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Initial Subgingival Colonization of ‘Pristine’ Pockets
1 Department of Periodontology, Correspondence: * *corresponding author, Marc.Quirynen{at}med.kuleuven.ac.be
The treatment of periodontitis/peri-implantitis involves the reduction/eradication of periopathogens. After therapy, beneficial and pathogenic species recolonize the subgingival area. The dynamics of recolonization and especially the role of the supragingival environment in this process are still not well-understood. This prospective, split-mouth study followed the early colonization of ‘pristine’ pockets created during implant surgery (16 partially edentulous patients), to record the time needed before a complex subgingival flora could be established with the supragingival area as the single source. Four subgingival plaque samples were taken from shallow and medium pockets around implants (test), and neighboring teeth (undisturbed microbiota as reference) 1, 2, and 4 wks after abutment connection. Checkerboard DNA-DNA hybridization and culture data revealed a complex microbiota (including several pathogenic species) in the pristine pockets within a wk, with a minimal increase in counts up to 4 wks. Analysis of these data demonstrated that, even with the supragingival environment as the single source for colonizing bacteria, a complex subgingival microbiota can develop within 1 wk.
Key Words: biofilm • colonization • dental plaque • peri-implantitis • microbiology • plaque growth • periodontitis • clinical dental implants
Actinobacillus actinomycetemcomitans, Tannerella forsythensis, and Porphyromonas gingivalis are considered key pathogens in periodontitis and peri-implantitis. Other species have been suggested as possible pathogens for these conditions including: Prevotella intermedia, Campylobacter rectus, Peptostreptococcus micros, Fusobacterium nucleatum, Eubacterium nodatum, Streptococcus intermedius, and spirochetes. Re-establishment of these pathogens after periodontal treatment is associated with a negative clinical outcome (Renvert et al., 1998; Cugini et al., 2000; Quirynen et al., 2002; Socransky and Haffajee, 2002). The dynamics of the microbiological shifts during the first weeks after periodontal therapy are not well-understood. Some early studies, where culture techniques were used, examined the changes within the subgingival microbiota during the first wk after mechanical debridement and reported an immediate reduction around 3 logs, followed by a re-growth toward nearly pre-treatment levels (– 0.5 log) within 7 days (Harper and Robinson, 1987; Goodson et al., 1991; Maiden et al., 1991). The fast recolonization was explained by several factors. A critical review of the effectiveness of subgingival debridement, for instance, revealed that a high proportion of treated tooth surfaces (from 5 to 80%) still harbored plaque and/or calculus (for review, see Petersilka et al., 2002). These remaining bacteria were considered the primary source for the subgingival recolonization. Other pathogens penetrate the soft tissues (Rudney et al., 2001) or the dentinal tubules (Giuliana et al., 1997), and may escape instrumentation. The impact of the supragingival environment (saliva, supragingival plaque, oral soft tissues) on subgingival recolonization after periodontal therapy is still controversial (Petersilka et al., 2002). Debridement without proper plaque control is clinically not very effective in comparison, for example, with repeated professional supragingival plaque control (for review, see Socransky and Haffajee, 2002). Thus, it may be speculated that part of the subgingival recolonization originates from the supragingival environment.
Oral implants provide a unique opportunity for the observation of initial subgingival colonization patterns, since one is starting with a ‘pristine’ bacteria-free surface/pocket. In the present investigation, we recorded the development of the ‘initial’ subgingival biofilm on implants with shallow (
Subjects Sixteen Caucasians (from 35 to 76 yrs of age; mean, 54.8 yrs; eight females; two smokers) volunteered for this prospective, single-blind, split-mouth study. They had previously received at least 2 two-stage Brånemark system® implants (Nobel Biocare®, Gothenburg, Sweden) to treat their partial edentulism. All subjects were in good general health, and none had used any antimicrobials during the 3 mos prior to the study. Most patients had received a mechanical debridement and thorough oral hygiene instructions prior to implant insertion. Some of them even went through surgical pocket elimination to achieve periodontal health. All participants signed informed consent. The protocol had been approved by the Clinical Trials Committee of the University Hospital of the Catholic University Leuven.
Experimental Design
Periodontal Parameters The teeth sites were selected on the basis of the probing data from the last follow-up visit (some mos prior to implant surgery). Their exact pocket depth was scored at the end of the four-week study period.
Microbiological Parameters Paperpoints for the checkerboard were placed in separate Eppendorf tubes containing 0.15 mL TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.6) to which 0.15 mL of 0.5 M NaOH was added for fixation. The DNA-DNA hybridization was completed at The Forsyth Institute. [For details about the technology, see Socransky et al.(1994).] The paperpoints for culturing were dispersed in Reduced Transport Fluid (Syed and Loesche, 1973), homogenized by vortexing for 30 sec, and transferred to the microbiology laboratory and processed within less than 24 hrs. Dilutions 10–1 to 10–5 were plated in duplicate onto non-selective blood agar plates (Blood Agar Base II®, Oxoid, Basingstoke, UK), supplemented with hemin (5 mg/L), menadione (1 mg/L), and 5% sterile horse blood. After 7 days of anaerobic (80% N2, 10% CO2, and 10% H2) and aerobic incubation at 37°C, respectively, the total numbers of colony-forming units (CFU/mL) were counted. All microbiological evaluations were performed blind. Details are summarized in a previous paper (Quirynen et al., 1995).
Data Analysis
Clinical Parameters The mean probing depth for the sampled sites around teeth was 2.7 mm for shallow and 4.7 mm for the moderate pockets, respectively. The average distance between bone level and the gingival margin around the abutments was, respectively, 3.7 and 4.6 mm, for thin and thick gingivae. At a recent follow-up visit, the probing depths of the selected peri-implant pockets were found to be 2.3 and 3.2 mm, respectively. Thus, moderate pockets around the implants were shallower than the moderate pockets around the teeth.
Detection Frequencies
Bacterial Counts At 1, 2, and 4 wks, the red and orange complex species exhibited higher mean counts in samples from the teeth, particularly those with > 4-mm pockets, when compared with the implants (Fig. 2  ). After adjustment for multiple comparisons, significant differences among sample locations were observed for F. nucleatum subspecies, P. micros, P. nigrescens, T. forsythensis, and P. gingivalis at some or all time-points (Fig. 2). When only the shallow implant and teeth pockets were considered, the significance of differences disappeared. There was no significant change in the counts of any of the test species over time at each of the 4 sample locations.
The numbers of CFU/mL in the aerobic and anaerobic cultures (week 2) were 6.4 x 105 and 1.8 x 106 for implants vs. 3.1 x 105 and 1.1 x 106 for teeth with shallow pockets, respectively. For the medium pockets, the data were 2.2 x 106 and 3.2 x 106 for implants, and 1.2 x 106 and 7.6 x 106 for teeth, respectively.
The present investigation demonstrated that a complex subgingival microbiota can be established in 1 wk. This microbiota appeared at peri-implant sites whether they were of shallow or intermediate depth. This finding is of considerable interest, since the experimental design eliminated possible sources of infection that confound recolonization studies performed on natural tooth surfaces. For example, there were no dentinal tubules harboring potential recolonizing species, no species resident within the adjacent pocket epithelium, and no bacterial cells left behind after the tooth surface was scaled. Since these sources of infection were ruled out in the ‘pristine’ pockets of the newly inserted abutment, one must conclude that the source of infecting bacteria was the saliva (representing the microbial load in the remaining dentition) or supragingival plaque that had accumulated on the implant surfaces. One of the most intriguing aspects of this study was the speed by which colonization occurred, and the diversity of species that could be detected within 1 wk. Species were detected in similar frequencies from the one-week microbiota around implants and in the undisturbed subgingival plaque of shallow tooth sites. The numbers (i.e., the DNA probe counts) of many red and orange complex species, however, were higher for tooth sites, particularly those with pockets > 4 mm. This suggests that species of these 2 communities may take longer to establish than the other species that were examined. This might indicate that some taxa are able to colonize a ‘pristine’ site without prior colonization of other species, while others, such as those of the red and orange complexes, may require the establishment of appropriate conditions by pioneer species to become established and eventually predominate. The rapid rate at which a subgingival microbiota can be established with the supragingival environment as the single source of colonizing species makes it reasonable to expect that it will also play a significant role in the recolonization of periodontal pockets—for example, after mechanical debridement or chemical disinfection. This hypothesis is supported by several recent observations, including the significant clinical and microbiological benefits of a one-stage, full-mouth disinfection during non-surgical therapy of periodontitis (for review, see Quirynen et al., 2001) or prior to guided pocket regeneration (Nowzari et al., 1996) or the local application of antibiotics (Mombelli et al., 1997). The fact that this recolonization could occur while subjects rinsed with a 0.2% chlorhexidine digluconate rinse is surprising, since this antiseptic is considered the ‘gold standard’ for mouthrinses. This may have occurred because chlorhexidine failed to reach the subgingival area, and organisms gaining entrance to this area were not affected. The latter seems to indicate that mouthrinsing gives only temporary (not 24-hours/day) protection, and that a subgingival plaque can be established without obvious supragingival plaque formation. Since there was no control group whose members did not rinse with chlorhexidine, we cannot know whether the chlorhexidine rinses slowed colonization of pristine sites. Another observation in this study was the low levels of Actinomyces species compared with other species in the samples from all locations. Actinomyces species are commonly the most dominant species in both supra- and subgingival plaque samples from both periodontally healthy and periodontitis subjects (Ximenez-Fyvie et al., 2000). It has recently been recognized that there are differences in microbial profiles in subgingival samples from subjects in different geographic locations (Haffajee et al., 2004). Thus, the low levels of Actinomyces species might be accounted for by different subject populations. Another possibility is that the low levels of Actinomyces reflect differences in the sampling technique. Most studies using the checkerboard hybridization technique use a scaler to take the samples. The current investigation used paperpoints to minimize damage to the peri-implant area. This sampling technique may enrich the unattached or epithelial attached plaque in which red and orange complex species predominate. Scaler samples may enrich the tooth-associated species, particularly the Actinomyces. The differences between recoveries using these 2 techniques are being directly tested. The present observation of colonization by a complex microbiota within days of a sterile hard surface being placed subgingivally was unexpected. High numbers of organisms were detected by both culture and DNA probe techniques. The reason for the rapid re-colonization in the ‘pristine’ environment is not clear. It is possible that the blood coagulum at the fresh implant sites may favor the colonization and growth of oral species in a fashion similar to that which might occur after mechanical debridement of periodontal pockets. Alternatively, the large numbers of organisms in saliva and on the oral soft tissues, particularly the tongue, and the rapid multiplication rate of bacteria may be sufficient for many species to establish and reach sizeable numbers in the absence of the competing microbiota. The factors that ‘regulate’ recolonization are not fully understood and deserve further investigation.
This study was supported by a grant from the ITI Research foundation (Switzerland), a grant from the Catholic University Leuven (OT/03/52), and by research grant DE14368 from the National Institute of Dental and Craniofacial Research, USA. Received for publication June 29, 2004. Revision received November 24, 2004. Accepted for publication January 13, 2005.
Journal of Dental Research, Vol. 84, No. 4,
340-344 (2005) This article has been cited by other articles:
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ABSTRACT
INTRODUCTION
3 mm) and moderate (> 3 mm) pockets, to estimate the time needed before a complex subgingival flora could be established with the supragingival area as the single source. The undisturbed subgingival microbiota of neighboring teeth in the same individuals served as controls. 
