Abstract
Primers were designed from 16S rRNA sequences of Prevotella intermedia sensu stricto and Prevotella nigrescens and were used to discriminate these two species by PCR. The results were compared with those from the PCR technique using primers designed from arbitrarily primed PCR products by Guillot and Mouton (E. Guillot and C. Mouton, J. Clin. Microbiol. 35:1876–1882, 1997). The specificities of both assays were studied by using P. intermedia ATCC 25611, P. nigrescens ATCC 33563, 174 clinical isolates of P. intermedia sensu lato, and 59 reference strains and 58 clinical isolates of other Prevotella species and/or common oral flora. In addition, the usefulness and reliability of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the differentiation of the two species were examined by comparing the results with those from PCR assays. The controversial lipase test for distinguishing these species was also carried out. Unambiguous differentiation was made by both PCR assays, and the results matched each other. The SDS-PAGE assay was found to misidentify a few strains tested, compared with the results of PCR assays. The lipase test was positive for both species, including the reference strains of P. intermedia and P. nigrescens. We conclude that both PCR assays are simple, rapid, reliable, and specific methods which could be used in clinical studies and that the lipase test is not valuable in the differentiation. The reliable discrimination of the two species by SDS-PAGE is questionable.
Prevotella intermedia, which is an obligately anaerobic black-pigmented gram-negative bacillus, is one of the microorganisms frequently implicated in the development of periodontitis. Intraspecies heterogeneity of P. intermedia has been demonstrated as two DNA homology groups, in an early study by Johnson and Holdeman (9). Shah and Gharbia confirmed the existence of these two groups and proposed a new pigmented species, Prevotella nigrescens, and separated it from the species P. intermedia (13).
Several methods have been utilized during the past few years to distinguish P. intermedia from P. nigrescens, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has also been used along with other methods by many researchers in this field. Although SDS-PAGE is an accepted technique used in species identification studies of many organisms and is also described as a simple and convenient method for the identification of P. intermedia and P. nigrescens to species level, the banding patterns revealed by different laboratories seemed to be different, as there is no reproducible band(s) observed by the researchers (2, 3, 4, 16). Therefore, the usefulness and reliability of this method in the differentiation of P. intermedia and P. nigrescens are yet to be assessed.
Biochemical tests and gas-liquid chromatography are of no use in the differentiation of these two genospecies. The suggested lipase activity test (13), which could differentiate the two species, has not been reproduced in other laboratories, and controversial results were observed between researchers. Dahlén et al. (3) reported that both species were negative for lipase reaction, whereas Pearce et al. (12) reported that both species showed positive reactions, except the reference strain of P. nigrescens. However, the bizarre behavior of the strains needs further investigation.
To establish the association of P. intermedia and P. nigrescens with health and disease status, one needs simple, reliable, and reproducible diagnostic techniques which are applicable to clinical studies. In this context, PCR is in principle a simple and rapid method for the detection and identification of microorganisms.
In this study we designed for the PCR assay the specific primers for P. intermedia sensu stricto designated Pi R1 and Pi R2 and primers for P. nigrescens designated Pn R1 and Pn R2, from the 16S rRNA sequences (11), as primers and probes derived from 16S rRNA have been widely used in species identification for various organisms (1, 8, 18, 19). Among the isolates tested by PCR with these primers, two clinical isolates were not differentiated into either of the two species. Thus, we used the primers designated Pi 754-1 and Pi 754-2 for P. intermedia sensu stricto and Pn 1100-1 and Pn 1100-2 for P. nigrescens, which are designed from arbitrarily primed PCR (AP-PCR) products described by Guillot and Mouton in a recent study (6), for clarification of the PCR results by comparison. The specificities of PCR with these primers were studied by using the organisms which are commonly isolated in the mouth and/or belong to the genus Prevotella. The lipase activities of the two species were also studied. In addition we compared the SDS-PAGE method with the PCR assays and evaluated the reliability of SDS-PAGE in distinguishing these two species.
MATERIALS AND METHODS
Bacterial strains.
Clinical isolates (174 strains) which were identified in our laboratory as P. intermedia sensu lato by using a combination of standard methods (14) and diagnostic kits, the RapID ANA II system (Innovative Diagnostic Systems, Norcross, Ga.) or Rapid ID 32 A system (bioMérieux, Marcy l’Étoile, France), were used in this study. A total of 61 reference strains (Table 1) and the following clinical isolates were also used: four strains of Prevotella bivia, three strains of Prevotella buccae, seven strains of Prevotella buccalis, eight strains of Prevotella corporis, three strains of Prevotella denticola, three strains of Prevotella loescheii, four strains of Prevotella melaninogenica, four strains of Prevotella oralis, two strains of Prevotella oris, seven strains of other Prevotella species, a single strain each of Prevotella disiens, Prevotella ruminicola, and Prevotella zoogleoformans, three strains of Porphyromonas asaccharolytica, four strains of Porphyromonas gingivalis, two strains of the Porphyromonas group, and a single strain of Porphyromonas endodontalis. The isolates were identified by using conventional methods and the RapID ANA II system. All strains were cultured on prereduced brucella HK agar (Kyokuto Pharmaceutical, Tokyo, Japan) supplemented with 5% laked sheep blood at 37°C in an anaerobic chamber (Hirasawa, Tokyo, Japan) in an atmosphere consisting of 82% N2, 10% CO2, and 8% H2.
TABLE 1.
Reference strains used in this study
| Species | Straina |
|---|---|
| Actinobacillus actinomycetemcomitans | JCM 2434 |
| Actinomyces odontolyticus | GAI 91002 |
| Actinomyces viscosus | JCM 8353 |
| Bacteroides distasonis | ATCC 8503 |
| Bacteroides eggerthii | ATCC 27754 |
| Bacteroides fragilis | ATCC 25285 |
| Bacteroides ovatus | ATCC 8483 |
| Bacteroides thetaiotaomicron | ATCC 29741 |
| Bacteroides uniformis | ATCC 8492 |
| Bacteroides ureolyticus | NCTC 10941 |
| Bacteroides vulgatus | ATCC 8482 |
| Bifidobacterium adolescentis | ATCC 15703 |
| Bifidobacterium bifidum | JCM 1255 |
| Bifidobacterium breve | ATCC 15700 |
| Bifidobacterium longum | ATCC 15707 |
| Bifidobacterium pseudolongum | ATCC 25526 |
| Bilophila wadsworthia | WAL 7959 |
| Campylobacter gracilis | JCM 8538 |
| Capnocytophaga ochracea | GAI 5586 |
| Clostridium clostridioforme | NCTC 11224 |
| Clostridium ramosum | ATCC 25582 |
| Desulfomonas pigra | DSM 749 |
| Eubacterium lentum | ATCC 25559 |
| Fusobacterium necrophorum | ATCC 25286 |
| Fusobacterium nucleatum | ATCC 25586 |
| Fusobacterium varium | ATCC 8501 |
| Gemella morbillorum | ATCC 27824 |
| Lactobacillus acidophilus | JCM 1132 |
| Lactobacillus brevis | JCM 1059 |
| Lactobacillus casei subsp. casei | JCM 1134 |
| Lactobacillus fermentum | JCM 1173 |
| Lactobacillus plantarum | JCM 1149 |
| Lactobacillus reuteri | JCM 1112 |
| Lactobacillus salivarius subsp. salivarius | JCM 1231 |
| Peptostreptococcus anaerobius | ATCC 27337 |
| Peptostreptococcus asaccharolyticus | WAL 3218 |
| Peptostreptococcus indolicus | GAI 0915 |
| Peptostreptococcus magnus | ATCC 29328 |
| Peptostreptococcus micros | VPI 5464-1 |
| Peptostreptococcus prevotii | ATCC 9321 |
| Porphyromonas asaccharolytica | ATCC 25260 |
| Porphyromonas endodontalis | JCM 8526 |
| Porphyromonas gingivalis | ATCC 33277 |
| Propionibacterium acnes | ATCC 11828 |
| Prevotella bivia | ATCC 29303 |
| Prevotella buccae | ATCC 33574 |
| Prevotella denticola | JCM 8528 |
| Prevotella heparinolytica | ATCC 35895 |
| Prevotella intermedia | ATCC 25611 |
| Prevotella loescheii | JCM 8530 |
| Prevotella melaninogenica | GAI 5490 |
| Prevotella nigrescens | ATCC 33563 |
| Prevotella oralis | ATCC 33269 |
| Prevotella oris | ATCC 33573 |
| Staphylococcus saccharolyticus | ATCC 14953 |
| Streptococcus constellatus | ATCC 27823 |
| Streptococcus intermedius | ATCC 27335 |
| Atopobium parvulum | VPI 0546 |
| Suterella wadsworthensis | ATCC 51579 |
| Veillonella dispar | ATCC 17748 |
| Veillonella parvula | ATCC 10790 |
ATCC, American Type Culture Collection. DSM, Deutsche Sammlung von Mikroorganismen. GAI, Gifu Anaerobic Institute. NCTC, National Collection of Type Cultures. VPI, Virginia Polytechnic Institute. WAL, Wadsworth Anaerobic Laboratory. JCM, Japan Collection of Microorganisms.
DNA extraction.
One or two colonies of bacterial strains on an agar plate were suspended in 50 μl of Tris-EDTA-saline (pH 8.0). The bacterial suspension was incubated for 10 min at 95°C and centrifuged at 18,600 × g for 2 min to obtain the DNA supernatant. DNA was stored at −80°C until used, except those of P. intermedia sensu lato strains; the DNA extract of the species was used immediately after the extraction for PCR amplification, as DNA degradation was noticed in this species on long waits.
PCR assay with primers designed from 16S rRNA sequences (16S primers).
The primers derived from the 16S rRNA sequences of P. intermedia sensu stricto and P. nigrescens (accession no. L16468 and L16471, respectively) were used in this assay. These primers were referred to as Pi R1 (5′-GCATCTGACGTGGACCAA-3′) and Pi R2 (5′-CCGCTTTACTCCCCAACAA-3′) for P. intermedia and Pn R1 (5′-GGCCTNATACCCGATGTGTT-3′) and Pn R2 (5′-ACACGTGCAATTTATTCCCA-3′) for P. nigrescens.
PCR was done with 1 μl of DNA extract in a total volume of 30 μl of a suspension containing 1.5 μl of each primer, 0.15 μl of Taq DNA polymerase (Pharmacia, Uppsala, Sweden), 3 μl of 10× reaction buffer (Pharmacia; 100 mM Tris-HCl [pH 9.0], 500 mM KCl, 15 mM MgCl2), 0.6 μl of 50× deoxynucleoside triphosphate (Pharmacia), and 0.1 μl of 100 mM MgCl2 (final concentration, 2.5 mM MgCl2) in distilled water. The mixture was overlaid with 1 drop of mineral oil. PCR amplification was performed with a Perkin-Elmer DNA thermal cycler (Perkin-Elmer Corporation, Norway, Conn.) programmed for 35 cycles as one cycle at 95°C for 20 s followed by 65°C for 2 min with an elongation cycle of 74°C for 5 min as a final cycle. Amplification products were electrophoresed on 5% polyacrylamide gels in Tris-borate-EDTA buffer. The molecular mass standard 100-bp DNA ladder (Gibco BRL, Burlington, Canada) was included in each gel. The gel was stained with ethidium bromide, and it was visualized and photographed under a UV transilluminator.
PCR assay with primers designed from products of AP-PCR (AP primers).
The primers designed by Guillot and Mouton (6) were used. The primers for P. intermedia were referred to as Pi 754-1 (5′-CAGCACCCACAACGATATGA-3′) and Pi 754-2 (5′-TTCCATCTTCTCTGCCTGTC-3′), and the primers for P. nigrescens were referred to as Pn 1100-1 (5′-TTATGTTACCCGTTATGATGGAAG-3′) and Pn 1100-2 (5′-ATGGCGAAATAGGAATGAAAGTTA-3′).
The same procedure described for the above PCR assay was used, except for the annealing temperature of 50°C which was selected carefully after experimenting with series of different temperatures.
Multiplex PCR assay for P. intermedia and P. nigrescens.
Multiplex PCR to amplify simultaneously specific DNAs of P. intermedia and P. nigrescens in the same reaction was carried out by using 16S primers and AP primers.
The PCR amplification was performed in the same manner as that described above. The annealing temperatures for 16S primers and AP primers were 65 and 50°C, respectively.
SDS-PAGE analysis.
Fifty clinical isolates identified in our laboratory as P. intermedia sensu lato were used in this assay. Type strains ATCC 25611 and ATCC 33563 were used as reference strains for P. intermedia and P. nigrescens, respectively. All strains were grown on prereduced brucella HK agar (Kyokuto) supplemented with 5% laked sheep blood, at 37°C in the anaerobic chamber in an atmosphere consisting of 82% N2, 10% CO2, and 8% H2.
Organisms were inoculated into glass tubes containing 10 ml of prereduced brucella HK broth (Kyokuto) and incubated for 24 to 48 h in an anaerobic chamber at 37°C. Purities of the broth cultures were checked by inoculating the broth cultures onto prereduced brucella HK agar supplemented with 5% laked sheep blood for anaerobic incubation, before proceeding to protein extraction. A cell pellet was obtained by centrifugation at 6,000 × g for 5 min, washed twice in phosphate-buffered saline (pH 7.4), suspended in 200 μl of 10% SDS, and left for 1 h at room temperature for cellular protein extraction. Then, the suspension was centrifuged at 18,600 × g for 2 min, and the supernatant of the cell extract was taken and stored at 20°C until used. At the time of use, the cell extract was thawed completely at room temperature, and 5 μl of extract was boiled with 15 μl of a loading buffer (4% SDS, 2% glycerol, 1% 2-mercaptoethanol, 0.01% bromophenol blue in 125 mM Tris-HCl [pH 6.8]) for 5 min (7). Once it had cooled, 17.5 μl of the boiled sample was loaded onto a gel, 1.5 mm in thickness (5% stacking gel and 12% running gel), and electrophoresed overnight at 50 V. The gel was stained with Coomassie brilliant blue for 20 min and destained in a solution containing 25% methanol and 10% acetic acid in distilled water, until the bands were clearly visible to the naked eye. The molecular marker MW-SDS-200 kit (Sigma Chemical Company, St. Louis, Mo.) was included in each run. The gel was dried in a gel dryer, and the bands were observed with the naked eye.
The organisms which showed proteolysis upon protein extraction, as described above, underwent the following procedure. Before the addition of 10% SDS for cellular protein extraction, the cells were treated with 100 μl of 10% trichloroacetic acid and incubated for 1 h at 25°C (17). The cell suspension was centrifuged at 18,600 × g for 2 min, and the supernatant was discarded. After this, the above-mentioned protein extraction procedure was carried out as described above.
Lipase test.
Lipase production was observed by the reaction of the strains on egg yolk agar (EYA). EYA was prepared according to the method of Summanen et al. (14), and the plates were prereduced in an anaerobic chamber prior to use.
Fifty strains used in the SDS-PAGE assay and the reference strains of P. intermedia and P. nigrescens were used for this assay. Frozen aliquots of strains were subcultured twice on prereduced brucella HK agar supplemented with 5% laked sheep blood, before inoculating them on EYA. The inoculated EYA plates were incubated anaerobically in the anaerobic chamber, and the positive lipase reaction was seen as an iridescent sheen on the surface of the agar when observed under oblique light. The plates were observed for a positive reaction for 10 days.
RESULTS
PCR amplification with two 16S primer sets generated expected PCR products of 267 bp for P. intermedia ATCC 25611 (Fig. 1A, lane 2) and 301 bp for P. nigrescens ATCC 33563 (Fig. 1B, lane 2), although none of the other related species tested gave positive PCR results (Fig. 1, lanes 4 to 10). Of 174 P. intermedia strains identified by biochemical tests, the PCR assay identified 97 strains as P. intermedia sensu stricto and 75 as P. nigrescens, with two unidentified; by the two PCR assays, one strain was positive in both and the other was negative in both.
FIG. 1.
PAGE of PCR products with 16S primers. (A) Lane 1, 100-bp DNA ladder; lane 2, P. intermedia ATCC 25611; lane 3, P. nigrescens ATCC 33563; lane 4, P. loescheii JCM 8530; lane 5, P. buccae ATCC 33574; lane 6, P. oris ATCC 33573; lane 7, P. oralis ATCC 33269; lane 8, P. bivia ATCC 29303; lane 9, P. asaccharolytica ATCC 25260; lane 10, negative control. The arrow indicates the 267-bp amplicon. (B) Lane 1, 100-bp size marker; lane 2, P. nigrescens ATCC 33563; lane 3, P. intermedia ATCC 25611; lanes 4 to 10 correspond to lanes 4 to 10 in panel A. The arrow indicates the 301-bp amplicon.
PCR with two AP primer pairs amplified PCR products of approximately 900 bp for P. intermedia ATCC 25611 (Fig. 2A, lane 2) and 1,100 bp for P. nigrescens ATCC 33563 (Fig. 2B, lane 2); none of the other related species tested gave positive PCR results (Fig. 2, lanes 4 to 10). In the study with 174 clinical strains, PCR assays with two AP primer pairs gave the same results as PCR with two 16S primer sets.
FIG. 2.
PAGE of PCR products with AP primers. (A) Lane 1, 100-bp DNA ladder; lane 2, P. intermedia ATCC 25611; lane 3, P. nigrescens ATCC 33563; lane 4, P. loescheii JCM 8530; lane 5, P. buccae ATCC 33574; lane 6, P. oris ATCC 33573; lane 7, P. oralis ATCC 33269; lane 8, P. bivia ATCC 29303; lane 9, P. asachcarolytica ATCC 25260; lane 10, negative control. The arrow indicates the 900-bp DNA product. (B) Lane 1, 100-bp DNA ladder; lane 2, P. nigrescens ATCC 33563; lane 3, P. intermedia ATCC 25611; lanes 4 to 10 correspond to lanes 4 to 10 in panel A. The arrow indicates the 1,100-bp product.
Multiplex PCR for the identification of P. intermedia and P. nigrescens in the same reaction was performed successfully with AP primers but not 16S primers (Fig. 3). Multiplex PCR with 16S primers amplified products for P. intermedia but not P. nigrescens. When primer concentrations for P. nigrescens were increased threefold, multiplex PCR generated products for both P. intermedia and P. nigrescens, but a PCR product for P. nigrescens was apparently found in concentrations lower than that for P. intermedia.
FIG. 3.
PAGE of multiplex PCR products with AP primers. Arrows indicate the 900- and 1,100-bp amplicons. Lane 1, 100-bp DNA ladder; lane 2, P. intermedia ATCC 25611; lane 3, P. nigrescens ATCC 33563; lane 4, P. intermedia ATCC 25611 and P. nigrescens ATCC 33563; lane 5, negative control.
SDS-PAGE analysis showed a protein band of 60 kDa for P. intermedia ATCC 25611 and 61- and 58-kDa proteins for P. nigrescens ATCC 33563 (Fig. 4). On this basis, 50 clinical strains identified by the PCR assay established in this study were subjected to SDS-PAGE analysis. Of 30 strains of P. intermedia, SDS-PAGE analysis identified 26 strains as P. intermedia and 4 as P. nigrescens, while all 20 P. nigrescens strains tested were identified as P. nigrescens.
FIG. 4.
SDS-PAGE analysis of cell surface protein extracts. Lane 1, molecular mass standards (in kilodaltons); lane 2, P. intermedia ATCC 25611; lane 3, P. nigrescens ATCC 33563; lanes 4 and 8, clinical isolates of P. nigrescens; lanes 5 to 7, clinical isolates of P. intermedia. Arrows indicate 58-, 60-, and 61-kDa proteins, from bottom to top.
The lipase activity test showed that all strains, including the reference strains of P. intermedia and P. nigrescens, gave positive results after 24 h of incubation, except for one strain which was negative even after 10 days of incubation; it was the strain which gave negative results by PCR assays.
DISCUSSION
A simple, rapid, and reliable method for the differentiation of P. intermedia and P. nigrescens has been the focus of several studies in the last few years, and various methods have been utilized. Such a method would be of value to research and reference laboratories involved in studies of these organisms. In this context the PCR assay is a simple and rapid method which allows in vitro amplification of target DNA to a detectable level within a matter of hours. Although it is possible to identify P. intermedia and P. nigrescens by DNA-DNA hybridization, serological methods, enzyme electrophoresis of malate and glutamate dehydrogenases, and SDS-PAGE (2, 4, 5, 13), the time and technology involved in these methods prevent their common usage in a clinical setting.
In this study, there were no discrepancies found between the results of the PCR assays, and unambiguous differentiation was made by both, with the 16S rRNA derived primer set, Pi R1-Pi R2 and Pn R1-Pn R2, as well as with Pi 754-1–Pi 754-2 and Pn 1100-1–Pn 1100-2 primer pairs, except for two clinical isolates which could not be differentiated into either category by either PCR assay. Of these two isolates, one was positive with the primer pairs of P. intermedia and P. nigrescens in both PCR assays, whereas the other was negative. These discordant findings, which were also noted by Guillot and Mouton (6), could be attributed to the possible existence of another species in the genus Prevotella which is closely related to P. intermedia or P. nigrescens. Heterogeneity among P. intermedia strains was also suggested by Moncla et al., to explain the four distinct protein patterns seen in SDS-PAGE among the strains tested (10). These PCR findings further indicate that each organism should be tested with both primer pairs of P. intermedia and P. nigrescens to make a reliable identification.
The array of organisms tested as negative controls, which are related to the genus Prevotella or organisms that could be isolated from the oral cavity, did not give the specific bands given by both PCR assays. This indicates the highly specific nature of the primers tested and also confirms the specificity of the primer pair reported by Guillot and Mouton (6). Therefore, it is suggested that these PCR assays can be used on the black-pigmented organisms seen on a primary plate of oral specimens, even though the colonies are mixed with those of other organisms, for the identification of P. intermedia and P. nigrescens. This helps to avoid the tedious and time-consuming process of purification of black-pigmented colonies before employing any identification methods.
In addition it was found that when the primers for P. intermedia and P. nigrescens were mixed together for the PCR detection with the primer pair Pi 754-1–Pi 754-2 and Pn 1100-1–Pn 1100-2, it was possible to get the specific band of the organism tested and it was also possible to get both specific bands when both species are present in the sample. In the case of 16S rRNA-derived primers, it was found that when both organisms were found in the sample in the mixed-primer PCR, the detection was possible only for P. intermedia strains and P. nigrescens was not identified. This may be due to the fact that the primer pair of P. nigrescens is preferentially binding to P. intermedia strains in the presence of P. intermedia, and thereby no amplification product of P. nigrescens was found. Therefore with 16S rRNA primers, it is not possible to detect the presence of both organisms by PCR, but still it is possible to identify them if only P. intermedia or P. nigrescens is present in the sample. These findings ease the preparation procedure for PCR.
Although SDS-PAGE has been successful in the differentiation of P. intermedia and P. nigrescens in the hands of many researchers (2, 3, 4, 15, 16), in the present study we could not differentiate a few strains (3 of 50 strains) due to the poor resolution of protein bands, and the cells had to be treated with 10% trichloroacetic acid for better results. It could be due to the loss and denaturation of proteins by the proteolytic activity exhibited by these strains after treatment with SDS (17). We also had difficulties distinguishing the banding patterns, as the bands which differentiated P. intermedia from P. nigrescens were close to each other (60 kDa for P. intermedia and 58 and 61 kDa for P. nigrescens). Although this possibility was suggested by Susan et al., they did not encounter this problem in their study (15). On comparing the SDS-PAGE method with PCR assays, a discrepancy was found between the results in the differentiation of P. intermedia and P. nigrescens. On the basis of the results obtained by PCR assays, four strains of P. intermedia were identified as P. nigrescens by SDS-PAGE. The 58- and 61-kDa bands were clearly visible, and the 60-kDa band was not found with these strains. The strains that showed proteolysis belonged to the species P. intermedia. All the strains of P. nigrescens were identified correctly as P. nigrescens by SDS-PAGE. This observation tends to suggest that P. nigrescens strains receive a correct identification by the SDS-PAGE method, and the fact that the extraction of surface proteins is easier and/or proteolytic activity is not seen after treatment with SDS can also be deduced. In addition it can be said that since heterogeneity of banding patterns is seen among P. intermedia strains (9, 10), one cannot rely only on one or two different bands for differentiation from P. nigrescens. Therefore, the reliability of this method is questionable, as the reproducibility of the banding pattern was not seen among the results obtained by various researchers. One reason could be the difference in the methodologies used for cell surface extraction, but as heterogeneity was reported among the strains, the reproducibility of the banding patterns should be considered as a fact in the differentiation.
The lipase activity test could not differentiate the two species, as all strains tested were positive, including the reference strains of P. intermedia and P. nigrescens, but one clinical isolate was negative even after 10 days of follow-up. It is notable that this strain could not be identified by either of the PCR assays, and it might belong to another novel species within the genus Prevotella.
In conclusion, both PCR assays described here are simple, rapid, specific, and reliable methods which could be used in clinical studies to differentiate P. intermedia and P. nigrescens. This also confirms the conclusion of Guillot and Mouton (6). The lipase test cannot discriminate these two species. We question the usage of SDS-PAGE as a reliable method for differentiation of these species, since misidentification could not be ruled out.
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