontal pockets offered the opportunity to follow the development of subgingival bacterial £ora as bio¢lm and to consider the spatial arrangement of di¡erent bacterial morphotypes. Additionally the speci¢c coaggregation of di¡erent bacterial species, which is visible in Figs. 5 and 6, could be analyzed. While electron microscopical methods demonstrated the organization of subgingival bio¢lm build up with di¡erent morphotypes such as cocci, rods or spirochetes, other methods were needed to carry out a detailed identi¢cation of bacteria.
lowed exact orientation within the sample and revealed di¡erent bacterial morphotypes in cross-sectioned subgingival bio¢lm of a RPP patient (Fig. 7). The thickness of the bio¢lm could be measured with 40^45 Wm. At higher magni¢cation, di¡erent morphotypes of e.g. cocci or rods ^ some organized as microcolonies ^ could clearly be di¡erentiated (Fig. 8). Moreover, simultaneous hybridization with probe TRE I-Cy3 revealed some large spirochetes interspersed between other bacteria. The higher magni¢cation (Fig. 8) of this bio¢lm illustrates the opportunity to obtain information about the length of the treponemes or their undulation. To our knowledge, this is the ¢rst time that the numbers and the spatial arrangement of spirochetes within the periodontal bio¢lm could be demonstrated. In this part of the bio¢lm, treponemes were rather separately localized, whereas in other areas high numbers of group I treponemes were spread in the bio¢lm as also shown by electron microscopy (Fig. 6).
3.2. Identi¢cation of bacteria by FISH
4. Discussion
Since the direct CLSM analysis of bio¢lm carriers such as gold, dentin or e-PTFE was hampered by light re£ection and auto£uorescence, semi-thin sections of Technovitembedded material were used. The EUB-FITC probe al-
Here we present a method that allows the removal of subgingival plaque as widely undisturbed bio¢lm. The advantage of this method is that neither insertion nor removal of carriers requires teeth or crown removal. The precise
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Fig. 7. CLSM micrograph of a subgingival plaque taken from a RPP patient after 6 days; showing the simultaneous hybridization using probes EUBFITC (green) and TRE I-Cy3 (red). Di¡erent green-colored morphotypes are often arranged in microcolonies. While EUB-338 stains all bacteria, the probe TRE I reveals the presence of spirochetes (red).
¢xed localization of the carriers allows the association of distinct bacterial morphotypes or species with the depth of periodontal pockets. In addition, information about the time-dependent formation of bio¢lm can be monitored. Electron microscopical analysis of di¡erent carriers revealed that the nature of the carrier material does not seem to have a great in£uence on the colonization. This is in agreement with a previous observation emphasizing that as compared to supragingival conditions, the surface characteristics were less important for subgingival plaque [15], showing almost no di¡erences in subgingival plaque composition on hydrophilic or hydrophobic polymers [16]. A possible explanation for this similar colonization pattern may be the salivary pellicle on the surface of each carrier rendering the colonization conditions alike [17^ 19]. Formation of dental plaque on epoxy resin crowns has been described earlier [20,21]. However, this epoxy resin crown model is only of limited use for studying periodontitis, as any information on deep periodontal pockets is missing. High numbers of spirochetes and the presence of Gram-negative rods and coccoid cells were observed only on those parts of the carriers localized in the deepest zone of the periodontal pockets. In another ultrastructural study, Gram-positive cocci were shown as part of the apical border plaque [5]. In our study, we found Gram-positive cocci only on the most coronal segments of the plaque carriers obviously exposed to a higher oxygen tension. Modern determination of bacterial £ora via cluster analysis examining the relationship among di¡erent taxa revealed that some species do not exist separately in the periodontal pockets but rather form complexes [4]. Elec-
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tron microscopy allowed to study the complexity of human plaque bio¢lm showing structural or spatial arrangements of di¡erent bacterial morphotypes (Fig. 6), described as `corncob' or `bristle-brush' formations
Fig. 8. Higher magni¢cation of a part of Fig. 7 (white frame) with large yellow/red colored treponemes (white arrows). Some microcolonies of di¡erent morphotypes (green colored) are marked (white arrow heads).
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[5,22]. The coaggregation and coadhesion of oral bacteria was summarized recently [23], however, there is doubt whether this in vitro study re£ects the situation in vivo. In contrast, the ultrastructural bio¢lm analysis enables the characterization of di¡erent bacterial morphotypes and their spatial arrangements. Further taxonomic characterizations are only possible with additional methods such as immuno-electron microscopy and/or in situ hybridization using speci¢c oligonucleotide probes. The advantage of using 16S rRNA-directed oligonucleotide probes for the identi¢cation and visualization of spatial distribution of di¡erent bacterial species was shown convincingly for environmental bio¢lms [7,8]. While the direct analysis of carriers with CLSM was hindered by re£ection and auto£uorescence of the carriers, the use of Technovit sections overcame these problems and allowed both the localization and identi¢cation of microorganisms. Detailed information on bio¢lm structure in habitats such as subgingival plaque is still lacking. FISH using di¡erent probes simultaneously may reveal the exact composition and architecture of particular bio¢lm. Thus, this novel technique presented here may contribute to an understanding of bio¢lm formation and perhaps the role of single bacterial species in the etiopathogenesis of periodontal infections. References [1] Socransky, S.S. and Ha¡ajee, A.D. (1991) Microbial mechanisms in the pathogenesis of destructive periodontal diseases : a critical assessment. J. Periodont. Res. 26, 195^212. [2] Marsh, P.D. and Bradshaw, D.J. (1997) Physiological approaches to the control of oral bio¢lms. Adv. Dent. Res. 11 (1), 176^185. [3] Burne, R.A., Quivey Jr., R.G. and Marquis, R.E. (1999) Physiologic homeostasis and stress responses in oral bio¢lms. Methods Enzymol. 310, 441^460. [4] Socransky, S.S., Ha¡ajee, A.D., Cugini, M.A., Smith, C. and Kent Jr., R.L. (1998) Microbial complexes in subgingival plaque. J. Clin. Periodontol. 25, 134^144. [5] Vrahopoulos, T.P., Barber, P.M. and Newman, H.N. (1995) The apical border plaque in severe periodontitis. An ultrastructural study. J. Periodontol. 66, 113^124. [6] Wood, S.R., Kirkham, J., Marsh, P.D., Shore, R.C., Natress, B. and Robinson, C. (2000) Architecture of intact natural human plaque bio¢lms studied by confocal laser scanning microscopy. J. Dent. Res. 79, 21^27. [7] Manz, W., Amann, R., Szewzyk, R., Szewzyk, U., Stenstro«m, T.-A., Hutzler, P. and Schleifer, K.-H. (1995) In situ identi¢cation of Legionellaceae using 16S rRNA-targeted oligonucleotide probes and confocal laser scanning microscopy. Microbiology 141, 29^39.
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[8] Amann, R., Snaidr, J., Wagner, M., Ludwig, W. and Schleifer, K.-H. (1996) In situ visualization of high genetic diversity in a natural microbial community. J. Bacteriol. 178, 3496^3500. [9] Moter, A., Leist, G., Rudolph, R., Schrank, K., Choi, B.-K., Wagner, M. and Go«bel, U.B. (1998) Fluorescence in situ hybridization shows spatial distribution of as yet uncultured treponemes in biopsies from digital dermatitis lesions. Microbiology 144, 2459^2467. [10] Moter, A., Hoenig, C., Choi, B.-K., Riep, B. and Go«bel, U.B. (1998) Molecular epidemiology of oral treponemes associated with periodontal disease. J. Clin. Microbiol. 36, 1399^1403. [11] Wecke, J., Wolf, V., Fath, S. and Bernimoulin, J.-P. (1995) The occurence of treponemes and their spherical bodies on polytetra£uoroethylene membranes. Oral Microbiol. Immunol. 10, 278^283. [12] Page, R.C., Altman, L.C., Ebersole, J.L., Vandersteen, G.E., Dahlberg, W.-H., Williamsa, P.L. and Osterberg, S.K. (1983) Rapidly progressive periodontitis ^ A distinct clinical condition. J. Periodontol. 54, 197^209. [13] Karnovsky, M.J. (1965) A formaldehyde^glutaraldehyde ¢xative of high osmolality for use in electron microscopy. J. Cell Biol. 26, 137A. [14] Amann, R.I., Binder, B.J., Olson, R.J., Chisholm, S.W., Devereux, R. and Stahl, D.A. (1990) Combination of 16S rRNA-targeted oligonucleotide probes with £ow cytometry for analyzing mixed microbial populations. Appl. Environ. Microbiol. 56, 1919^1925. [15] Quirynen, M. and Bollen, C.M.L. (1995) The in£uence of surface roughness and surface-free energy on supra- and subgingival plaque formation in man. J. Clin. Periodontol. 22, 1^14. [16] Busscher, H.J. and Van der Mei, H.C. (1997) Physico-chemical interactions in initial microbial adhesion and relevance for bio¢lm formation. Adv. Dent. Res. 11 (1), 24^32. [17] Newman, H.N. and Barber, P.M. (1995) Dental plaque structure in vivo. In: The Life and Death of Bio¢lm. Contributions Made at the Second Meeting of the British Bio¢lms Club Powys (Wimpenny, J., Handley, P., Gilbert, P. and Lappin-Scott, H., Eds.), pp. 27^32. Bio Line. [18] Glantz, P.O., Baier, R.E. and Christersson, C.E. (1996) Biochemical and physiological considerations for modelling bio¢lms in the oral cavity: A review. Dent. Mater. 12, 208^214. [19] Ellen, R.P., Le¨pine, G. and Nghiem, P.M. (1997) In vitro models that support adhesion speci¢city in bio¢lms of oral bacteria. Adv. Dent. Res. 11 (1), 33^42. [20] Listgarten, M.A., Mayo, H.E. and Tremblay, R. (1975) Development of dental plaque on epoxy resin crowns in man. A light and electronmicroscopy study. J. Periodontol. 46, 10^26. [21] Listgarten, M.A. (1994) The structure of dental plaque. Periodontology 2000 (5), 52^65. [22] Westergaard, J., Frandsen, A. and Slots, J. (1978) Ultrastructure of the subgingival micro£ora in juvenile periodontitis. Scand. J. Dent. Res. 86, 421^429. [23] Kolenbrander, P.E., Anderson, R.N., Clemans, D.L., Whittaker, C.J. and Klier, C.M. (1999) Potential role of functionally similar coaggregation mediators in bacterial succession. In: Dental Plaque Revisited ^ Oral Bio¢lms in Health and Disease (Newman, H.N. and Wilson, M., Eds.), pp. 171^186. Eastman Dental Institute University College, London.
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