Real-Time PCR Quantification of Red Complex Bacterial DNA and Periodontal Status in Removable Partial Denture Wearers

Summary

Real-time polymerase chain reaction (PCR) was used to quantify red complex bacteria DNA in removable partial denture (RPD) wearers. This study promises a novel approach to future implant prosthetic treatment strategies for these patients, considering the role of these bacteria in the development of peri-implantitis.

Abstract

The insertion of removable partial dentures (RPDs) in the oral cavity is associated with changes in the oral microflora over time. However, there is a paucity of literature examining the presence of red complex bacteria (RCB) in these patients. Given RCB's established role in the development of periodontitis, peri-implantitis, and various systemic diseases, investigating the impact of RPDs on these bacteria is crucial. This study aims to quantify the subgingival burden of RCB-Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola-in abutment teeth of partially edentulous patients rehabilitated with RPDs, using real-time polymerase chain reaction (RT-PCR). A secondary objective was to assess the periodontal status of abutment and non-abutment teeth and to determine the correlations between microbial load and clinical periodontal indices.

Thirty participants requiring RPDs were enrolled. Deoxyribonucleic acid (DNA) was isolated from bacterial samples obtained from abutment teeth before (T0) and 3 months post-insertion (T3). RCB quantification was performed via RT-PCR and expressed as "Lg (genome equivalents/sample)". Clinical parameters-plaque index (PI), gingival index (GI), probing depth (PD), and tooth mobility (TM)-were recorded for abutment and non-abutment teeth at both timepoints.

Abutment teeth demonstrated statistically significant increases in RCB loads across all three target species from T0 to T3: P. gingivalis (1.99 ± 2.01 vs 3.64 ± 2.21, p = 0.00003), T. forsythia (2.20 ± 2.17 vs 3.56 ± 2.33, p = 0.00009), and T. denticola (0.82 ± 1.41 vs 2.35 ± 2.21, p = 0.0001). While periodontal parameters were elevated in abutment versus non-abutment teeth post-treatment, differences did not reach statistical significance. Among pathogens, T. forsythia exhibited the strongest positive correlation with periodontal indices.

Short-term use of RPDs is associated with a significant increase in RCB colonization of abutment teeth, suggesting early microbial and inflammatory shifts that may precede clinically evident periodontal breakdown.

Introduction

The oral cavity harbors a dynamic microbiome whose ecological balance is critical to maintaining periodontal health¹. Disruption of this microbial homeostasis, due to endogenous and exogenous factors, can disrupt the microbial homeostasis, creating favorable conditions for the growth of pathogenic bacteria, including periodontal pathogens such as Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola, collectively termed the red complex bacteria (RCB)². These pathogens are pivotal in the pathogenesis of periodontitis and peri-implantitis, working synergistically to evade host defenses, promote inflammation, and facilitate the destruction of periodontal tissues, frequently leading to tooth loss^3,4. Tooth loss adversely affects an individual's ability to eat and speak, and it contributes to a diminished sense of confidence and well-being, thereby increasing the need for prosthetic compensation. Among the most common methods employed, particularly in the elderly population due to various local and general conditions, is prosthetic treatment with removable partial dentures (RPDs). However, over time, RPDs⁵ may lead to a variety of problems.

Robust evidence suggests that the use of RPDs can result in dental plaque accumulation and the development of inflammation of the gingiva and periodontal tissues, particularly involving abutment teeth^6,7,8,9,10. However, the occurrence of such oral disorders also depends on the patient's commitment to oral hygiene as well as the care and maintenance of the denture^11,12,13,14. Bassi et al. emphasized in their research the importance of controlling dental plaque. They examined patients who had worn RPDs for a period of 6-12 years and found that the periodontal condition of abutment teeth was similar to that of non-abutment teeth in cases with regular oral hygiene. In contrast, the periodontal condition worsened in the group of patients who did not attach importance to the follow-up and maintenance of oral hygiene¹³.

Despite the functional advantages of RPDs, their insertion into the oral cavity hinders the free flow of saliva, predisposing the formation of a pellicle layer on the surface of the prosthesis that may lead to bacterial colonization. Different types of harmful bacteria have been identified in PRPD patients¹⁵. Recent studies have shown that the levels of pathogens causing infections associated with dental caries, such as Streptococcus mutans¹⁶, as well as those of Staphylococcus aureus¹⁷, may increase significantly after the placement of dentures. In addition to functioning as an indicator of the status of the oral mucosa, changes in the level of bacteria resulting from the use of RPDs can also lead to the development of dental disease in the remaining teeth¹⁸. Thus, if patients neglect to maintain their RPDs properly, anaerobic conditions can be created under the denture base, which may result in the excessive growth of pathogenic bacteria. Certain types of periodontal pathogens can enter the general blood circulation through the oropharyngeal portal, representing a considerable risk factor for the development of various systemic diseases^19,20.

However, there is still a paucity of studies on microbiological changes in the gingival sulcus of abutment teeth in patients with RPDs^21,22,23,24. Among the few studies on this subject, conventional methods for the detection of periodontal pathogens, including the BANA-ZymeTM test and the culture method, have been used. Considering that most periodontal pathogens are facultative anaerobes, the use of such methods may result in difficulties in their cultivation, since in this context conditions of high reducing intensity and for which oxygen is toxic are required²⁵. Moreover, the available dental literature has not yet satisfactorily addressed the relationship between such pathogens and the different periodontal indices within this perspective.

Therefore, to overcome the limitations of conventional microbial culture and to improve diagnostic accuracy, this study employed real-time polymerase chain reaction (RT-PCR) to quantify red complex bacterial (RCB) loads in abutment teeth of RPD wearers. In parallel, the periodontal status of both abutment and non-abutment teeth was assessed using standardized clinical indices. The study further investigated correlations between bacterial burden and periodontal parameters to better elucidate the biological interplay between prosthesis use and microbial dysbiosis.

Protocol

This study included 30 partially edentulous patients who sought prosthodontic care at the Clinic of Dental Prosthetics, University Dentistry Clinical Center of Kosovo, between September 2021 and March 2022. Ethical approval was obtained from the institutional review board (Approval No. 378/19), and all procedures adhered to the principles of the Declaration of Helsinki. Written informed consent was secured from all participants.

The sample size was determined based on prior research, which indicates that 11 to 30 participants are sufficient to obtain significant results in microbial and periodontal parameters among RPD wearers^21,23. Assuming a two-tailed paired t-test with an alpha level of 0.05, the power analysis indicated that a sample size of 28 participants would provide 80% power to detect a statistically significant difference of this magnitude. To account for potential dropouts, we enrolled 30 participants. Furthermore, a follow-up period of three months was adequate to observe an increase in bacterial load in these patients²⁴.

Eligible patients were first-time users of prosthetic appliances, presenting with both adjacent and antagonistic natural dentition. Exclusion criteria included: probing depths >4mm, immunocompromised status (e.g., chemotherapy), antibiotic use within the past 90 days, heavy smoking (>25 cigarettes/day), and cognitive impairment impeding comprehension of RPD procedures.

All participants received clasp-retained RPDs fabricated with a cobalt-chromium metal framework and acrylic resin (polymethyl methacrylate). The design of each RPD was customized based on the classification of edentulism, quality of residual ridge, and biomechanical considerations.

Sample collection procedure

Sample collection was exclusively performed on the abutment teeth of removable partial dentures (RPDs) between time points T0 and T3. Participants were first asked to gargle thoroughly with water to eliminate any remaining food debris. Before sample collection, the selected abutment tooth was isolated using cotton rolls to maintain a dry working environment. Two sterile paper points (no. 35; 04 tapered) were inserted into the gingival sulcus on the mesial and distal aspects of the buccal surface of the tooth and left in place for 1 min. The paper points were then transferred to a sterile microtube containing 1.5 mL of normal saline solution and transported to a designated microbiological laboratory. The samples were stored at -20°C until the DNA isolation process.

Bacterial DNA preparation and amplification:

The Parodontoscreen test ( Table of Materials) was employed to identify T. denticola, T. forsythia, and P. gingivalis using RT-PCR²⁶. The test consists of: DNA preparation; real-time PCR amplification using specific reagents (a mixture for universal bacterial amplification, a mixture for opportunistic bacterial amplification, and a mixture for human genomic DNA amplification); and the recording and interpretation of amplification results.

DNA extraction was carried out using a DNA extraction kit (Table of Materials), following the manufacturer's guidelines to ensure optimal DNA quality. The test utilizes real-time PCR qualitative analysis with a paraffin-sealed PCR-mix. This mix includes an Internal Control to verify the validity of the PCR run and a Sample Intake Control (SIC) to assess extraction quality and confirm sufficient DNA for accurate amplification.

PCR amplification was performed using specialized strips and caps, with reagents including Taq-polymerase solution, master mix under paraffin layer, DNA sample, and mineral oil. The PCR program is the following: (i) initial denaturation at 80 °C for 30 s, (ii) denaturation at 94 °C for 1 min 30 s, (iii) 5 cycles of 30 s at 94 °C (denaturation), 15 s at 64 °C (primer binding and elongation, detection of fluorescence), (iv) 45 cycles of 10 s at 94 °C (denaturation), 15 s at 64°C (primer binding and elongation, detection of fluorescence), (v) 5 s at 94 °C (final denaturation).

Fluorescence detection was performed on the Fam and Hex detection channels, except for tube №5, which contained the marker (where fluorescence was detected on the Fam and Rox channels). The amplification process was conducted using real-time thermal cyclers (Table of Materials). The Software RealTime for the instrument (version 7.9) was used for the automatic registration, interpretation, and quantitative analysis of the PCR amplification data, enabling precise pathogen load estimation.

Microbial loads were represented as "Lg (genome equivalents/sample)". The results were presented in three ranges on the Parodontoscreen test report for each patient, based on bacterial load levels as follows: P. gingivalis (normal <5.0; moderate ≥5.0; severe >6.0); T. forsythia (normal <5.0; moderate ≥5.0; severe >5.5); and T. denticola (normal <3.5; moderate ≥3.5; severe >5.0).

Clinical examination

The clinical examination included a detailed examination of the periodontal condition of abutment and non-abutment teeth at T0 and T3. The plaque index (PI) was evaluated using a dental probe according to the Silness-Loe criteria (0-3)²⁷. The gingival condition was evaluated at six points on the tooth surface using the gingival index (GI) according to the following criteria: 0, no inflammation; 1, mild inflammation; 2, inflammation with gingival bleeding during probing; 3, severe inflammation with possible spontaneous gingival bleeding²⁸.

Probing depth (PD) was measured using the Williams probe from the gingival margin to the end of the clinical pocket depth²⁹. Tooth mobility (TM) was determined by bimanual palpation in the horizontal and vertical directions, according to Miller's criteria³⁰.

All study participants received adequate nonsurgical periodontal treatment prior to clinical examination, including removal of dental plaque and calculus using ultrasonic scaling. Subjects were also instructed in oral hygiene procedures, such as brushing teeth and cleaning dentures, as well as receiving advice on removing dentures at night.

Statistical analysis

Data were analyzed using Microsoft Excel and IBM SPSS Statistics for Windows. Descriptive statistics (mean, standard deviation, and range) were calculated for age, microbial loads, and periodontal indices, including PI, GI, PD, and TM. Changes in clinical and microbiological parameters between before (T0) and 3 months post-insertion (T3) were assessed using the Wilcoxon signed-rank test for paired non-parametric data. The Fisher's exact test (Monte Carlo simulation, two-sided) was used to evaluate categorical data related to microbial load classification. Spearman's rank correlation coefficient (ρ) was employed to assess associations between microbial loads and periodontal parameters. A p-value < 0.05 was considered statistically significant for all analyses.

Results

Participant demographic

Table 1 presents the distribution of demographic parameters. Among the 30 participants, 16 were male (53.3%), and 14 were female (46.7%). The average age of participants was 64.6 years, with a range from 48 to 76 years. Additional details regarding the social demographics are provided in Table 1.

Clinical examination

Table 2 shows ...

Discussion

Maintaining optimal oral hygiene becomes increasingly challenging with age, due to diminished immune responsiveness and the mechanical limitations imposed by prosthetic appliances such as clasp-retained RPDs. These conditions favor the accumulation of pathogenic biofilms, particularly around abutment teeth, which serve as critical load-bearing structures but are also vulnerable to periodontal deterioration. This study sought to address these concerns by quantifying red complex bacteria (RCB) loads and assessing associate...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 mL Microcentrifuge Tubes with Safety Lock	Biotek	MT1.5/100
Dental Tweezers	Bader	17/4021-1
DTprime Real-Time Thermal Cyclers	DNA-Technology		Real-Time Thermal Cyclers
IBM SPSS	IBM Corp	Version 21.0
Microsoft Excel	Microsoft Office 2010
Paper points (no. 35; 04 tapered)	DiaDent	MP210-602
ParodontoScreen REAL-TIME PCR Detection Kit	DNA-Technology	R1-P808-S3/5EU
Periodontal probe	MEDESY	548/9PT
PREP-GS PLUS	DNA-Technology	P-003/2EU	DNA extraction kit
RealTime software	DNA-Technology	Version 7.9
Sodium Chloride 0.9%	VIOSER