Enterococcus faecalis leakage of root canal sealers: an ex vivo study

The aim of this ex vivo study was to evaluate bacterial penetration after filling root canals using 3 different techniques. Three experimental groups of 25 teeth each, obturated with lateral-warm-vertical condensation of gutta-percha, Microseal technique and EndoREZ (R) system, respectively, were tested in a split chamber model system using Enterococcus faecalis and monitored for 180 days to determine bacterial penetration. A statistical analysis was performed using the Kaplan-Meier method. Median survival time was 25 days for Microseal system, 41 for lateral-warm-vertical condensation and 81 for EndoREZ (R). Significant differences were demonstrated between Microseal and EndoREZ (R) (p<0.001) and between Microseal and lateral-warm-vertical condensation technique (p<0.05). No statistically significant differences were observed between EndoREZ (R) and lateral-warm-vertical condensation. After 180 days of assessment, 20% of the EndoREZ (R) samples resisted bacterial penetration and furthermore, the EndoREZ (R) system has the potential to be a filler system compatible with other currently used systems.

Nodule ultrastructure and initial growth of Anadenanthera peregrina (L.) Speg. var. falcata (Benth.) Altschul plants infected with rhizobia

The anatomy and ultrastructure of root nodules of Anadenanthera peregrina var. falcata (Leguminosae-Mimosoideae) were analysed, as was plant growth. To ensure that nodules developed, seedlings were inoculated with a mixture of six strains of rhizobia. Nodules were produced that differed in appearance-and probably also effectiveness-but their structure was similar and they showed characteristics typical of indeterminate nodules, such as persistent meristematic tissue and a gradient of cells at different stages of development. Many starch grains were present in inner cortex cells and interstitial cells of infected tissue. Infected cells were densely packed with bacteroids, which contained many poly-beta-hydroxybutyrate granules. The high incidence of these granules, together with high levels of starch accumulation in interstitial cells, suggested low N-2-fixation efficiency of the rhizobia isolates used for inoculation. In the symbiosomes of early-senescent infected cells, reticulum-like structures, small vesicles and a fibrillar material were observed; these may be related to bacteroid degradation. In the cytoplasm of late-senescent infected cells, many vesicles and membrane-like structures were observed, probably associated with membrane degradation of bacteroids and peribacteroids. The total biomass of plants inoculated with rhizobia was low and their xylopodia and shoots had low levels of N compared with non-inoculated plants fertilized with ammonium nitrate. However, inoculated plants did not show N-deficiency symptoms and grew better than non-inoculated plants without N fertilization. These growth results, together with ultrastructural observations of nodules, suggest that nitrogen fixation of rhizobia isolates associated with Anadenanthera peregrina var. falcata roots is poor. (C) 2002 Annals of Botany Company.

Identification of bacteria in endodontic infections by sequence analysis of 16S rDNA clone libraries

A significant proportion of oral bacteria are unable to undergo cultivation by existing techniques. In this regard, the microbiota from root canals still requires complementary characterization. The present study aimed at the identification of bacteria by sequence analysis of 16S rDNA clone libraries from seven endodontically infected teeth. Samples were collected from the root canals, subjected to the PCR with universal 16S rDNA primers, cloned and partially sequenced. Clones were clustered into groups of closely related sequences (phylotypes) and identification to the species level was performed by comparative analysis with the GenBank, EMBL and DDBJ databases, according to a 98 % minimum identity. All samples were positive for bacteria and the number of phylotypes detected per subject varied from two to 14. The majority of taxa (65(.)2 %) belonged to the phylum Firmicutes of the Gram-positive bacteria, followed by Proteobacteria (10(.)9 %), Spirochaetes (4(.)3 %), Bacteroidetes (6(.)5 %), Actinobacteria (2(.)2 %) and Deferribacteres (2(.)2 %). A total of 46 distinct taxonomic units was identified. Four clones with low similarity to sequences previously deposited in the databases were sequenced to nearly full extent and were classified taxonomically as novel representatives of the order Clostridiales, including a putative novel species of Mogibacterium. The identification of novel phylotypes associated with endodontic infections suggests that the endodontium may still harbour a relevant proportion of uncharacterized taxa.

Effects of a lectin-like protein isolated from Acacia farnesiana seeds on phytopathogenic bacterial strains and root-knot nematode

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.

Radiographic and microbiologic evaluation of posttreatment apical and periapical repair of root canals of dogs' teeth with experimentally induced chronic lesion

The objective of the present study was to evaluate radiographically and bacteriologically apical and periapical repair in dogs' teeth with induced chronic periapical lesions with the use of two different operative techniques (techniques 1 and 2). The study was conducted on 40 root canals of upper and lower premolars from two dogs aged approximately 12 months. Periapical lesions were induced by leaving the root canals exposed to the oral environment for 5 days and then sealing them with zinc oxide-eugenol for 45 days. After this period, radiographic examination revealed the occurrence of a radiolucent lesion and endodontic treatment was started. The two techniques did not differ in terms of chemomechanical preparation, final filling, or type of cement, but differed in terms of irrigating solution and the presence of an antibacterial dressing. Thus 4% to 6% hypochlorite and hydrogen peroxide (10 volumes) were used in technique 1 during chemomechanical preparation and an antibacterial dressing based on calcium hydroxide was applied between sessions, whereas Dakin's fluid (0.5% sodium hypochlorite solution) and a final filling with no antibacterial dressing were used in technique 2. After chemomechanical preparation, the root canals were filled with gutta-percha cones and Sealapex (Sealapex-Sybron, Kerr, Sao Paulo, Brazil), and the animals were killed 270 days after the final filling. Blocks were cut into 6-μm sections and stained by the Brown and Brenn method. Radiographic, histomicrobiologic and statistical analysis permitted us to conclude the following: radiographically there was a marked reduction or even the disappearance of the radiolucent area present before treatment with greater success in the group treated with technique 1 (group I) than in the group treated with technique 2 (group II); the extent of bacterial invasion of dentinal tubules was greater and more intense in group II than in group I; and the amount of microorganisms detected in the ramifications of the apical delta and in the lumen of the root canal was intense in group II and mild or absent in group I. © 1994.

Distribution of Xylella fastidiosa in citrus rootstocks and transmission of citrus variegated chlorosis between sweet orange plants through natural root grafts

To study translocation of Xylella fastidiosa to citrus rootstocks, budsticks from citrus variegated chlorosis (CVC)-affected cv. Pera sweet orange (Citrus sinenesis (L.) Osb.) were top grafted on 15 citrus rootstocks. Disease symptoms were conspicuous 3 months later on all 15 rootstocks tested. The presence of X. fastidiosa was confirmed by light microscopy, double-antibody sandwich enzyme-linked immunosorbent assays, and polymerase chain reaction in rootlets and main roots of CVC-symptomatic Pera sweet orange in 11 of the 15 rootstocks tested. These results suggest that bacterial translocation from the aerial plant parts to the root system occurs but is not essential for X. fastidiosa to induce symptoms in the aerial parts. Bacterial translocation to the roots was not correlated with CVC leaf-symptom severity in the Pera scion. To determine if CVC disease could be transmitted by natural root grafts, two matched seedlings of each of four sweet orange cultivars (Pera, Natal, Valencia, and Caipira) were transplanted into single pots. One seedling rootstock of each pair was inoculated by top grafting with a CVC-contaminated budstick while the other seedling rootstock was cut but not graft inoculated. Transmission of X. fastidiosa from an inoculated plant to a noninoculated plant sharing the same pot was observed in all four sweet orange cultivars tested. Transmission was confirmed by observation of natural roots grafts between the two plants, presence of X. fastidiosa in the root grafts, and disease development in the uninoculated plants. This is the first report of transmission of CVC disease through natural root grafts.

Histomicrobiologic aspects of the root canal system and periapical lesions in dogs' teeth after rotary instrumentation and intracanal dressing with Ca(OH)2 pastes

OBJECTIVE: The purpose of this study was to evaluate the distribution of microorganisms in the root canal system (RCS) and periapical lesions of dogs' teeth after rotary instrumentation and placement of different calcium hydroxide [Ca(OH)2]-based intracanal dressings. MATERIALS AND METHODS: Chronic periapical lesions were experimentally induced in 80 premolar roots of four dogs. Instrumentation was undertaken using the ProFile rotary system and irrigation with 5.25% sodium hypochlorite. The following Ca(OH) 2-based pastes were applied for 21 days: group 1 - Calen (n=18); group 2 - Calen+CPMC (n=20); group 3 - Ca(OH)2 p.a. + anaesthetic solution (n=16) and group 4 - Ca(OH)2 p.a.+ 2% chlorhexidine digluconate (n=18). Eight root canals without endodontic treatment constituted the control group. Histological sections were obtained and stained with Brown & Brenn staining technique to evaluate the presence of microorganisms in the main root canal, ramifications of the apical delta and secondary canals, apical cementoplasts, dentinal tubules, areas of cemental resorption and periapical lesions. The results were analyzed statistically by the Mann-Whitney U test (p<0.05). RESULTS: The control group showed the highest prevalence of microorganisms in all sites evaluated. Gram-positive cocci, bacilli and filaments were the most frequent morphotypes. Similar microbial distribution patterns in the RCS and areas of cementum resorption were observed in all groups (p>0.05). The percentage of RCS sites containing microorganisms in groups 1, 2, 3, 4 and control were: 67.6%, 62.5%, 78.2%, 62.0% and 87.6%, respectively. CONCLUSION: In conclusion, the histomicrobiological analysis showed that the rotary instrumentation and the different calcium hydroxide pastes employed did not effectively eliminate the infection from the RCS and periapical lesions. However, several bacteria seen in the histological sections were probably dead or were inactivated by the biomechanical preparation and calcium hydroxide-based intracanal dressing.

Periapical tissue healing after post space preparation with or without use of a protection plug and root canal exposure to the oral environment. Study in dogs

The purpose of this study was to evaluate the influence of coronal leakage on the healing of dogs' periapical tissues after root canal filling, post space preparation and protection or not with a temporary sealer plug. Forty root canals of dogs' teeth were instrumented and filled by the lateral condensation technique with gutta-percha points and Endomethasone or CRCS sealers. After post space preparation, the remaining filling material was protected or not with a plug of temporary Coltosol sealer and exposed to the oral environment for 90 days. Thereafter, the animals were sacrificed and the specimens were removed and prepared for histomorphological and histobacteriological analysis. The findings revealed 35% of microbial leakage in the groups without plugs and 15% of leakage in the groups with plugs. Statistical analysis showed that the use of a Coltosol plug improved significantly the histomorphological results regardless of the type of root canal sealer (p=0.05) and that CRCS and Endomethasone sealers showed similar results (p>0.05).

Microbial distribution in the root canal system after periapical lesion induction using different methods

The aim of this study was to evaluate the microbial distribution in the root canal system after periapical lesion induction in dogs' teeth using different methods. Fifty-two root canals were assigned to 4 groups (n=13). Groups I and II: root canals were exposed to the oral cavity for 180 days; groups III and IV: root canals were exposed for 7 days and then the coronal openings were sealed for 53 days. The root apices of groups I and III were perforated, while those of groups II and IV remained intact. After the experimental periods, the animals were euthanized and the anatomic pieces containing the roots were processed and stained with the Brown & Brenn method to assess the presence and distribution of microorganisms. The incidence of microorganisms at different sites of the roots and periapical lesions was analyzed statistically by the chi-square test at 5% significance level. All groups presented microorganisms in the entire root canal system. A larger number of microorganisms was observed on the root canal walls, apical delta and dentinal tubules (p<0.05), followed by cementum and cemental resorption areas. In spite of the different periods of exposure to the oral environment, the methods used for induction of periapical periodontitis yielded similar distribution of microorganisms in the root canal system.

Ex-vivo Effect of Intracanal Medications Based on Ozone and Calcium Hydroxide in Root Canals Contaminated with Enterococcus faecalis

This ex vivo study evaluated the antibacterial effect of intracanal medications in root canals contaminated with Enterococcus faecalis. Fifty single-rooted human teeth were contaminated with E. faecalis (ATCC 29212) and incubated at 37°C for 21 days. The specimens were randomly divided into 5 groups according to the intracanal medication used: OZ-PG: ozonized propylene glycol; CH/CPMC: calcium hydroxide/camphorated paramonochlorophenol; OZ-PG/CH ozonized PG/CH; PC: positive control group (no medication); and NC: negative control group (no contamination). The samples were collected after 7 days (post-medication) and 14 days (final). Bacterial growth was checked by counting the colony-forming units (CFU). OZ-PG and CH/CPMC reduced significantly the CFU counts compared with PC in the post-medication and final samples, with no statistically significant differences among them. On the other hand, OZ-PG/CH did not reduce significantly the number of bacteria compared with PC. In conclusion, among the evaluated medications OZ-PG and CH/CPMC were the most effective against E. faecalis.

In vitro antimicrobial activity of auxiliary chemical substances and natural extracts on Candida albicans and Enterococcus faecalis in root canals

Objective: The aim of this study was to evaluate the antimicrobial activity of auxiliary chemical substances and natural extracts on Candida albicans and Enterococcus faecali's inoculated in root canals. Material and Methods: Seventy-two human tooth roots were contaminated with C. albicans and E. faecalis for 21 days. The groups were divided according to the auxiliary chemical substance into: G1) 2.5% sodium hypochlorite (NaOCl), G2) 2% chlorhexidine gel (CHX), G3) castor oil, G4) glycolic Aloe vera extract, g5) glycolic ginger extract, and G6) sterile saline (control). The samples of the root canal were collected at different intervals: confirmation collection, at 21 days after contamination; 1st collection, after instrumentation; and 2nd collection, seven days after instrumentation. Microbiological samples were grown in culture medium and incubated at 37°C for 48 hours. Results: The results were submitted to the Kruskal-Wallis and Dunn (5%) statistical tests. NaOCl and CHX completely eliminated the microorganisms of the root canals. Castor oil and ginger significantly reduced the number of CFU of the tested bacteria. Reduction of CFU/mL at the 1st and 2nd collections for groups G1, G2, G3 and G4 was greater in comparison to groups G5 and G6. Conclusion: It was concluded that 2.5% sodium hypochlorite and 2% chlorhexidine gel were more effective in eliminating C. albicans and E. faecalis, followed by the castor oil and glycolic ginger extract. The Aloe vera extract showed no antimicrobial activity.

Plant growth promoting bacteria in Brachiaria brizantha

Brachiaria brizantha is considered one of the preferred fodders among farmers for having high forage yield and large production of root mass. The association of beneficial bacteria with these grasses can be very valuable in the recovery of the pasture areas with nutritional deficiency. With the aim of studying this possibility, we carried out the sampling of soil and roots of B. brizantha in three areas (Nova Odessa-SP, So Carlos-SP and Campo Verde-MT, Brazil). Seventy-two bacterial strains were isolated and used in tests to evaluate their biotechnological potential. Almost all isolates presented at least one positive feature. Sixty-eight isolates produced analogues of indole-3-acetic acid, ten showed nitrogenase activity when subjected to the method of increasing the concentration of total nitrogen (total N) in the culture medium and sixty-five isolates showed nitrogenase activity when subjected to acetylene reduction technique. The partial sequencing of 16S rRNA of these isolates allowed the identification of seven main groups, with the prevalence of those affiliated to the genus Stenotrophomonas (69 %). At the end, this work elected the strains C4 (Pseudomonadaceae) and C7 (Rhodospirillaceae) as promising organisms for the development of inoculants due to their higher nitrogenase activity.

Clinical investigation of bacterial species and endotoxin in endodontic infection and evaluation of root canal content activity against macrophages by cytokine production

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Endodontic retreatment: clinical comparison of reciprocating systems versus rotary system in disinfecting root canals

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)