Simultaneous detection and evaluation of Prevotella intermedia, Prevotella nigrescens, Prevotella loescheii, and Prevotella melaninogenica in subgingival plaque samples of chronic periodontitis and healthy individuals through multiplex polymerase chain reaction

Abstract

Background:

Prevotella is a Gram-negative anaerobic bacilli. The phenotypic characteristics of the various species of Prevotella are similar, which often makes it difficult in routine differentiation and identification of all the species.

Aim:

The purpose of the study was to detect and compare presence of Prevotella intermedia, Prevotella nigrescens, Prevotella melaninogenica, and Prevotella loescheii in subgingival plaque samples of chronic periodontitis and healthy individuals.

Materials and Methods:

Two hundred and thirty-six subjects were considered consisting of chronic periodontitis (128) and healthy (108) individuals. Subgingival plaque sample was collected in reduced transport fluid and analyzed. DNA extraction and polymerase chain reaction (PCR) were performed for genus Prevotella followed by positive samples were considered for the detection of selected species through multiplex PCR using specific primers.

Results:

Out of 236 samples, 94.1% were positive for genus Prevotella. Out of 222 cases P. nigrescens showed the highest number of cases positive (59.5%) followed by P. melaninogenica (57.2%), P. intermedia (55.4%), and P. loescheii (40.1%). Species were analyzed individually between chronic periodontitis and healthy, P. intermedia, P. nigrescens, and P. loescheii showed greater positivity in healthy compared to chronic periodontitis. Positivity for P. melaninogenica was high in chronic periodontitis compared to healthy.

Conclusion:

The number of positive cases for species, when correlated with clinical parameters showed an increase in mean score for all clinical parameters assessed, suggesting the presence of variation in the prevalence of Prevotella species and geographic variation do exist in oral microflora. Findings suggest that they can be normal commensals and opportunistic.

INTRODUCTION

Bacteroides melaninogenicus originally classified as a black-pigmented Gram-negative anaerobic rod which was categorized into three different genera as Bacteroides, Prevotella, and Porphyromonas. Bacteroides are present only in animals, whereas Prevotella and Porphyromonas is indigenous flora of the oral cavity of humans.[1] Prevotella species form a major portion of the microflora of the oral cavity compared to Porphyromonas.[2] Prevotella belong to the periodontal pathogen. Similarity in phenotypic characteristics of these species often makes it onerous in routine differentiation and identification.[3]

Fifty species were discriminated through phylogenetic analysis. Many Prevotella species are contributing pathogens in polymicrobial oral infections, such as periodontal disease, deep dental caries, and mixed infection abscesses.[4] Identification of these organisms by culture produces both pigmented and nonpigmented colonies. Biochemical analysis for the categorization of species is difficult because of the overlapping of phenotypic characteristics, such as Prevotella melaninogenica, Prevotella loescheii, and P. denticola show phenotypic overlapping. Similarly, Prevotella intermedia and Prevotella nigrescens overlap, making identification difficult.[2,3] Studies focused on the characterization of these pigmented organisms individually through different methods of isolation techniques such as DNA fingerprinting, Random Amplified Polymorphic DNA polymerase chain reaction (PCR),[5] and DNA-DNA hybridization.[6] Among these one method which is reliable, less time-consuming, economical and helps in identifying more than one organism at a time is through multiplex PCR, and identification of multiple organisms is done in a single step. Yoshida et al., first studied the utility of multiplex PCR in identifying pigmented Prevotella species and they selected P. intermedia, P. nigrescens, P. melaninogenica, and P. loescheii. They detected these organisms through multiplex PCR and confirmed the results by running PCR for each species and matched the results. Concluding that multiplex PCR assay is highly sensitive and specific diagnostic tool for the identification of oral black-pigmented Prevotella species and suggested that the role of these black-pigmented Prevotella species in periodontitis is still unclear and needs further evaluation.[2] Thus the present study was designed to evaluate and compare the presence of P. intermedia, P. nigrescens, P. melaninogenica, and P. loescheii in the subgingival plaque samples of periodontally healthy individuals and chronic periodontitis individuals. Furthermore, revalidate the efficacy of multiplex PCR in detecting P. intermedia, P. nigrescens, P. melaninogenica, and P. loescheii.

MATERIALS AND METHODS

Two hundred and thirty-six subjects were included in the study, 128 subjects were chronic periodontitis and 108 were periodontally healthy. Ethical clearance was obtained from the Institutional Ethical Review Board. Patients were selected from the outpatient department of the institute between the age group of 20 and 70 years. Informed consent was obtained from the subjects, who fulfilled the inclusion and exclusion criteria and enrolled in the study. The inclusion criteria for healthy controls were probing depth ≤3 mm, no clinical signs of inflammation, no clinical attachment loss, and no tooth mobility. For chronic periodontitis subjects, inclusion criteria were clinical attachment loss ≥3 mm, bleeding on probing, and probing depth ≥5 mm in at least four or more teeth. Exclusion criteria for both groups were patients with any systemic diseases/medically compromised patients, patients who have received periodontal therapy, antibiotics/antimicrobials within 3 months before sampling, pregnant and lactating women, subjects using smoke and smokeless tobacco. After recording the required periodontal indices,[7] subgingival plaque samples were collected after stripping off the supragingival plaque using sterile Gracey Curettes from at least four teeth in healthy controls and four deepest pockets in the chronic periodontitis patients. Samples were transported to the laboratory in the reduced transport fluid. DNA extraction was done using the modified proteinase K method from the plaque sample.

DNA extraction was done by centrifuging samples at 5000 rpm for 3–5 min at room temperature, and supernatant was discarded leaving the sediment in the pellet. Six µl of extraction solution and 20 µl of proteinase K were added to the sediment and vortexed. The pellets were incubated in the water bath for 2 h at 60°C. 4 µl of ribonucleic acid-free DNA was added and incubated at 37°C for 5–10 min. Two hundred µl of protein precipitation solution was added and vortexed, followed by centrifugation at 10,000 rpm for 3–5 min at room temperature. The supernatant was transferred to a fresh vial without disturbing the precipitate. 600 µl of isopropanol was added allowing for precipitation of DNA and centrifuged for 1 min at high speed at room temperature. The supernatant was carefully discarded without disturbing the pellet. Pellet was washed with 1 ml of 70% ethanol and centrifuged again for 1 min at high speed. The supernatant was removed and discarded, leaving the pellet to dry for 5–10 min at room temperature. One hundred µl of distilled water was added and stored at − 20°C until further use.

Following DNA extraction, PCR was performed for the detection of Prevotella genus with a conserved region of 16S rDNA as the target. The primers used to target Prevotella conserved region are: Forward primer-5’- CCAGCCAAGTAGCGTTGCA-3’ and Reverse primer-5’TGGACCTTCCGTATTACCGC-3’.[4]

Samples which were positive for Prevotella species were subjected to multiplex PCR to detect P. intermedia, P. nigrescens, P. melaninogenica, and P. loescheii. Primers used to target conserved region for the selected organism is given in Table 1.

Table 1.

Oligonucleotide primers for black pigmented Prevotella species[2]

Oligonucleotide primers	Sequence	Amplicon size (bp)	Gene
Pi phoC 51F Pi phoC 768R	5’- GAC TTT TGC ACA GAA TGC A-3’ 5’- CTT GGC AAC CTT GCC TTC-3’	718	phoC
Pn 16S 188F Pn 16S 1012R	5’- TTT CAT TGA CGG CAT CCG-3’ 5’- CAC GTC TCT GTG GGC AG-3’	825	16S rRNA
Pm phyA 132F Pm phyA 520R	5’- CGT CAT GAA GGA GAT TGG-3’ 5’- ATA GAA CCG TCA ACG CTC-3’	389	phyA 1
Pl plaA 186F Pl plaA 822R	5’- TGC CAA CTC CCG ATT TC-3’ 5’- TAC ACC AAG GTT TTC CCC-3’	637	plaA

PCR reaction was carried out in total 25 µl final volume in a veriti 96-well thermal cycler (Applied Biosystems, USA). Taq DNA polymerase master mix (Ampliqon, Odense, Denmark) was used for the preparation of reaction mixture which contained Tris-HCL pH 8.5, (NH₄) ₂SO₄, 3 mM MgCl₂, 0.2% Tween 20, 0.4 mM of each dNTP, 0.2 units/µl Ampliqon Taq DNA Polymerase, Inert red dye and stabilizer. The reaction mixture was prepared as per the manufacturer’s instructions with optimized concentrations of primers (2.5 pmole/µl). 25 µl reaction mixture consisted of 12.5 µl of Taq DNA polymerase master mix, 2.5 pmole/µl of each primer, and 3 µl (Approx. 100 ng) of DNA template. Thermal cycling condition was used for carrying out the reaction at 95°C for 5 min, followed by 94°C for 30 s, 54°C for 90 s, and 72°C for 90 s.

Amplified products were separated on 2% agarose by electrophoresis. 100 bp DNA ladder (Genetix Biotech Asia Pvt Ltd., Mumbai, India) was added simultaneously with the samples. After electrophoresis, agarose gel was stained with 0.5 µg/ml of ethidium bromide and observed under UV transilluminator.[2] Sizes of bands were determined by comparing with known base pairs of DNA ladder. Gel images were captured using a Gel documentation system (Major Sciences, Saratoga, USA).

Statistical analysis

All the analysis was carried out using SPSS software 14.0 version,SPSS Inc.Chicago,Illinois(USA). P ≤ 0.05 is considered statistically significant

RESULTS

Two hundred and thirty-six subjects were considered in the study which included 133 (56.4%) females and 103 (43.6%) males. The chronic periodontitis group had 58.6% (75 cases) females, whereas 41.4% (53 cases) were male. In healthy groups, females were 53.7% (58 cases) and males were 46.3% (50 cases). In both groups, females were more in number than males. For all 236 samples, PCR was performed to determine the genus Prevotella. In the chronic periodontitis group, 95.3% (122 cases) of positivity was observed for Prevotella genus and 4.7% (6 cases) was negative, whereas in healthy group, 92.6% (100 cases) of cases showed positivity for the genus Prevotella and only 7.4% (8 cases) of cases were negative. Overall positivity for genus was 94.1% [Table 2].

Table 2.

Cases positive for genus Prevotella in chronic periodontitis and healthy individuals

Category	PCR positivity for genus Prevotella	Number of cases (%)
CP	Negative	6 (4.7)
	Positive	122 (95.3)
	Total	128 (100.0)
Healthy	Negative	8 (7.4)
	Positive	100 (92.6)
	Total	108 (100.0)
Overall	Negative	14 (5.9)
	Positive	222 (94.1)
	Total	236 (100.0)

CP – Chronic periodontitis; PCR – Polymerase chain reaction

Positive for genus Prevotella were considered for multiplex PCR for determining the select species. Out of 222 positive cases for genus Prevotella multiplex PCR was negative for P. intermedia, P. nigrescens, P. melaninogenica, and P. loescheii in 39 individuals (17.5%). In chronic periodontitis out of 122 positive cases, 22 (22.1%) of them were negative for selected species and in healthy it was 12 cases (12%) negative for selected species. Out of 222 cases, P. nigrescens showed the highest number of cases positive with 59.5% (132 cases) followed by P. melaninogenica 57.2% (127 cases), P. intermedia 55.4% (123 cases), and P. loescheii 40.1% (89 cases) [Table 3 and Figure 1]. Species were analyzed individually between groups, Pintermedia, P nigrescens, and P. loescheii showed greater positivity in healthy individuals compared to chronic periodontitis. In P. intermedia, the difference was minimal and the difference was statistically insignificant, whereas in P. nigrescens and P. loescheii, the difference was statistically significant. Positivity for P. melaninogenica was marginally high in the chronic periodontitis group compared to healthy and the difference was statistically insignificant [Table 3].

Table 3.

Number cases positive for species in chronic periodontitis and healthy individuals

Microorganism	Category	Multiplex PCR positivity	Number of cases (%)	Fisher’s exact test
Prevotella intermedia	CP	Negative	55 (45.1)	0.893 (NS)
		Positive	67 (54.9)
		Total	122 (100.0)
	Healthy	Negative	44 (44.0)
		Positive	56 (56.0)
		Total	100 (100.0)
	Overall	Negative	99 (44.6)
		Positive	123 (55.4)
		Total	222 (100.0)
Prevotella nigrescens	CP	Negative	57 (46.7)	0.041 (S)
		Positive	65 (53.3)
		Total	122 (100.0)
	Healthy	Negative	33 (33.0)
		Positive	67 (67.0)
		Total	100 (100.0)
	Overall	Negative	90 (40.5)
		Positive	132 (59.5)
		Total	222 (100.0)
Prevotella melaninogenica	CP	Negative	51 (41.8)	0.786 (NS)
		Positive	71 (58.2)
		Total	122 (100)
	Healthy	Negative	44 (44.0)
		Positive	56 (56.0)
		Total	100 (100.0)
	Overall	Negative	95 (42.8)
		Positive	127 (57.2)
		Total	222 (100.0)
Prevotella loescheii	CP	Negative	85 (69.7)	0.001 (S)
		Positive	37 (30.3)
		Total	122 (100.0)
	Healthy	Negative	48 (48.0)
		Positive	52 (52.0)
		Total	100 (100.0)
	Overall	Negative	133 (59.9)
		Positive	89 (40.1)
		Total	222 (100.0)

P<0.01. CP – Chronic periodontitis; PCR – Polymerase chain reaction; NS – Nonsignificant; S – Significant; P <0.01

Figure 1.

Photograph Showing Positive bands for P. intermedia, P. nigrescens, P. melaninogenica and P. loeschii for sample 1, 2, 3, 4, 7, 9. Sample 5, 10 is positive for P. nigrescens and P. melaninogenica, Sample 8 for P. intermedia, P. nigrescens, and P. loeschii, Sample 6 is positive for only P. intermedia. Sample 11 is negative for all the species. P. intermedia – Prevotella intermedia, P. nigrescens – Prevotella nigrescens, P. melaninogenica – Prevotella melaninogenica, P. loeschii – Prevotella loeschii

Microorganisms were analyzed in groups and then compared with clinical parameters in chronic periodontitis patients to check the association. When all four species were positive, the mean plaque index, gingival index, and clinical attachment loss were high, when compared to group of three, two or one species. Suggesting, more disease progression when all four species are involved than individual. Increase in the mean score of plaque index, gingival index, and clinical attachment loss with the number of cases positive for all four species. However, the difference was statistically insignificant [Table 4]. Analysis was done for each species and correlated with clinical parameters. The mean score increased for all the parameters assessed with the positivity of the species and it was less in negative cases. However, the difference was statistically insignificant for most of the parameter assessed except for clinical attachment loss in P. intermedia and P. nigrescens and plaque index in P. loescheii [Table 5].

Table 4.

Comparison of clinical parameters with species individually and in group in chronic periodontitis individuals

Clinical parameters	Species in group	Number of cases	Mean	SD	95% CI for mean		ANOVA
					Lower bound	Upper bound	F ratio	P	Significance
Plaque index	Negative for any species studied	27	3.00	1.10	2.57	3.44	1.559	0.190	NS
	One	17	3.12	1.01	2.60	3.64
	Two	33	2.97	0.99	2.62	3.32
	Three	23	3.38	1.81	2.60	4.17
	All four species	22	3.71	1.09	3.23	4.20
	Total	122	3.21	1.24	2.99	3.43
Gingival index	Negative for any species studied	27	2.93	1.10	2.49	3.37	1.652	0.166	NS
	One	17	2.79	1.17	2.19	3.39
	Two	33	3.00	1.38	2.51	3.49
	Three	23	3.10	1.17	2.60	3.61
	All four species	22	3.67	1.16	3.15	4.18
	Total	122	3.10	1.23	2.87	3.32
Clinical attachment loss	Negative for any species studied	27	4.68	2.80	3.57	5.79	2.458	0.050	NS
	One	17	4.69	2.08	3.62	5.76
	Two	33	5.51	2.31	4.69	6.33
	Three	23	5.08	2.47	4.00	6.15
	All four species	22	6.66	2.52	5.54	7.79
	Total	122	5.34	2.53	4.88	5.79

P<0.01. NS – Nonsignificant; CI – Confidence interval; SD – Standard deviation; P <0.01

Table 5.

Comparison of clinical parameters with each species of Prevotella in chronic periodontitis

Group	Clinical parametes	Multiplex PCR	Number of cases	Mean	SD	t	df	P	Significance
Prevotella intermedia	Plaque index	Negative	55	2.99	1.04	–1.760	120	0.081	NS
		Positive	67	3.39	1.36
	Gingival index	Negative	55	2.89	1.10	–1.649	120	0.102	NS
		Positive	67	3.26	1.31
	Clinical attachment loss	Negative	55	4.59	2.49	–3.071	120	0.003	S
		Positive	67	5.95	2.40
Prevotella nigrescens	Plaque index	Negative	57	3.14	1.44	–0.560	120	0.577	NS
		Positive	65	3.27	1.04
	Gingival index	Negative	57	2.93	1.19	–1.418	120	0.159	NS
		Positive	65	3.24	1.25
	Clinical attachment loss	Negative	57	4.82	2.48	–2.163	120	0.033	S
		Positive	65	5.80	2.49
Prevotella melaninogenica	Plaque index	Negative	51	3.04	1.02	–1.252	120	0.213	NS
		Positive	71	3.33	1.37
	Gingival index	Negative	51	2.96	1.18	–0.997	120	0.321	NS
		Positive	71	3.19	1.26
	Clinical attachment loss	Negative	51	5.15	2.66	–0.676	120	0.500	NS
		Positive	71	5.47	2.44
Prevotella loescheii	Plaque index	Negative	85	3.04	1.01	–2.367	120	0.020	S
		Positive	37	3.61	1.60
	Gingival index	Negative	85	2.95	1.25	–1.938	120	0.055	S
		Positive	37	3.42	1.13
	Clinical attachment loss	Negative	85	5.12	2.40	–1.445	120	0.151	NS
		Positive	37	5.84	2.76

P<0.01. NS – Nonsignificant, S – Significant, df – Degrees of freedom, PCR – Polymerase chain reaction; SD – Standard deviation; P <0.01

DISCUSSION

Black-pigmented Prevotella being the dominant bacteria in the gingival sulcus has been implicated in the progress of periodontal disease and some are associated with healthy plaque like P. nigrescens.[2] Initially, periodontal disease involves an imbalance between the growth of periodonto-pathogenic bacteria and host defense mechanism. Immediate environment factors, pocket depth, pH, and oxygen concentration also influence the growth and virulence of periodontal pathogens are responsible for periodontitis.[8-12] Periodontitis being a polymicrobial disease, anaerobic microorganisms play a role in the progression of disease, mainly black-pigmented Gram-negative anaerobic bacilli such as Porphyromonas gingivalis and Prevotella species. Prevotella species has been implicated in early and advanced periodontitis.[13,14] Black-pigmented Prevotella, selected in this study such as P. intermedia, P. nigrescens, P. melaninogenica, and P. loescheii are associated strongly with oral infection and also isolated as normal oral commensals. However, their role in the progression of periodontitis remains controversial. Several studies in the literature have been carried out to identify these pigmented Prevotella species to determine the site specificity and also to find the geographic variation or to find the suitable and reliable technique to isolate and identify these species. Only few studies establish their role in periodontitis and apart from this these species have been studied either individually or in combination of two or three species and or in association with other species such as P. gingivalis, Tannerella forsythus, and Treponema denticola.[1-6,8] We found only one study in the literature where isolation and identification of P. intermedia, P. nigrescens, P. melaninogenica, and P. loescheii has been carried out mainly to determine the reliability of multiplex PCR in the identification of these species.[2] This is the first study where identification and comparison of these species are made between healthy and chronic periodontitis and also determined the association of these species with clinical parameters in periodontitis individual.

In our study, 94.1% (222 cases) cases showed positivity for genus Prevotella. High positivity can be correlated with the finding of Okuda et al. who found P. intermedia in all the samples analyzed (with or without periodontal disease) and found 100% positivity for the species.[15]

Among the four species analyzed P. intermedia and P. nigrescens belong to the member of “orange complex”[13,16] and are known for causing odontogenic infections and also associated with early periodontitis along with P. gingivalis, T. forsythus, and T. denticola.[8,17] In the present study, P. intermedia, P. loescheii, and P. nigrescens showed higher positivity in healthy individuals compared to chronic periodontitis. Literature evidence shows that P. intermedia are more likely associated with periodontal sites and P. nigrescens are more frequently associated with healthy sites. Controversy exists between the two species regarding site specificities.[2,18-21] This suggests that these species are either opportunistic pathogens or they are heterogeneous species with respect to virulence.[2,22,23] Yoshida et al. through multiplex analysis observed P. nigrescens and P. melaninogenica in higher number and in subgingival plaque samples, compared to other sites of the oral cavity. P. loescheii was seen in two tongue samples and was negative in subgingival plaque. They found increased positivity for P. melanonogenica, followed by P. nigrescens, P. intermedia, and P. loescheii.[2] We found the highest positivity for P. negrescens followed by P. melaninogenica and P. intermedia. Our study showed less percentage of positivity for P. loescheii when compared to other species.

Haffajee et al. studied subgingival plaque samples from periodontally healthy and minimally diseased subjects by checker board hybridisation method for 40 different bacterial species. They performed cluster analysis to group subjects based on their subgingival microbial diversity and they created 8 microbial profiles. One cluster P. nigrescens and P. melaninogenica was found to be higher in numbers and in another cluster P. intermedia was dominant members. They established presence of Prevotella species in periodontally healthy controls and also with minimal disease.[24] We found P. melaninogenica marginally high in chronic periodontitis individuals and P. intermedia, P. nigrescens and P. loescheii was high in healthy group, in our study.

Haffajee et al. demonstrated the role of some Prevotella species in periodontal infections from four different countries and found variation in the isolation of these oral microorganism. P. intermedia were predominantly found in Brazil and the USA population whereas P. melaninogenica was found in larger proportion in Chile. In Sweden, Prevotella species was a minor component. The study clearly established variation in microbiota according to different geographic location.[6]

Socransky et al. showed P. intermedia and P. nigrescens detected early in the disease process of periodontitis proceeding and co-existing with later colonizing periopathogens.[25] Nadkarni et al.[4] found 89% positivity for P. melaninogenica, 72% for P. intermedia, 67% for P. nigrescens, 47% for P. loescheii from healthy sites, whereas from diseased site 100% positivity for P. melaninogenica, 93% for P. nigrescens, 91% for P. intermedia, and 54% for P. loescheii. They also found that P. melaninogenica consistently colonized in subginigival pockets in Aborigines. P. intermedia and P. nigrescens were commonly found and were strong colonizers of the aboriginal community. P. melaninogenica and P. intermedia were seen significantly associated with healthy gingival site, as well as increasing probing depth.[4] In our study, we found P. melaninogenica in greater number in chronic periodontitis followed by P. intermedia, P. nigrescens, and P. loescheii and in healthy group we found P. nigrescens in greater percentage followed by P. melaninogenica, P. intermedia, and P. loescheii. Similar to the findings of Nadkarni et al., we observed P. melaninogenica higher in chronic periodontitis.

We found a good correlation between clinical parameters and positivity of species. The mean score increased for all the parameters assessed with the number of cases positive for the species and was less in negative cases, especially clinical loss of attachment which determines the progression of disease. We also found a positive correlation when a combination of species was assessed for clinical parameters. This finding suggests that these species are inter related and have a role in disease progression as a whole.

CONCLUSION

Comparing the findings of our study to other studies in literature we found variation in the prevalence of selected Prevotella species, suggesting geographic variation do exist in the prevalence of oral microflora. This is the first study done on the Indian population to isolate P. intermedia, P. nigrescens, P. melaninogenica, and P. loescheii. We also found that multiplex PCR was a sensitive, specific, and reliable method in detecting the microorganism and a credible diagnostic tool for simultaneous detection of P. intermedia, P. nigrescens, P. melaninogenica, and P. loescheii. We found association of these microorganisms in periodontal disease and found good positivity in healthy individuals, suggesting that these can be normal commensals and are also opportunistic in nature when the oral environment becomes conducive resulting in organism become pathogenic in nature. Further studies are to be carried out in the Indian population to elucidate the role of Prevotella species along with other commensals to understand the pathophysiology of disease progression and their symbiotic relationship in the oral cavity in health and disease.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.