Antimicrobial effect of endodontic solutions used as final irrigants on a dentine biofilm model

Aim To evaluate the residual biovolume of live bacterial cells, the mean biofilm thickness and the substratum coverage found in mixed biofilms treated with different endodontic irrigant solutions. Methodology Twenty-five bovine dentine specimens were infected intraorally using a removable orthodontic device. Five samples were used for each irrigant solution: 2% chlorhexidine, 1% sodium hypochlorite (NaOCl), 10% citric acid, 17% EDTA and distilled water. The solutions were used for 5 min. The samples were stained using the Live/Dead technique and evaluated using a confocal microscope. Differences in the amount of total biovolume (mu m3), number of surviving cells (mu m3), mean biofilm thickness (mu m) and substratum coverage (%) of the treated biofilms were determined using nonparametric statistical tests (P < 0.05). Results Similar values of biovolume total, biovolume of live subpopulations and substratum coverage were found in 2% chlorhexidine, 10% citric acid, 17% EDTA and distilled water-treated biofilms (P > 0.05). The lower values of the studied parameters were found in 1% NaOCl-treated dentine (P < 0.05) with the exception of the mean biofilm height criteria that did not reveal significant differences amongst the irrigant solutions (P > 0.05). Conclusions One per cent sodium hypochlorite was the only irrigant that had a significant effect on biofilm viability and architecture.

Nonculture-based identification of bacteria in milk by protein fingerprinting

Traditional methods for bacterial identification include Gram staining, culturing, and biochemical assays for phenotypic characterization of the causative organism. These methods can be time-consuming because they require in vitro cultivation of the microorganisms. Recently, however, it has become possible to obtain chemical profiles for lipids, peptides, and proteins that are present in an intact organism, particularly now that new developments have been made for the efficient ionization of biomolecules. MS has therefore become the state-of-the-art technology for microorganism identification in microbiological clinical diagnosis. Here, we introduce an innovative sample preparation method for nonculture-based identification of bacteria in milk. The technique detects characteristic profiles of intact proteins (mostly ribosomal) with the recently introduced MALDI SepsityperTM Kit followed by MALDI-MS. In combination with a dedicated bioinformatics software tool for databank matching, the method allows for almost real-time and reliable genus and species identification. We demonstrate the sensitivity of this protocol by experimentally contaminating pasteurized and homogenized whole milk samples with bacterial loads of 10(3)-10(8) colony-forming units (cfu) of laboratory strains of Escherichia coli, Enterococcus faecalis, and Staphylococcus aureus. For milk samples contaminated with a lower bacterial load (104 cfu mL-1), bacterial identification could be performed after initial incubation at 37 degrees C for 4 h. The sensitivity of the method may be influenced by the bacterial species and count, and therefore, it must be optimized for the specific application. The proposed use of protein markers for nonculture-based bacterial identification allows for high-throughput detection of pathogens present in milk samples. This method could therefore be useful in the veterinary practice and in the dairy industry, such as for the diagnosis of subclinical mastitis and for the sanitary monitoring of raw and processed milk products.

Bactericidal effects of two parameters of Er:YAG laser intracanal irradiation: ex-vivo study

The success of endodontic treatment depends on the complete elimination of microorganisms from the root canal system, thus the search for new procedures to eliminate them is justified. The aim of this study was to assess bacterial reduction after intracanal irradiation with the Er:YAG laser. The canals of 70 extracted human maxillary canines were prepared up to file #40 using 1% NaOCl, irrigated with 17% EDTA, and then washed with physiological solution activated by ultrasound. The roots were sterilized by autoclaving, inoculated with 10 mu l of a suspension containing 1.5 x 10(8) CFU/ml of Enterococcus faecalis ATCC 29212 and incubated at 37A degrees C for 72 h. The canals were irradiated with the Er:YAG laser using two energy settings: 60 mJ and 15 Hz, and 100 mJ and 10 Hz. The remaining bacteria were counted immediately and 48 h after laser irradiation. The results showed a high bacterial reduction at both time points. With 60 mJ and 15 Hz there was an immediate reduction of 99.73% and the reduction was 77.02% after 48 h, and with 100 mJ and 10 Hz there was an immediate reduction of 99.95% and the reduction was 84.52% after 48 h. Although the best results were observed with 100 mJ of energy, the difference between the two settings was not statistically significant. The count performed 48 h after irradiation showed that E. faecalis were able to survive, and can grow even from small numbers.

Salivary IgA antibody responses to Streptococcus mitis and Streptococcus mutans in preterm and fullterm newborn children

Objectives: The intensities and specificities of salivary IgA antibody responses to antigens of Streptococcus mutans, the main pathogen of dental caries, may influence colonization by these organisms during the first 1.5 year of life. Thus, the ontogeny of salivary IgA responses to oral colonizers continues to warrant investigation, especially with regard to the influence of birth conditions, e.g. prematurity, on the ability of children to efficiently respond to oral microorganisms. In this study, we characterised the salivary antibody responses to two bacterial species which are prototypes of pioneer and pathogenic microorganisms of the oral cavity (Streptococcus mitis and Streptococcus mutans, respectively) in fullterm (FT) and preterm (PT) newborn children. Methods: Salivas from 123 infants (70 FT and 53 PT) were collected during the first 10 h after birth and levels of IgA and IgM antibodies and the presence of S. mutans and S. mitis were analysed respectively by ELISA and by chequerboard DNA-DNA hybridization. Two subgroups of 24 FT and 24 PT children were compared with respect to patterns of antibody specificities against S. mutans and S. mitis antigens, using Western blot assays. Cross-adsorption of 10 infant's saliva was tested to S. mitis, S. mutans and Enterococcus faecalis antigens. Results: Salivary levels of IgA at birth were 2.5-fold higher in FT than in PT children (Mann-Whitney; P < 0.05). Salivary IgA antibodies reactive with several antigens of S. mitis and S. mutans were detected at birth in children with undetectable levels of those bacteria. Adsorption of infant saliva with cells of S. mutans produced a reduction of antibodies recognizing S. mitis antigens in half of the neonates. The diversity and intensity of IgA responses were lower in PT compared to FT children, although those differences were not significant. Conclusion: These data provide evidence that children have salivary IgA antibodies shortly after birth, which might influence the establishment of the oral microbiota, and that the levels of salivary antibody might be related to prematurity. (C) 2011 Elsevier Ltd. All rights reserved.

Antimicrobial Photodynamic Therapy for the Treatment of Teeth with Apical Periodontitis: A Histopathological Evaluation

Introduction: This study evaluated the in vivo response of apical and periapical tissues of dogs' teeth with apical periodontitis after one-session endodontic treatment with and without antimicrobial photodynamic therapy (aPDT). Methods: Sixty root canals with experimentally induced apical periodontitis were instrumented and assigned to 4 groups receiving aPDT and root canal filling (RCF) or not: group aPDT+/RCF- (n = 20): aPDT (photosensitizer phenothiazine chloride at 10 mg/mL for 3 minutes and diode laser [2 = 660 nm, 60 mW/cm(2)] for 1 minute) and RCF in the same session; group aPDT+/RCF (n = 10); group aPDT /RCF+ (n = 20), and group aPDT /RCF (n = 10). Teeth were restored, and the animals were killed after 90 days. Sections from the maxillas and mandibles were stained with hematoxylin-eosin and Mallory trichrome and examined under light microscopy. Descriptive (ie, newly formed apical mineralized tissue, periapical inflammatory infiltrate, apical periodontal ligament thickness, and mineralized tissue resorption) and quantitative (ie, periapical lesion size and number of inflammatory cells) microscopic analysis was performed. Quantitative data were analyzed by the Kruskal-Wallis and Dunn tests (alpha =.05). Results: In the aPDT-treated groups, the periapical region was moderately/severely enlarged with no inflammatory cells, moderate neoangiogenesis and fibrogenesis, and the smallest periapical lesions. Conclusions: Although apical closure by mineralized tissue deposition was not achieved, the absence of inflammatory cells, moderate neoangiogenesis, and fibrogenesis in the periapical region in the groups treated with aPDT indicate that this can be a promising adjunct therapy to cleaning and shaping procedures in teeth with apical periodontitis undergoing one-session endodontic treatment. (J Endod 2012;38:360-366)

Response of mice connective tissue to intracanal dressings containing chlorhexidine

Substances containing chlorhexidine (CHX) have been studied as intracanal medicaments. The aim of the present study was to characterize the response of mouse subcutaneous connective tissue to CHX-containing medications by conventional optical microscopy. The tissue response was evaluated by implanting polyethylene tubes containing one of the substances evaluated: Calen paste + 0.5% CHX, Calen + 2% CHX, 2% CHX gel, and Calen paste (control). After experimental periods of 7, 21, and 63 days, the implants (n = 10) were removed along with the subcutaneous connective tissue. Tissue samples were subjected to histological processing, and sections were stained with hematoxylin and eosin. Qualitative and quantitative analyses of the number of inflammatory cells, blood vessels, and vascularized areas were performed. Results were analyzed by ANOVA and Tukey tests with the significance level set at 5%. We concluded that Calen + 0.5% CHX led to reparative tissue response in contrast with Calen + 2% CHX and 2% CHX gel, which induced persistent inflammatory response, pointing to the aggressive nature of this mixture. When Calen + 2% CHX and 2% CHX gel were compared, the latter induced more intense inflammatory response. Microsc. Res. Tech., 2012. (C) 2012 Wiley Periodicals, Inc.

Effect of Ultrasonic Activation on pH and Calcium Released by Calcium Hydroxide Pastes in Simulated External Root Resorption

Introduction: The purpose of this study was to analyze the influence of ultrasonic activation of calcium hydroxide (CH) pastes on pH and calcium release in simulated external root resorptions. Methods: Forty-six bovine incisors had their canals cleaned and instrumented, and defects were created in the external middle third of the roots, which were then used for the study. The teeth were externally made impermeable, except for the defected area, and divided into the following 4 groups containing 10 samples each according to the CH paste and the use or not of the ultrasonic activation: group 1: propylene glycol without ultrasonic activation, group 2: distilled water without ultrasonic activation, group 3: propylene glycol with ultrasonic activation, and group 4: distilled water with ultrasonic activation. After filling the canals with the paste, the teeth were restored and individually immersed into flasks with ultrapure water. The samples were placed into other flasks after 7, 15, and 30 days so that the water pH level could be measured by means of a pH meter. Calcium release was measured by means of an atomic absorption spectrophotometer. Six teeth were used as controls. The results were statistically compared using the Kruskal-Wallis and Mann-Whitney U tests (P < .05). Results: For all periods analyzed, the pH level was found to be higher when the CH paste was activated with ultrasound. Calcium release was significantly greater (P < .05) using ultrasonic activation after 7 and 30 days. Conclusions: The ultrasonic activation of CH pastes favored a higher pH level and calcium release in simulated external root resorptions. (J Endod 2012;38:834-837)

Chemical resistance of the gram-negative bacteria to different sanitizers in a water purification system

Abstract Background Purified water for pharmaceutical purposes must be free of microbial contamination and pyrogens. Even with the additional sanitary and disinfecting treatments applied to the system (sequential operational stages), Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas alcaligenes, Pseudomonas picketti, Flavobacterium aureum, Acinetobacter lowffi and Pseudomonas diminuta were isolated and identified from a thirteen-stage purification system. To evaluate the efficacy of the chemical agents used in the disinfecting process along with those used to adjust chemical characteristics of the system, over the identified bacteria, the kinetic parameter of killing time (D-value) necessary to inactivate 90% of the initial bioburden (decimal reduction time) was experimentally determined. Methods Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas alcaligenes, Pseudomonas picketti, Flavobacterium aureum, Acinetobacter lowffi and Pseudomonas diminuta were called in house (wild) bacteria. Pseudomonas diminuta ATCC 11568, Pseudomonas alcaligenes INCQS , Pseudomonas aeruginosa ATCC 15442, Pseudomonas fluorescens ATCC 3178, Pseudomonas picketti ATCC 5031, Bacillus subtilis ATCC 937 and Escherichia coli ATCC 25922 were used as 'standard' bacteria to evaluate resistance at 25°C against either 0.5% citric acid, 0.5% hydrochloric acid, 70% ethanol, 0.5% sodium bisulfite, 0.4% sodium hydroxide, 0.5% sodium hypochlorite, or a mixture of 2.2% hydrogen peroxide (H2O2) and 0.45% peracetic acid. Results The efficacy of the sanitizers varied with concentration and contact time to reduce decimal logarithmic (log10) population (n cycles). To kill 90% of the initial population (or one log10 cycle), the necessary time (D-value) was for P. aeruginosa into: (i) 0.5% citric acid, D = 3.8 min; (ii) 0.5% hydrochloric acid, D = 6.9 min; (iii) 70% ethanol, D = 9.7 min; (iv) 0.5% sodium bisulfite, D = 5.3 min; (v) 0.4% sodium hydroxide, D = 14.2 min; (vi) 0.5% sodium hypochlorite, D = 7.9 min; (vii) mixture of hydrogen peroxide (2.2%) plus peracetic acid (0.45%), D = 5.5 min. Conclusion The contact time of 180 min of the system with the mixture of H2O2+ peracetic acid, a total theoretical reduction of 6 log10 cycles was attained in the water purified storage tank and distribution loop. The contact time between the water purification system (WPS) and the sanitary agents should be reviewed to reach sufficient bioburden reduction (over 6 log10).

Identification of bacteria in drinking and purified water during the monitoring of a typical water purification system

Abstract Background A typical purification system that provides purified water which meets ionic and organic chemical standards, must be protected from microbial proliferation to minimize cross-contamination for use in cleaning and preparations in pharmaceutical industries and in health environments. Methodology Samples of water were taken directly from the public distribution water tank at twelve different stages of a typical purification system were analyzed for the identification of isolated bacteria. Two miniature kits were used: (i) identification system (api 20 NE, Bio-Mérieux) for non-enteric and non-fermenting gram-negative rods; and (ii) identification system (BBL crystal, Becton and Dickson) for enteric and non-fermenting gram-negative rods. The efficiency of the chemical sanitizers used in the stages of the system, over the isolated and identified bacteria in the sampling water, was evaluated by the minimum inhibitory concentration (MIC) method. Results The 78 isolated colonies were identified as the following bacteria genera: Pseudomonas, Flavobacterium and Acinetobacter. According to the miniature kits used in the identification, there was a prevalence of isolation of P. aeruginosa 32.05%, P. picketti (Ralstonia picketti) 23.08%, P. vesiculares 12.82%,P. diminuta 11.54%, F. aureum 6.42%, P. fluorescens 5.13%, A. lwoffi 2.56%, P. putida 2.56%, P. alcaligenes 1.28%, P. paucimobilis 1.28%, and F. multivorum 1.28%. Conclusions We found that research was required for the identification of gram-negative non-fermenting bacteria, which were isolated from drinking water and water purification systems, since Pseudomonas genera represents opportunistic pathogens which disperse and adhere easily to surfaces, forming a biofilm which interferes with the cleaning and disinfection procedures in hospital and industrial environments.

Influence of radiopacifying agents on the solubility, pH and antimicrobial activity of portland cement

The aim of this study was to evaluate the interference of the radiopacifiers bismuth oxide (BO), bismuth carbonate (BC), bismuth subnitrate (BS), and zirconiun oxide (ZO) on the solubility, alkalinity and antimicrobial properties of white Portland cement (WPC). The substances were incorporated to PC, at a ratio of 1:4 (v/v) and subjected to a solubility test. To evaluate the pH, the cements were inserted into retrograde cavities prepared in simulated acrylic teeth and immediately immersed in deionized water. The pH of the solution was measured at 3, 24, 72 and 168 h. The antimicrobial activity was evaluated by a radial diffusion method against the microorganisms S. aureus (ATCC 25923), P. aeruginosa (ATCC 27853), E. faecalis (ATCC 29212) and C. albicans (ATCC 10231). The zone of microbial growth inhibition was measured after 24 h. The addition of BS and BC increased the solubility of the cement. The pH values demonstrated that all materials produced alkaline levels. At 3 h, BS showed lower pH than WPC (p<0.05). At 168 h, all materials showed similar pHs (p>0.05). The materials did not present antimicrobial activity for S. aureus, P. aeruginosas and E. faecalis (p>0.05). With regards to C. albicans, all materials formed an inhibition zone, mainly the mixture of WPC with ZO (p<0.05). The type of radiopacifier incorporated into WPC interfered with its physical and antimicrobial properties. ZO was found to be a viable radiopacifier that can be used with WPC.