Real-Time PCR Quantification of Methanobrevibacter oralis in Periodontitis

Abstract

A real-time PCR assay developed to quantify Methanobrevibacter oralis indicated that its inoculum significantly correlated with periodontitis severity (P = 0.003), despite a nonsignificant difference in prevalence between controls (3/10) and patients (12/22) (P = 0.2, Fisher test). The M. oralis load can be used as a biomarker for periodontitis.

TEXT

Periodontitis is an anaerobic infection possibly leading to loss of teeth (1–3). It likely results from infection with microbial complexes comprising methanogens (4, 5), mostly Methanobrevibacter oralis (6–8). In this study, we correlated the M. oralis load as measured by real-time PCR in subgingival plaque with the severity of periodontitis.

All patients diagnosed with periodontitis from October 2011 to June 2012 were included. The control individuals with generally healthy gingiva were volunteers. All patients underwent interviews for medical and dental history, intraoral examination to determine bleeding on probing (BOP), probing depth (PD), and plaque index (PI), and radiographs (9); periodontal status was also scored as described previously (10) (Table 1). All individuals signed an informed-consent form, and the ethics committee of the IFR 48, University of Aix-Marseille, approved the protocol.

Table 1.

M. oralis cell numbers and C_T values of two internal controls^a

Sample	CT value		No. of M. oralis cells	BOP	PD (mm)	PI (%)	Clinical score
	Stdint	All bacteria
P1	21.9	33.3		Localized	6	32	19
P2	22.6	23.1	3.125E+04	Generalized	8	63	28
P3	22.4	22.6	7.962E+05	Localized	8	34	30
P4	21.2	21.4	8.965E+05	Generalized	6	60	27
P5	24.0	27.9	6.325E+03	Generalized	5	65	27
P6	20.9	35.1		Localized	5	28	16
P7	22.8	37.8		Generalized	6	56	18
P8	23.4	31.5		Generalized	7	34	23
P9	24.5	21.6	6.125E+06	Generalized	6	38	27
P10	23.3	22.2	3.896E+05	Localized	6	54	21
P11	21.8	37.6		Generalized	9	40	29
P12	22.2	27.2	2.005E+04	Generalized	7	70	29
P13	24.3	28.5	2.654E+03	Generalized	6	55	26
P14	241	26.3	9.652E+02	Localized	6	59	27
P15	23.3	35.2		Generalized	7	59	24
P16	22.2	37.2		Generalized	6	62	28
P17	23.4	24.2	5.324E+03	Localized	5	29	27
P18	23.9	35.6		Localized	10	85	30
P19	22.2	34.3		Localized	7	75	26
P20	23.6	23.9	1.956E+05	Localized	7	12	28
P21	24.2	26.8	5.785E+03	Generalized	7	33	28
P22	21.9	32.7		Generalized	7	22	27
C23	24.0	35.4					4
C24	23.6	35.2	4.862E+02				8
C25	22.3	37.2					5
C26	23.5						7
C27	23.2	37.3					5
C28	23.5	35.2					7
C29	22.7	32.3	5.329E+02				4
C30	24.1	33.5	5.974E+02				6
C31	23.3						5
C32	24.2	36.2					4
T–
T–
T+	22.2	18.3	10E+09
T+	23.4	17.5	10E+09

^aThe total bacterial DNA was tested by real-time PCR, and a quantification plasmid (Stdint) was introduced as an internal control to monitor the absence of PCR inhibition. P, patient; C, controls; BOP, bleeding on probing; PD, probing depth; PI, plaque index. Clinical score was determined as previously reported (10).

Subgingival plaque specimens (50 μl) collected from 3- to 12-mm periodontal pockets in both patients and controls were suspended in 1 ml Tris-HCl (0.05 M, pH 7.5) buffer. After homogenization, a 250-μl aliquot was shaken with 0.3 g of acid-washed beads (≤106 mm; Sigma, Saint-Quentin-Fallavier, France) in a FastPrep-24 instrument (MP Biomedical Europe, Illkirch, France) at a speed of 6.5 m/s (full speed) for 90 s. The supernatant was incubated overnight at 56°C with 180 ml of lysis buffer and 25 μl proteinase K (20 mg/ml) in the Qiagen EZ1 DNA tissue kit (Qiagen, Courtaboeuf, France). After a second cycle of mechanical lysis, the supernatant was incubated for 10 min at 100°C, and total DNA was then extracted using the same kit. Negative controls consisting of sterile DNA-free water were introduced in all the manipulations. The specificity of the M. oralis-cnp602P probe (6-carboxyfluorescein [FAM]-5′AGCAGTGCACCTGCTGATATGGAAGG-3′) (Applied Biosystems, Courtaboeuf, France) and the primer pair M. oralis-cnp602F (5′-GCTGGTGTAATCGAACCTAAACG-3′) and M. oralis-cnp602R (5′-CACCCATACCCGGATCCATA-3′) (Eurogentec, Seraing, Belgium) was verified in silico using the BLAST program at NCBI (http://www.ncbi.nlm.nih.gov/BLAST) and further experimentally ensured by incorporating DNA extracted from Methanobrevibacter smithii ATCC 35061^T, Methanosphaera stadtmanae ATCC 43021^T, Methanomassiliicoccus luminyensis CSURP135^T, Escherichia coli, Salmonella enterica, Staphylococcus aureus, and Treponema denticola ATCC 35405^T. Real-time PCR assays were performed with a CFX96 Touch real-time PCR detection system (Bio-Rad, Marnes-la-Coquette, France) using the Mastermix PCR kit (Eurogentec) with 5 pmol of each primer and probe and 5 μl of about 2 μg of DNA into a 20-μl final volume. M. oralis DSMZ 7256^T DNA, used as a positive control, and one negative extraction control were included in each reaction plate. Results were expressed as the number of M. oralis cell/ml of specimen (Table 1). A quantification synthetic plasmid was used as an internal control to monitor PCR inhibition, and the total bacterial load was measured as previously described (11). The PCR program was 95°C for 5 min, followed by 40 cycles of 95°C for 1 s, 60°C for 35 s, and 45°C for 30 s. A calibration curve was done by measuring the cycle threshold (C_T) value of a serial dilution of M. oralis (10E+01 to 10E+09). As for clinical specimens tested in duplicate, the real-time PCR was regarded as positive for any C_T value of <40.

Ten male and twelve female periodontitis patients of ages between 38 and 86 years and ten age-matched controls were prospectively enrolled. The clinical score varied from 4 to 8 for controls and from 16 to 30 for patients (P < 10⁻³, Student t test). In all PCR-based experiments, negative controls remained negative, whereas the quantification plasmid and the M. oralis control yielded positive amplifications in 100% of specimens. Total bacterial DNA was positive in 8/10 (80%) controls and in 22/22 (100%) periodontitis patients (Table 1). M. oralis DNA was detected in 3/10 (30%) controls, with an average cell number of 5.39E+02 ± 5.58E+01, and in 12/22 (54%) patients, with an average cell number of 7.06E+05 ± 1.74E+06 (P = 0.2, Student t test). M. oralis detection was negative in 3/3 (100%) patients with a clinical score of <20 and positive in 12/19 (63%) patients with a clinical score of >20. The M. oralis cell number correlated significantly with the periodontal clinical score, with an r value of 0.527 (n = 30) and a P value of 0.003. The significance, analysis of variance (ANOVA) test, or the Kruskal-Wallis test (2-tailed) correlation was 0.003.

PCR systems previously used to detect methanogens in the subgingival plaque (6, 8, 12, 13) were not completely specific for M. oralis. Indeed, other methanogens, including Methanobrevibacter smithii (14), Methanosphaera stadtmanae (6), Methanobacterium curvum/Methanobacterium congolense, and Methanosarcina mazeii (15, 16), have been detected in this situation. Here, we developed a real-time PCR assay from the perspective of its routine use in clinical microbiology laboratories. In silico analyses indicated that the cnp60 gene system reported here was M. oralis specific. Accordingly, no amplification was obtained by incorporating seven other archaea and bacteria. Furthermore, amplification of the plasmid control demonstrated the lack of PCR inhibitors in all specimens, and this system was calibrated. Total bacterial control indicated that for a C_T value of >35.5, M. oralis detection was not interpretable. Based on these controls, the detection of M. oralis DNA in healthy individuals (C_T ≤ 35.5) was possible, in contrast to findings of previous studies (12, 13, 17, 18), and a significant correlation was observed between the M. oralis load and periodontitis severity.

Because of its sensitivity and specificity, the use of this real-time PCR system simplifies the molecular method-based detection of M. oralis in clinical specimens, rendering this task compatible with a routine diagnosis activity. Data herein reported indicate that it is possible to measure the load of M. oralis in the subgingival plaque by using real-time PCR. This study further established the proof of concept that the M. oralis load in the periodontal pockets correlates to a standardized severity score of periodontitis. The increased amount of M. oralis in healthy and diseased patients may indicate its use as a biomarker of altered microbiota. Monitoring the M. oralis load by using real-time PCR could be useful for the diagnosis and staging of patients with periodontitis and treatment follow-up. It could complement clinical evaluation in detecting individuals at higher risk of developing severe periodontitis and be useful in evaluating the efficacy of antibiotic treatment, since M. oralis is highly resistant to antibiotics, except for metronidazole (19).