Pattern of Disinfection of Root Canal Dentin by Alternated Acid-Base Irrigating Regimen.

OBJECTIVE: To quantify Enterococcus faecalis density in root canal dentin after chemomechanical preparation (CMP) using alternated irrigating regimen. METHODOLOGY: Root canals (RC) were contaminated with E. faecalis (ATCC 19433) for 3 weeks and evident biofilms were obtained. After initial sampling (S1), the CMP was aided by irrigants: saline solution (control; n=12), a conventional regimen (CR) (group 1; n=12) using 5.25% NaOCl and a final rinse with 17% EDTA, and an alternating regimen (AR) of intercalated use of NaOCl and EDTA (group 2, n=12), followed by a second sampling (S2). After 2 weeks, S3 was obtained. Two roots were analyzed by scanning electron microscopy. Each root was divided into cervical, mild, and apical segments and sampling of the superficial (n=90) and deep (n=90) dentin layers was obtained using Gates-Glidden burs. The E. faecalis density (CFU/mg) in log10 was categorized as residual (0 > 0.2), moderate (0.2 ≥ 0.5), or elevated (> 0.5). The prevalence of positive samples in BHI and BHI-A was analyzed by Pearson's chi-square test. The data were normalized by a log10 transformation of CFU and were analyzed by one-way ANOVA and Tukey's tests. RESULTS: Biofilms were observed only in the control root canal walls. Topographically, the controls and CR showed similar distributions of E. faecalis in the dentin. Microbiologically positive root canals harbored much E. faecalis in the adjacent dentin (p < 0.05). Irrigating saline provided moderate density of E. faecalis in the dentin while CR and AR resulted in a residual density of microorganisms (p < 0.05). CONCLUSIONS: The Enterococcus faecalis density in dentin was influenced by the irrigating regimen and the microbiological status of the root canal. The CMP aided by the alternating regimen interfered with the recolonization of the root canal and topographic distribution of Enterococcus in root dentin.

Investigating cross-contamination by yeast strains from dental solid waste to waste-handling workers by DNA sequencing.

Trying to widen the discussion on the risks associated with dental waste, this study proposed to investigate and genetically compare yeast isolates recovered from dental solid waste and waste workers. Three samples were collected from workers' hands, nasal mucosa, and professional clothing (days 0, 30, and 180), and two from dental waste (days 0 and 180). Slide culture, microscopy, antifungal drug susceptibility, intersimple sequence repeat analysis, and amplification and sequencing of internal transcribed spacer regions were performed. Yeast strains were recovered from all waste workers' sites, including professional clothes, and from waste. Antifungal susceptibility testing demonstrated that some yeast recovered from employees and waste exhibited nonsusceptible profiles. The dendrogram demonstrated the presence of three major clusters based on similarity matrix and UPGMA grouping method. Two branches displayed 100% similarity: three strains of Candida guilliermondii isolated from different employees, working in opposite work shifts, and from diverse sites grouped in one part of branch 1 and cluster 3 that included two samples of Candida albicans recovered from waste and the hand of one waste worker. The results suggested the possibility of cross-contamination from dental waste to waste workers and reinforce the need of training programs focused on better waste management routines.

Monitoring the effectiveness of photodynamic therapy with periodic renewal of the photosensitizer on intracanal Enterococcus faecalis biofilms.

BACKGROUND AND OBJECTIVE: Photodynamic therapy (PDT) can eliminate microorganisms in a root canal. However, the parameters for disinfection remain undefined. This study assessed the effectiveness of a PDT protocol against intracanal Enterococcus faecalis biofilms. MATERIALS AND METHODS: Root canals were contaminated with E. faecalis for 21 days. The instrumentation was associated to irrigation with 0.85% saline or an alternate irrigation (AI) with 5.25% NaOCl and 17% EDTA. Complementary treatments included saline/PDT and AI/PDT. Four PDT cycles were performed using a diode laser (660nm, 40mW) delivered through a tapered optical fiber. In each cycle, the root canal was filled with 1.56µM/mL methylene blue and irradiated for 150s. Microbiological samples were collected before (S1) and after (S2) instrumentation; after PDT (S3); and daily over the course of 14 days (S4-S17). Colony-forming units (CFUs) were counted, positive cultures verified, and data subjected to parametric and proportion's tests. RESULTS: The highest bacterial load reduction was observed in S2. In regard to S3, Saline/PDT reduced 1.3 log(10) CFU counts (p=0.000 for S2) and no CFUs were recovered after AI/PDT treatment. All canals were CFU-free on the 14th day for saline/PDT, AI and AI/PDT. Positive cultures were observed in 60% of saline-irrigated canals on the 14th day, whereas the saline/PDT, AI and AI/PDT treatments resulted in germ-free canals after 10, 5 and 2 days, respectively. CONCLUSION: Our findings suggest immediate and delayed antibacterial effects using the PDT protocol tested.

Sub-Inhibitory Concentration of Piperacillin-Tazobactam May be Related to Virulence Properties of Filamentous Escherichia coli.

Sub-inhibitory concentrations of antibiotics are always generated as a consequence of antimicrobial therapy and the effects of such residual products in bacterial morphology are well documented, especially the filamentation generated by beta-lactams. The aim of this study was to investigate some morphological and pathological aspects (virulence factors) of Escherichia coli cultivated under half-minimum inhibitory concentration (1.0 µg/mL) of piperacillin-tazobactam (PTZ sub-MIC). PTZ sub-MIC promoted noticeable changes in the bacterial cells which reach the peak of morphological alterations (filamentation) and complexity at 16 h of antimicrobial exposure. Thereafter the filamentous cells and a control one, not treated with PTZ, were comparatively tested for growth curve; biochemical profile; oxidative stress tolerance; biofilm production and cell hydrophobicity; motility and pathogenicity in vivo. PTZ sub-MIC attenuated the E. coli growth rate, but without changes in carbohydrate fermentation or in traditional biochemical tests. Overall, the treatment of E. coli with sub-MIC of PTZ generated filamentous forms which were accompanied by the inhibition of virulence factors such as the oxidative stress response, biofilm formation, cell surface hydrophobicity, and motility. These results are consistent with the reduced pathogenicity observed for the filamentous E. coli in the murine model of intra-abdominal infection. In other words, the treatment of E. coli with sub-MIC of PTZ suggests a decrease in their virulence.

Solid Lipid Nanoparticles Loaded with Retinoic Acid and Lauric Acid as an Alternative for Topical Treatment of Acne Vulgaris.

Topical therapy is the first choice for the treatment of mild to moderate acne and all-trans retinoic acid is one of the most used drugs. The combination of retinoids and antimicrobials is an innovative approach for acne therapy. Recently, lauric acid, a saturated fatty acid, has shown strong antimicrobial activity against Propionibacterium acnes. However, topical application of retinoic acid is followed by high incidence of side-effects, including erythema and irritation. Solid lipid nanoparticles represent an alternative to overcome these side-effects. This work aims to develop solid lipid nanoparticles loaded with retinoic acid and lauric acid and evaluate their antibacterial activity. The influence of lipophilic stearylamine on the characteristics of solid lipid nanoparticles was investigated. Solid lipid nanoparticles were characterized for size, zeta potential, encapsulation efficiency, differential scanning calorimetry and X-ray diffraction. The in vitro inhibitory activity of retinoic acid-lauric acid-loaded solid lipid nanoparticles was evaluated against Propionibacterium acnes, Staphylococcus aureus and Staphylococcus epidermidis. High encapsulation efficiency was obtained at initial time (94 ± 7% and 100 ± 4% for retinoic acid and lauric acid, respectively) and it was demonstrated that lauric acid-loaded-solid lipid nanoparticles provided the incorporation of retinoic acid. However, the presence of stearylamine is necessary to ensure stability of encapsulation. Moreover, retinoic acid-lauric acid-loaded solid lipid nanoparticles showed growth inhibitory activity against Staphylococcus epidermidis, Propionibacterium acnes and Staphylococcus aureus, representing an interesting alternative for the topical therapy of acne vulgaris.

Proteomic changes in Bacteroides fragilis exposed to subinhibitory concentration of piperacillin/tazobactam.

Bacteroides fragilis is the anaerobe most frequently isolated from clinical specimens and piperacillin/tazobactam is among the drugs that can be used to treat polymicrobial infections in which this bacteria is often involved. During antibiotic therapy, inhibitory concentrations of antibiotics are always followed by subinhibitory concentrations which can generate phenotypic changes in bacteria. So, in this study we aimed to evaluate changes in the proteomic profile of B. fragilis grown in a sub-MIC of PTZ, using 2-D electrophoresis followed by matrix-assisted laser desorption/ionization time-of-flight/time of-flight. Analysis of the 2-DE gels showed 18 spots with significantly different volume percentages between experimental conditions and 12 were successfully identified by MS/MS. Two proteins with decreased abundance in sub-MIC condition were involved in the glycolysis (glyceraldehyde-3-phosphate dehydrogenase and triose phosphate isomerase), others two involved in amino acid metabolism (Oxoacyl-(acyl-carrier protein) synthase II and dihydrodipicolinate reductase), and finally, one protein involved in fatty acid metabolism (UDP-N-acetylglucosamine acyltransferase). Among the proteins with increased abundance, we founded three ATP synthase (alpha, beta, and alpha type V), which could be involved in antibiotic bacterial resistance by efflux pump, one protein involved in glycolysis (enolase), and one involved in protein degradation (aminoacyl-histidine dipeptidase). In conclusion, our data show overall changes in the proteome of B. fragilis conducted by sub-MIC of PTZ, whose consequences on bacterial physiology deserve further investigation.

Count, identification and antimicrobial susceptibility of bacteria recovered from dental solid waste in Brazil.

In Brazil, few studies on microbial content of dental solid waste and its antibiotic susceptibility are available. An effort has been made through this study to evaluate the hazardous status of dental solid waste, keeping in mind its possible role in cross-infection chain. Six samples of solid waste were collected at different times and seasons from three dental health services. The microbial content was evaluated in different culture media and atmospheric conditions, and the isolates were submitted to antibiotic susceptibility testing. A total of 766 bacterial strains were isolated and identified during the study period. Gram-positive cocci were the most frequent morphotype isolated (48.0%), followed by Gram-negative rods (46.2%), Gram-positive rods (5.0%), Gram-negative-cocci (0.4%), and Gram-positive coccobacillus (0.1%). Only two anaerobic bacteria were isolated (0.3%). The most frequently isolated species was Staphylococcus epidermidis (29.9%), followed by Stenotrophomonas maltophilia (8.2%), and Enterococcus faecalis (6.7%). High resistance rate to ampicillin was observed among Gram-negative rods (59.4%) and Gram-positive cocci (44.4%). For Gram-negative rods, high resistance was also noted to aztreonam (47.7%), cefotaxime (47.4%), ceftriaxone and cefazolin (43.7%), and ticarcillin-clavulanic acid (38.2%). Against Gram-positive cocci penicillin exhibit a higher resistance rate (45.0%), followed by ampicillin, erythromycin (27.2%), and tetracycline (22.0%). The present study demonstrated that several pathogenic bacteria are present in dental solid waste and can survive after 48 h from the waste generation time and harbor resistance profiles against several clinical recommended antibiotics.

Proteomic analysis of Escherichia coli with experimentally induced resistance to piperacillin/tazobactam.

The worldwide emergence of antibiotic-resistant bacteria poses a serious threat to human health. In addition to the difficulties in controlling infectious diseases, the phenotype of resistance can generate metabolic changes which, in turn, can interfere with host-pathogen interactions. The aim of the present study was to identify changes in the subproteome of a laboratory-derived piperacillin/tazobactam-resistant strain of Escherichia coli (minimal inhibitory concentration [MIC] = 128 mg/L) as compared with its susceptible wild-type strain E. coli ATCC 25922 (MIC = 2 mg/L) using 2-D fluorescence difference gel electrophoresis (2D-DIGE) followed by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF MS). In the resistant strain, a total of 12 protein species were increased in abundance relative to the wild-type strain, including those related to bacterial virulence, antibiotic resistance and DNA protection during stress. Fourteen proteins were increased in abundance in the wild-type strain compared to the resistant strain, including those involved in glycolysis, protein biosynthesis, pentose-phosphate shunt, amino acid transport, cell division and oxidative stress response. In conclusion, our data show overall changes in the subproteome of the piperacillin/tazobactam-resistant strain, reporting for the first time the potential role of a multidrug efflux pump system in E. coli resistance to piperacillin/tazobactam.

Composition analysis of dental solid waste in Brazil.

When developing proper waste management strategies, it is essential to characterize the volume and composition of solid waste. The aim of this work was to evaluate the composition of dental waste produced by three dental health services in Belo Horizonte, Minas Gerais State, Brazil. Two universities, one public and one private, and one public dental health service were selected. Waste collection took place from March to November 2007. During this period, three samples were collected from each dental health service. The total amount of dental waste produced in one day of dental work was manually separated into three categories: infectious and potentially infectious waste, accounting for 24.3% of the total waste; non-infectious waste, accounting for 48.1%; and domestic-type waste, accounting for 27.6% (percentages are for mean weights of solid waste). Our results showed that most of the waste considered as biomedical may be misclassified, consequently making the infectious waste amount appear much larger. In addition, our results suggest that the best waste minimization method is recycling, and they help to define an appropriate waste management system in all three of the dental health services involved in this study.

Actinomycetemcomitin: a new bacteriocin produced by Aggregatibacter (Actinobacillus) actinomycetemcomitans.

Aggregatibacter (Actinobacillus) actinomycetemcomitans P(7-20) strain isolated from a periodontally diseased patient has produced a bacteriocin (named as actinomycetemcomitin) that is active against Peptostreptococcus anaerobius ATCC 27337. Actinomycetemcomitin was produced during exponential and stationary growth phases, and its amount decreased until it disappeared during the decline growth phase. It was purified by ammonium sulphate precipitation (30-60% saturation), and further by FPLC (mono-Q ionic exchange and Phenyl Superose hydrophobic interaction) and HPLC (C-18 reversed-phase). This bacteriocin loses its activity after incubation at a pH below 7.0 or above 8.0, following heating for 30 min at 45 degrees C, and after treatment with proteolytic enzymes such as trypsin, alpha-chymotrypsin, and papain. Actinomycetemcomitin has a molecular mass of 20.3 KDa and it represents a new bacteriocin from A. actinomycetemcomitans.

Antimicrobial susceptibility of microorganisms recovered from intraabdominal infections at Belo Horizonte, Brazil.

We evaluated antimicrobial susceptibility patterns of microorganisms isolated from intraabdominal infection of Brazilian patients, by agar dilution, agar diffusion, and E test. Among the strictly anaerobes, 57.7% were resistant to penicillin, 28.2% to clindamycin, and 9.9% to metronidazole. The majority of Escherichia coli and Staphylococcus were sensitive and resistant to almost all drugs, respectively. Half of Candida samples were resistant to itraconazole. Our data reinforce the importance of this kind of study to support rational antimicrobial therapy.