Determination of para-Chloroaniline and Reactive Oxygen Species in Chlorhexidine and Chlorhexidine Associated with Calcium Hydroxide

The aim of this study was to determine whether para-chloroaniline (PCA) and/or reactive oxygen species (ROS) are generated by chlorhexidine (CHX) alone or after CHX is mixed with calcium hydroxide at different time points. Mass spectrometry was performed to detect PCA in samples of 0.2% CHX and Ca(OH)2 mixed with 0.2% CHX. High-performance liquid chromatography was used to confirm the presence of CHX in the mixture with Ca(OH)2. The samples were analyzed immediately after mixing and after 7 and 14 days. During the intervals of the experiment, the samples were maintained at 36.5 degrees C and 95% relative humidity. PCA was detected in the 0.2% CHX solution after 14 days. The mixture of CHX with Ca(CH)2 liberated ROS at all time points, but no traces of CHX were present in the mixture as a result of immediate degradation of the CHX. (J Endod 2008;34:1508-1514)

In vitro antibacterial action of Tetraclean, MTAD and five experimental irrigation solutions

P>Aim To investigate the antibacterial effect of Tetraclean, MTAD and five experimental irrigants using both direct exposure test with planktonic cultures and mixed-species in vitro biofilm model. Methodology Tetraclean, MTAD and five experimental solutions that were modifications of existing formulae including MTAD + 0.01% cetrimide (CTR), MTAD + 0.1% CTR, MTAC-1 (Tween 80 replaced by 0.01% CTR in MTAD), MTAC-2 (Tween 80 replaced by 0.1% CTR) and MTAD-D (MTAD without the Tween 80 and no CTR added) were used as disinfectants in the experiments. In the direct exposure test, a suspension of Enterococcus faecalis was mixed with each of the solutions. After 0.5, 1, 3 and 10 min, an inactivator was added and the number of surviving bacteria was calculated. A mixed-species biofilm from subgingival plaque bacteria was grown in brain heart infusion broth in anaerobic conditions on synthetic hydroxyapatite discs. Two-week-old biofilms were exposed to the solutions for 0.5, 1 and 3 min. The samples were observed by confocal laser scanning microscopy after bacterial viability staining. The scans were quantitatively analysed, and the volume of killed cells of all cells was calculated for each medicament. Results Tetraclean and MTAC-2 (0.1% CTR) killed planktonic E. faecalis in < 30 s. Complete killing of bacteria required 1 min by MTAC-1, 3 min by MTAD + 0.1% CTR and 10 min by MTAD, MTAD-D and MTAD + 0.01% CTR. In the biofilm test, there were significant differences in microbial killing between the different solutions and times of exposure (P < 0.005). MTAC-2 showed the best performance, killing 71% of the biofilm bacteria in 3 min, followed by MTAC-1 and Tetraclean. MTAD and the three MTAD modifications demonstrated the lowest antibacterial activity. Conclusion Tetraclean was more effective than MTAD against E. faecalis in planktonic culture and in mixed-species in vitro biofilm. CTR improved the antimicrobial properties of the solutions, whereas Tween 80 seemed to have a neutral or negative impact on their antimicrobial effectiveness.

Fungicidal effect of photodynamic therapy against fluconazole-resistant Candida albicans and Candida glabrata

P>Although photodynamic therapy (PDT) has shown great promise for the inactivation of Candida species, its effectiveness against azole-resistant pathogens remains poorly documented. This in vitro study describes the association of Photogem (R) (Photogem, Moscow, Russia) with LED (light emitting diode) light for the photoinactivation of fluconazole-resistant (FR) and American Type Culture Collection (ATCC) strains of Candida albicans and Candida glabrata. Suspensions of each Candida strain were treated with five Photogem (R) concentrations and exposed to four LED light fluences (14, 24, 34 or 50 min of illumination). After incubation (48 h at 37 degrees C), colonies were counted (CFU ml-1). Single-species biofilms were generated on cellulose membrane filters, treated with 25.0 mg l-1 of Photogem (R) and illuminated at 37.5 J cm-2. The biofilms were then disrupted and the viable yeast cells present were determined. Planktonic suspensions of FR strains were effectively killed after PDT. It was observed that the fungicidal effect of PDT was strain-dependent. Significant decreases in biofilm viability were observed for three strains of C. albicans and for two strains of C. glabrata. The results of this investigation demonstrated that although PDT was effective against Candida species, fluconazole-resistant strains showed reduced sensitivity to PDT. Moreover, single-species biofilms were less susceptible to PDT than their planktonic counterparts.

Effectiveness of calcium hydroxide-based intracanal medicaments against Enterococcus faecalis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Microbial leakage of MTA, Portland cement, Sealapex and zinc oxide-eugenol as root-end filling materials

Objective: The aim of this study was to compare the microbial leakage of mineral trioxide aggregate (MTA), Portland cement (PC), Sealapex and zinc oxide-eugenol (ZOE) as root-end filling materials.Study design: An in vitro microbial leakage test (MLT) with a split chamber was used in this study. A mixture of facultative bacteria and one yeast (S. aureus + E. faecalis + P. aeruginosa + B. subtilis + C. albicans) was placed in the upper chamber and it could only reach the lower chamber containing Brain Heart Infusion broth by way of leakage through the root-end filling. Microbial leakage was observed daily for 60 days. Sixty maxillary anterior human teeth were randomly assigned to different groups - MTA and PC (gray and white), Sealapex + zinc oxide and ZOE, control groups and subgroups to evaluate the influence of EDTA for smear layer removal. These materials were further evaluated by an agar diffusion test (ADT) to verify their antimicrobial efficacy. Data were analyzed statistically by Kruskal-Wallis and Mann-Whitney test.Results: In the MLT, Sealapex + zinc oxide and ZOE did not show evidence of microbial leakage over the 60-day experimental period. The other materials showed leakage from the 15th day. The presence of smear layer influenced microbial leakage. Microbial inhibition zones were not observed in all samples tested by ADT.Conclusion: Sealapex + zinc oxide and ZOE did not show microbial leakage over the experimental period, whereas it was verified within 15 to 45 days in MTA and Portland cement.

Desenvolvimento e caracterização de sistemas nanoestruturados bioadesivos contendo peptídeo análogo à adesina do streptococcus mutans

Pós-graduação em Ciências Farmacêuticas - FCFAR

Efeito de scaffolds de nanofibras incorporados com antibióticos sobre biofilmes formados por bactérias presentes nos canais radiculares

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Influence of Ozone Gas and Ozonated Water Application to Dentin and Bonded Interfaces on Resin-Dentin Bond Strength

Purpose: To assess the influence of ozone gas and ozonated water application to prepared cavity and bonded interfaces on the resin/dentin bond strength of two-step etch-and-rinse adhesive systems (Adper Single Bond 2 [SB2] and XP-Bond [XP]). Materials and Methods: Sixty extracted human third molars were sectioned perpendicularly to their long axes to expose flat occlusal dentin surfaces. In experiment 1, dentin was treated with ozone before the bonding procedure, while in experiment 2, ozone was applied to resin/dentin bonded interfaces. In experiment 1, dentin surfaces were treated either with ozone gas (2100 ppm), ozonated water (3.5 ppm), or distilled water for 120 s, and then bonded with SB2 or XP according to manufacturers' instructions. Hybrid composite buildups were incrementally constructed and the teeth were sectioned into resin-dentin sticks (0.8 mm(2)). In experiment 2, dentin surfaces were first bonded with SB2 or XP, composite buildups were constructed, and bonded sticks obtained. The sticks were treated with ozone as previously described. Bonded sticks were tested under tensile stress at 1 mm/min. Silver nitrate impregnation along the resin/dentin interfaces was also evaluated under SEM. Results: Two-way ANOVA (adhesive and ozone treatment) detected no significant effect for the cross-product interaction and the main factors in the two experiments (p > 0.05), which was confirmed by the photomicrographs. Conclusion: Ozone gas and ozonated water used before the bonding procedure or on resin/dentin bonded interfaces have no deleterious effects on the bond strengths and interfaces.

Honey - a potential agent against Porphyromonas gingivalis: an in vitro study.

BACKGROUND Honey has been discussed as a therapeutic option in wound healing since ancient time. It might be also an alternative to the commonly used antimicrobials in periodontitis treatment. The in-vitro study was aimed to determine the antimicrobial efficacy against Porphyromonas gingivalis as a major periodontopathogen. METHODS One Manuka and one domestic beekeeper honey have been selected for the study. As a screening, MICs of the honeys against 20 P. gingivalis strains were determined. Contents of methylglyoxal and hydrogen peroxide as the potential antimicrobial compounds were determined. These components (up to 100 mg/l), propolis (up to 200 mg/l) as well as the two honeys (up to 10% w/v) were tested against four P. gingivalis strains in planktonic growth and in a single-species biofilm. RESULTS 2% of Manuka honey inhibited the growth of 50% of the planktonic P. gingivalis, the respective MIC50 of the German beekeeper honey was 5%. Manuka honey contained 1.87 mg/kg hydrogen peroxide and the domestic honey 3.74 mg/kg. The amount of methylglyoxal was found to be 2 mg/kg in the domestic honey and 982 mg/kg in the Manuka honey. MICs for hydrogen peroxide were 10 mg/l - 100 mg/l, for methylglyoxal 5 - 20 mg/l, and for propolis 20 mg/l - 200 mg/l. 10% of both types of honey inhibited the formation of P. gingivalis biofilms and reduced the numbers of viable bacteria within 42 h-old biofilms. Neither a total prevention of biofilm formation nor a complete eradication of a 42 h-old biofilm by any of the tested compounds and the honeys were found. CONCLUSIONS Honey acts antibacterial against P. gingivalis. The observed pronounced effects of Manuka honey against planktonic bacteria but not within biofilm can be attributed to methylglyoxal as the characteristic antimicrobial component.

In vitro activity of photoactivated disinfection using a diode laser in infected root canals

OBJECTIVE To investigate the lethal activity of photoactivated disinfection (PAD) on Enterococcus faecalis (ATCC 29212) and mixed populations of aerobic or anaerobic bacteria in infected root canals using a diode laser after the application of a photosensitizer (PS). MATERIALS AND METHODS First, the bactericidal activity of a low power diode laser (200 mW) against E. faecalis ATCC 29212 pre-treated with a PS (toluidine blue) for 2 min were examined after different irradiation times (30 s, 60 s and 90 s). The bactericidal activity in the presence of human serum or human serum albumin (HSA) was also examined. Second, root canals were infected with E. faecalis or with mixed aerobic or anaerobic microbial populations for 3 days and then irrigated with 1.5% sodium hypochlorite and exposed to PAD for 60 s. RESULTS Photosensitization followed by laser irradiation for 60 s was sufficient to kill E. faecalis. Bacteria suspended in human serum (25% v/v) were totally eradicated after 30 s of irradiation. The addition of HSA (25 mg/ml or 50 mg/ml) to bacterial suspensions increased the antimicrobial efficacy of PAD after an irradiation time of 30 s, but no longer. The bactericidal effect of sodium hypochlorite was only enhanced by PAD during the early stages of treatment. PAD did not enhance the activity of sodium hypochlorite against a mixture of anaerobic bacteria. CONCLUSIONS The bactericidal activity of PAD appears to be enhanced by serum proteins in vitro, but is limited to bacteria present within the root canal.

Studies on enhanced surface disinfection and skin antisepsis using chlorhexidine and eucalyptus oil

Healthcare associated infections may arise from many sources, including patient?s own skin flora and the clinical environment, and inflict a significant burden within the health service. Adequate and effective skin antisepsis and surface disinfection are therefore essential factors in infection control. Current EPIC guidelines recommend 2 % chlorhexidine (CHG) in 70 % isopropyl alcohol (IPA) for skin antisepsis however poor penetration has been reported. Eucalyptus oil (EO) is a known permeation enhancer, producing synergistic antimicrobial activity when combined with CHG. In this current study, the antimicrobial efficacy of EO and its main constituent 1,8-cineole were assessed against a panel of clinically relevant microorganisms, alone and in combination with CHG. The superior antimicrobial efficacy of EO compared with 1,8-cineole, and synergistic effects with CHG against planktonic and biofilm cultures, confirmed its suitability for use in subsequent studies within this thesis. Impregnation of EO, CHG and IPA onto prototype hard surface disinfectant wipes demonstrated significantly improved efficacy compared with CHG/IPA wipes, with clear reductions in the time required to eliminate biofilms. Optimisation of the EO/CHG/IPA formulation resulted in the development of Euclean® wipes, with simulated-use and time kill studies confirming their ability to remove microbial surface contamination, prevent cross contamination and eliminate biofilms within 10 minutes. The employment of isothermal calorimetry provided additional information on the type and rate of antimicrobial activity possessed by Euclean® wipes. A clinical audit of the Euclean® wipes at Birmingham Children?s Hospital, Birmingham, U.K. revealed divided staff opinion, with the highest cited advantage and disadvantage concerning the odour. Finally, skin penetration and cell toxicity studies of EO/CHG biopatches and Euclean® solution developed during this study, revealed no permeation into human skin following biopatch application, and no significant toxicity. These current studies enhance the knowledge regarding EO and its potential applications.

Studies of skin antisepsis and enhanced penetration of chlorhexidine

Current evidence-based guidelines recommend that 2% (w/v) chlorhexidine digluconate (CHG), preferentially in 70% (v/v) isopropyl alcohol (IIPA), is used for skin antisepsis prior to incision of the skin. In this current study, the antimicrobial efficacy of CHG, six essential oils [tea tree oil (TTO), thymol, eucalyptus oil (EO), juniper oil, lavender oil and citronella] and novel benzylidenecarboxamidrazone and thiosemicarbazone compounds were determined against a panel of microorganisms commonly associated with skin infection (Staphylococcus epidermidis, S. aureus, meticillin-resistant S. aureus, Propionibacterium acnes, Acinetobacter spp., Pseudomonas aeruginosa and Candida albicans) The results demonstrated synergistic activity of CHG in combination with EO against biofilm cultures of S. epidermidis, with significantly reduced concentrations of CHG and EO required to inhibit biofilm growth compared to CHG or EO alone. Skin permeation of CHG was subsequently investigated using an in vitro human skin model (Franz cell) and the penetration profile was determined by serial sectioning of the full thickness human skin. Two percent (w/v) CHG in aqueous solution and in 70% (v/v) IPA demonstrated poor skin permeation; however, the skin permeation was significantly enhanced in combination with 5% - 50% (v/v) EO. Detectable levels of CHG did not permeate through full thickness skin in 24 h. Skin permeation of 2% (w/v) CHG in 70% (v/v) IPA in the presence of 10% (v/v) EO was subsequently studied. The results demonstrated a significantly enhanced skin penetration of CHG after a 2 min application, with CHG detected at significant levels to a depth of 600 m with CHG in combination with EO and IPA compared to 100 m with IPA alone. Combination antisepsis comprising CHG and EO may be beneficial for skin antisepsis prior to invasive procedures to reduce the number of microorganisms on and within the skin due to enhanced skin penetration of CHG and improved efficacy against S. epidermidis in a biofilm mode of growth.

Digluconate and isopropyl alcohol biocide formulation

Effective surface disinfection is a fundamental infection control strategy within healthcare. This study assessed the antimicrobial efficacy of novel biocide formulations comprising 5% and 2% eucalyptus oil (EO) combined with 2% chlorhexidine digluconate (CHG) and 70% isopropyl alcohol (IPA) contained within a wipe. The efficacy of this novel antimicrobial formulation to remove and eliminate methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli and Candida albicans from steel surfaces was investigated. Adpression studies of pre-contaminated wipes were also utilised to assess their potential to induce cross-contamination between hard surfaces. Furthermore, the bactericidal nature of the EO-formulation was established in addition to time-kill. The EO-containing formulations demonstrated bactericidal antimicrobial efficacy against all microorganisms and did not induce surface cross-contamination. There was no significant difference (p < 0.05) between the 5% and 2% EO formulations in their ability to remove microorganisms from steel surfaces, however both significantly (p < 0.05) removed more than the control formulations. Microbial biofilms were eliminated within 10 min (p < 0.05) when exposed to the EO formulations. Our novel EO-formulation demonstrated rapid antimicrobial efficacy for potential disinfection and elimination of microbial biofilms from hard surfaces and may therefore be a useful adjunct to current infection control strategies currently employed within healthcare facilities.

Chlorhexidine and the prevention of surgical site infection

Surgical site infections (SSI) are a prevalent health care-associated infection (HAl). Prior to the mid-19th century, surgical sites commonly developed postoperative wound complications. It was in the 1860's, after Joseph Lister introduced carbolic acid and the principles of antisepsis that postoperative wound infection significantly decreased. Today, patient preoperative skin preparation with an antiseptic agent prior to surgery is a standard of practice. Povidone-iodine and chlorhexidine gluconate are currently the most commonly used antimicrobial agents used to prep the patient's skin. In this current study, the epidemiology, diagnosis, surveillance and prevention of SSI with chlorhexidine were investigated. The antimicrobial activity of chlorhexidine was assessed. In in-vitro and in-vivo studies the antimicrobial efficacy of 2% (w/v) chlorhexidine gluconate (CHG) in 70% isopropyl alcohol (IPA) and 10% povidoneiodine (PVP-I) in the presence of 0.9% normal saline or blood were examined. The 2% CHG in 70% IPA solutions antimicrobial activity was not diminished in the presence of 0.9% normal saline or blood. In comparison, the traditional patient preoperative skin preparation, 10% PVP-I antimicrobial activity was not diminished in the presence of 0.9% normal saline, but was diminished in the presence of blood. In an in-vivo human volunteer study the potential for reduction of the antimicrobial efficacy of aqueous patient preoperative skin preparations compromised by mechanical removal of wet product from the application site (blot) was assessed. In this evaluation, 2% CHG and 10% povidone-iodine (PVP-I) were blotted from the patient's skin after application to the test site. The blotting, or mechanical removal, of the wet antiseptic from the application site did not produce a significant difference in product efficacy. In a clinical trial to compare 2% CHG in 70% IPA and PVP-! scrub and paint patient preoperative skin preparation for the prevention of SSI, there were 849 patients randomly assigned to the study groups (409 in the chlorhexidine-alcohol and 440 in the povidone-iodine group) in the intention-to-treat analysis. The overall surgical site infection was significantly lower in the 2% CHG in 70% IPA group than in the PVP-I group (9.5% versus 16.1 %, p=0.004; relative risk, 0.59 with 95% confidence interval of 0.41 to 0.85). Preoperative cleansing of the patient's skin with chlorhexidine-alcohol is superior to povidone-iodine in preventing surgical site infection after clean-contaminated surgery.