Colonization dynamics of subgingival microbiota in recently installed dental implants compared to healthy teeth in the same individual: a 6-month prospective observational study ^*

Abstract

Objectives

To evaluate the colonization dynamics of subgingival microbiota established over six months around newly installed dental implants in periodontally healthy individuals, compared with their corresponding teeth.

Methodology

Seventeen healthy individuals assigned to receive single dental implants participated in the study. Subgingival biofilm was sampled from all implant sites and contralateral/ antagonist teeth on days 7, 30, 90, and 180 after implant installation. Microbiological analysis was performed using the Checkerboard DNA-DNA hybridization technique for detection of classical oral taxa and non-oral microorganisms. Significant differences were estimated by Mann-Whitney and Friedman tests, while associations between implants/teeth and target species levels were assessed by linear regression analysis (LRA). Significance level was set at 5%.

Results

Levels of some species were significantly higher in teeth compared to implants, respectively, at day 7 ( V.parvula , $6\times {10}^5$ vs $3\times 105$ ; Milleri streptococci , $2\times {10}^6$ vs $6\times {10}^5$ ; Capnocytophaga spp., $2\times {10}^6$ vs $9\times {10}^5$ ; E.corrodens , $2\times {10}^6$ vs $5\times {10}^5$ ; N. mucosa , $2\times {10}^6$ vs $5\times {10}^5$ ; S.noxia , $2\times {10}^6$ vs $3\times {10}^5$ ; T.socranskii , $2\times {10}^6$ vs $5\times {10}^5$ ; H.alvei , $4\times {10}^5$ vs $2\times {10}^5$ ; and Neisseria spp., $6\times {10}^5$ vs $4\times {10}^4$ ), day 30 ( V.parvula , $5\times {10}^5$ vs 10 ⁵ ; Capnocytophaga spp., $1.3\times {10}^6$ vs $6.8\times {10}^4$ ; F.periodonticum , $2\times {10}^6$ vs 10 ⁶ ; S.noxia , $6\times {10}^5$ vs $2\times {10}^5$ ; H.alvei , $8\times {10}^5$ vs $9\times {10}^4$ ; and Neisseria spp., $2\times {10}^5$ vs 10 ⁶ ), day 120 ( V.parvula , $8\times {10}^5$ vs $3\times {10}^5$ ; S.noxia , $2\times {10}^6$ vs 0; and T.socranskii , $3\times {10}^5$ vs $8\times {10}^4$ ), and day 180 ( S.enterica subsp. enterica serovar Typhi, $8\times {10}^6$ vs $2\times {10}^6$ ) (p<0.05). Implants showed significant increases over time in the levels of F.nucleatum , Gemella spp., H.pylori , P.micra , S.aureus , S.liquefaciens , and T.forsythia (p<0.05). LRA found that dental implants were negatively correlated with high levels of S. noxia and V. parvula (β=-0.5 to -0.3; p<0.05).

Conclusions

Early submucosal microbiota is diverse and only a few species differ between teeth and implants in the same individual. Only 7 days after implant installation, a rich microbiota can be found in the peri-implant site. After six months of evaluation, teeth and implants show similar prevalence and levels of the target species, including known and new periodontopathic species.

Dental implants; Microbiota; Molecular diagnostic techniques; DNA probes

Introduction

The increasing number of individuals living with dental implants has brought clinical challenges for patients and professionals alike as eventually most patients may develop peri-implant mucositis or peri-implantitis, depending on several local and systemic factors related to the individual’s susceptibility.¹ , ² Peri-implant infections have as a primary etiological factor the overgrowth and persistence of pathogenic subgingival biofilm.¹ , ³ , ⁴ Peri-implant microbial colonization occurs soon after implant installation,⁵ whereas late implant failures are related to a dysbiotic bacterial biofilm.⁶ , ⁷ Since the severity of peri-implantitis is associated with the established bacterial dysbiosis,⁸ maintaining balance between a bacterial biofilm and the host around dental implants is important to preserve peri-implant health.⁹ This biofilm-host balance becomes increasingly relevant in patients with a history of periodontitis, since periodontal pockets are a reservoir for periodontal pathogens, and their translocation to submucosal sites is more likely to occur.⁴ , ¹⁰ - ¹³

Previous studies have described the bacterial profile in early colonization around dental implants by DNA probes⁵ , ¹⁴ or standard culture techniques¹⁵ targeting usual periodontal pathogens, showing that red and orange complexes species have a similar detection frequency around implants and teeth with comparable probing depths.⁵ Overall, bacterial counts increase in the submucosal site a few weeks after implant installation.¹⁴ , ¹⁶ Comparisons between periodontitis and peri-implantitis have also found similarities in the subgingival and submucosal colonization by putative periodontal pathogens.¹⁷ , ¹⁸ Conversely, the microbial composition of healthy implants seems to differ significantly from peri-implantitis cases.¹⁹

Advances in culture-independent techniques for studying the subgingival microbiota have indicated novel microbial taxa that may play a role in periodontal or peri-implant disease pathogenesis, such as Dialister and Filifactor. ¹⁶ - ¹⁸ , ²⁰ , ²¹ Importantly, most of the genomic sequencing data related to subgingival biofilm around implants are presented at the phyla and genera levels and not at the species level.¹⁶ , ²¹ , ²² A systematic review showed that available evidence in the literature is insufficient to provide a final answer concerning the microorganisms colonizing dental implants.²³ Moreover, there is limited information on the colonization dynamics over the first months after implant installation regarding candidate periodontal pathogens and other bacteria of medical importance. Understanding the first stages of bacterial colonization for those new periodontal pathogens might be essential for developing effective strategies to prevent peri-implant diseases. Hence, we hypothesized that novel periodontal pathogens can colonize the submucosa around dental implants at early stages after installation. This study evaluated the kinetics of the submucosal microbiota colonization in newly installed dental implants and their corresponding teeth in periodontally healthy individuals for six months, investigating the frequency and levels of putative periodontal species, as well as potential novel periodontal taxa of medical importance.

Methodology

Study population

This study included 19 healthy partially edentulous individuals assigned to receive single dental implants, who were recruited and treated from March to December 2016 at the Post-graduation Clinics, Universidade do Grande Rio. All participants signed an informed consent form after being informed about the study objectives. In addition to following STROBE guidelines, this study was conducted in full accordance with the revised Helsinki Declaration and approved by the Research Ethics Committee of the Universidade do Grande Rio (#1359026).

Prior to entering the study, all participants underwent a full periodontal examination. Inclusion criteria consisted of individuals >18 years of age with a correspondent tooth for the dental implant installed, <20% of sites with visible dental biofilm and <10% of sites with bleeding on probing, and no attachment loss due to periodontitis. Persisting bleeding after probing depth and clinical attachment level measurements was considered positive bleeding on probing. Visual inspection under relative isolation and proper illumination of the dental surface determined the presence of visible dental biofilm. Study exclusion criteria included: history of periodontitis; known diseases of the immune system (e.g., HIV-positive); diabetes; pregnancy or breastfeeding; necessity of chemoprophylaxis for dental care; antimicrobial use in the six months prior to the study; smoking; and periodontal treatment in the last year. Moreover, they should not be users of removable prosthesis or orthodontic appliances. Participants received no prophylactic antibiotics previously to implant placement surgery. To guarantee adequate oral hygiene control throughout the study, participants received personalized instructions on toothbrush (i.e., conventional, interproximal, etc.) and dental floss use at every visit.

Sample size calculation

Sample size was calculated based on two standard deviations (5.27 and 5.41 counts x10⁶) of the mean counts of a bacterial species in samples from the same healthy individuals at two different timepoints.²⁴ Detection of a $6\times {10}^6$ difference required a minimum of 13 individuals with approximately 95% CI for the difference between means (D) D - 4.240 to D + 4.240. Sample size calculation was performed using a free software (WinPep, http://www.brixtonhealth.com/pepi4windows.html).

Dental implant installation

Titanium alloy implants, manufactured according to ASTM standard F136, were installed in the posterior maxilla and mandibula using a one-stage open-flap technique, without surgical guide, with torques ranging from 35 to 60 N. All procedures were late installations with no graft addition as all surgical sites presented adequate keratinized mucosa width. Torques were measured using a manual torquemeter available on the surgical kit (Neodent^®, Curitiba, PR, Brazil). A healing abutment was applied immediately after implant placement (SlimFit, Neodent^®), followed by suture using Mononylon 5-0 (Mononylon^®, J&J, São José dos Campos, SP, Brazil). All installed implants had an external hexagon connection (Neodent^®), which are made of type 2 titanium alloy and have a surface treated by abrasive blasting followed by acid etching. Each participant received only one implant. We instructed all participants to use chlorhexidine at 0.12% twice a day for 7 days after the procedure and prescribed only analgesics. On day 7, we installed provisional crowns and checked occlusion by asking patients to bite the occlusion tape in all directions. Occlusion adjustments were made with a diamond bur at high rotation when necessary. The provisional crowns were kept until final evaluation. All provisional crowns were manufactured in acrylic resin by the same prosthetic laboratory using an addition silicone impression mold. The screw-retained prosthesis was obtained on a regular platform. We provided all participants with detailed and personalized hygiene instructions regarding dental brush, dental floss, and interdental brush, and made reinforcements at every visit when necessary.

Microbiologic analysis

Subgingival biofilm samples were collected by the same trained examiner at 7, 30, 120, and 180 days after implant installation⁵ , ¹⁴ , ²⁵ from all sites around the implant and its homologous tooth, when present, or its antagonist, which should not present bleeding on probing, in relative isolation using titanium sterile Gracey curettes (Hu-Friedy; Chicago, IL, USA) after cleaning the area of interest with sterile gauze. Samples were always collected first from the implant and then the tooth. Two pooled biofilm samples, one from each site, were obtained per patient. Sampling was performed before removal of the healing abutment on day 7, and with the provisional crowns in place at the remaining timepoints. The sampled pools were placed in individual microtubes containing 150 μL of tris-EDTA buffer (TE, 10 mM Tris HCl, 1 mM EDTA, pH 7.6), which were then filled with 150 μL of 0.5 M NaOH.

Microbiota composition was determined using checkerboard DNA-DNA hybridization.²⁶ Briefly, the samples were lysed and fixed onto individual lanes on a nylon membrane (Molecular Dynamics GE Healthcare LifeSciences, Piscataway, NJ, USA) using a specific apparatus (Minislot 30, Immunetics, Cambridge, MA, USA), and hybridized against whole genomic digoxigenin-labeled probes by a hybridization apparatus (Miniblotter 45, Immunetics). DNA from Aggregatibacter actinomycetemcomitans ( Aa ) serotypes a, b and c, and Cutibacterium acnes I and II were pooled into two separate probes. Probes for other closely related species were pooled in equal concentrations, including Actinomyces spp., Campylobacter spp., Capnocytophaga spp., Enterics spp., Eubacterium spp., Fusobacterium nucleatum , Gemella spp., Lactobacillus spp., Neisseria spp., Milleri streptococci, Mitis streptococci, Mutans streptococci, Prevotella spp., and Serratia spp. ( Supplemental Table 1S ). We analyzed 85 species in total. Bound probes were detected by a digoxigenin phosphatase-conjugated antibody (Roche Applied Science, São Paulo, SP, Brazil) and fluorescence (AttoPhos®, Promega Corporation, Madison, WI, USA). Membrane signals captured and analyzed by an imaging system (Storm TM 860 and ImageQuant^® version 5.2, Molecular Dynamics GE Healthcare LifeSciences) were compared to the standards for the tested species, present on the same membrane at 100,000 and 1,000,000 bacterial cell counts. Signals were classified as: 0, not detected; 1, <100,000 cells; 2, approximately 100,000; 3, 100,000 to 1,000,000; 4, approximately 1,000,000; and 5, >1,000,000 cells. Assay sensitivity was adjusted to enable the detection of 10,000 cells of a given species by fixing each DNA probe concentration. This procedure was performed to provide the same detection sensitivity for each species. Failure to detect a signal was recorded as zero, although counts in the 1 to 1,000 ranges may have been present.

The microbiological analysis was performed by a blinded examiner.

Data analysis

Statistical analyses were performed using a statistical software package (IBM SPSS Statistics Version 19, IBM, Armonk, USA). Mean levels (cell counts in a sample) and detection frequency of each target species were calculated for implants and teeth at days 7, 30, 120, and 180. Significant differences between teeth and implants were estimated by Mann-Whitney test, whereas changes in the microbiota over time were analyzed by Friedman test. Associations between implants or teeth and the levels of the species, which differed significantly between groups at the observation timepoints, were evaluated by linear regression analysis using the stepwise method. Significance level was set at 5%. Each group received a code to avoid bias during statistical analysis.

Results

A total of 19 eligible individuals, ten women (45 ± 10.8 years) and nine men (49 ± 15.5 years), participated in this study. During follow-up, two male patients dropped out (one moved away and another gave no explanation), thus, the current analysis considered only the individuals who took part in all periods of observation (n = 17), as illustrated in Figure 1 . We present the list of the dental implant location and the respective control tooth in a supplemental file ( Supplemental Table 2S ).

Figure 1
Flowchart of participant inclusion

Figure 2 summarizes the detection frequency of the target species. Data from 07 and 30 days show that Hafnia alvei was detected in significantly higher frequency in teeth (70.8% and 72.2%, respectively) compared with implants (36% and 27.8%, respectively) (Mann-Whitney test; p<0.05). At 30 and 120 days, other species such as Capnocytophaga spp., F. nucleatum, Filifactor alocis , Selenomonas noxia , Treponema socranskii and V. parvula were also significantly more frequently detected in teeth compared with implants. We found no differences between implants and teeth at 180 days (p>0.05). Analysis over time showed that detection frequency increased significantly for F. nucleatum, Helicobacter pylori, P. micra, and Staphylococcus aureus in implant samples (Friedman test; p<0.05).

Figure 2
Detection frequency of the studied species in teeth and implant samples. (a) tooth samples; (b) implant samples; species: S. enterica ssp. enterica sorv , Salmonella enterica subsp. enterica serovar Typhi; *Significant difference between teeth and implants at 7 days, p <0.05, Mann-Whitney test; †Significant difference between teeth and implants at 30 days; ‡Significant difference between teeth and implants at 120 days; #Significant difference within group over time, Friedman test.

Figure 3 shows that some studied oral species presented levels significantly higher (p<0.05) in teeth compared to implants at day 07 ( Capnocytophaga spp., E. corrodens, H. alvei, Milleri streptococci, N. mucosa, Neisseria spp. , S. noxia, T. socranskii , and V. parvula ), day 30 ( V. parvula , Capnocytophaga spp., Fusobacterium periodonticum , S. noxia, and H. alvei ), day 120 ( S. noxia, T. socranskii, and V. parvula ), and day 180 ( Salmonella enterica subsp . enterica serovar Typhi). We found a significant increase in the levels of some species over time in teeth and implant samples ( F. nucleatum , H. pylori, P. micra , and S. aureus). Conversely , other species showed a level increase only in teeth ( C. acnes and Salmonella enterica subsp . enterica serovar Typhi) or in implants ( Gemella spp., Serratia liquefaciens and Tannerella forsythia ). Only one studied species decreased significantly over time in teeth samples ( Peptostreptococcus anaerobius ). We present the levels of the detected species grouped according to their phylum in a supplemental file ( Supplemental Figure 1S ; Supplemental Table 3S ).

Figure 3
Levels of the studied species in teeth and implant samples. (a) tooth samples; (b) implant samples; species: S. enteric a ssp., Salmonella enterica subsp. enterica serovar Typhi; *Significant difference between teeth and implants at 7 days, p <0.05, Mann-Whitney test; †Significant difference between teeth and implants at 30 days; ‡Significant difference between teeth and implants at 120 days; #Significant difference between teeth and implants at 180 days; §Significant difference within group over time, Friedman test.

Final linear regression models for each observation period showed that dental implants had significant negative correlations with the levels of S. noxia and V. parvula after 7 days ( Table 1 ). Only S. noxia remained in the final model 1, showing a standardized β coefficient (β) of -0.331 (p=0.02). In model 2, S. noxia (β=-0.288 ; p= 0.035 ) and V. parvula (β=-0.288; p=0.035) presented significant negative correlations with implants. In the 30-day analysis, only S. noxia showed a significant negative correlation with implants (β=-0.472; p=0.004). In model 2, however, S. noxia (β=-0.401 ; p= 0.009 ) and V. parvula (β= -0.354 ; p= 0.020) showed significant negative correlations with dental implants. At 120 days only V. parvula remained in the model, presenting a significant negative correlation with dental implants (β=-0.500; p=0.009). On day 180, only S. enterica subsp . enterica serovar Typhi had a significant negative correlation with dental implants (β=-0.639; p=0.001). LRA indicates that these species were less likely to be found in implant samples compared with teeth samples. As shown in these adjusted models, fewer species stood out at the observation periods, differently from the direct statistical comparison, in which a greater number of species presented significant differences.

Table 1
Linear regression analysis of the association between level of studied species and dental implant on the 6-month evaluation period

Predictor species included in the final model	Standardized β coefficient	P value	Adjusted R2
7 days*
Model 1
S. noxia	-0.331	0.020	0.091
Model 2
S. noxia	-0.348	0.012	0.157
V. parvula	-0.288	0.035
30 days†
Model 1
S. noxia	-0.472	0.004	0.199
Model 2
S. noxia	-0.401	0.009	0.303
V. parvula	-0.354	0.020
120 days‡
V. parvula	-0.500	0.009	0.218
180 days§
S. enterica subsp. enterica serovar Typhi	-0.639	0.001	0.380

• *Species included in the step 1 for the analysis of day 7: Capnocytophaga spp., E. corrodens , Milleri streptococci , N. mucosa , S. noxia , T. socranskii , V. parvula , H. alvei , and Neisseria spp.; † Species included in the step 1 for the analysis of day 30: C. sputigena , F. periodonticum , S. noxia , V. parvula , and H. alvei . ‡Species included in the step 1 for the analysis of day 120: S. noxia , T. socranskii , and V. parvula ; §Species included in the step 1 for the analysis of day 180: S. enterica subsp. enterica serovar Typhi.

Discussion

Our study evaluated the kinetics of subgingival microbiota colonization, analyzing classical bacterial complexes and novel periodontal pathogens established over time around newly installed implants in periodontally healthy individuals compared to their corresponding teeth. The levels of some species differed significantly between tooth and implant at one week after implant installation, whereas only Salmonella enterica subsp . enterica serovar Typhi showed higher levels in teeth compared to implants at six months. These results indicate that peri-implant sites promptly acquire a similar microbiota to the one found in the remaining teeth. Interestingly, the levels of these species leveled up over time between teeth and implants, corroborating data from other studies.⁵ , ¹⁶ , ²⁷ After only one week in place, submucosal sites are already colonized by a diverse microbiota comparable to the one found in the subgingival healthy site, in line with other findings.¹⁶

According to the literature, any source or reservoir of potential pathogens (e.g., periodontal pockets) should be eradicated before dental implant installation to avoid microbiota dissemination to other areas,³ , ⁷ , ²¹ , ²⁸ , ²⁹ indicating that sites with dysbiotic subgingival biofilm might work as a reservoir for submucosal colonization. A previous study noted that implants with bacterial levels above 10,000 cells for the species T. forsythia, Treponema denticola, Capnocytophaga rectus, T. socranskii, Porphyromonas gingivalis, S. aureus, Campylobacter gracilis, and Prevotella intermedia had a significantly higher risk to present peri-implantitis with an odds ratio ranging from 3.1 to 4.6.³⁰ Most of these species were detected above 10,000 cells in the present research, suggesting a potential risk of peri-implant disease onset even in periodontally healthy individuals since they can become periodontal/peri-implant diseased patients as they age. Moreover, current findings pointed out that periodontal pathogenic species show significant increase in levels over time, including F. nucleatum and T. forsythia, members of the orange and red complexes, respectively.³¹ Interestingly, colonization occurred even in patients who kept good dental/implant hygiene throughout the study period.

Our findings show that some species not usually associated with the subgingival site, such as Filifactor alocis , H. pylori, and S. aureus , are common in teeth and dental implants. Regarding periodontal disease, the implication of F. alocis in periodontal disease severity is well-described in clinical studies.³² , ³³ Moreover, previous research has pointed out a potential role in periodontal disease pathogenesis for some species detected in the subgingival biofilm that are not usually associated with periodontal diseases, such as Acinetobacter baumannii and Pseudomonas aeruginosa .³⁴ , ³⁵ However, few studies explore the role of these bacteria and other “non-periodontal” species in peri-implant health or peri-implantitis.³⁰ , ³⁶ , ³⁷ Relatively high levels of Haemophilus influenzae, H. pylori, and S. aureus may be detected in healthy dental implants or those presenting peri-implantitis.³⁰ Another study detected F. alocis in 80%, Staphylococcus epidermidis in 29%, and S. aureus in 7.9% of the implant samples collected.³⁶ Interestingly, some bacteria may be implant-specific, including Propionibacteria, Paludibacter, Staphylococci, Filifactor , and Mogibacterium .³⁸ At the species level, another study found that Rothia aeria, Streptococcus sanguinis, Streptococcus gordonii, and V. parvula were the species most commonly associated with healthy submucosal sites . ¹³ Comparisons between the microbial profile of healthy and peri-implantitis sites show that their microbiota differs significantly, with peri-implantitis microbiota presenting greater diversity,²⁰ including a wider list of statistically significant species related to peri-implant diseases.²³ Our results demonstrate that the submucosal microenvironment is promptly colonized by a wide variety of species, even when considering periodontally healthy individuals, suggesting that a preventive program for individuals receiving dental implants should be established promptly.

The linear regression analysis showed that oral species S. noxia and V. parvula were essential for the colonization differences found from day 07 to 120. An early colonizer, V. parvula is considered a beneficial species and is clustered in the purple complex, as described by Socransky and Haffajee.³⁹ S. noxia , in turn, does not cluster within any bacterial complex³⁹ and seems to be more associated with healthy peri-implant than peri-implantitis sites.⁴ Another study also found a negative correlation between S. noxia and clinical parameters in peri-implant evaluation.¹⁷ Moreover, its detection increases over time in the peri-implant site of newly installed implants.²⁷ Additional interesting data concerns the significant negative association between S. enterica subsp . enterica serovar Typhi and dental implants at 180 days. A previous research showed that the detection frequency and counts of this species tend to be higher in healthy sites compared to periodontitis sites, despite no significant differences detected.³⁵ S. enterica subsp . enterica serovar Typhi is a gram-negative diarrheic enteropathogen with a virulence factor that can be found in other bacteria, such as A. actinomycetemcomitans , the cytolethal distending toxin.⁴⁰ Most of the non-periodontal species analyzed here might be considered opportunistic pathogens and not oral cavity resident members. However, their clinical relevance and association with severe systemic infections justify further research on their interaction with periodontal and peri-implant health and disease.³⁵

Following previous studies,⁵ , ²⁵ we chose observation timepoints that would allow us to examine early submucosal colonization. At the 7-day timepoint, we were able to examine the immediate response of the host’s microbiota to the implant. By the 30-day timepoint, biofilm development is usually well underway, allowing us to investigate its composition and dynamics. Lastly, at the 120-day and 180-day timepoints we could glimpse the long-term effects of the implant on the microbiota. Biofilm is usually well-established by these later timepoints, and chronic interactions between the microbiota and the host have already occurred. Thus, these long-term observations can help us assess biofilm stability and evaluate any potential changes in microbial composition over time.²⁵ But differently from those studies, the set of microorganisms analyzed here included species that are not commonly associated with periodontal/peri-implant diseases. As oral microbiological knowledge and technology advances, new relevant species are unveiled. Since our study included a great variety of species related to diseases/conditions in other parts of the human organism, the current findings can be used by future research to help target species that may present a higher risk for peri-implant diseases but not yet associated due to a lack of studies and evidence.

Despite the well-established causal relationship between the presence of pathogenic biofilm in peri-implant sites and the onset of peri-implant diseases,¹ , ¹² , ²⁰ , ²³ its colonization mechanisms require better understanding, including the interaction between the biomaterial and the biofilm and inflammatory response.²² Regarding implant material, research shows that zirconia and titanium are equally colonized by the bacteria present in the remaining teeth.²² Importantly, we used specific genomic probes to investigate only target species, which does not exclude the possibility of other bacteria being also associated with dental implants.

Most of the cited studies, such as the present research, included non-smokers, which represents a very restricted portion of the population with dental implants. Moreover, our study population included only individuals with periodontal health and good plaque or dental biofilm control. Thus, future studies should investigate not only the microorganism-biomaterial interaction but also how this is modulated by host risk factors. Another study limitation concerns the impossibility of blinding the examiner and the individuals to the intervention. To minimize bias, members of our group without knowledge of the sample codes performed all microbiological and statistical analyses.

Overall, our findings corroborate the literature,⁵ , ²⁵ showing that submucosal sites are promptly colonized, even in individuals with good biofilm control. Hence, clinicians should provide proper care to the remaining teeth before considering dental implant installation and periodically monitor dental implants.

Conclusion

Early submucosal microbiota is diverse and only a few species differ from teeth and implants in the same individual. Only seven days after installation, a rich microbiota can be found in the peri-implant site. After a 6-month evaluation, teeth and implants show similar prevalence and levels of the target species, including known and new periodontopathic species.

Acknowledgments

This study was partially supported by the National Council for Scientific and Technological Development (CNPq), and the Coordination for the Improvement of Higher Education Personnel (CAPES), Brasilia, Brazil; and the Rio de Janeiro Research Support Foundation (FAPERJ; #E-26/202.775/2015), Rio de Janeiro, Brazil.

References

• ¹
  - Herrera D, Berglundh T, Schwarz F, Chapple I, Jepsen S, Sculean A, et al. Prevention and treatment of peri-implant diseases-The EFP S3 level clinical practice guideline. J Clin Periodontol. 2023;50 Suppl 26:4-76. doi: 10.1111/jcpe.13823
  » https://doi.org/10.1111/jcpe.13823
• ²
  - Berglundh T, Armitage G, Araujo MG, Avila-Ortiz G, Blanco J, Camargo PM, et al. Peri-implant diseases and conditions: Consensus report of workgroup 4 of the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions. J Clin Periodontol. 2018;45 Suppl 20:S286-S291. doi: 10.1111/jcpe.12957
  » https://doi.org/10.1111/jcpe.12957
• ³
  - Aoki M, Takanashi K, Matsukubo T, Yajima Y, Okuda K, Sato T, et al. Transmission of periodontopathic bacteria from natural teeth to implants. Clin Implant Dent Relat Res. 2012;14(3):406-11. doi: 10.1111/j.1708-8208.2009.00260.x
  » https://doi.org/10.1111/j.1708-8208.2009.00260.x
• ⁴
  - Shibli JA, Melo L, Ferrari DS, Figueiredo LC, Faveri M, Feres M. Composition of supra- and subgingival biofilm of subjects with healthy and diseased implants. Clin Oral Implants Res. 2008;19(10):975-82. doi: 10.1111/j.1600-0501.2008.01566.x
  » https://doi.org/10.1111/j.1600-0501.2008.01566.x
• ⁵
  - Quirynen M, Vogels R, Peeters W, van Steenberghe D, Naert I, Haffajee A. Dynamics of initial subgingival colonization of ‘pristine’ peri-implant pockets. Clin Oral Implants Res. 2006;17(1):25-37. doi: 10.1111/j.1600-0501.2005.01194.x
  » https://doi.org/10.1111/j.1600-0501.2005.01194.x
• ⁶
  - Kumar PS, Mason MR, Brooker MR, O’Brien K. Pyrosequencing reveals unique microbial signatures associated with healthy and failing dental implants. J Clin Periodontol. 2012;39(5):425-33. doi: 10.1111/j.1600-051X.2012.01856.x
  » https://doi.org/10.1111/j.1600-051X.2012.01856.x
• ⁷
  - Mombelli A, van Oosten MA, Schurch E Jr, Land NP. The microbiota associated with successful or failing osseointegrated titanium implants. Oral Microbiol Immunol. 1987;2(4):145-51. doi: 10.1111/j.1399-302x.1987.tb00298.x
  » https://doi.org/10.1111/j.1399-302x.1987.tb00298.x
• ⁸
  - Kröger A, Hulsmann C, Fickl S, Spinell T, Huttig F, Kaufmann F, et al. The severity of human peri-implantitis lesions correlates with the level of submucosal microbial dysbiosis. J Clin Periodontol. 2018;45(12):1498-509. doi: 10.1111/jcpe.13023
  » https://doi.org/10.1111/jcpe.13023
• ⁹
  - Bauman GR, Rapley JW, Hallmon WW, Mills M. The peri-implant sulcus. Int J Oral Maxillofac Implants. 1993;8(3):273-80.
• ¹⁰
  - Jepsen S, Ruhling A, Jepsen K, Ohlenbusch B, Albers HK. Progressive peri-implantitis: incidence and prediction of peri-implant attachment loss. Clin Oral Implants Res. 1996;7(2):133-42. doi: 10.1034/j.1600-0501.1996.070207.x
  » https://doi.org/10.1034/j.1600-0501.1996.070207.x
• ¹¹
  - Karoussis IK, Salvi GE, Heitz-Mayfield LJ, Bragger U, Hammerle CH, Lang NP. Long-term implant prognosis in patients with and without a history of chronic periodontitis: a 10-year prospective cohort study of the ITI Dental Implant System. Clin Oral Implants Res. 2003;14(3):329-39. doi: 10.1034/j.1600-0501.000.00934.x
  » https://doi.org/10.1034/j.1600-0501.000.00934.x
• ¹²
  - Renvert S, Roos-Jansaker AM, Lindahl C, Renvert H, Persson GR. Infection at titanium implants with or without a clinical diagnosis of inflammation. Clin Oral Implants Res. 2007;18(4):509-16. doi: 10.1111/j.1600-0501.2007.01378.x
  » https://doi.org/10.1111/j.1600-0501.2007.01378.x
• ¹³
  - Sanz-Martin I, Doolittle-Hall J, Teles RP, Patel M, Belibasakis GN, Hammerle CH, et al. Exploring the microbiome of healthy and diseased peri-implant sites using Illumina sequencing. J Clin Periodontol. 2017;44(12):1274-84. doi: 10.1111/jcpe.12788
  » https://doi.org/10.1111/jcpe.12788
• ¹⁴
  - Quirynen M, Vogels R, Pauwels M, Haffajee AD, Socransky SS, Uzel NG, et al. Initial subgingival colonization of ‘pristine’ pockets. J Dent Res. 2005;84(4):340-4. doi: 10.1177/154405910508400409
  » https://doi.org/10.1177/154405910508400409
• ¹⁵
  - Stokman MA, van Winkelhoff AJ, Vissink A, Spijkervet FK, Raghoebar GM. Bacterial colonization of the peri-implant sulcus in dentate patients: a prospective observational study. Clin Oral Investig. 2017;21(2):717-24. doi: 10.1007/s00784-016-1941-x
  » https://doi.org/10.1007/s00784-016-1941-x
• ¹⁶
  - Shimogishi M, Watanabe T, Shibasaki M, Shiba T, Komatsu K, Nemoto T, et al. Patient-specific establishment of bacterial composition within the peri-implant microbiota during the earliest weeks after implant uncovering. J Periodontal Res. 2021;56(5):964-71. doi: 10.1111/jre.12898
  » https://doi.org/10.1111/jre.12898
• ¹⁷
  - Maruyama N, Maruyama F, Takeuchi Y, Aikawa C, Izumi Y, Nakagawa I. Intraindividual variation in core microbiota in peri-implantitis and periodontitis. Sci Rep. 2014;4:6602. doi: 10.1038/srep06602
  » https://doi.org/10.1038/srep06602
• ¹⁸
  - Polymeri A, van der Horst J, Buijs MJ, Zaura E, Wismeijer D, Crielaard W, et al. Submucosal microbiome of peri-implant sites: a cross-sectional study. J Clin Periodontol. 2021;48(9):1228-39. doi: 10.1111/jcpe.13502
  » https://doi.org/10.1111/jcpe.13502
• ¹⁹
  - Zheng H, Xu L, Wang Z, Li L, Zhang J, Zhang Q, et al. Subgingival microbiome in patients with healthy and ailing dental implants. Sci Rep. 2015;5:10948. doi: 10.1038/srep10948
  » https://doi.org/10.1038/srep10948
• ²⁰
  - Gazil V, Bandiaky ON, Renard E, Idiri K, Struillou X, Soueidan A. Current data on oral peri-implant and periodontal microbiota and its pathological changes: a systematic review. Microorganisms. 2022;10(12):2466. doi: 10.3390/microorganisms10122466
  » https://doi.org/10.3390/microorganisms10122466
• ²¹
  - Yu XL, Chan Y, Zhuang L, Lai HC, Lang NP, Leung WK, et al. Intra-oral single-site comparisons of periodontal and peri-implant microbiota in health and disease. Clin Oral Implants Res. 2019 Aug; 30(8):760-776. doi: 10.1111/clr.13459.
  » https://doi.org/10.1111/clr.13459
• ²²
  - Freitas AR, Silva TS, Ribeiro RF, Albuquerque RF Junior, Pedrazzi V, Nascimento C. Oral bacterial colonization on dental implants restored with titanium or zirconia abutments: 6-month follow-up. Clin Oral Investig. 2018;22(6):2335-43. doi: 10.1007/s00784-018-2334-0
  » https://doi.org/10.1007/s00784-018-2334-0
• ²³
  - Retamal-Valdes B, Formiga MC, Almeida ML, Fritoli A, Figueiredo KA, Westphal M, et al. Does subgingival bacterial colonization differ between implants and teeth? A systematic review. Braz Oral Res. 2019;33(suppl 1):e064. doi: 10.1590/1807-3107bor-2019.vol33.0064
  » https://doi.org/10.1590/1807-3107bor-2019.vol33.0064
• ²⁴
  - Hartenbach F, Silva-Boghossian CM, Colombo AP. The effect of supragingival biofilm re-development on the subgingival microbiota in chronic periodontitis. Arch Oral Biol. 2018;85:51-7. doi: 10.1016/j.archoralbio.2017.10.007
  » https://doi.org/10.1016/j.archoralbio.2017.10.007
• ²⁵
  - van Winkelhoff AJ, Goene RJ, Benschop C, Folmer T. Early colonization of dental implants by putative periodontal pathogens in partially edentulous patients. Clin Oral Implants Res. 2000;11(6):511-20. doi: 10.1034/j.1600-0501.2000.011006511.x
  » https://doi.org/10.1034/j.1600-0501.2000.011006511.x
• ²⁶
  - Socransky SS, Smith C, Martin L, Paster BJ, Dewhirst FE, Levin AE. “Checkerboard” DNA-DNA hybridization. Biotechniques. 1994;17(4):788-92.
• ²⁷
  - Fürst MM, Salvi GE, Lang NP, Persson GR. Bacterial colonization immediately after installation on oral titanium implants. Clin Oral Implants Res. 2007;18(4):501-8. doi: 10.1111/j.1600-0501.2007.01381.x
  » https://doi.org/10.1111/j.1600-0501.2007.01381.x
• ²⁸
  - Mombelli A. Microbiology and antimicrobial therapy of peri-implantitis. Periodontol 2000. 2002; 28:177-89. doi: 10.1034/j.1600-0757.2002.280107.x.
  » https://doi.org/10.1034/j.1600-0757.2002.280107.x
• ²⁹
  - Schaumann S, Staufenbiel I, Scherer R, Schilhabel M, Winkel A, Stumpp SN, et al. Pyrosequencing of supra- and subgingival biofilms from inflamed peri-implant and periodontal sites. BMC Oral Health. 2014;14:157. doi: 10.1186/1472-6831-14-157
  » https://doi.org/10.1186/1472-6831-14-157
• ³⁰
  - Persson GR, Renvert S. Cluster of bacteria associated with peri-implantitis. Clin Implant Dent Relat Res. 2014;16(6):783-93. doi: 10.1111/cid.12052
  » https://doi.org/10.1111/cid.12052
• ³¹
  - Socransky SS, Haffajee AD, Cugini MA, Smith C, Kent RL Jr. Microbial complexes in subgingival plaque. J Clin Periodontol. 1998;25(2):134-44. doi: 10.1111/j.1600-051x.1998.tb02419.x
  » https://doi.org/10.1111/j.1600-051x.1998.tb02419.x
• ³²
  - Araujo LL, Lourenco TG, Colombo AP. Periodontal disease severity is associated to pathogenic consortia comprising putative and candidate periodontal pathogens. J Appl Oral Sci. 2023;31:e20220359. doi: 10.1590/1678-7757-2022-0359
  » https://doi.org/10.1590/1678-7757-2022-0359
• ³³
  - Goncalves C, Soares GM, Faveri M, Perez-Chaparro PJ, Lobao E, Figueiredo LC, et al. Association of three putative periodontal pathogens with chronic periodontitis in Brazilian subjects. J Appl Oral Sci. 2016;24(2):181-5. doi: 10.1590/1678-775720150445
  » https://doi.org/10.1590/1678-775720150445
• ³⁴
  - Souto R, Silva-Boghossian CM, Colombo AP. Prevalence of Pseudomonas aeruginosa and Acinetobacter spp. in subgingival biofilm and saliva of subjects with chronic periodontal infection. Braz J Microbiol 2014;45(2):495-501. doi: 10.1590/S1517-83822014000200017
  » https://doi.org/10.1590/S1517-83822014000200017
• ³⁵
  - Colombo AP, Magalhães CB, Hartenbach FA, Souto RM, Silva-Boghossian CM. Periodontal-disease-associated biofilm: a reservoir for pathogens of medical importance. Microb Pathog. 2016;94:27-34. doi: 10.1016/j.micpath.2015.09.009
  » https://doi.org/10.1016/j.micpath.2015.09.009
• ³⁶
  - Charalampakis G, Leonhardt A, Rabe P, Dahlen G. Clinical and microbiological characteristics of peri-implantitis cases: a retrospective multicentre study. Clin Oral Implants Res. 2012;23(9):1045-54. doi: 10.1111/j.1600-0501.2011.02258.x
  » https://doi.org/10.1111/j.1600-0501.2011.02258.x
• ³⁷
  - Tamrakar AK, Murali G, Singh S, Shakila R. Evaluation of subgingival microbiota around single tooth implants. J Oral Biol Craniofac Res. 2020;10(2):180-3. doi: 10.1016/j.jobcr.2020.03.012
  » https://doi.org/10.1016/j.jobcr.2020.03.012
• ³⁸
  - Sousa V, Nibali L, Spratt D, Dopico J, Mardas N, Petrie A, et al. Peri-implant and periodontal microbiome diversity in aggressive periodontitis patients: a pilot study. Clin Oral Implants Res. 2017;28(5):558-70. doi: 10.1111/clr.12834
  » https://doi.org/10.1111/clr.12834
• ³⁹
  - Socransky SS, Haffajee AD. Periodontal microbial ecology. Periodontol 2000. 2005;38:135-87. doi: 10.1111/j.1600-0757.2005.00107.x
  » https://doi.org/10.1111/j.1600-0757.2005.00107.x
• ⁴⁰
  - Smith JL, Bayles DO. The contribution of cytolethal distending toxin to bacterial pathogenesis. Crit Rev Microbiol. 2006;32(4):227-48. doi: 10.1080/10408410601023557
  » https://doi.org/10.1080/10408410601023557

• *
  This study is derived from a Master’s thesis of the Postgraduate Program in Dentistry at UNIGRANRIO.

• Data availability statement

  The datasets generated and analyzed during the current study are available in the SciELO Data repository, https://doi.org/10.48331/scielodata.CIQAKP

Edited by

Editor: Linda Wang

Data availability

The datasets generated and analyzed during the current study are available in the SciELO Data repository, https://doi.org/10.48331/scielodata.CIQAKP

Publication Dates

• Publication in this collection
  15 Sept 2023
• Date of issue
  2023

History

• Received
  13 Apr 2023
• Reviewed
  5 July 2023
• Accepted
  7 Aug 2023