1 Institute of Oral Biology, Dental Faculty, University of Oslo, PB 1052 Blindern, 0316 Oslo, Norway
2 Institut für Mikrobiologie und Hygiene, Universitätsklinikum Charité, Humboldt-Universität zu Berlin, Dorotheenstrasse 96, D-10117 Berlin, Germany
3 Abteilung für Pathologie, Ruhr-Universität Bochum, Universitätsstrasse 150, D-44780 Bochum, Germany
4 Universitätsklinik für Mund-, Kiefer- und Plastische Gesichtschirurgie, Knappschafts-Krankenhaus Bochum-Langendreer, In der Schornau 23-25, D-44892 Bochum, Germany
Correspondence
Pia T. Sunde
titterud{at}odont.uio.no
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ABSTRACT |
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INTRODUCTION |
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In situ hybridization has proved to be a useful method for detection and identification of bacteria within their natural environment (for a review see Moter & Göbel, 2000). Recently, fluorescence in situ hybridization (FISH) has been applied to detect bacteria in tissue embedded in cold polymerizing resin (Moter et al., 1998a
; Hammer et al., 2001
). In these plastic sections, visualization of bacteria and excellent histological conservation were achieved. Furthermore, confocal laser scanning microscopy (CLSM) has been established as a valuable tool for obtaining high-resolution images and three-dimensional reconstructions of a variety of biological samples (Wagner et al., 1994
; Manz et al., 1995
; Moter et al., 1998a
; Wecke et al., 2000
).
The aim of the present study was to visualize and identify bacteria directly within periapical lesions by means of FISH in combination with epifluorescence and CLSM.
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METHODS |
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Processing of tissue specimens.
The enucleated periapical lesions from the 39 patients were washed with sterile saline, fixed directly with 3·7 % (v/v) formaldehyde in PBS (pH 7·4) and kept at 4 °C. The embedding procedure, utilizing cold polymerizing resin (Technovit 8100; Kulzer), was performed according to the manufacturer's instructions. The specimens were washed overnight in PBS containing 6·8 % (v/v) sucrose, dehydrated in acetone for 1 h and infiltrated with the plastic solution (Technovit 8100 base-liquid with hardener I) for at least 6 h. After adding hardener II, the periapical lesions were aligned properly in the wells of Histoform S (Kulzer), sealed with cover foil and placed in the refrigerator. After polymerization, histoblocs were mounted on the specimens using Technovit 3040. The blocks were stored at 4 °C prior to sectioning.
The blocks were sectioned on a rotary microtome (Medim, Type DDM 0036) using steel knives with hard metal edges. Tissue sections (3 µm) were straightened on sterile water, placed on silanized slides [Super Frost (Medim); 3-aminopropyltrimethoxysilane (Sigma)] and stored at 4 °C. Three tissue sections from different parts of each lesion were hybridized. The sections were examined prior to the FISH procedure for autofluorescence.
Oligonucleotide probes.
Probe EUB 338, which is complementary to a portion of the 16S rRNA gene conserved in the domain Bacteria, was used to visualize the entire bacterial population in the specimens (Amann et al., 1990). The group-specific probes TRE I, TRE II and TRE IV (Choi et al., 1997
; Moter et al., 1998b
) were applied to the detection of most phylotypes of oral treponemes of Group I (including Treponema vincentii and Treponema medium as cultivable members), Group II (including Treponema denticola) and Group IV (including Treponema maltophilum and Treponema lecithinolyticum), respectively. For the demonstration of Streptococcus spp. a genus-specific probe, STREP, was used (Trebesius et al., 2000
). For visualization of Fusobacterium spp. a genus-specific probe, FUSO, was designed, and species-specific probes were designed for Actinobacillus actinomycetemcomitans (ACTACT), Tannerella forsythensis [B(T)AFO], Porphyromonas gingivalis (POGI), Prevotella intermedia (PRIN) and Veillonella parvula (VEPA) (Table 1
). All probes have been deposited in ProbeBase (Loy et al., 2003
); an online resource for rRNA-targeted oligonucleotide probes where probe difference alignments are available (http://www.microbial-ecology.de/probebase/index.html).
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Besides A. actinomycetemcomitans, probe ACTACT also matches Pasteurella sp. MCCM02539, a sequence submitted while this work was in progress. Therefore, we could not exclude hybridization with this species. The closest negative control for ACTACT was Haemophilus influenzae with a single mismatch as determined by sequencing, that was not detected by the probe. Similarly, probe POGI has none or one single mismatch to the recently published species Porphyromonas gulae, isolated from the gingival sulcus of various animal hosts (Fournier et al., 2001). Therefore, we cannot exclude cross-hybridization with this species that so far has not been detected in humans. V. dispar was the closest negative control for probe VEPA and yielded only weak signals when hybridized. Probe B(T)AFO detects 13 sequences deposited as Bacteroides forsythus in the GenBank and EMBL databases. However, it has seven mismatches to the recently published Bacteroides spp. BU063 that is associated with health (Leys et al., 2002
). Therefore, no cross-hybridization with this species would be expected. Campylobacter rectus with one mismatch to probe FUSO allowed optimization of the respective probe. Probes B(T)AFO and PRIN had more than three mismatches to the closest available phylogenetic relative and were therefore optimized using the above-mentioned panel of oral species.
All probes used for FISH were synthesized commercially and 5' end-labelled with fluorescein isothiocyanate (FITC) providing a green signal or with fluorochrome Cy3 (indocarbocyanine; Thermo Hybaid Interactiva division) giving a bright orange signal. The group-, genus- and species-specific probes labelled with the Cy3 fluorescent dye were applied simultaneously with the probe EUB 338-FITC. Some samples were tested in parallel with NON 338-Cy3, a probe complementary to EUB 338, in order to control non-specific binding of the EUB 338 probe (Wallner et al., 1993). Alternatively, some samples were stained with DAPI (4',6'-diamidino-2-phenylindole), which detects DNA of bacteria, fungi and host cells. DAPI staining was applied simultaneously with two specific probes, using the Vit-dental system (Vermicon) according to the manufacturer's instructions.
FISH.
The hybridization buffer contained 0·9 M NaCl, 20 mM Tris/HCl, pH 7·3, and 0·01 % SDS. The stringency was adjusted by varying the formamide concentration from 0 to 30 % (v/v), depending on the oligonucleotide probe used. Pre-warmed hybridization buffer (20 µl) was mixed with approximately 5 pmol of the appropriate oligonucleotide probe and carefully applied to the tissue sections. After incubation for 3·5 h in a dark humid chamber at 46 °C, each of the slides were rinsed with sterile double-distilled water, air-dried in the dark and mounted with Citifluor AF 1 (Chemical Laboratory, University of Kent).
Epifluorescent microscopy and CLSM.
An epifluorescence microscope (Axioskop; Zeiss) was used to view the bacteria in hybridized sections processed for FISH. The microscope was equipped with a 50 W high-pressure mercury lamp (HB050; Osram) and x10, x40 and x100 objectives (Zeiss). Narrow band filter sets HQ-F41-007 and HQ-F41-001 (AHF; Analysentechnik) were used to analyse the DAPI, FITC and Cy3 signals, respectively at a magnification of x1000. Photomicrographs were taken using a Kodak Ektachrome HC 400 film. A Zeiss confocal laser scanning microscope 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 SLM version 1.6). Reconstructed and processed images were produced on slide film (Kodak Professional, HC 100).
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RESULTS |
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Most often rods, cocci and especially spirochaetes of different sizes hybridized with the EUB 338 probe only. These bacteria were often seen spread among cells and fibres in the tissue (Fig. 1a). Simultaneously, hybridization with the probe EUB 338-FITC and the control probe NON 338-Cy3 showed several bacteria with the FITC filter while no signals could be seen with the Cy3 filter (Fig. 1b
).
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In three lesions, areas of tissue necrosis were apparent and micro-organisms of different morphologies were observed (Fig. 1d). In some lesions the EUB 338 probe detected co-aggregated bacteria of different morphologies within microcolonies (Fig. 1e
). In addition, single colonies of EUB 338-detected cocci were also seen.
A distinct morphotype of large, curved bacterial rods was detected with the EUB 338 probe in several lesions (Fig. 2a).
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DISCUSSION |
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By using the control probe NON 338 and DAPI staining, non-specific binding could be excluded, which gave confidence to the results achieved, especially when unusual and large morphotypes of bacteria were observed. In addition, autofluorescence was excluded by viewing the sections prior to the FISH procedure. No bacteria were observed at the borders of the lesions, indicating that contamination had been prevented. This was consistent with the results of a previous methodological study of extraradicular infection where our surgical technique proved to be adequate for microbial sampling of periapical lesions (Sunde et al., 2000a).
In most cases, bacteria were detected with the eubacterial probe EUB 338 only. The organisms were spread between tissue cells and fibres in the lesion or were co-aggregated, forming microcolonies. The bright signal intensities of the bacteria indicated a high amount of rRNA, which is evidence for physiological activity of the cells at the time of sampling (Kemp et al., 1993; Wallner et al., 1993
).
In all lesions where bacteria were observed, they seemed to colonize localized parts of the lesions while other parts seemed to be bacteria-free. Co-aggregation and accumulation of bacteria may suggest that a synergistic interaction is taking place between the organisms involving use of food chains and consorted degradation of complex host and bacterial exopolymers. Positive interactions between different species occur in the periodontal pocket (van Winkelhoff et al., 1987; Socransky et al., 1998
) and in the root canal (Fabricius et al., 1982
; Sundqvist 1992
), and it is likely that positive interactions also exist between bacteria living in periapical lesions.
In one lesion, Group I treponemes were observed. Treponema vincentii and/or Treponema vincentii-related organisms, which are Group I treponemes, have been associated with human periodontal diseases (Choi et al., 1994; Moter et al., 1998b
; Willis et al., 1999
; Dewhirst et al., 2000
), but have to our knowledge not previously been reported in the root canal or in periapical lesions. In a previous epidemiologic study on oral treponemes, the parallel use of oligonucleotide probes specific for cultivable and as-yet-uncultivable organisms showed a great discrepancy between cultivable and as-yet-uncultivable treponemes of Group I, II and IV (Moter et al., 1998b
), suggesting the presence of novel yet unknown organisms at a high frequency. In the present study several spirochaete-like organisms of different sizes were detected with the universal probe EUB 338, but no specific signals could be obtained with the treponeme-specific probes. The explanation may be that the probes did not enable detection of all treponemes present, emphasizing the considerable genetic diversity of this group of organisms (Choi et al., 1994
; Dewhirst et al., 2000
). Previously, our group has described a large uncultivable spirochaete-like organism of 180 µm inside the root canal (Dahle et al., 1993
), and in a previous study with transmission and scanning electron microscopy spiral-form bacteria were commonly seen in sulfur granules' from periapical lesions (Sunde et al., 2002
). The detection of these strict anaerobic bacteria suggested that both the root canal and periapical lesion contain highly reduced microenvironments. Because spirochaetes are difficult or even sometimes seemingly impossible to cultivate, their prevalence in endodontic infections is probably underestimated.
Other noteworthy observations in the present study were the positive signals obtained from Porphyromonas gingivalis, Prevotella intermedia and Tannerella forsythensis appearing in three different lesions. These organisms have been associated with periodontal disease (Ashimoto et al., 1996; Socransky et al., 1998
), similar to oral treponemes. Their presence in periapical lesions is consistent with recent observations from the root canal and periapical lesions based on molecular methods (Conrads et al., 1997
; Gatti et al., 2000
; Sunde et al., 2000b
; Jung et al., 2001
; Roças et al., 2001
).
Streptococci were observed in microcolonies in several lesions. They are considered important pathogens in dental caries and have often been isolated from the root canal and periapical lesions.
Most of the probes used did not show specific reaction with any of the observed bacteria. One possibility of non-detection is interference from elastin, collagen and blood cells causing bright autofluorescence. This could have decreased the signal-to-noise ratio and it cannot be excluded that some specific fluorescent signals were masked, particularly from small spirochaetes.
It is also possible that some strict anaerobic bacteria may not be detected by the FISH technique due to their low metabolic activity and the small number of rRNA copies, resulting in low signal intensity and false negative results. However, many of the detected micro-organisms seem to be not yet cultured isolates or are not so-called oral pathogens.
In conclusion, direct visualization of bacteria with the FISH technique provided additional support to the notion that bacteria are present in periradicular tissue of asymptomatic teeth with apical periodontitis, that the bacteria here constitute a consortium of different species and that some of them are probably as yet uncultivable. This technique may also have a potential value in elucidating the aetiology/pathogenesis of extraradicular infection. However, more FISH studies combined with epifluorescence and CLSM should be done with a battery of specific probes for visualization and identification of additional bacteria, including uncultivable ones. No doubt, the endodontic microflora needs to be revisited.
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ACKNOWLEDGEMENTS |
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Received 28 October 2002;
revised 10 February 2003;
accepted 17 February 2003.