Abstract
Objectives
1. To provide information about the subgingival microbiota around single tooth implants.2. To assess the subgingival microbial flora around the teeth adjacent to single tooth implants.3. To clinically evaluate the gingival health surrounding the single-tooth implants.
Methods
Patients undergoing the single-tooth implant replacements, were selected as subjects for the study. The natural teeth adjacent to implant sites were taken as control sites. Clinically each peri-implant gingival tissue health was evaluated. Subgingival plaque samples were removed with sterile curette and evaluated for microbial flora, by microscopic examinations. Bacterial cultures of samples studied. The similar procedure was followed for the control sites also. Finally the data collected were statistically analyzed and interpreted.
Results
The subgingival microbiota around single tooth implants was cultured and studied. Enterobacter species, Klebsiella pneumonia, Pseudomonas aeruginosa and Streptococcus species were predominantly found. Klebsiella pneumonia and Pseudomonas aeruginosa were found more frequently around implant sites than control sites. Anaerobic Bacteroides species were found in only one case around the implant site.
Conclusions
Prevention and control of bacterial infection in the peri-implant region are among the key factors in determining the long term success or failure of dental implant therapy. The thorough knowledge about the subgingival microbiota around the healthy and diseased peri-implant mucosa is needed to determine the overall outcome of implant therapy.
Keywords: Peri-implant subgingival microbiota, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter sp., Streptococcus sp., Bacteroides sp
1. Introduction
Bacterial infection in the peri-implant region can jeopardize the long term success and survival of dental implant therapy. The information about the peri-implant subgingival microbial flora is needed to determine the outcome of implant therapy. While several factors are considered vital and crucial in determining the long-term success/survival or failure of dental implants, very little thought is given to the microbiological standing of the peri-implant tissues.1 This clinical study was done mainly to focus on the significance of microbiological status of the peri-implant sulcus in determination of success/survival criteria in single-tooth implant therapy.
2. Materials and methods
Prior approval was obtained from the Ethical Committee and Research Committee of the institution. Informed written consents were obtained from all the participants in this study.
Armamentarium: Armamentarium used for the study is shown in the [Fig. 1].Patients undergoing the single-tooth implant replacements, in the Department of Prosthodontics, Mahatma Gandhi Post-Graduate Institute of Dental Sciences - Puducherry were selected as subjects for this study [Fig. 2].
Fig. 1.
Armamentarium used for clinical examination and subgingival plaque sample collection.
Fig. 2.
Single anterior tooth implant case [replacing missing 11-maxillary right central incisor].
Inclusion Criteria: 1. Patient should have undergone single-tooth implant therapy replacing the missing tooth, in Department of Prosthodontics, Mahatma Gandhi Postgraduate Institute of Dental Sciences, Puducherry. 2. Patient should be free of any systemic diseases which include diseases of endocrine origin, cardiovascular origin, hematological origin and any other debilitating diseases.
Diagnostic Phase: Medical history and dental history were taken. All the cases chosen for the study had a single-tooth implant replacing, the missing tooth. The radiographic investigation that was conducted in this study included periapical radiography, panoramic radiography and radiovisuography. Blood investigation were done as it might influence the implant surgery protocol or long term success rate.
Microbial Sample Collection and Culture Procedures: Clinically each peri-implant gingival tissue health was evaluated, by using gingival index modified and plaque index modified. Then presence of any supragingival plaque was detected and removed with help of Hoe Scaler. Later, subgingival plaque samples were removed with sterile implant curette from implant surfaces [Fig. 3] and placed in sterile normal saline placed on the microscopic glass slides. The subgingival bacterial plaque samples were placed in the anaerobic culture medium – Robertson Cooked Meat (RCM) medium in a screw capped tight glass container. Care was taken while collecting the sample and while transferring it to the medium in such a way that it was done as quickly as possible to avoid the exposure of the anaerobic organism to the air. The microscopic slides were stained by gram staining in the microbiological laboratory of the Department of Clinical Microbiology, Pondicherry Institute of Medical Sciences. These slides were evaluated for microbial flora, by microscopic examinations. The adjacent natural teeth to dental implants were taken as controls. Similar procedures were followed for the microbial sample collection for the adjacent natural teeth also [Fig. 4]. Then both aerobic and anaerobic bacterial cultures [Fig. 5] were done from this collected sample and bacterial culture growths (colonies) were studied and identified. Thus the subgingival microbiota around the immediately placed single tooth implants in anterior maxilla, around the conventionally placed single tooth implants in posterior mandible and around the natural teeth adjacent to the single tooth implants were evaluated and finally the data collected were statistically analyzed.
Fig. 3.
Subgingival Plaque Collection from implant site using implant curette.
Fig. 4.
Subgingival Plaque Collection from control site [adjacent tooth] using implant curette.
Fig. 5.
Materials used for anaerobic bacterial culture.
3. Results
Various variables in the present study, for the statistical evaluation are age of the patients, region of dental implant placement in the oral cavity, loading time considerations of dental implants, duration of time from implant placement to observation, gingival health of peri-implant area and adjacent natural tooth.
Comparison of the Aerobic and Anaerobic Bacteriological Culture from Sub-gingival plaque (Microbial Colonies Grown) between Implant sites and Control sites was made.
The main statistical tool used in the present study is Pearson's Chi-Square test. This statistical tool is used to find any correlations or associations between two or more variables. The same tool was used to determine Sensitivity, Specificity, Positive predictive value and Negative predictive value of presence of a particular microbial organism at implant site and at control site.
In the present study subgingival microflora associated with dental implants had been shown to be similar to that associated with natural teeth, which was in accordance with findings of previous studies.2, 3, 4
In the present study, subgingival microbial flora around single tooth implants was cultured and studied. Enterobacter species, Klebsiella pneumoniae, Pseudomonas aeruginosa and Streptococcus species were predominantly found.3,4 Anaerobic Bacteroides species grown only one case out of twenty-two samples (eleven cases).
Microbial flora was correlated between immediately placed single tooth implants in anterior maxilla and around the conventionally loaded single tooth implants. No significant difference noted.
It was observed that there was no statistically significant association between the region in the oral cavity and the gingival health when it was correlated in terms of gingival index modified as well as in terms of plaque index modified (Table 1 & Table 2).
Table 1.
Association between region and gingival health relative to gingival index modified (GIM). Figure in brackets are percentages to row total. Chi-square is not significant (p > 0.05).
| REGION | Gingival Index Modified (GIM) |
||
|---|---|---|---|
| Healthy | Mild | Moderate | |
| Anterior | 1(20.0) | 4(80.0) | 0(0.0) |
| Posterior | 4(66.7) | 1(16.7) | 1(16.7) |
| Both | 5(45.5) | 5(45.5) | 1(9.1) |
| Pearson Chi-Square | 4.55 | p = 0.10299 | |
Table 2.
Association between Region and Gingival Health Relative to Plaque Index Modified (PIM). Figure in brackets are percentages to row total. Chi-square is not significant (p > 0.05).
| REGION | Plaque Index Modified (PIM) |
||
|---|---|---|---|
| Healthy | Mild | Moderate | |
| Anterior | 1(9.1) | 3(60.0) | 1(20.0) |
| Posterior | 4(66.7) | 1(16.7) | 1(16.7) |
| Both | 5(45.5) | 4(36.4) | 2(18.2) |
| Pearson Chi-Square | 2.73 | p = 0.25518 | |
Microbial flora was compared between implant site and adjacent natural teeth (control site). Klebsiella pneumonia (Table 3) and Pseudomonas aeruginosa (Table 4) were found more frequently around implant sites than control sites.
Table 3.
Correlates the identification of Klebsiella pneumonie between control site (adjacent natural tooth) and implant sites. It was observed that there was statistical significant difference in identification of Klebsiella pneumonie between control site and implant sites. Chi-square is significant (p < 0.05).
| Control Site |
Total (Implant) | |||
|---|---|---|---|---|
| Identified | Not Identified | |||
| Implant Site | Identified | 3 | 2 | 5 |
| Not Identified | 0 | 6 | 6 | |
| Total (Control) | 3 | 8 | 11 | |
| Sensitivity | 100% |
| Specificity | 75% |
| Positive predicted value (Identified) | 60% |
| Negative predicted value (Not identified) | 100% |
Pearson Chi-square 4.95 p = 0.02610.
Table 4.
Correlates the identification of Pseudomonas aeruginosa between control site (adjacent natural tooth) and implant sites. It was observed that there is statistical significant difference in identification of Pseudomonas aeruginosa between control site and implant sites. Chi-square is significant (p < 0.05).
| Control Site |
Total (Implant) | |||
|---|---|---|---|---|
| Identified | Not Identified | |||
| Implant Site | Identified | 4 | 1 | 5 |
| Not Identified | 0 | 6 | 6 | |
| Total (Control) | 4 | 7 | 11 | |
| Sensitivity | 100.0% |
| Specificity | 85.7% |
| Positive predicted value (Identified) | 80.0% |
| Negative predicted value (Not identified) | 100.0% |
Pearson Chi-square 7.54 p = 0.00603.
The statistical evaluation of the data obtained from the present study shown that there was no significant association between the region in the oral cavity and the gingival health as well as loading time of single tooth implants and the gingival health when it was correlated in terms of gingival index modified as well as in terms of plaque index modified. No significant difference in the gingival health across different time duration (time from implant placement to observation).
4. Discussion
In the present study the single tooth implant cases were classified in terms of anterior and posterior teeth region of the oral cavity. The subgingival microbial flora was studied and compared between immediately placed single tooth implant in anterior maxilla and around the conventionally loaded single tooth implant in posterior mandible. Also this was correlated with the gingival health.
The oral hygiene status of all the cases studied was good to excellent (as measured through plaque index modified), which reflected upon gingival and peri-implant tissue health (gingival index modified) and this subsequently resulted in disease free subgingival area around single tooth implants and control teeth.
But the presence of Enterics like Enterobacter species, Klebsiella pneumoniae and Pseudomonas aeruginosa should be given attention. Enterics such as Escherichia coli, Enterobacter species, Klebsiella spp. and Pseudomonas spp. are infrequently found in the oral cavity and are related to periodontitis as well as peri-implantitis cases.4,5 Few species of Enterics have developed resistance towards antiseptics as well as antibiotics.5
In the present study majority of the micro-organisms cultured were Gram negative, namely Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter species. The Gram positive oraganisms grown in bacterial culture were Streptococcus species (Streptococcus viridans also called as α-hemolytic streptococci).4
The microbial diversity found in the present study should therefore be considered in the long term maintenance of dental implants. These findings should be considered to prevent any kind of infection in the peri-implant tissues and also in treatment strategy of peri-implantitis.
Healthy peri-implant areas have smaller amounts of bacteria dominated by facultative Gram-positive cocci and rods, whereas diseased sites have a microbiota consists predominantly Gram-negative anaerobic rods and black-pigmented Bacteroides.6, 7, 8, 9, 10, 11
The key factor in the bacterial colonization of the internal part of screw retained implant fixed dental prostheses is the microbial leakage through the gap between the suprastructure and the abutment.12
Characteristics of microbiota around peri-implantitis cases have also been studied in depth by various researchers, which concluded that the peri-implantitis cases are result of a mixed anaerobic infection.13,14
5. Conclusion
This clinical study was done mainly to focus on the significance and importance of microbiological status of the peri-implant sulcus in determination of success of single-tooth implant therapy. In the present study, subgingival microbial flora around single tooth implants was cultured and studied. The subgingival microbial flora was studied and compared between immediately placed single tooth implant in anterior maxilla and around the conventionally loaded single tooth implant in posterior mandible. Also this was correlated with the gingival health.
Source of funding
This research did not receive any specific grant from funding agencies in the public, commercial or not for profit sector.
Declaration of competing interest
The authors declare that they have no conflicts of interest.
Acknowledgement
The authors are grateful for the help from the staff of Department of Prosthodontics, Mahatma Gandhi Postgraduate Institute of Dental Sciences (Govt. of Puducherry Institution) Puducherry-605006, India; as well as the staff of Department of Clinical Microbiology, Pondicherry Institute of Medical Sciences (A Unit of Madras Medical Mission), Kalapet, Puducherry-605014, India for all the help rendered during the course of study.
References
- 1.Rams T.E., Roberts T.W., Tatum H.J., Keyes P.H. The subgingival microbial flora associated with human dental implants. J Prosthet Dent. 1984 Apr;51(4):529–534. doi: 10.1016/0022-3913(84)90309-3. [DOI] [PubMed] [Google Scholar]
- 2.Silverstein L.H., Kurtzman D., Garnick J.J., Schuster G.S., Steflik D.E., Moskowitz M.E. The microbiota of the peri-implant region in health and disease. Implant Dent. 1994;3:170–174. doi: 10.1097/00008505-199409000-00006. [DOI] [PubMed] [Google Scholar]
- 3.Lee K.H., Maiden M.F., Tanner A.C., Weber H.P. Microbiota of successful osseointegrated dental implants. J Periodontol. 1999;70:131–138. doi: 10.1902/jop.1999.70.2.131. [DOI] [PubMed] [Google Scholar]
- 4.Leonhardt A., Renvert S., Dahlen G. Microbial findings at failing implants. Clin Oral Implants Res. 1999;10:339–345. doi: 10.1034/j.1600-0501.1999.100501.x. [DOI] [PubMed] [Google Scholar]
- 5.Renvert S., Lessem J., Dahlén G., Renvert H., Lindahl C. Mechanical and repeated antimicrobial therapy using a local drug delivery system in the treatment of peri-implantitis: a randomized clinical trial. J Periodontol. 2008;79:836–844. doi: 10.1902/jop.2008.070347. [DOI] [PubMed] [Google Scholar]
- 6.Apse P., Ellen R.P., Overall C.M., Zarb G.A. Microbiota and crevicular fluid collagenase activity in the osseointegrated dental implant sulcus: a comparison of sites in edentulous and partially edentulous patients. J Periodontal Res. 1989 Mar;24(2):96–105. doi: 10.1111/j.1600-0765.1989.tb00863.x. [DOI] [PubMed] [Google Scholar]
- 7.Sanz M., Newman M., Nachnani S., Holt R., Stewart R., Flemmig T. Characterization of the subgingival microbial flora around endosteal sapphire dental implants in partially edentulous patients. Int J Oral Maxillofac Implants. 1990 Fall;5(3):247–253. [PubMed] [Google Scholar]
- 8.Heydenrijk K., Meijer H.J., van der Reijden W.A., Raghoebar G.M., Vissink A., Stegenga B. Microbiota around root-form endosseous implants: a review of the literature. Int J Oral Maxillofac Implants. 2002 Nov-Dec;17(6):829–838. [PubMed] [Google Scholar]
- 9.Quirynen M., De Soete M., van Steenberghe D. Infectious risks for oral implants: a review of the literature. Clin Oral Implants Res. 2002 Feb;13(1):1–19. doi: 10.1034/j.1600-0501.2002.130101.x. [DOI] [PubMed] [Google Scholar]
- 10.Romeo E., Ghisolfi M., Carmagnola D. Peri-implant diseases. A systematic review of the literature. Minerva Stomatol. 2004 May;53(5):215–230. [PubMed] [Google Scholar]
- 11.Botero J.E., Gonzalez A.M., Mercado R.A., Olave G., Contreras A. Subgingival microbiota in peri-implant mucosa lesions and adjacent teeth in partially edentulous patients. J Periodontol. 2005 Sep;76(9):1490–1495. doi: 10.1902/jop.2005.76.9.1490. [DOI] [PubMed] [Google Scholar]
- 12.Keller W., Bragger U., Mombelli A. Peri-implant microflora of implants with cemented and screw retained suprastructures. Clin Oral Implants Res. 1998 Aug;9(4):209–217. doi: 10.1034/j.1600-0501.1998.090401.x. [DOI] [PubMed] [Google Scholar]
- 13.Mombelli A., Décaillet F. The characteristics of biofilms in peri-implant disease. J Clin Periodontol. 2011 Mar;38(Suppl 11):203–213. doi: 10.1111/j.1600-051X.2010.01666.x. [DOI] [PubMed] [Google Scholar]
- 14.Rakic M., Grusovin M.G., Canullo L. The microbiologic profile associated with peri-implantitis in humans: a systematic review. Int J Oral Maxillofac Implants. 2016 Mar-Apr;31(2):359–368. doi: 10.11607/jomi.4150. Epub 2015 Oct 6. [DOI] [PubMed] [Google Scholar]