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

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.

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.

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 the results obtained from the periodontal indices. In abutment teeth, almost all indices except for TM were significantly higher after 3 months of the RPDs insertion. On the other hand, PI and GI values were found to be significantly higher in non-abutment teeth, whereas no significant differences were observed for TM and PD.

Differences in periodontal indices between abutment and non-abutment teeth

The difference in periodontal index values between abutment and non-abutment teeth before and 3 months after insertion of PRPD is presented in Table 3. Before treatment with RPDs, there were no significant differences in the indices between abutment and non-abutment teeth. However, 3 months after treatment with RPDs, the values were slightly higher in abutment teeth than in non-abutment teeth, but still without statistical significance (p>0.05).

Microbiological examination

Table 4 shows the microbial loads of all periodontal pathogens evaluated before and 3 months after the RPD insertion. The loads of all three bacteria were significantly higher after 3 months of denture insertion.

In addition, as shown in Table 5, before the RPD insertion, one patient (3.33%) had a severe prevalence of P.gingivalis (>6.0 Lg), two (6.67%) a moderate prevalence (>5.0 Lg), while 27 (90.00%) were in the normal range (<5.0 Lg). One patient with severe prevalence of P. gingivalis before the RPDs insertion evolved to a moderate prevalence (>5.0 Lg) after 3 months of denture insertion. Of the two patients with moderate prevalence of P. gingivalis (>5.0 Lg) before the RPDs insertion, one (100.0%) evolved to severe prevalence (>6.0 Lg) while the other (100.0%) remained in the same range (>5.0 Lg) after 3 months of denture insertion. Of the 27 patients in the normal range (<5.0 Lg) before the RPDs insertion, three (11.1%) had severe prevalence of P. gingivalis (>6.0 Lg), five (18.5%) had moderate prevalence (>5.0 Lg), while 19 (70.4%) were in the normal range (<5.0 Lg) considering the period of 3 months of denture insertion.

However, in the cross-tabulation of the microbial loads before and 3 months after treatment with RPDs, there were significant differences for P. gingivalis (Fisher's Exact Test = 7.984 and p < 0.05 (p = 0.048) / Monte Carlo Sig (2 sided) / 0.043-0.054 /) and T. denticola ( Fisher's exact test = 9240 and p < 0.01 (p = 0.006 ) / Monte Carlo Sig. (2 sided) / 0.004-0.008 /), while the same was not observed for T. forsythia (Fisher's exact test = 4.490 and p> 0.05 (p = 0.265) / Monte Carlo Sig. (two-sided) / 0.254-0.277.

Correlations between microbial loads and periodontal indices

The results presented in Table 6 show the correlations between each periodontal index and microbial loads before and 3 months after the RPDs insertion. A strong correlation was found between T.forsythia and all periodontal indices studied.

DATA AVAILABILITY:
The raw data used to calculate representative results is available in Supplementary File 1.

Figure 1: Impact of Removable Partial Denture on the microbiome of abutment teeth. A schematic representation illustrating how removable partial dentures (RPD) may alter the microbiological environment of abutment teeth, resulting in an increase in red complex bacteria. Created in BioRender. https://BioRender.com/vexty3l Please click here to view a larger version of this figure.

Table 1: Study population demographics. Abbreviations: SD = Standard deviation.

Table 2: Periodontal indices in abutment and non-abutment teeth before and 3 months after RPD insertion. Abbreviations: CI = confidence interval; Z and P = statistical variables; T = time of sampling; SD = standard deviation; AT = abutment teeth; Non AT = non-abutment teeth; Min = minimum; Max = maximum; mm = millimeter.

Table 3: Differences in periodontal indices between abutment and non-abutment teeth. Abbreviations: U, Z, and P = statistical variables; AT = abutment teeth; Non-AT = non-abutment teeth; mm = millimeter.

Table 4: Bacterial loads of periodontal pathogens before and 3 months after RPD insertion. Abbreviations: CI = confidence interval; Z and P = statistical variables; T = time of sampling; SD = standard deviation; Min = minimum; Max = maximum; lg = logarithm of the number of genome equivalents per sample.

Table 5: Prevalence of periodontal pathogens before and 3 months after RPDs insertion.

Table 6: Correlation between periodontal indices and bacterial loads of the three periodontal pathogens studied before and 3 months after RPD insertion. Abbreviations: R and P = statistical variables; T = time of sampling.

Supplementary File 1: Raw data for representative results. Please click here to view a larger version of this figure.

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 associated periodontal changes over a 3-month period following RPD insertion in older adults. While prior studies have evaluated the clinical impact of RPDs on periodontal health using indices such as PI, GI, and PD, few have investigated the microbial profile in parallel-particularly with the sensitivity and specificity of RT-PCR. This dual-pronged approach identifies the subclinical microbial shifts that may predispose patients to long-term peri-abutment inflammation and prosthesis-related complications. However, the microbial increase is a potential early biomarker of future deterioration since these are subclinical changes that might precede clinical disease.

According to our findings, overall, the treatment with RPDs resulted in an increase in PI, GI, and PD values; however, without significant differences between abutment and non-abutment teeth. The impact of RPDs on periodontal health has been investigated in different studies. Our results are consistent with some of them^31,32. Chandler and Brudvik evaluated in their study the clinical condition of RPD patients and patients without dentures. After a follow-up period of 8 to 9 years, no significant differences were found in sulcus depth, tooth mobility, and alveolar bone loss in the comparison between abutment and non-abutment teeth in both study groups³². However, this is not a consensus in the literature. Other researchers have reported that the insertion of RPDs may increase the values of periodontal indices in abutment teeth compared to non-abutment teeth^33,34. Also, in contrast to our results, Dula et al. found a greater periodontal risk in abutment teeth during the same follow-up period considered for the present study⁷. This difference can probably be attributed to the smaller number of teeth analyzed in our research.

Other important factors associated with periodontal condition are residence and education. In our study, 26.7% of participants lived in rural areas, and 40% had only completed primary school. Malakaret al. demonstrated that residents of rural areas may have a worse periodontal status compared to those living in urban areas³⁵. Moreover, Boillotet al. found that patients with a lower level of education are more likely to develop periodontitis in comparison to those with a higher level³⁶. However, to better understand these differences, other social determinants such as income, transportation, and sanitation must also be considered.

Here, more specifically, when determining the microbial loads of the three periodontal pathogens studied, we found the presence of RCB at T0, although the abutment teeth had shallow pockets. This can be explained by the inclusion of smokers (<25 cigarettes per day) in the study^37,38. Jiang et al. evaluated how smoking can affect the subgingival flora, modifying the periodontal status from healthy to periodontitis. Their findings suggest that smoking, by affecting the microbial functions of the pathogen-host cell interaction, facilitates the early colonization of periodontal pathogens³⁸. On the other hand, periodontal pathogens were also found by other researchers to be common members of the oral microbiota of healthy children, as well as in the oral cavities of newborns and edentulous patients^39,40,41. Furthermore, another study employing molecular genetic analysis detected high levels of periodontal pathogens when assessing their prevalence, including RCB in young patients (<35 years) with healthy periodontium. This then leads us to the conclusion that such microorganisms must colonize the oral cavity even of individuals with healthy periodontium⁴², regardless of the depth of periodontal pockets.

We also observed a significant increase in the microbial loads of the three evaluated pathogens after 3 months of the RPDs insertion (Figure 1). This finding can initially be attributed to the placement of RPDs in the oral cavity, which, by limiting the cleaning action of the tongue and lips, may affect the quality and quantity of certain types of microorganisms, thus predisposing to a favorable environment for the growth of pathogenic bacteria^43,44.

Many of our results in this context are also in agreement with the findings of other previous studies. In a study conducted by Tanaya et al., which evaluated the microbial flora of RPDs wearers at three intervals, there was an increase in the number of microbial colonies, including colonies of P. gingivalis⁴⁵. Similarly to that, Mineet al. found a significant increase in the loads of T.forsythia, P.gingivalis, and T.denticola in abutment teeth after 6 months of RPDs insertion²². Moreover, another study reported an increase in the total microbial genome count after 180 days of RPDs insertion, however, without significant differences between abutment and non-abutment teeth²¹. However, it is worth noting that the maintenance of RPDs was not controlled over time in these three studies. In contrast to them, Vanzeveren et al. analyzed microbiological and periodontal parameters in abutment teeth of RPD wearers who received oral health instructions, professional dental prophylaxis, and denture hygiene control compared to patients with no callback. The authors observed a few differences between the groups during the 2 years of follow-up²³. This finding, in turn, reinforces the importance of receiving guidance on oral hygiene and the proper use of dentures, as well as on the positive impacts of routine dental examinations performed throughout life.

The RPD framework is also an important factor that may affect the levels of periodontal pathogens. Mengattoet al. investigated the biofilm on RPD metal clips and observed an increase in the composition of the biofilm over time. The authors concluded that the framework of RPDs can serve as a reservoir of pathogenic microorganisms⁴⁶. In the present study, patients were instructed to clean their teeth and dentures twice a day, and that the dentures should be placed in water overnight. Even so, the results of our microbiological analysis showed a large increase in the microbial loads over the 3-month follow-up period. This may be related to inappropriate oral hygiene habits in elderly patients and the lack of monthly follow-up, which certainly implies the need to encourage more oral hygiene practices in patients treated with prosthetic devices.

Another key objective of this investigation was to explore correlations between microbial profiles and clinical periodontal parameters. Among the red complex bacteria assessed, Tannerella forsythia demonstrated the strongest association with worsening plaque index PI, GI, PD, and TM. Given its role in periodontal pathogenesis-mediated through glycosylated S-layer proteins and synergistic interactions with other pathogens-T. forsythia may serve as a microbial marker of early periodontal deterioration in RPD wearers⁴⁷. These findings demonstrate their potential clinical relevance in both diagnostics and targeted intervention strategies.

One of the study's strengths is its contribution to addressing a gap in the literature by combining clinical periodontal indices with quantitative microbiological analysis, which represents a novel approach. Additionally, the application of molecular methods, specifically RT-PCR, provides a sensitive and high-throughput analysis of bacterial load. In contrast, nearly all previous studies have employed alternative methods for identifying these bacteria, which, as facultative anaerobes, present challenges in cultivation.

Another strength of this work is its focused investigation of RCB, which are known to play a crucial role in the pathogenesis of periodontal and peri-implant diseases. Consequently, this study introduces an innovative approach to implant-prosthetic planning for patients with partial edentulosis who have utilized RPDs. This approach underscores the potential risks for clinicians in managing these patients, particularly concerning the risk of future peri-implantitis development.

This study also acknowledges several limitations. First, it did not statistically account for prosthesis-related variables such as connector design, clasp configuration, or Kennedy classification of edentulism-factors that may modulate plaque accumulation and microbial colonization. Second, although microbial analysis via RT-PCR was rigorously conducted on abutment teeth, comparative data from non-abutment sites were not collected. Third, the inclusion of non-heavy smokers (<26 cigarettes/day), individuals with diabetes, and the modest sample size may restrict the generalizability of the findings. Additionally, the study was conducted during the COVID-19 pandemic, which may also impact its applicability. Despite these constraints, the study provides a valuable foundation for future research, particularly longitudinal investigations that incorporate broader microbial panels and other factors relevant to dentures.

This study suggests that the use of RPDs is associated with a significant increase in the microbial load of red complex bacteria in abutment teeth within three months of insertion. While clinical indices also increased, differences between abutment and non-abutment teeth remained statistically nonsignificant. Among the pathogens, T. forsythia showed the strongest correlation with periodontal deterioration, suggesting its potential as an early biomarker. These findings support individualized maintenance protocols and the use of molecular diagnostics to detect subclinical changes. Further longitudinal studies are necessary to clarify the long-term effects of RPDs on periodontal health, as well as to assess the impact of various RPD designs and maintenance protocols on the microbiological levels in non-abutment teeth.