Dentin inhibits the antibacterial effect of new and conventional endodontic irrigants.

INTRODUCTION: This in vitro study aimed to compare the antibacterial effect of a new irrigant, QMiX, with that of conventional irrigation solutions in the presence or absence of dentin powder. METHODS: Dentin powder was prepared from bovine incisors and sterilized. The following irrigants were tested against Enterococcus faecalis (ATCC 29212): 6% sodium hypochlorite (NaOCl), 1% NaOCl, QMiX, 2% chlorhexidine gluconate (CHX), and 17% EDTA. Sterilized saline solution was used as negative control. Survival of bacteria exposed to the irrigants in the presence or absence of dentin was monitored under planktonic conditions. Colony-forming units were counted, and log-transformed numbers were analyzed by using Kruskal-Wallis and Mann-Whitney tests. P values less than .05 were considered significant. RESULTS: In the absence of dentin, after 10 seconds of contact with the bacterial suspension, 6% NaOCl showed the lowest bacterial count; the difference to the negative control was significant. After 30 seconds, 6% NaOCl displayed 0 colony-forming units per milliliter, whereas 1% NaOCl and QMiX showed reduced number of colonies in comparison with the negative control. After 1 minute, both concentrations of NaOCl presented no bacterial growth and QMiX reduced the number of colonies, but EDTA and CHX had bacterial counts similar to the negative control. Dentin had a significant inhibitory effect on 6% NaOCl (10 seconds), 1% NaOCl (10 seconds and 1 minute), and QMiX (10 seconds and 1 minute). After 6 hours, both concentrations of NaOCl, QMiX, and CHX killed all bacteria, regardless of the presence of dentin. CONCLUSIONS: Six percent NaOCl was the most effective irrigant against E. faecalis. Saline and EDTA had no measurable antibacterial effect. Dentin delayed the antibacterial activity of NaOCl and QMiX but did not completely prevent their action.

Pitfalls in efficacy testing--how important is the validation of neutralization of chlorhexidine digluconate?

BACKGROUND: Effective neutralization of active agents is essential to obtain valid efficacy results, especially when non-volatile active agents like chlorhexidine digluconate (CHG) are tested. The aim of this study was to determine an effective and non-toxic neutralizing mixture for a propan-1-ol solution containing 2% CHG. METHODS: Experiments were carried out according to ASTM E 1054-02. The neutralization capacity was tested separately with five challenge microorganisms in suspension, and with a rayon swab carrier. Either 0.5 mL of the antiseptic solution (suspension test) or a saturated swab with the antiseptic solution (carrier test) was added to tryptic soy broth containing neutralizing agents. After the samples were mixed, aliquots were spread immediately and after 3 h of storage at 2 - 8 degrees C onto tryptic soy agar containing a neutralizing mixture. RESULTS: The neutralizer was, however, not consistently effective in the suspension test. Immediate spread yielded a valid neutralization with Staphylococcus aureus, Staphylococcus epidermidis and Corynebacterium jeikeium but not with Micrococcus luteus (p < 0.001) and Candida albicans (p < 0.001). A 3-h storage period of the neutralized active agents in suspension resulted in significant carry-over activity of CHG in addition against Staphylococcus epidermidis (p < 0.001) and Corynebacterium jeikeium (p = 0.044). In the carrier test, the neutralizing mixture was found to be effective and non toxic to all challenge microorganisms when spread immediately. However, after 3 h storage of the neutralized active agents significant carry-over activity of CHG against Micrococcus luteus (p = 0.004; Tukey HSD) was observed. CONCLUSION: Without effective neutralization in the sampling fluid, non-volatile active ingredients will continue to reduce the number of surviving microorganisms after antiseptic treatment even if the sampling fluid is kept cold straight after testing. This can result in false-positive antiseptic efficacy data. Attention should be paid during the neutralization validation process to the amount of antiseptic solution, the storage time and to the choice of appropriate and sensitive microorganisms.

Thymosin beta4 is cytoprotective in human gingival fibroblasts.

Thymosin beta4 (Tbeta(4)) is a naturally occurring, ubiquitous, non-toxic protein with documented wound-healing, anti-inflammatory, anti-apoptotic, and tissue-repair properties in skin, the ocular surface, and the heart. The ability of Tbeta(4) to demonstrate similar protective properties in cells of the oral cavity was analyzed using an in vitro model of cultured human gingival fibroblasts. Thymosin beta 4 significantly suppressed the secretion of interleukin-8 (IL-8) following stimulation with tumor necrosis factoralpha (TNF-alpha), suggesting that it may suppress the inflammatory response initiated by pro-inflammatory cytokines. By contrast, Tbeta(4) was not effective in protecting fibroblasts from challenge with lipopolysaccharide purified from Porphyromonas gingivalis or Escherichia coli. Thymosin beta 4 was able to protect gingival fibroblasts against the known cytotoxic effects of chlorhexidine digluconate, a mouthrinse containing chlorhexidine digluconate, and carbamide peroxide. Additionally, Tbeta(4) was able to protect gingival fibroblasts from the apoptosis that is induced by stimulation with TNF-alpha or by exposure to chlorhexidine. Because of its multifunctional roles in protecting cells against damage, Tbeta(4) may have significant potential for use as an oral heathcare aid with combined antimicrobial, anti-inflammatory, anti-apoptotic, and cytoprotective properties.

Effect of a neutralising agent on the evaluation of the antimicrobial activity of chlorhexidine on the bacterial salivary flora.

OBJECTIVE: To test the efficacy of a previously described neutralising agent to counteract any antimicrobial activity of 0.2% of chlorhexidine (CHX) mouthrinse on the salivary flora, which is only exhibited after sampling of surviving bacteria, resulting in false positive efficacy data. METHODS: Unstimulated salivary samples were collected of 20 volunteers under basal conditions and at 30s and 1h after of a single mouthrinse of 0.2% CHX. Each salivary sample was divided into 2 equal aliquots; one was mixed with neutralising agent (3% Tween 80, 0.3% lecithin and 0.1% cysteine) and the other with a control solution. The colony forming units (cfu/mL) were determined and expressed as logarithms (log(10)cfu/mL). RESULTS: At baseline, the total bacterial concentrations were similar, independently of the addition of neutralising solution or control solution (8.419+/-0.346log(10)cfu/mL and 8.462+/-0.474log(10)cfu/mL, respectively, p=0.440). At 30s performing the CHX mouthrinse, the bacterial load reduction was statistically significant between both sampling methods (1.917+/-1.275log(10)cfu/mL, p<0.001). One hour after performing the CHX mouthrinse, the bacterial load reduction was statistically significant between both sampling methods (0.537+/-0.706log(10)cfu/mL, p=0.003). CONCLUSIONS: Neutralising agent was not toxic to the bacterial salivary flora and effectively deactivated the "residual antimicrobial activity" of the 0.2% CHX (after exposure and during processing of samples). We propose the use of this neutralising agent when evaluating the antibacterial activity of CHX mouthrinses.

Thimerosal neurotoxicity is associated with glutathione depletion: protection with glutathione precursors.

Thimerosol is an antiseptic containing 49.5% ethyl mercury that has been used for years as a preservative in many infant vaccines and in flu vaccines. Environmental methyl mercury has been shown to be highly neurotoxic, especially to the developing brain. Because mercury has a high affinity for thiol (sulfhydryl (-SH)) groups, the thiol-containing antioxidant, glutathione (GSH), provides the major intracellular defense against mercury-induced neurotoxicity. Cultured neuroblastoma cells were found to have lower levels of GSH and increased sensitivity to thimerosol toxicity compared to glioblastoma cells that have higher basal levels of intracellular GSH. Thimerosal-induced cytotoxicity was associated with depletion of intracellular GSH in both cell lines. Pretreatment with 100 microM glutathione ethyl ester or N-acetylcysteine (NAC), but not methionine, resulted in a significant increase in intracellular GSH in both cell types. Further, pretreatment of the cells with glutathione ethyl ester or NAC prevented cytotoxicity with exposure to 15 microM Thimerosal. Although Thimerosal has been recently removed from most children's vaccines, it is still present in flu vaccines given to pregnant women, the elderly, and to children in developing countries. The potential protective effect of GSH or NAC against mercury toxicity warrants further research as possible adjunct therapy to individuals still receiving Thimerosal-containing vaccinations.

Inhibition of the anti-staphylococcal activity of the antiseptic polihexanide by mucin.

The antiseptic Lavasept (LS), containing the polymeric biguanide polihexanide (CAS 28757-48-4), possesses microbicidal activity against a broad spectrum of bacteria including Staphylococcus aureus. It is used for antiseptic wound care in concentrations corresponding to 0.2-0.4 mg polihexanide per ml. To obtain basic data on its ability to eradicate S. aureus colonizing the nasal mucosa, the influence of mucin on the anti-staphylococcal activity was investigated. A disk agar-diffusion method was applied. Two reference strains of S. aureus (methicillin-sensitive S. aureus ATCC 29213 and methicillin-resistant S. aureus ATCC 33591) and 20 fresh clinical isolates were used. In the absence of mucin, the growth of all strains was inhibited by polihexanide concentrations of 0.1 mg/ml. In the presence of 0.25% mucin in the test medium, a concentration of 0.4 mg/ml was necessary to inhibit all strains. Mucin concentrations of 0.5% and 1%, that are even lower than the mucin concentrations in healthy nasal secretions, abolished the activity of the therapeutic concentrations of polihexanide. It is concluded that the inactivation of LS by mucin obstructs a reliable clearance of nasal S. aureus carriage.

An effective method of inactivating chlorhexidine.

OBJECTIVE: The purpose of this study was to find an effective inactivating agent for chlorhexidine that would facilitate removal of all residual antimicrobial effect, which may cause false-negative results during microbiologic culturing. STUDY DESIGN: L-alpha-lecithin, Tween 80, and sodium thiosulfate were used in different proportions to prepare 6 potential inactivating solutions. Nine mL of each inactivating solution was mixed with 1 mL of 2% chlorhexidine solution. After 5 minutes of equilibration, 0.1 mL of bacterial cell suspension containing 2 x 10(4) viable cell of Enterococcus faecalis was added to the mixture. At 10 and 60 minutes, 0.1-mL aliquots were withdrawn and spread over blood agar plates and incubated at 37 degrees C for 72 hours. The number of colony-forming units on the blood agar plates was determined and recorded. RESULTS: The combination of 3% Tween 80 and 0.3% L-alpha-lecithin was found to be the most effective inactivating agent, allowing full recovery of the test organisms in the presence of chlorhexidine. CONCLUSION: The present study demonstrated a method to predictably inactivate chlorhexidine.

The effect of toothpaste on the propensity of chlorhexidine and cetyl pyridinium chloride to produce staining in vitro: a possible predictor of inactivation.

BACKGROUND/AIMS: Cationic antiseptics such as chlorhexidine (CHX) and cetyl pyridinium chloride (CPC) interact with dietary chromogens to produce extrinsic stain, and this can be used as a measure of activity of products. The aim of these studies in vitro was to determine if toothpaste influenced the tea staining effects of CHX and CPC as a predictor of action in vivo. METHOD: Clear acrylic specimens were soaked in pooled human saliva followed by sequential 2-min soaks in pairs of agents, namely 0.05% CHX, 0.05% CPC, 0.2% CHX, water (W) and toothpaste slurry (TP). The combinations were; TP/CHX, CHX/TP, TP/CPC, CPC/TP, W/CHX, CHX/W, W/CPC, CPC/W, TP/W, W/TP, W/W. These treatments were followed by a 60 min soak in tea. Optical density readings were taken at baseline and after each of 8 cycles. RESULTS: In the separate CHX and CPC studies by comparison with W/CHX, TP/0.05% CHX had little effect on staining, but TP/0.2% CHX showed a reduction in staining of 18%. 0.05% CHX/TP reduced staining by >40%, and 0.2% CHX by >78%. TP/CPC reduced staining by >26% and CPC/TP by 80%. Water after 0.2% CHX, 0.05% CHX and CPC reduced staining by 18%, 13% and 17% respectively. Little staining was seen with TP and W combinations. The data for CHX are in agreement with a study in vivo except TP followed by CHX reduced the activity of CHX. CONCLUSION: Toothpaste appears to adversely affect the activity of CHX and CPC particularly if used immediately after the antiseptics. The data further supports the concept of separating the use of antiseptics until sometime after the use of toothpaste, and the idea of developing mouthwash friendly toothpastes.

Inactivation of root canal medicaments by dentine, hydroxylapatite and bovine serum albumin.

AIM: This study examined and compared the inhibition of the antibacterial effect of saturated calcium hydroxide solution, chlorhexidine acetate and iodine potassium iodide by dentine, hydroxylapatite and bovine serum albumin. METHODOLOGY: Enterococcus faecalis strain A197A prepared to a suspension of 3 x 10(8) cells per ml in 0.5% peptone water was used. Fifty microL of saturated calcium hydroxide solution, 0.05% chlorhexidine acetate or 0.2/0.4% iodine potassium iodide were incubated at 37 degrees C with 28 mg dentine powder (DP), hydroxylapatite (HA) or bovine serum albumin (BSA) in 50 microL water for 1 h before adding 50 microL of the bacterial suspension. Samples for bacterial culturing were taken from the suspension 1 and 24 h after adding the bacteria. In further experiments, the amount of dentine was stepwise reduced from 28 mg 150 microL-1 to 2.8 mg 150 microL-1. RESULTS: Calcium hydroxide was totally inactivated by the presence of 28 mg of DP, HA or BSA. Chlorhexidine (0.05%) was strongly inhibited by BSA and slowed down by dentine. However, HA had little or no inhibitory effect on chlorhexidine. The antibacterial effect of 0.2/0.4% iodine potassium iodide on E. faecalis was totally inhibited by dentine (28 mg), but was practically unaffected by HA or BSA. A stepwise reduction of dentine from 28 mg 150 microL-1 to 2.8 mg 150 microL-1 was followed by a similar reduction of the inhibition of the antibacterial activity of chlorhexidine. Iodine potassium iodide was not inhibited at all with dentine amounts less than 28 mg. However, the effect of saturated calcium hydroxide solution was totally eliminated by dentine, in all four concentrations. CONCLUSION: Inhibition by dentine of the antibacterial activity of calcium hydroxide, chlorhexidine and iodine potassium iodide occurs by different mechanisms. Different components of dentine may be responsible for the inhibition of these three medicaments. Calcium hydroxide was particularly sensitive to inhibition by both inorganic and organic compounds.

Inactivation of local root canal medicaments by dentine: an in vitro study.

AIMS: The aim of the study was to investigate the inactivation by dentine of the antibacterial activity of various commonly used local root canal medicaments. METHODOLOGY: The medicaments tested were saturated calcium hydroxide solution, 1% sodium hypochlorite, 0.5% and 0.05% chlorhexidine acetate, and 2/4% and 0.2/0.4% iodine potassium iodide. Dentine was sterilized by autoclaving and crushed into powder with a particle size of 0.2-20 microns. Aliquots of dentine suspension were incubated with the medicaments in sealed test tubes at 37 degrees C for 24 h or 1 h before adding the bacteria. In some experiments bacteria were added simultaneously with dentine powder and the medicament. Enterococcus faecalis A197A was used as a test organism. Samples for bacterial culturing were taken from the suspensions at 5 min, 1 h and 24 h after adding the bacteria. RESULTS: Dentine powder had an inhibitory effect on all medicaments tested. The effect was dependent on the concentration of the medicament as well as on the length of the time the medicament was preincubated with dentine powder before adding the bacteria. The effect of calcium hydroxide on E. faecalis was totally abolished by the presence of dentine powder. Similarly, 0.2/0.4% iodine potassium iodide lost its effect after preincubation for 1 h with dentine before adding the bacteria. The effect of 0.05% chlorhexidine and 1% sodium hypochlorite on E. faecalis was reduced but not totally eliminated by the presence of dentine. No inhibition could be measured when full strength solutions of chlorhexidine and iodine potassium iodide were used in killing E. faecalis. CONCLUSIONS: The dentine powder model appears to be an efficient tool for the study of interactions between local endodontic medicaments, dentine, and microbes.

Development of resistance to chlorhexidine diacetate and cetylpyridinium chloride in Pseudomonas stutzeri and changes in antibiotic susceptibility.

Strains of Pseudomonas stutzeri developed stable resistance to chlorhexidine diacetate (CHA) or cetylpyridinium chloride (CPC) when exposed to gradually increasing concentrations of either antibacterial agent. Such strains showed reduced sensitivity to other non-antibiotics, including triclosan, and to some antibiotics, although this varied from strain to strain. Resistant strains were inactivated less readily by CHA or CPC and were less sensitive to sodium dodecyl sulphate. Some CHA-resistant and some CPC-resistant strains were more hydrophobic than the parent strains. Alterations in the cell envelope are likely to be responsible for non-specific changes in sensitivity to several antibacterial agents. Attempts to transfer CHA or CPC resistance by conjugation were unsuccessful. DNA from some CHA- or CPC-resistant strains could transform Ps. stutzeri strain JM 302, a histidine auxotroph, to prototrophy.

Does the nature of the solvent affect the anti-inflammatory capacity of triclosan? An experimental study.

The anti-inflammatory properties of triclosan have been revealed in several recent studies, including an effect on histamine-induced inflammation. In other studies, the nature of the solvent has been shown to be of importance for the plaque inhibiting as well as the antibacterial potential of triclosan. This study was aimed at examining whether the nature of the solvent also may influence the anti-inflammatory capacity of triclosan and further to study a possible dose/ response relationship. The study was performed as 3 separate, double-blind experiments, comprising 10, 11 and 12 healthy females. In all 3 experiments, 5 sites on the lower part of the back of the volunteers were intradermally exposed to one drop of 1% histamine dihydrochloride for 15 min. The size of the resulting wheals was recorded before and after 40 min of triclosan treatment. In experiment 1, 4 different concentrations of triclosan in 2-fold dilutions in absolute alcohol (0.125%-1%) were applied on the histamine-induced wheals. In experiments 2 and 3, 4 different solutions containing 0.5% triclosan and a saline solution as negative control were used. The solvents in experiment 2 were as follows: (1) absolute alcohol (positive control), (2) propylene glycol (PG), (3) polyethylene glycol (PEG), (4) olive oil, and in experiment 3: (1) absolute alcohol (positive control), (2) Tween 80, (3) sodium carbonate, (4) soy oil. The results showed a dose/ response effect of triclosan and further that the solvent may be of importance for its anti-inflammatory potential.

[The dynamics of the antiseptic resistance of the causative agents of soft-tissue suppurative-inflammatory diseases in children]. / Dinamika ustoichivosti k antiseptikam vozbuditelei gnoino-vospalitel'nykh zabolevanii miagkikh tkanei u detei.

Opening of purulent foci in children with pyo-inflammatory diseases (PID) of soft tissues often leads to detection of staphylococcus aureus in the monoculture and different associations of this microbe with gram-negative bacteria in amounts exceeding the "critical level" responsible for the development of PID. Repeated examinations revealed a 2 times decreased frequency of detection of staphylococcus monocultures and 1.5 time less diversity of the association of microorganisms. Most efficient antiseptics among the investigated ones in relation to bacteria isolated in the opened purulent foci were found to be iodopyron, pervomur, boric acid, resorcinum, dioxidine.

[Antiseptic sensitivity of clinical strains of Pseudomonas aeruginosa]. / Chuvstvitel'nost' k antiseptikam u klinicheskikh shtammov Pseudomonas aeruginosa.

MICs, the frequency of clinical and statistic resistance and the antiseptic activity index were studied in complex on out-of-hospital and hospital ecovars of P. aeruginosa. The forms resistant to a number of antiseptics, i.e. chloramine B, chlorhexidine, decamethoxine and dioxidine whose frequency eventually increased were shown to be widely distributed. The antiseptic sensitivity spectrum was more narrow and more heterogeneous than that of other bacteria, the heterogeneity level being dependent on the antiseptic type and bacterial ecovar. The activity of pervomur, phenol, resorcin and boric acid was higher against the clinical strains of P. aeruginosa while iodopyrin, sulfacetamide sodium and dioxidine were less active. The P. aeruginosa strains had natural resistance to cetylpyridinium chloride, rokkal, ethonium, sodium laurate and laurylsulfate and rivanol. It was recommended to assay antiseptic sensitivity of agents causing purulent inflammatory infections and to control circulation of antiseptic resistant variants of bacteria in hospitals.

[Antiseptic resistance of clinical strains of Staphylococcus aureus]. / Ustoichivost' k antiseptikam klinicheskikh shtammov Staphylococcus aureus.

To evaluate the activity of antiseptics and the sensitivity-resistance of bacteria to antiseptics, a number of characteristics has been used, including the minimum inhibiting concentration for different strains, the frequency of statistical and clinical resistance, the antiseptic activity index. A wide spread of S. aureus strains isolated from patients with hospital infections has been revealed. Differences in the resistance of bacterial strains have been established, depending on the type of the antiseptic and the ecovar of bacteria: among hospital ecovars, resistant strains occur more frequently and can resist a wider range of antibiotics. In staphylococcal hospital ecovars the occurrence and level of resistance to a number of widely used antiseptics increase with time. In connection with a wide spread of staphylococcal hospital strains resistant to antiseptics, measures on the control of the circulation of such strains should be introduced into hospitals, and the data thus obtained should be used for the periodic reevaluation of antiseptics used in medical practice and for the choice of preparations to be used for individual therapeutic and prophylactic antisepsis.

[Verification of the effectiveness of neutralization in the in vivo study of antiseptics]. / Vérification de la validité de la neutralisation dans l'étude des antiseptiques in vivo.

Well defined controls of the effectiveness of antiseptic neutralizing agents in vitro already exist (Afnor Standards for antiseptics and disinfectants). Conversely, there is currently no standard method for verifying how effectively such agents neutralize antiseptics which have been applied to the skin. We describe a method in which the neutralizing solution is first applied to the antiseptic-treated skin and then filtered to remove skin flora. The effect of the neutralizing agent is then checked using a test organism (Staphylococcus epidermidis ATCC 14990) in parallel with four controls: viability of the test organism (T1), activity of the untreated antiseptic (T2), non-bactericidal effect of the neutralizing agent (T3) and of skin secretions (4). Skin sampling was carried out using Gaschen's bag method for the hands and the Williamson and Kligman method modified by Fleurette for other sites. Twelve soaps and/or antiseptics were studied. In each case, activity of the antiseptic (T2) was confirmed, except when the product under study was a non-bactericidal soap. Bacterial counts obtained in controls T3 and T4 and in the neutralization test itself were always greater than 80% of the control value (T1). Occasionally, the counts in the neutralization test exceeded 100% of the T1 value, probably because of the dispersal effect of the triton X100 present in the neutralizing solution. This method thus offers a means of standardizing the study of antiseptics in vivo. In addition, the test organism, Staphylococcus epidermidis, is more representative of the skin flora than the bacterial strains recommended by Afnor for in vitro studies.

Development and validation of a neutralizer system for in vitro evaluation of some antiseptics.

A neutralizer system was developed and validated for use in the in vitro bactericidal evaluation of three commonly used antiseptics, namely, Hibiclens (4% [wt/vol] chlorhexidine gluconate), Betadine (7.5% [wt/vol] povidone-iodine), and pHisoHex (3% [wt/vol] hexachlorophene). The neutralizer finally selected after a screening of 12 potential candidates consisted of 3% Asolectin, 10% Tween 80, and 0.3% sodium thiosulfate in the recovery agar. This neutralizer system was tested and validated for its neutralizing capacity for the three antiseptics, as well as for its lack of inherent bactericidal action against Staphylococcus aureus and a number of gram-negative bacteria of clinical significance, With no more than a 10-fold dilution of the antiseptic, the selected neutralizer system was 100% effective in neutralizing all the bacteriostatic carry-over of the three antiseptics and was also completely without any inherent bactericidal action against all the test organisms used. Sodium sulfite (considered to be a potential inactivator for iodophores such as Betadine), even in concentrations as low as 0.1%, was found to be ineffective or inherently bactericidal, whereas 0.3% sodium thiosulfate, in combination with Asolectin and Tween 80, was adequate (effective as well as non-bactericidal) and was considered to be essential for the neutralization of the three test antiseptics, namely, Hibiclens, Betadine, and pHisoHex.