Influence of the length of remaining root canal filling and post space preparation on the coronal leakage of Enterococcus faecalis

This study evaluated the sealing ability of different lengths of remaining root canal filling and post space preparation against coronal leakage of Enterococcus faecalis. Forty-one roots of maxillary incisors were biomechanically prepared, maintaining standardized canal diameter at the middle and coronal thirds. The roots were autoclaved and all subsequent steps were undertaken in a laminar flow chamber. The canals of 33 roots were obturated with AH Plus sealer and gutta-percha. The root canal fillings were reduced to 3 predetermined lengths (n=11): G1=6 mm, G2=4 mm and G3=2 mm. The remaining roots served as positive and negative controls. Bacterial leakage test apparatuses were fabricated with the roots attached to Eppendorf tubes keeping 2 mm of apex submerged in BHI in glass flasks. The specimens received an E. faecalis inoculum of 1 x 107 cfu/mL every 3 days and were observed for bacterial leakage daily during 60 days. Data were submitted to ANOVA, Tukey's test and Fisher's test. At 60 days, G1 (6 mm) and G2 (4 mm) presented statistically similar results (p>0.05) (54.4% of specimens with bacterial leakage) and both groups differed significantly (p<0.01) from G3 (2 mm), which presented 100% of specimens with E. faecalis leakage. It may be concluded that the shortest endodontic obturation remnant leaked considerably more than the other lengths, although none of the tested conditions avoids coronal leakage of E. faecalis.

Photodynamic therapy for root canals infected with Enterococcus faecalis

Objective: The aim of this study was to investigate the effects of photodynamic therapy (PDT) on endodontic pathogens by evaluating the decrease in numbers of Enterococcus faecalis colonies in the canals of extracted human teeth. Background Data: Failure in endodontics is usually related to inadequate cleaning and disinfection of the root canal system. This is due to the establishment of microorganisms in areas where the instruments and chemical agents used during root canal preparation cannot eliminate them. PDT is a complementary therapeutic method that could be used to eliminate these remaining bacteria. PDT is a process in which radiation acts on a dye that is applied to the target organism, resulting in bacterial death. Materials and Methods: Forty-six uniradicular teeth had their canals contaminated with bacteria and were incubated for 48 h at 35 degrees C. After that, the teeth were divided into a control group (CG) and a test group (TG). The 23 CG teeth did not undergo any intervention, whereas in the TG the teeth received a solution of 0.0125% toluidine blue for 5 min followed by irradiation using a 50-mW diode laser (Ga-Al-As) at a wavelength of 660 nm. Bacterial samples were taken before and after irradiation. In each of the samples, the number of colony-forming units (CFU) was counted. Results: The mean decrease in CFU was 99.9% in the TG, whereas in the CG an increase of 2.6% was observed. Conclusion: PDT was effective as a bactericidal agent in Enterococcus faecalis-contaminated root canals.

Characterisation of an antiviral pediocin-like bacteriocin produced by Enterococcus faecium

The bacteriocin-producing strain Enterococcus faecium ST5Ha was isolated from smoked salmon and identified by biomolecular techniques. Ent. faecium ST5Ha produces a pediocin-like bacteriocin with activity against several lactic acid bacteria, Listeria spp. and some other human and food pathogens, and remarkably against HSV-1 virus. Bacteriocin ST5Ha was produced at high levels in MRS broth at 30 degrees C and 37 degrees C, reaching a maximum production of 1.0 x 10(9) AU/ml, checked against Listeria ivanovii ATCC19119 as target strain and surrogate of pathogenic strain Listeria monocytogenes. The molecular weight of bacteriocin ST5Ha was estimated to be 4.5 kDa according to tricine-SDS-PAGE data. Ent. faecium ST5Ha harbors a 1.044 kb chromosomal DNA fragment fitting in size to that of pediocin PA-1/AcH. In addition, the sequencing of bacteriocin ST5Ha gene indicated 99% of DNA homology to pediocin PA-1/AcH. The combined application of low levels (below MIC) of ciprofloxacin and bacteriocin ST5Ha resulted in a synergetic effect in the inhibition of target strain L ivanovii ATCC19119. Bacteriocin ST5Ha displayed antiviral activity against HSV-1, an important human pathogen, with a selectivity index of 173. To the best of our knowledge, this is the first report on Ent. faecium as a potential producer of pediocin-like bacteriocin with antiviral activity. (C) 2010 Elsevier Ltd. All rights reserved.

Prevalence and characterization of Enterococcus spp. isolated from Brazilian foods

Enterococci can be used in the food industry as starter or probiotic cultures. However, enterococci are also implicated in severe multi-resistant nosocomial infections. In this study, the prevalence of enterococci in selected Brazilian foodstuffs (raw and pasteurized milk, meat products, cheeses and vegetables) was evaluated. Phenotypic and PCR protocols were used for species identification. Tests for production of gelatinase, haemolysin, bacteriocin and bile salt hydrolysis were done with all enterococci isolates, whereas molecular determination of virulence markers (genes esp, gel, ace, as, efaA, hyl and cylA) and antibiotic resistance was checked only for Enterococcus faecium and Enterococcus faecalis isolates. The antibiotic-resistant isolates were assayed for biofilm formation and adhesion to mammalian cells. From the 120 food samples analyzed, 52.5% were positive for enterococci, meat and cheese being the most contaminated. E. faecium was the predominant species, followed by E. faecalis, E. casseliflavus and Enterococcus gallinarum. Phenotypic tests indicated that 67.7% of isolates hydrolyzed bile salts, 15.2% produced bacteriocin, 12.0% were beta-hemolytic and 18.2% produced gelatinase. Antibiotic resistance (gentamicin, tetracycline and erythromycin) and genes encoding for virulence traits were more frequent in E. faecalis than in E. faecium. Three E. faecium isolates were resistant to vancomycin. Among antibiotic-resistant isolates, 72.4% of E. faecalis were able to form biofilm and 13.8% to adhere to Caco-2 cells. Antibiotic-resistant E. faecalis and E. faecium isolates were grouped by RAPD-PCR and a scattered distribution was noted, indicating that resistance was not related to a particular clone. The spread of virulence/resistance traits in isolates of the two species and different RAPD-types suggest the pathogenic potential of both species. By contrast, the recovery of bacteriocinogenic E. faecium isolates with no virulence traits suggests their potential for biotechnological applications. In conclusion, our results showed that enterococci from Brazilian foods present important dualist aspects for food safety. (C) 2008 Elsevier Ltd. All rights reserved.

Changes in vancomycin-resistant Enterococcus faecium causing outbreaks in Brazil

Enterococci have been implicated in severe human infections as a consequence of associated determinants of virulence and antimicrobial resistance. The majority of vancomycin-resistant Enterococcus faecium (VRE(fm)) connected to outbreaks worldwide pertains to the clonal complex 17 (CC17). In Brazil, the majority of VRE(fm) involved in outbreaks reported so far are not related to CC17. VRE(fm) strains responsible for an outbreak and sporadic cases in hospitals located in the city of Campinas, Brazil, were compared to other VRE(fm) strains in the country. Twenty-two out of 23 E. faecium were vancomycin-resistant and harboured the vanA gene. One vancomycin-susceptible E. faecium (VSE(fm)) strain was included in this study because it was isolated from a patient who one week later harboured a VRE(fm). All strains, except VSE, showed the same alteration in the VanA element characterised by deletion of the left extremity of the transposon and insertion of IS1251 between the vanS and vanH genes. Genes codifying virulence factors such as collageneadhesin protein, enterococcal surface protein and hyaluronidase were detected in the VRE(fm) and VSE(fm) studied. Both pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) revealed that VRE(fm) and VSE(fm) strains have a clonal relationship. New sequence types (STs) were identified by MLST as ST447, ST448, ST478 and ST412 but all belonged to the CC17. The present study revealed that VRE(fm) outbreaks in Brazil were caused by strains that did not share a common evolutionary history, and that VRE(fm) strains belonging to CC17 could be predominant in Brazil as in other countries. (C) 2011 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Outbreak of vancomycin-resistant enterococci in a tertiary hospital: The lack of effect of measures directed mainly by surveillance cultures and differences in response between Enterococcus faecium and Enterococcus faecalis

To describe the effect of active surveillance to control vancomycin-resistant enterococci (VRE) after an outbreak, 549 surveillance rectal cultures were performed in 308 patients (35% positive). An educational intervention to prevent transmission was implemented. Infection and colonization by VR-Enterococcus faecalis decreased, but Enterococcus faecium persisted despite control measures. Infections by VR-E faecalis fell to zero in 2008. We observed difficulties in controlling colonization with measures directed mainly by surveillance cultures and differences between responses of E faecium and E faecalis.

Antimicrobial Effects of Calcium Hydroxide and Chlorhexidine on Enterococcus faecalis

Introduction: Endodontic treatment is commonly based on nonspecific elimination of intraradicular micro-organisms. Although some authors prefer single-visit root canal operations for endodontic treatment, several studies have shown the importance of intracanal medication between sessions to kill microorganisms that biomechanical preparations alone cannot achieve. The purpose of this study was to evaluate the efficacy of calcium hydroxide Ca(OH)2 and chlorhexidine gel on the elimination of intratubular Enterococcus faecalis. Methods: Human uniradicular teeth contaminated with E. faecalis were treated with Ca(OH)(2), 2% chlorhexidine gel, Ca(OH)(2) plus 2% chlorhexidine gel, or saline (0.9% NaCl) as a negative control. Samples obtained at a depth of 0 to 100 mu m and 100 to 200 mu m from these root canal preparations were analyzed for bacterial load by counting the number of colonyforming units (CFUs) and bacterial viability using fluorescence microscopy. Results: A significant decrease in the number of CFUs and the percentage of viable E. faecalis was observed after treatment with either Ca(OH)(2) or chlorhexidine when compared with the control group. Additionally, chlorhexidine gel had a significantly higher antimicrobial efficacy as measured by the number of CFUs and the percentage of viable cells than Ca(OH)(2). No differences were observed between the antimicrobial properties of chlorhexidine gel with and without the addition of Ca(OH)(2). Conclusion: Both Ca(OH)(2) and chlorhexidine have antimicrobial effects on E. faecalis. Chlorhexidine had increased antimicrobial activity when compared with Ca(OH)(2.) Ca(OH)(2) combined with chlorhexidine showed similar antimicrobial activity to chlorhexidine alone. (J Endod 2010;36:1389-1393)

Antibacterial efficacy of endodontic irrigating solutions and their combinations in root canals contaminated with Enterococcus faecalis

Objective. The objective of this study was to evaluate the antibacterial efficacy of irrigating solutions and their combinations against Enterococcus faecalis. Study design. One hundred ten single-rooted human teeth were inoculated with E. faecalis and incubated for 21 days. Teeth were divided according to the irrigant: Group I (GI), 2.5% sodium hypochlorite solution (NaOCl); GII, 2.5% NaOCl + 10% citric acid; GIII, 2.5% NaOCl + apple cider vinegar; GIV, apple cider vinegar; GV, 2% chlorhexidine solution; GVI, 1% peracetic acid; GVII, saline solution. Microbiological samples were taken after root canal preparation and 7 days later. Data were submitted to ANOVA (5%). Results. All solutions promoted reduction of E. faecalis after instrumentation, but bacterial counts were higher in the final sample. GI, GV, and GVI had lower bacterial counts than the other groups. Conclusions. The irrigating solutions may present activity but do not eradicate E. faecalis in the root canal system. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011; 112:396-400)

Heat-killed Enterococcus faecalis Alters Nitric Oxide and CXCL12 Production but not CXCL8 and CCL3 Production by Cultured Human Dental Pulp Fibroblasts

Introduction: Fibroblasts are the most abundant cells in dental pulp. To investigate their capacity to produce the chemokines CCL3, CXCL8, and CXCL12 as well as nitric oxide (NO), we evaluated the production of these mediators in supernatants of cultured human dental pulp fibroblasts (HDPF) stimulated by heat-killed Enterococcus faecalis (HKEF). Methods: Primary cultures of HDPF were stimulated with medium alone or HKEF (1:1, 10:1, or 100:1 bacteria:fibroblast) for 1, 6, and 24 hours. Chemokines and NO were assessed through enzyme-linked immunosorbent assay and Griess reaction, respectively. Statistical analysis was performed by using analysis of variance and Tukey post test. Results: CCL3 was not detected, whereas constitutive CXCL8 was not affected. Production of CXCL12 was increased at 1 and 6 hours, and NO was increased at the concentration of 1:1 bacteria:fibroblast at 24 hours. Viability and proliferation assays did not reveal cell number differences. Conclusions: These findings demonstrate that heat-killed E. faecalis is able to increase production of CXCL12 and NO by HDPF. (J Endod 2010;36:91-94)

Confocal laser scanning microscopy is appropriate to detect viability of Enterococcus faecalis in infected dentin

The purpose of this study was to explore the potential of confocal laser scanning microscopy (CLSM) for in situ identification of live and dead Enterococcus faecalis in infected dentin. Eight cylindrical dentin specimens were infected with Enterococcus faecalis in BHI for 21 days. After the experimental period, the specimens were stained with fluorescein diacetate (FDA) and propidium iodide (PI) or acridine orange (0.01 %) and analyzed by CLSM. Two noninfected dentin specimens were used as negative controls. CLSM analysis shows that the discrimination between viable (green) and dead (red) bacteria in infected dentinal tubules could be observed after staining with FDA/PI. Acridine orange was able to show metabolic activity of the E. faecalis cells inside the dentinal tubules showed by its red fluorescence. The viability of bacteria in infected dentin can be determined in situ by CLSM. FDA/PI and acricline orange are useful for this technique.

The relationship of a clonal outbreak of Enterococcus faecium vanA to methicillin-resistant Staphylococcus aureus incidence in an Australian hospital

Australian isolates of vancomycin-resistant enterococci (VRE) have been widely scattered geographically, predominantly polyclonal and of the VanB phenotype. Forty-nine VRE were isolated from 47 patients in our hospital from October 1996 to December 1999. Forty-four of these VRE were Enterococcus faecium with a vanA glycopeptide resistance genotype. Four isolates were pathogenic. Thirty-five VRE were from an outbreak in the Renal and Infectious Diseases Units over a four-month period. Pulsed-field gel electrophoresis (PFGE) demonstrated that 41 of the 49 VRE were indistinguishable or closely related. Enhanced environmental cleaning, strict contact isolation of colonized patients and reducing inpatient admissions terminated the epidemic. Cohorting of methicillin-resistant Staphylococcus aureus (MRSA)-positive patients was restricted because VPE patients occupied the isolation facilities. This resulted in a statistically significant increase in MRSA infections across the hospital. VRE epidemics have the ability to influence the epidemiology of other nosocomial pathogens when infection control resources are exhausted. (C) 2001 The Hospital Infection Society.

Desenvolvimento e avaliação da acurácia do sistema de filtração (BacFil 6.0) na detecção de bacilos álcool-ácido resistentes em amostras de lavado broncoalveolar de pacientes com suspeita de tuberculose

A baciloscopia é o principal método diagnóstico da tuberculose (TB) em razão do seu baixo custo, rapidez e facilidade de execução. Porém sua sensibilidade é baixa, principalmente nas formas paucibacilares da doença. Nesse sentido, novas metodologias que resultem no aumento da sensibilidade da baciloscopia tornam-se absolutamente necessárias. Neste estudo, nos propusemos a aprimorar e padronizar o sistema de filtração BacFil e avaliar sua performance e acurácia em amostras paucibacilares (lavado broncoalveolar). Para alcançar esses objetivos desenvolvemos o estudo em duas fases. Na primeira fase, foram realizados ensaios para definir: 1) utilização de membranas brancas para visualização de BAAR corados por Auramina-O; 2) aumento do volume de NALC, utilização de pérolas de vidro no processo de digestão das amostras e mudança no sistema do suporte do pré-filtro; 3) meios de fixação da membrana de filtração na lâmina de microscopia; 4) limite de detecção da técnica; 5) aumento do volume de NaOCl e a influência desse aumento na visualização de BAAR na membrana. Verificou-se que: a membrana de policarbonato branca tem a mesma eficiência da membrana de policarbonato preta na visualização de BAAR fluorescentes; o método modificado utilizando maior concentração de NALC, pérolas de vidro e sistema de pré-filtro baseado em orifícios obteve 98% de passagem das amostras pelo sistema; a melhor forma de fixação da membrana na lâmina foi obtida por intermédio da utilização de fita dupla face; o limite de detecção da técnica de filtração utilizando o sistema BacFil versão 6.0 foi de 2 a 9 BAAR por mililitro de suspensão bacteriana com D.O de 0,250 ± 0,01 a 625nm; o aumento da concentração de NaOCl para 10% ajuda na passagem de um maior volume de amostra pelo sistema, porém ocasiona a diminuição da quantidade BAAR na membrana de filtração após o período necessário para todo o processo. Na segunda fase foi avaliado acurácia da técnica padronizada em 101 amostras de lavado broncoalveolar (LBA) de pacientes com suspeita de TB provenientes do Hospital Universitário Cassiano Antônio de Moraes. Observou-se que a sensibilidade da técnica de filtração (BacFil versão 6.0) foi de 76% e especificidade de 95%. A sensibilidade obtida foi significativamente maior do que a observada na baciloscopia após centrifugação que foi de 59% (p = 0,001). No entanto, constatou-se uma diminuição de especificidade na técnica de filtração (95%) em relação a técnica de 8 centrifugação (99%). Concluímos que as modificações realizadas nos sistema de filtração permitiu sanar os problemas técnicos apresentados nas versões anteriores e aumentar a sensibilidade da baciloscopia de amostras paucibacilares (lavado broncoalveolar) para o diagnóstico da TB pulmonar.

Características físico-quimicas, análise sensorial e conservação de frutos de cultivares de bananeira resistentes à sigatoca-negra

O cultivar Prata Anã é o mais plantada no Norte de Minas Gerais, o que demonstra estar a bananicultura regional baseada numa variedade suscetível à sigatoca-negra. Apesar de existirem cultivares resistentes em fase de recomendação para o cultivo na região, ainda são escassos os trabalhos relacionados a metodologias de conservação pós-colheita. Assim, o objetivo deste trabalho foi avaliar o período de armazenamento e as características físico-químicas e sensoriais dos cultivares de bananeira Fhia - 02 e Precioso, ambos resistentes à sigatoca-negra, e o efeito da atmosfera modificada associada à refrigeração. Foram realizados três experimentos, o primeiro em temperatura ambiente, o segundo a 12 ºC e o terceiro a 15 ºC. Esses experimentos foram instalados no delineamento em blocos casualizados em esquema de parcelas subdivididas no tempo, tendo nas parcelas um fatorial 2 (com e sem embalagem) x 2 (Fhia - 02 e Precioso) e nas subparcelas as avaliações em intervalo de seis e dois dias para frutos armazenados nas temperaturas de 12, 15 e 25 ºC, com quatro repetições e três frutos por unidade experimental. Após o tratamento, os frutos foram dispostos em bandejas de poliestireno expandido (sem embalagem) e embalagens de 10 e 16 µm e umidade relativa de 90%. As características avaliadas foram: tamanho, diâmetro, cor, firmeza, perda de matéria fresca, sólidos solúveis totais (SST), acidez total titulável (ATT), SST/ATT, pH e análise sensorial. O uso da embalagem associada à refrigeração permitiu armazenar os frutos por 28 dias, mantendo as características físico-químicas.

Herbicidas alternativos para o controle de Conyza sumatrensis (Retz.) E. H. Walker resistentes aos inibidores da ALS e EPSPs

RESUMO A buva (Conyza sumatrensis), uma das principais plantas daninhas já identificadas no sul do Brasil, vem apresentando controle insatisfatório, em resposta à aplicação dos herbicidas chlorimuron-ethyl e glyphosate. Por esta razão, o objetivo deste estudo foi avaliar herbicidas alternativos, visando ao controle de biótipos de C. sumatrensis, com resistência de nível baixo ao herbicida chlorimuron-ethyl e resistentes ao herbicida glyphosate. O experimento foi realizado em casa de vegetação, entre abril e agosto de 2012, no município de Passo Fundo, RS. O delineamento experimental foi o inteiramente casualizado, com quatro repetições, sendo avaliados 15 tratamentos com herbicidas, além de uma testemunha sem aplicação. As variáveis consideradas foram controle percentual, aos 14, 21 e 28 dias após a aplicação dos tratamentos (DAT) e a matéria seca da parte aérea, aos 28 DAT. Como resultados, observou-se que os biótipos foram 100% controlados, aos 28 DAT, pelos tratamentos alternativos de paraquat + diuron; ammonium glufosinate; glyphosate + 2,4-D; glyphosate + ammonium glufosinate; 2,4-D; tembotrione e tembotrione + atrazine. O biótipo 17 evidenciou menor sensibilidade aos herbicidas inibidores da ALS e os biótipos 05, 17 e 20 não foram controlados pelo herbicida glyphosate.