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Guiding Periodontal Pocket Recolonization: a Proof of Concept
1 Catholic University Leuven, Research Group for Microbial Adhesion, Department of Periodontology, Kapucijnenvoer 7, 3000 Leuven, Belgium; Correspondence: * corresponding author, Wim.Teughels{at}med.kuleuven.be
The complexity of the periodontal microbiota resembles that of the gastro-intestinal tract, where infectious diseases are treatable via probiotics. In the oropharyngeal region, probiotic or replacement therapies have shown some benefit in the prevention of dental caries, otitis media, and pharyngitis, but their effectiveness in the treatment of periodontitis is unknown. Therefore, this study addressed the hypothesis that the application of selected beneficial bacteria, as an adjunct to scaling and root planing, would inhibit the periodontopathogen recolonization of periodontal pockets. Analysis of the data showed, in a beagle dog model, that when beneficial bacteria were applied in periodontal pockets adjunctively after root planing, subgingival recolonization of periodontopathogens was delayed and reduced, as was the degree of inflammation, at a clinically significant level. The study confirmed the hypothesis and provides a proof of concept for a guided pocket recolonization (GPR) approach in the treatment of periodontitis.
Key Words: probiotics • periodontitis • therapy • microbial interference • replacement therapy • treatment
Periodontitis painlessly destroys the supporting tissues around teeth, and increases the risk for atherosclerosis (Engebretson et al., 2005) or low-birthweight babies (Lopez et al., 2002). It develops when the equilibrium between microbial load and host defense mechanisms is disturbed in the periodontal tissues (Taubman et al., 2005). Given the risk of serious side-effects associated with altering the host response (e.g., COX-2 inhibitors), treatment of periodontitis focuses on the reduction of the bacterial threat (Salvi and Lang, 2005). Conventional treatment involves mechanical subgingival debridement, which results in 2-to 3-log10 reductions in the total subgingival microbiota (Maiden et al., 1991; Rhemrev et al., 2006). However, recolonization toward pre-treatment levels, primarily by bacteria less strongly implicated as periodontopathogens, occurs within weeks (Harper and Robinson, 1987), and re-establishment of a more pathogenic microbiota occurs within months (Magnusson et al., 1984). Adjunctive use of local or systemic antibiotics and antiseptics improves the outcome of periodontal therapy only temporarily (Quirynen et al., 2002). Thus, a life-long need for (re)treatment arises, creating a serious socio-economic problem. Additionally, increasing levels of antibiotic-resistant bacteria favor the development of approaches that do not rely on antibiotics. This study tested the hypothesis that the subgingival application of beneficial bacteria after mechanical debridement would enhance the microbial shift away from periodontopathogens. The beneficial bacteria used were selected because of their ability to prevent colonization of hard tissues and epithelial cells by periodontopathogens (Teughels et al., 2007; Van Hoogmoed et al., 2007), and their ability to prevent other mucosal infections, including pharyngitis (Falck et al., 1999) and acute otitis media (Roos et al., 2001) in vivo.
Subjects Eight male beagle dogs with an average age of 3.08 (± 0.37) yrs were used in this split-mouth, double-blind, randomized trial. The protocol was approved by the University’s Ethical Committee for Animal Experimentation. Surgical pocket creation, scaling, and root planing were performed after the animals were anesthetized by an intravenous injection of thiopental sodium (Abbott Laboratories, Leuven, Belgium), and carprofen (Pfizer, Luxembourg, Belgium) was given as a painkiller. All other manipulations were performed with the animals under sedation by medetomidine hydrochloride (Pfizer, Belgium) and ketamine (Eurovet Animal Health, Bladel, The Netherlands). Buprenorfine hydrochloride (Schering-Plough, Luxembourg, Belgium) was administrated as a painkiller. None of the dogs received antibiotics prior to or during the course of the study.
Periodontal Pockets
Experimental Design
Bacterial Mixture
Microbiological Parameters The samples were cultured under aerobic (3 days) and anaerobic conditions (10% H2, 80% N2, and 10% CO2; 7 days) in non-selective (aerobic and anaerobic counts, black-pigmented bacteria, Porphyromonas gulae, and Prevotella intermedia) and selective media [Hammond medium for the detection of Campylobacter rectus (Hammond and Mallonee, 1988), Trypticase-Soy-Bacitracin-Vancomycin medium for Aggregatibacter actinomycetemcomitans (Slots, 1982), and Crystal-Violet-Erythromycin medium for the detection of Fusobacterium nucleatum (Walker et al., 1979)]. Specific colony morphologies suggestive for P. gulae and P. intermedia were pure-cultured and verified via a series of biochemical tests (diagnostic tablets for microbial identification, International Medical, Brussels, Belgium). A pilot study on 100 dogs with naturally occurring periodontitis indicated that the species resembling Porphyromonas gingivalis in reality (after DNA sequencing) represents P. gulae [i.e., the canine form of P. gingivalis (Harvey et al., 1995)]. Details concerning the growth conditions, colony selection, and final identification of specific species have been summarized previously (Quirynen et al., 1999). All microbiological evaluations were performed blind.
Periodontal Parameters
Statistical Analyses
Microbiological Parameters There were no significant inter-group differences in total anaerobic (6.8 ± 0.1 log10 CFU/mL), black-pigmented bacteria (5.9 ± 0.1 log10 CFU/mL) and aerobic (5.3 ± 0.1 log10 CFU/mL) counts at baseline. After treatment, the shift in anaerobic species (p < 0.0001) and black-pigmented bacteria (p < 0.001) differed among strategies (Fig., A,B,C  ). Nc showed negligible changes, whereas all other treatments reduced (p < 0.001) the anaerobic species and black-pigmented bacteria. Rp reduced the levels of anaerobic species and black-pigmented bacteria, but a re-emergence reaching baseline levels at week 12 took place. Rpsingle demonstrated a lesser tendency for reemergence of anaerobic species when compared with Rp. Rpmulti showed the most pronounced reduction in anaerobic species and black-pigmented bacteria, and maintained the reduced levels over the entire study period. The Rpmulti strategy was superior to both Rp for anaerobic species and black-pigmented bacteria (p = 0.002 and p < 0.001) and Rpsingle (p = 0.03 and p < 0.001). The changes in the total number of aerobic species (Fig., C), generally regarded as host-compatible species, were minimal in all treatments, although they tended to be elevated at week 12 for Rpsingle and Rpmulti.
At baseline, there were no significant inter-group differences for P. gulae (5.8 ± 0.1 log10 CFU/mL), P. intermedia (5.4 ± 0.1 log10 CFU/mL), and C. rectus (5.2 ± 0.1 log10 CFU/mL). In the Nc sites, the changes in bacterial counts were negligible (Fig., D,E,F  ), with only a temporary slight reduction in C. rectus. Rp resulted in small, temporary reductions of P. gulae (p = 0.001) and P. intermedia (p = 0.001), but not for C. rectus (p = 0.17). Rpsingle and Rpmulti resulted in significant reductions for all 3 species (p < 0.001, P. gulae and P. intermedia; and p = 0.001, C. rectus). In comparison with Rp, Rpsingle resulted in minor additional reductions (p > 0.05), whereas Rpmulti showed clearly reduced levels of P. gulae (p < 0.001) and P. intermedia (p = 0.02). Compared with Rpsingle, Rpmulti resulted in additional reductions for P. gulae (p = 0.002) and P. intermedia (p = 0.05). F. nucleatum, A. actinomycetemcomitans, and T. forsythia were never detected.
Periodontal Parameters
’Replacement therapy’, also known as ’probiotic therapy’, originated over a century ago, but was largely abandoned after the discovery of antibiotics (Wilson, 2005). However, the emerging problem of antibiotic resistance has led to renewed interest in bacterial replacement. The approach is recognized in the management of gastro-intestinal diseases, and encouraging reports have shown possibilities in the oto-oropharyngeal area (Falck et al., 1999; Roos et al., 2001). Studies on replacement therapy for caries prevention (for review, see Meurman, 2005) have revealed promising results with the use of a genetically modified Streptococcus mutans strain (Hillman, 2002). Our study is the first to test the concept of bacterial replacement therapy in the treatment of plaque-related periodontal disease. This study assessed quantitative changes in the subgingival microbiota after root planing when beneficial bacteria were applied adjunctively. To challenge the hypothesis of a prolonged beneficial microbial shift after mechanical debridement by deliberate application of beneficial species, we established a reservoir for recolonization (Quirynen et al., 2001), by leaving teeth that were not included in the study unaffected, and by incorporating a negative control. To promote recolonization of periodontopathogens, no oral hygiene was performed (Magnusson et al., 1984). Because ethical considerations imply the performance of oral hygiene after periodontal therapy in humans, a comparison between the obtained microbiological data in this study and the current literature is difficult. The present findings confirmed a rapid recolonization of debrided periodontal pockets, often reaching pre-treatment levels at week 12 (Magnusson et al., 1984). Although application of beneficial bacteria did not exclude pathogen recolonization, it did delay the recolonization process significantly. Inoculation of beneficial bacteria immediately after root planing, and especially with additional inoculations during the recolonization process, significantly lowered bacterial counts for all monitored pathogens. The differences between Rp and Rpmulti, at week 12 for anaerobic, P. gulae, and P. intermedia species, were identical or exceeded differences reported in similar, human, split-mouth studies where local antiseptics or antibiotics were used as adjuncts to root planing (Quirynen et al., 2002). It is known that the subgingival microbial profile is related to pocket depth (Socransky and Haffajee, 2005). Therefore, in some of these studies, microbiological differences among treatments might have originated from differences in the space available for recolonization due to differences in pocket depth reduction. The absence of significant differences in pocket depth reduction in our study excludes pocket depth differences as a confounding factor for the interpretation of the microbial changes. This makes the microbiological results even more interesting. It can be criticized that no placebo control group was included in this study. However, there was, in our opinion, no correct placebo for the pure bacterial pellets used, as long as the exact mode of interference for the species was unknown. Additionally, because sampling the subgingival microbiota implied a disruption of the subgingival ecology (Mousques et al., 1980), it is conceivable that the repeated applications of the bacterial pellets after sample-taking in the Rpmulti group did not have a major additional impact on the microbial profiles. Moreover, even in the weeks following the last application (week 4), the microbiota in the Rpmulti group remained significantly different from that in the other groups. We cannot exclude the possibility that the beneficial bacteria applied translocated to control sites, or that their application induced an immunological response that interfered with the recolonization of the control sites. A recent investigation of the effects of gastro-intestinal probiotics has attributed the beneficial gastr-intestinal response to immunomodulation (Madsen, 2006). However, as evidenced by the colonization pattern of C. rectus in the Nc group in comparison with the Rp group, the beneficial effect appears to relate to a retarded recolonization. Clinically, it is well-known that the re-emergence of periodontopathogens is correlated with lack of clinical improvement and the risk for disease relapse (Haffajee et al., 1997). Although not statistically significant, the post-treatment attachment level was slightly lower for the treatments that included the application of beneficial species. The limited clinical improvement can be attributed to the absence of oral hygiene. Interestingly, this improved response also applied to the proportion of sites that exhibited bleeding on probing, a clinical marker for subgingival inflammation. The difference between Rp and Rpmulti for this latter variable was statistically significant. This observation supports the concept that the application of beneficial bacteria can lead to a more host-compatible subgingival microbiota. The questions of whether the applied species really colonized the subgingival habitat, and how the prolongation of the microbial shift was induced, remain unsolved. However, re-application of the beneficial bacteria seemed to improve the microbiological outcome of the treatment. In addition, it has been well-established for probiotics in the gastro-intestinal tract that they usually colonize for a short time, or even not at all (for review, see Wilson, 2005). Therefore, extrapolating probiotic colonization behavior on mucosal surfaces, such as in the intestine, to the periodontal pocket and its close association with the non-shedding, biofilm-prone root surface might be difficult or even impossible. The mechanisms behind the successful inhibition of periodontopathogen (re-)colonization remain hypothetical. Occupation or a physico-chemical alteration of the subgingival niche (Teughels et al., 2007), competition for essential nutrients (Sanders, 1969), inhibition of the viability or growth of pathogens (Wilson, 2005), and modification of the production or degradation of virulence factors of pathogens or immune responses are possible underlying mechanisms (Yasui et al., 1999). In summary, these results show that application of beneficial bacteria as an adjunct to root planing is a valid, non-antibiotic treatment approach for periodontitis. Given the emergence of antibiotic resistance and the lack of non-antibiotic treatment options, this Guided Pocket Recolonization approach may provide a valuable addition or alternative to the armamentarium of treatment options for periodontitis.
This study was supported by the NIDCR (Bethesda, MD, USA), grant DE015360 (Principal Investigator Marc Quirynen), by the National Fund for Scientific Research (Brussels, Belgium), grant G0240.04, and by the Catholic University Leuven (Leuven, Belgium), grant OT03/52. This paper is based on a thesis submitted to the Catholic University of Leuven in partial fulfilment of the requirements for the PhD degree (W. Teughels). Received for publication December 2, 2006. Revision received June 7, 2007. Accepted for publication June 7, 2007.
Journal of Dental Research, Vol. 86, No. 11,
1078-1082 (2007) This article has been cited by other articles:
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