Institute for Oral Microbiology and General Immunology, Centre for Dental, Oral Medicine and Maxillofacial Surgery, University of Zürich, Plattenstrasse 11, CH-8028 Zürich, Switzerland1
Author for correspondence: Rudolf Gmür. Tel: +41 1634 3306. Fax: +41 1634 4310. e-mail: gmuer{at}zzmk.unizh.ch
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ABSTRACT |
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Keywords: fluorescent in situ hybridization, 16S rRNA, subgingival plaque, periodontitis
Abbreviations: FISH, fluorescent in situ hybridization; IF, indirect immunofluorescence
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INTRODUCTION |
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Phenotypically, the two species are very similar. Biochemical or serological differentiation is difficult, laborious and sometimes unreliable (Gmür & Wyss, 1985 ; Shah & Gharbia, 1992
; Gharbia et al., 1994
; Frandsen et al., 1995
; Dahlén et al., 1996
; Debelian et al., 1996
; Mättö et al., 1996b
; Paquet & Mouton, 1997
; Baumgartner et al., 1999
; Lie et al., 2001
). Therefore, investigations now employ semi-quantitative checkerboard DNA probe assays (Socransky et al., 1994
) or PCR-based assays, of which multiple variations have been described, to differentiate between P. intermedia and P. nigrescens. So far, PCR-based assays have yielded only qualitative data, that is they were used either to type clonal black-pigmented colonies derived from primary cultures (Mättö et al., 1996a
, b
; Conrads et al., 1997
; Guillot & Mouton, 1997
; Baumgartner et al., 1999
; Haraldsson & Holbrook, 1999
; Premaraj et al., 1999
; Robertson et al., 1999
) or to directly determine the presence or absence of the two species in clinical samples (Ashimoto et al., 1996
; Riggio et al., 1998
; Stubbs et al., 1999
; Mullally et al., 2000
; Siqueira et al., 2001
). To our knowledge, a quantitative real-time PCR assay has not yet been described, and fluorescent single-cell-specific assays are not available.
The motivation for the present study was our finding, in an ongoing investigation assessing selected microbiota in dental plaque from the gingival margins of Chinese patients with either necrotizing ulcerative gingivitis or gingivitis, that the former group had a significantly higher load of serologically detectable P. intermedia/P. nigrescens cells than the gingivitis group (data not shown). In light of the postulated different roles of the two species as periodontal pathogens, it seemed important to identify and quantify them differentially. Thus, the aim of the present study was to develop direct, quantitative, species-specific P. intermedia and P. nigrescens detection assays and to evaluate their performance in comparison with culture and indirect immunofluorescence (IF) applied, in parallel, to a set of subgingival plaque samples. Based on the available infrastructure and experience, we opted for fluorescent in situ hybridization (FISH) using DNA probes to species-specific 16S rRNA sequences to target the two species.
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METHODS |
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Subgingival plaque samples (n=39) were obtained, with paper points (Gmür et al., 1989 ), from periodontal pockets of 16 patients (14 female) with a mean age of 45·5 years (range 2567 years). The subjects had been referred to our institute for microbial testing because of refractory chronic periodontitis (Armitage, 1999
). The sampled pockets had a mean probing depth of 6·2±1·3 mm (range 510 mm). Within 1 h of harvesting, samples were vortexed for 30 s at the maximum setting and split into different aliquots. Aliquots selected for FISH were frozen at -20 °C until used. Aliquots for IF and cultivation were processed immediately. Those reserved for cultures were serially diluted from 10-1 to 10-5 in 0·9% NaCl. A 50 µl aliquot from each dilution was distributed on Columbia blood agar base (Oxoid) supplemented with 5% haemolysed human blood using a spiral diluter (Spiral System), and plates were incubated anaerobically for 96 h at 37 °C. Colony-forming units (c.f.u.) were counted using a stereomicroscope, selectively assessing plates with an optimum concentration of colonies.
Cell fixation and preparation of multiwell slides.
For FISH assays of cultured bacteria, strains were washed once in 0·9% NaCl, resuspended in sterile PBS at pH 7·4, and fixed (20 min, 4 °C) by mixing the cell suspension 1:10 (v/v) with 4% freshly prepared paraformaldehyde solution (Manz, 1999 ). Thereafter, the cells were pelleted by centrifugation at 10000 g, resuspended in 50% ethanol, and either directly spotted on precleaned 18- or 24-well microscope slides (Cel-Line Associates) or stored at -20 °C until used. Air-dried slides were dehydrated in 50, 80 and 100% ethanol (3 min each). For FISH assays of plaque samples, 10 µl diluted (1:5 or 1:10) plaque suspension per well was dropped onto 18- or 24-well slides, air-dried and then fixed immediately (20 min, 4 °C) by inserting the slide in a 50 ml centrifuge tube containing 4% paraformaldehyde. For IF assays, cultured bacteria or subgingival plaque samples were used to coat 18-or 24-well slides and fixed with methanol as described (Gmür et al., 1989
). Prior to the coating stage, subgingival plaque samples had been centrifuged at 10000 g for 5 min, resuspended for 3 min in 0·83% NH4Cl to lyse erythrocytes, recentrifuged, resuspended in 0·9% NaCl/0·02% NaN3/0·00025% cetyltrimethylammonium bromide and stored at -20 °C until used.
FISH.
Custom-synthesized oligonucleotide probes, labelled at the 5'-ends with Cy3 or 6-FAM, were purchased from Interactiva. Probes were designed according to the criteria described by Manz (1999) , using programs and rRNA sequence information from Ribosomal Database Project II (Maidak et al., 1999
) (http://rdp.cme.msu.edu/html/) and the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST/). The sequences of the probes employed for P. intermedia, P. nigrescens, Prevotella denticola and Prevotella pallens are listed in Table 1
. The probes were used at a final concentration of 5 ng µl-1, except for competitor probes, which were used at 50 ng µl-1. FISH was performed using a modification (Thurnheer et al., 2001
) of the method described by Manz (1999)
, except that the length of the hybridization period was set to 150 min. Optimum reactivity conditions for the probes with target and non-target strains were determined as described (Thurnheer et al., 2001
). The universal bacterial probe EUB338 (Amann et al., 1995
) served as a positive control. Fluorescence intensity was graded using an arbitrary five-step scale, where 0 (no fluorescence above background) and 1+ (very faint fluorescence) were considered negative signals, and 2+ (weak), 3+ (strong) and 4+ (brilliant fluorescence) were considered positive signals.
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Microscopy.
FISH- and IF-stained slides were read with an Olympus BX60 epifluorescence microscope [Olympus Optical (Schweiz)] equipped with phase-contrast, an HBO 103 W/2 mercury photo optic lamp (Osram) and Olympus filter sets U-MNIBA (6-FAM), U-MA41007 (Cy3) and BX-DFC5 (6-FAM/Cy3). The samples were evaluated for the number of fluorescent bacteria per ml of sample as described (Gmür & Guggenheim, 1994 ), monitoring either randomly selected fields that covered, in total, between 1/100 and 1/2 of a well or an entire well, depending on the density of positive cells. Colour micrographs were taken with a digital Olympus Camedia 3030 camera, transferred to a Macintosh G3 personal computer and processed using the standard software provided with the camera and PHOTOSHOP 6.0 (Adobe) without any qualitative changes to the raw images.
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RESULTS |
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Comparison of FISH data with IF and culture data
P. intermedia and P. nigrescens enumerations by FISH and IF are compared in Fig. 3. Because the two species are not discriminated by the IF assay, the IF data had to be plotted against the sum of P. intermedia and P. nigrescens as detected with probes Pint657 and Pnig657. The results demonstrated a satisfactory correspondence (r=0·710) between the two assays, except for five samples that were positive by IF but negative by FISH.
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DISCUSSION |
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The rapid and accurate identification and enumeration of black-pigmented anaerobes from highly complex microbial communities such as subgingival plaque are difficult tasks. Many of the available techniques for differentiation between P. intermedia and P. nigrescens rely on culture as the initial step; black-pigmented colonies are selected for and counted, and then a variety of secondary techniques are used to identify representative colonies (Dahlén et al., 1990 ; Mättö et al., 1996b
; van Steenbergen et al., 1997
; Conrads et al., 1997
; Debelian et al., 1997
; Haraldsson & Holbrook, 1999
; Robertson et al., 1999
). This approach has several disadvantages. First, it is expensive and time-consuming because of a lengthy incubation period. Second, data from this study (Fig. 4
) demonstrate clearly that culture substantially underestimates the cell numbers of P. intermedia and P. nigrescens in subgingival plaque. This is not surprising in the light of the overwhelming association of both species with dense mixed bacterial aggregates (Fig. 1
) that are not disrupted by brief sonication and lengthy vortexing (60 s). Similar evidence has been reported previously (Loesche et al., 1992
; Giertsen et al., 2000
); nevertheless, the problem that aggregation poses to quantitative culture analyses remains widely neglected. Techniques that do not require culturing and which differentiate between P. intermedia and P. nigrescens are available (various PCR-based assays, checkerboard DNADNA hybridization), but so far they have the disadvantage of yielding only qualitative (Ashimoto et al., 1996
; Riggio et al., 1998
; Stubbs et al., 1999
; Mullally et al., 2000
), or at best semi-quantitative, data (Socransky et al., 1994
; Tanner et al., 1998
). Clearly, there is a need for a rapid and objectively quantitative procedure for the direct quantification of the two species in clinical samples to investigate their postulated roles in oral health and disease in more detail. We feel that our FISH assay with new probes to species-specific stretches of 16S rRNA of P. intermedia, P. nigrescens, P. pallens and P. denticola, described in this work, provides such a tool. Unlike currently used IF assays, the FISH assay can differentiate between P. intermedia, P. nigrescens and P. pallens. Like IF, it offers sensitive, qualitative and quantitative monitoring of single fluorescent cells in clinical samples, and has a lower detection limit of slightly more than 103 cells ml-1 for samples containing approximately 108 bacteria. (Such cell densities require sample dilutions of 1:5 or 1:10 for the coating of 4 mm wells with 10 µl. If the minimum requirement for a positive score is set to at least three positive cells per well, it follows that the lower detection limit at a 1:10 dilution is 3x103 ml-1; Gmür, 1995
). Fluorescence assays for bacteria are always confronted with the possible presence of autofluorescence-emitting bacteria or of non-specifically fluorescent particles. In the present study, autofluorescence with the characteristic orange-red colour of the Cy3 label or the fluorescein-isothiocyanate-like colour of 6-FAM was not observed. Fluorescent particles were always present but could be recognized readily because of the extremely intensive fluorescence and the irregular shapes. A comparison of the performance of FISH and IF generally showed good correspondence (Fig. 3
), in particular if one considers that increased data variability had to be expected because cells were distributed unevenly (in aggregates) in the wells of the microscope slide and only a part of the entire well was monitored. Interestingly, five samples were positive for P. intermedia/P. nigrescens by IF but not by FISH. The most likely explanation for this observation is that mAbs 37BI6.1 and 39BI1.1.2 could bind to another, so far unidentified, species. We had noted recently that both mAbs labelled strains of the newly described species P. pallens (unpublished observation), but, according to the FISH data with probes Ppal186 and Ppal654 (+Ppal654comp), P. pallens was not present in the samples studied.
In designing probes for the FISH assay described, we preferentially targeted the highly variable 5' half of helix 23 of the 16S rRNA with nucleotides 645680 (Escherichia coli numbering; Brosius et al., 1981 ). This region has yielded numerous bright oligonucleotides with specificity at genus or species level (Amann et al., 1995
; Fuchs et al., 1998
) (our unpublished data). All five probes from this study against this region were usable at high formamide concentrations and yielded very strong fluorescence signals. In contrast, a P. intermedia-specific probe targeted to nucleotides 620638 gave completely negative results (data not shown). P. intermedia and P. pallens differ within the stretch from nucleotides 645680 only at position 658. Thus, probes Pint657 and Pint649 (both 19-mer oligonucleotides) each have this single mismatch with P. pallens, which, however, is located either peripherally or centrally. Not unexpectedly (Manz, 1999
), the peripheral mismatch location in probe Pint657 led to cross-reactivity with P. pallens at medium fluorescence intensities, whereas Pint649 with the mismatch in the centre of the probe proved to be P. intermedia-specific. Conversely, probe Ppal654 was only negative with P. intermedia if an unlabelled competitor probe was added in excess to prevent the hybridization of the fluorescent Ppal654 to P. intermedia. As all samples of this study appeared to be P. pallens-free (no bacteria positive with Ppal186 or Ppal654/Ppal654comp could be detected), one would expect FISH with Pint657 and Pint648 to result in approximately the same number of fluorescent bacteria. This was indeed the case with most samples, which underlines the credibility of this quantitative assay. However, five exceptional samples, three from one patient, were noted. In these samples, Pint657 labelled between 105 and 107 positive bacteria that remained unlabelled by Pint649. Positive cells had the typical morphology of plaque-derived P. intermedia. The data suggest that Pint657 may detect (in addition to normal P. intermedia) an unidentified variant or different taxa with at least one mismatch within the rRNA region (nucleotides 657667) covered by both probes.
In summary, the results presented in this investigation show good discrimination between P. intermedia and P. nigrescens by oligonucleotide probes, and demonstrate the advantages of the application of a FISH assay specific for these species in comparison to both culture and IF.
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ACKNOWLEDGEMENTS |
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Received 28 September 2001;
revised 7 January 2002;
accepted 28 January 2002.
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