A summary of representative experiments demonstrating that chemical approach failed to detach the biofilm structure.
Abstract
Antimicrobial measures, such as topical antiseptics and local drug delivery, have proven effective as complements to mechanical control. However, recent investigations have reported some adverse influences of antimicrobial strategy.
Keywords
- oral biofilm
- antimicrobial agent
- residual structure
- sub-MIC
- stress response
1. Introduction
Mechanical approach by procedures such as self-performed oral hygiene, scaling and root planning (SRP), or periodontal surgery is fundamental in the control of mature oral biofilms [1]. Chemical approaches such as topical antiseptics, local drug delivery, and systemic antibiotics are used with the expectation of producing an adjunctive effect [2‒5]. In fact, it has been demonstrated that adjunctive antimicrobials improve clinical parameters, including plaque index, gingival inflammation, and probing pocket depth [3, 5‒7]. It has also been reported that antiplaque biocides do not cause the microbial resistance and alterations of microbial flora [8].
However, recent investigations have demonstrated that antimicrobial compounds do not work as intended [9‒12. Especially in short-time exposure, the antimicrobials failed to penetrate into deeper area inside biofilm. Wakamatsu et al. have reported the penetration kinetics of mouthrinses into
This chapter is focusing to the studies demonstrating adverse influences of antimicrobial strategy against mature oral biofilm.
2. Adverse influences of antimicrobial strategy
2.1. Residual structure
Recent investigations have demonstrated that chemical disinfection for oral biofilm may leave intact biofilm structures. We performed a direct time-lapse microscopic observation throughout continuous exposure of commercial mouthrinses to an oral biofilm model [10]. Consequently, no removal of biomass was observed in control, ethanol (EtOH), 0.12% chlorhexidine gluconate (CHG), and Biotene, which contains lysozyme, lactoferrin, lactoperoxidase, glucose oxidase, and potassium thiocyanate, even after 20 min exposure. Treatments with CHG and EtOH resulted in only a slight contraction of the biofilm (Figure 3).
Davison et al. investigated the dynamic antimicrobial action of chlorine, a quaternary ammonium compound, glutaraldehyde, and nisin within biofilm cell clusters of
Bacterium | Experimental design | Incubation time | Antimicrobial agent |
Exposure time | Judgment | Reference |
---|---|---|---|---|---|---|
Multispecies ( |
Flow-cell | 20h | 11.6% EtOH 0.12% CHG Biotene |
20 min | Microscopic observation (transmission image) | [10] |
Multispecies ( |
Flow-cell | 20h | 40% EtOH 0.1% SLS 0.03% TRN 0.12% CHG 0.05% CPC 0.005% nisin |
60 min | Microscopic observation (transmission image) | [12] |
Glass-based dish | 24h | 0.12% CHG EO CPC IPMP |
5 min | Microscopic observation (transmission image) | [13] | |
Flow-cell | 24h | 0.14mM QAC 0.5mM Glutaraldehyde 14.9µM nisin |
60 min | Microscopic observation (transmission image) | [21] | |
Chambered coverglass | 24h | 0.05 to 0.2% CHG | 5min | Microscopic observation (transmission image), Quantitative analysis of protein and carbohydrate composition |
[22] |
In contrast, there are some reports that the biofilm structure has been successfully degraded by repeated exposures of mouthrinse [23‒25]. Although it is likely that biofilm reduction may be enhanced by repeated pulse of a mouthrinse, this approach may not always be effective. Pratten and Wilson have reported that anaerobic counts in dental plaque biofilm returned to pretreatment levels with altered bacterial composition after 4 days, despite the continuous pulsing of CHG [26].
Summarizing the above, these results suggest that chemical approach such as the mouthrinse, especially without repeated use, may not be sufficient to eradicate oral biofilm structure. Residual structure may cause adverse effects in oral environment, even if the microorganisms in the biofilm are completely killed.
2.1.1. Antigen and host inflammatory reaction
As the remaining biofilm matrix contains carbohydrates, proteins, polysaccharide, lipids, and nucleic acid [27], dead bacteria and biofilm components could work as antigens and induce inflammatory reactions.
For example,
In addition, even if the microorganisms in the biofilm are completely eradicated, various microbial components in the biofilm could play a role in disease pathogenesis. Augustin et al. reported that injection of dead components of
2.1.2. Calculus formation
The remaining dental biofilm structure will absorb calcium and phosphate from saliva for the formation of supragingival calculus and from crevicular fluid for the formation of subgingival calculus. Calculus formation begins with the deposition of kinetically favored precursor phases of calcium phosphate, octacalcium phosphate, and dicalcium phosphate dihydrate, which are gradually hydrolyzed and transformed into less soluble hydroxyapatite and whitlockite mineral phases [33].
The calculus surface may not in itself induce inflammation in the adjacent periodontal tissue [34, 35]. Jepsen et al. stated that periodontal healing occurs even in the presence of calculus as long as the bacteria is removed or disinfected [34]. For example, it has been reported that autoclaved calculus does not cause pronounced inflammation or abscess formation in connective tissues [36]. Listgarten et al. have demonstrated that a normal epithelial attachment can be formed on its structure when microorganisms on calculus surface were completely disinfected with CHG [37]. Johnson et al. investigated the clinical outcomes of treatment with locally delivered controlled-release doxycycline (DH) or SRP in adult periodontitis patients. Treatment with either DH or SRP resulted in significant statistical and clinical improvements in clinical attachment levels, pocket depth, and bleeding on probing. These clinical outcomes were equivalent regardless of the extent of subgingival calculus present at baseline, suggesting that positive clinical change depend on altering the subgingival biofilm rather than the removal of calculus [38].
However, calculus is known to be a plaque retention factor as well as a reservoir for toxic bacterial products and antigens. Histological section of a human tooth root showed that calculus is covered with viable bacterial plaque [34]. Nichols et al. reported that the dihydroceramide lipids produced by
2.1.3. Scaffold for secondary bacterial adhesion
Recent investigations revealed that residual structure would promote a secondary bacterial adhesion and biofilm redevelopment [22, 40]. Yamaguchi et al. compared the volume of
Our research group has demonstrated that residual structure of
The mechanism of
Thus, since a numerous and diverse range of microorganisms reside in our intraoral environment, the residual biofilm will contribute to biofilm redevelopment.
2.2. Antimicrobials-induced biofilm formation
Numerous studies have shown that subminimum inhibitory concentrations (sub-MICs) of various antibiotics and chemicals can inhibit biofilm formation. A representative example is the macrolide antibiotics. Although
In the field of dentistry, it has also been reported that sub-MICs of antimicrobial agents or compounds can inhibit bacterial attachment [54, 56, 57], biofilm formation [54, 55, 57, 58], and downregulate virulence genes [54, 56, 59, 60]. Moon et al. reported N-acetyl cysteine (NAC) that is an antioxidant possessing anti-inflammatory activities, showed a significant decrease of
In contrast to the inhibitory effects of sub-MIC antimicrobials against biofilm formation, recent studies have shown that some antibiotics at sub-MIC can significantly induce biofilm formation in a variety of bacterial species such as
This phenomenon may have clinical relevance because bacteria are exposed to sub-MIC of antibiotics at the beginning and end of a dosing regimen [63]. In addition, antimicrobials are retarded to diffuse within the biofilm matrix [14, 15]. In such cases, the bacteria in deeper areas are exposed to antimicrobials at sub-MICs.
As for oral biofilm, there are a few studies reported that sub-MICs of antimicrobial agents upregulate the genes related to EPS production and induce biofilm formation. Dong et al. evaluated the expression of genes related to
Bedran et al. investigated the effect of triclosan at sub-MICs on
Even in limited works with regard to oral biofilms, it is likely that short-time exposure of antimicrobial agents in oral cavity sometimes cause adverse influences because the survived microorganisms after exposure to the agents will alter gene expressions in a positive and negative way.
3. Conclusion
Although chemical agents provide some benefits in terms of controlling oral biofilms, they have the limitation of leaving biofilm structures that may induce adverse reactions such as biofilm regrowth. Furthermore, sub-MICs of certain antimicrobial agents might induce biofilm formation and upregulate pathogenic genes. Future strategies for the control of oral biofilms may therefore shift to the degradation and/or detachment of biofilm matrix.
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