Antimicrobial efficacy of chlorhexidine against bacteria in biofilms at different stages of development

Introduction

Detailed knowledge on the nature of the physiological and metabolic phases of biofilm development is important in combating resistant, disease-associated biofilms. The aim of this study was to examine the susceptibility of multispecies biofilms at different phases of growth to root canal irrigants.

Methods

The multispecies biofilms were grown from plaque bacteria on collagen-coated hydroxyapatite discs in brain-heart infusion broth for time periods ranging from 2 days to several months. Fresh nutrients were added weekly for the first 3 weeks, followed by a nutrient deprivation phase, by adding fresh brain-heart infusion broth medium only once a month. Biofilms of different age were subjected to 1-, 3-, or 10-minute exposure to 2% chlorhexidine (CHX) or CHX-Plus. After treatment, the volume ratio of dead bacteria in biofilms was assessed by confocal laser scanning microscopy by using a LIVE/DEAD viability stain. Biofilm structure was visualized by scanning electron microscopy.

Results

The thickness of biofilms increased from 57 μm (2 days) to 155 μm (3 weeks) during biofilm development. It reached a steady state under nutrient-limiting conditions, with thickness of 190 μm (6 weeks) to 201 μm (12 weeks). The proportion of killed bacteria in mature biofilms (3 weeks) was lower than in young biofilms (2 days, 1 and 2 weeks) after treatment with both CHX products (P < .01).The resistance of mature biofilms under the nutrient-limiting phase (6-12 weeks) to CHX remained stable and was similar to 3-week-old biofilm. However, treatment with CHX-Plus for 3 and 10 minutes did not lose effectiveness against biofilm bacteria in mature and nutrient-limited phases. CHX-Plus showed higher levels of bactericidal activity at all exposure times than 2% CHX (P < .01).

Conclusions

Bacteria in mature biofilms and nutrient-limited biofilms are more resistant to CHX killing than in young biofilms. The results emphasize the importance of standardization of factors such as biofilm age when studying the comparative effectiveness of disinfecting agents against biofilm bacteria.     

Comparison of the bisbiguanide antiseptics alexidine and chlorhexidine. II. Clinical and in vitro staining properties

Abstract A blind cross-over trial was carried out to compare the tooth and tongue staining associated with the use of a 0.035% alexidine and a 0.2% chlorhexidine mouthrinse. Twenty-two volunteers were divided into two groups termed “tea drinkers” and “non-tea drinkers”. All volunteers were requested to refrain from oral hygiene measures throughout two 10-day periods when they rinsed twice a day with the preparation randomly allocated for the respective period. During both periods the members of the groups excluded coffee, red wine and port from their diet. The tea drinking group consumed seven cups of tea per day. Tooth and tongue staining was recorded for extent and severity at the end of each period. The amount of stain accumulating in the two groups was similar following the use of chlorhexidine and alexidine. However, for both chlorhexidine and alexidine the extent and severity of tooth and tongue staining were significantly increased in the tea drinking group. An in vitro study of tea staining of perspex blocks exposed twice a day to 0.035% solutions of alexidine or chlorhexidine throughout a 5-day period demonstrated significantly more staining with alexidine when measured spectrophotometrically. Visually however, the differences in the specimens were minimal. Saliva treatment the perspex did not significantly alter the staining by alexidine or chlorhexidine. The results provide further evidence for a dietary aetiology to the staining associated with cationic antiseptics. However, alexidine at the concentration used offered no advantage in reducing the side effect of staining when compared with chlorhexidine.     

In vitro activity of chlorhexidine against enteric rods, pseudomonads and acinetobacter from human periodontitis

Fifty-eight periodontal isolates of the families Enterobacteriaceae and Pseudomonadaceae, and the genera Acinetobacter and Achromobacter were studied to determine their susceptibility to Peridex (0.12% chlorhexidine digluconate solution; Procter & Gamble). In an agar dilution assay, about 50% of the study strains grew in the presence of 70 micrograms/ml of chlorhexidine. Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas cepacia, and Serratia marcescens comprised the most resistant species. Studies are needed to determine the microbiological and clinical effects of chlorhexidine usage in patients infected with enteric rods and pseudomonads.     

Antimicrobial properties of human dentin impregnated with tetracycline HCI or chlorhexidine

Abstract Substantivity of tetracycline HCI and chlorhexidine digluconate to human root dentin was assessed in vitro. 51 extracted single-rooted teeth, their crowns removed, were assigned to 1 of 4 treatments in groups of 12. A control groups included 3 roots. Each group was divided into 3 subgroups to allow evaluation of drug exposure for 1, 3 or 5 min. The roots were immersed in tetracycline HCI (10 or 50 mg/ml) or chlorhexidine digluconate (0.12 or 0.2%) solutions following root planing. Control roots were immersed in sterile saline (0.9%). Following drug immersion, the roots were transferred to tubes containing 2 ml tris buffered saline. The tubes were incubated at room temperature for 22 days. Desorption media were replaced at 24-h intervals. Removed media were examined for antimicrobial activity using a microtiter assay in which bacterial growth was evaluated by optical density readings. Roots immersed in tetracycline HCl 50 mg/ml released antimicrobial activity to successive desorption media for 14 days. Tetracycline HCl 10 mg/ml activity lasted 4 days. Roots subjected to chlorhexidine digluconate released antimicrobial activity for 24 h only. Within each treatment, there were no differences between the 3 exposure intervals of 1, 3 or 5 min. Our findings suggest usage of the periodontally exposed instrumented root as a depot for sustained release of tetracycline HCI, but not chlorhexidine digluconate, to the subgingival environment. The substantiveness of tetracycline HCI seems related to drug concentration rather than the exposure interval. Clinical trials are needed to confirm the clinical significance of these in vitro observations.     

In vitro antibacterial effect of chlorhexidine added to glass-ionomer cements

Abstract— The aims of the present study were to investigate the antibacterial properties of glass-ionomer cements containing different concentrations of chlorhexidine and the possible release of chlorhexidine from the compound. Chlorhexidine gluconate or diacetate was mixed with glass-ionomer cements and tested in vitro against strains of Lactobacillus casei and mutans streptococci. A spectrophotometric test was used to monitor the release of chlorhexidine from the mixture. In a broth culture test, the higher the concentrations of chlorhexidine in the mixture, the longer the time of antibacterial effect on Streptococcus mutans and the greater the tendency of the material to deteriorate. Agar diffusion tests revealed bacterial inhibition in a dose-response manner. The tested bacterial strains were similarly inhibited and the antibacterial properties decreased with time. A minor portion of added chlorhexidine was released from the cement. The deterioration of the cements indicates that the material could be useful as a varnish-like chlorhexidine carrier.     

Antimicrobial activity of garlic,tea tree oil,and chlorhexidine against oral microorganisms

OBJECTIVE: To compare the antimicrobial activity of tea tree oil, garlic, and chlorhexidine solutions against oral microorganisms. METHOD: The five-week study consisted of thirty subjects. The first week was considered baseline. All subjects used a control solution (second week), and were randomly divided into the three groups (third week): G1-0.12% chlorhexidine; G2 - 2.5% garlic (Allium sativum, L.); and G3 - 0.2% tea tree oil (Melaleuca alternifolia). Dishes containing blood agar and Mitis Salivarius Bacitracin agar (MSB) were inoculated with the subjects' saliva (collected twice a week). Total microorganisms and mutans streptococci were counted in blood agar and MSB, respectively. RESULTS: Chlorhexidine and garlic groups showed antimicrobial activity against mutans streptococci, but not against other oral microorganisms. The tea tree oil group showed antimicrobial activity against mutans streptococci and other oral microorganisms. Maintenance of reduced levels of microorganisms was observed only for garlic and tea tree oil during the two consecutive weeks (fourth and fifth). Unpleasant taste (chlorhexidine 40%, tea tree oil 30%, garlic 100%), burning sensation (chlorhexidine 40%, tea tree oil 60%, garlic 100%), bad breath (chlorhexidine 40%, tea tree oil 20%, garlic 90%), and nausea (chlorhexidine 0%, tea tree oil 10%, garlic 30%) were reported. CONCLUSION: Garlic and tea tree oil might be an alternative to chlorhexidine.     

基于重水拉曼技术的氯己定对白色念珠菌 抑菌效能的研究

目的评价重水拉曼技术的普适性及氯己定(CHX)对白色念珠菌的抑菌效能。方法1)采用分光光度计测定吸光度值OD600,重水拉曼技术测定C?D ratio(重水峰所占比例)随时间的变化,探索重水对白色念珠菌生长的影响及该菌对重水的吸收规律,以评价重水拉曼技术的普适性。2)采用肉汤稀释法和重水拉曼技术,测定CHX对白色念珠菌的最低抑菌浓度(MIC)及最小代谢活性抑制浓度(MIC?MA),评价CHX对白色念珠菌生长及代谢的抑制效果。结果1)白色念珠菌在质量分数≤30%重水中,生长未受到明显抑制(P>0.05);白色念珠菌能活跃代谢重水并通过拉曼图谱检测,且C?D ratio与重水质量分数呈线性正相关关系(R^2=0.9894,P<0.05)。2)CHX对白色念珠菌的MIC为4μg·mL^-1,MIC?MA为8μg·mL^-1。在MIC下,白色念珠菌的生长受到完全抑制,但仍具有很高的代谢活性;只有达到2×MIC,即MIC?MA,该菌的代谢才能得到完全抑制。结论重水拉曼技术适用于评价药物对真菌代谢活性的影响,临床常用质量分数的CHX可完全抑制白色念珠菌的生长及代谢。     

洗必汰对体外菌斑代谢产酸作用的实验研究

目的：观察洗必汰对体外菌斑不同生长时期代谢产酸的作用。方法：对0．2g/L洗必汰溶液处理不同生长时期改良体外菌斑糖酵解模型,ORION电极检测产酸液pH。结果：洗必汰对生长48h的早期体外菌斑的代谢产酸具有明显的抑制作用。而对培养96h的成熟菌斑的代谢产酸没有明显的影响。结论：随着菌斑的发育成熟,洗必汰对体外菌斑代谢产酸的抑制作用减弱。     

洗必汰对不同生长期人工体外菌斑产酸代谢的作用

目的:观察洗必汰对不同生长期人工体外菌斑产酸代谢的作用。方法:以0.2 g/L洗必汰溶液处理不同生长期改良人工体外菌斑糖酵解模型,ORION电极检测产酸液pH。结果:洗必汰对生长48 h的早期人工体外菌斑的产酸代谢具有明显的抑制作用,对培养96 h的成熟人工体外菌斑的产酸代谢没有明显影响。结论:随着菌斑的发育成熟,洗必汰对菌斑产酸代谢的抑制作用减弱。     

Antimicrobial effect of chlorhexidine in a controlled release delivery system

Abstract Chlorhexidine is widely used as a mouth rinse in the prevention and treatment of periodontal diseases and dental caries. The purpose of the present study was to evaluate the in vitro antimicrobial effect of chlorhexidine in a controlled release delivery system. The controlled release dispenser comprised a polymeric inner core matrix containing the medicament with an outer vinyl membrane controlling the drug release. The effect on the following bacteria was studied: Actinobacillus actinomycetemcomitans, Actinomyces viscosus, Streptococcus mutans, Wolinella recta, Bacteroides gingivalis, Bacteroides intermedius, Eikenella corrodens, Pseudomonas aeruginosa, Enterobacter aerogenes, and Enterobacter cloacae. Chlorhexidine-containing vinyl patches with a diameter of 5.5 mm were placed on blood agar plates containing the various bacteria. The plates were incubated aerobically or anaerobically at 37°C for 24 h or longer, when appropriate, and examined for inhibition of bacterial growth. Distinct zones of inhibition were seen surrounding all vinyl patches on all plates with all bacteria. Thus, the vinyl dispenser appeared to be an effective vehicle for releasing chlorhexidine to a localized area such as the surface of a tooth, a periodontal pocket, or a root canal.     

Comparative staining in vitro and plaque inhibitory properties in vivo of 0.12% and 0.2% chlorhexidine mouthrinses

Abstract. The mere incorporation of an active ingredient in an oral hygiene product does not necessarily guarantee efficacy. As new formulations appear, it would seem prudent to at least screen for activity by comparison with an established product. The aim of this study was to compare a new 0.12% chlorhexidine containing rinse with a well researched 0.2% chlorhexidine rinse product. The rinses were firstly compared in vitro for dietary staining effects and in vivo for plaque inhibition. Both formulations in vitro produced progressive staining of acrylic specimens with increasing passages through a standard tea solution. The clinical investigation was a 3-treatment, randomised, double blind, crossover, 4-day plaque regrowth study, balanced for 1st and 2nd order residual effects and involving 24 volunteers. From a zero plaque baseline, volunteers suspended oral hygiene and rinsed 2×per day with the allocated rinse. The chlorhexidine doses were 18 mg and 20 mg per rinse for the 0.12% and 0.2% formulations, respectively. Plaque was scored by area and index on day 5. The results showed that the 2 chlorhexidine rinses were similar in efficacy by comparison with the control rinse. These studies in vitro and in vivo indicate that the chlorhexidine in the new preparation is available and active and the product could find use in those preventive applications suggested for other chlorhexidine formulations.     

Antimicrobial and physicochemical characterization of endodontic sealers after exposure to chlorhexidine digluconate

ObjectivesAssess the antibacterial, physical and chemical properties of AH Plus, BioRoot RCS and Pulp Canal Sealer (PCS) in contact with 2% chlorhexidine digluconate (CHX) used as final irrigant prior to root canal obturation.MethodsThe antimicrobial properties were investigated by direct contact tests for planktonic and biofilm growth of E. faecalis, S. mutans, S.epidermidis and S.aureus in vitro. The setting time, wettability, microhardness and surface roughness were also assessed. The sealers were studied in no contact, 1-minute (short-term) and continuous contact (long-term) with CHX. Chemical characterization of sealers was performed by scanning electron microscopy, X-ray diffraction analysis and Fourier-transform infrared spectroscopy after CHX or saline used as the last irrigant in an ex vivo tooth model and in endo training blocks.ResultsCHX increased the antibacterial activity of all the sealers investigated against planktonic bacteria and biofilms with PCS exerting the highest antimicrobial activity with and without the presence of CHX. The setting of AH Plus and BioRoot RCS was retarded, while for PCS accelerated in the presence of CHX. AH Plus and PCS were more hydrophilic after contact with CHX, whilst BioRoot RCS was hydrophobic in a time-dependent manner. The microhardness of sealers was compromised and the surface roughness increased after CHX exposure for AH Plus and BioRoot RCS, and decreased for PCS. CHX did not affect the sealers’ chemistry, but PCS that exhibited two extra phases.SignificanceCHX improved the antibacterial efficacy of endodontic sealers but further evidence is needed to confirm its suitability as a final irrigant prior to root canal obturation.     

The properties and applications of chlorhexidine in endodontics

Microorganisms and their by‐products are considered to be the major cause of pulp and periradicular pathosis. Hence, a major objective in root canal treatment is to disinfect the entire root canal system, which requires that all contents of the root canal system be eliminated as possible sources of infection. This goal may be accomplished using mechanical instrumentation and chemical irrigation, in conjunction with medication of the root canal system between treatment sessions. To reduce or eliminate bacteria, various irrigation solutions have been advocated. Chlorhexidine is a cationic molecule, which can be used during treatment. It has a wide range of antimicrobial activity. Its cationic structure provides a unique property named substantivity. The purpose of this paper is to review the structure and mechanism of action of CHX, its antibacterial and antifungal activity, its effect on biofilm, its substantivity (residual antibacterial activity), its tissue solvent ability, its interaction with calcium hydroxide and sodium hypochlorite, its anticollagenolytic activity, its effect on coronal and apical leakage of bacteria, its toxicity and allergenicity and the modulating effect of dentine and root canal components on its antimicrobial activity. A Medline search was performed from 1981 to the end of March 2008 and was limited to English‐language papers. The keywords searched on Medline were ‘chlorhexidine AND endodontics’, ‘chlorhexidine AND root canal therapy’, ‘chlorhexidine AND substantivity’ and ‘chlorhexidine AND toxicity’. The reference lists of each article were manually checked for additional articles of relevance.     

Interaction between chlorhexidine digluconate and sodium monofluorophosphate in vitro

Abstract — The aim of the present study was to investigate the compatibility of chlorhexidine digluconate and sodium monofluorophosphate since these agents are potential ingredients in future products in preventive dentistry. Varying combinations of chlorhexidine digluconate and sodium monofluorophosphate in water, covering the possible ranges of clinically relevant concentrations of both compounds, were made, incubated for 24 h and observed for precipitation of insoluble salts. The mixtures were analyzed for presence of free chlorhexidine and monofluorophosphate after incubation. The results showed that chlorhexidine digluconate and sodium monofluorophosphate are not compatible in clinically relevant concentrations. A chlorhexidine- monofluorophosphate salt of low solubility in water is presumably formed.     

Efficacy of chlorhexidine in disinfecting dentinal tubules in vitro

Abstract Solutions of 0.2% and 2% chlorhexidine, 0.2% and 2% sodium hypochlorite (NaOCl) and normal saline were tested for their efficacy in disinfecting dentinal tubules following root canal irrigation in vitro. Freshly extracted bovine incisor roots were prepared as cylindrical shapes, 4 mm high and 5 mm wide with a lumen 2.1 mm wide. After mechanical removal of the cementum and elimination of the smear layer on the dentine surface with EDTA and NaOCl, the root sections were autoclaved and the dentinal tubules infected with E. faecalis (NCTC 775) by incubating in yeast extract glucose broth for 1 week. The root canals were irrigated with 20 ml of an irrigant solution using a syringe. Each irrigant was used in six specimens. Dentine was removed from the canal wall by sterile burs of increasing diameter to give samples 100, 100–300 and 300–500 μm deep. The dentine samples were then cultured to determine the presence and quantity of remaining micro-organisms. The results indicated that chlorhexidine and NaOCl were equally effective antibacterial agents at similar concentrations against the test microorganism. They significantly reduced the bacterial counts in the first 100 μm of dentinal tubules, however up to 50% of dentine samples remained infected following use of both agents.     

Photographic assessment of the antiplaque properties of sanguinarine and chlorhexidine

The purpose of this study was to assess the effect of rinsing and topical application of sanguinarine on labial surface plaque accumulation, compared with topically applied water, and compared with rinsing with chlorhexidine, while refraining from all oral hygiene measures for 5 days. Color photographs of the disclosed plaque were taken at the end of each phase of treatment and blindly traced using an Apple II micro-computer graphic tablet digitizer. The plaque score for each tooth was calculated by dividing the computer reading of the labial plaque surface area by the total labial surface area of the tooth. Topically applied sanguinarine showed better plaque reduction than mouthrinsing with sanguinarine. There was a significant reduction in plaque accumulation after rinsing with chlorhexidine compared with topically applied sanguinarine, water, and mouthrinsing with sanguinarine. The results of this study indicate that chlorhexidine is a more superior antiplaque agent than sanguinarine. The use of an Apple II micro-computer graphic tablet digitizer provided a valuable method for accurate plaque assessments.     

Effect of chlorhexidine gel treatment on Streptococcus mutans population in human saliva and dental plaque

Abstract – The effect of topical application of a 1% chlorhexidine gel on the population of S.mutains in dental plaque and saliva was studied in five subjects highly infected with the organism. After treatment for 5 min daily for 14 d, S. mutans was not detected in three of the subjects and counts of 700 colony-forming units or less per ml of saliva were found in the other subjects. Reappearanceof S. mutans was slow and did not reach pretreatment level until 14 weeks or more after therapy. The proportion of S. saguis in plaque increased temporatrily after chlorhexidine treatment, whereas the population of lactobacilli was unaffected. These observations indicate that short-term use of chlorhexidine is of value in controlling oral infection of bumans by S. mutans.     

Comparison of 2 chlorhexidine mouthwashes on plaque regrowth in vivo and dietary staining in vitro

Abstract Until recently, the few available chlorhexidine mouthrinse products have been 0.2% formulations. However, concentrations of 0.12% chlorhexidine appear as effective as 0.2%, if the volume of the rinse is increased to 15 ml. Since the mere incorporation of chlorhexidine in a formulation does not guarentee availability of the antiseptic, it would seem reasonable to evaluate or compare all products. This is particularly the case when other ingredients, such as fluoride are added. The 1st study compared the effect of a 0.12% chlorhexidine rinse with a 0.12% chlorhexidine/0.022% sodium fluoride rinse for effects on plaque re-growth. The study was a 7-day, blind, randomised, 2-cell cross-over design with a baseline control run in period, in which 18 subjects participated. Both chlorhexidine products significantly reduced plaque compared to control but the chlorhexidine fluoride rinse was less effective than the chlorhexidine only rinse. The 2nd study assessed the propensity of the chlorhexidine rinses to induce dietary staining in vitro. For the chlorhexidine fluoride rinse, this was less than the other 0.12% rinse and a commonly used 0.2% product. The data in vivo and in vitro suggest reduced chlorhexidine availability from the chlorhexidine fluoride product which appears to cause some loss of efficacy.     

Influence of chlorhexidine on in vitro uptake of fluoride in dental enamel

ABSTRACT – Chlorhexidine gluconate and sodium fluoride were found to be compatible in the concentration range of interest in clinical use. Admixture of chlorhexidine to sodium fluoride solutions did not interfere with the fluoride uptake in clinically intact premolars in vitro .