Antimicrobial activity and enterococcus faecalis biofilm formation on chlorhexidine varnishes

Objective: To evaluate, in vitro, the antimicrobial activity and biofilm formation of three chlorhexidine varnishes in four Enterococcus faecalis strains: E. faecalis ATCC 29212, E. faecalis EF-D1 (from failed endodontic treatment), E. faecalis 072 (cheese) and E. faecalis U-1765 (nosocomial infection), and one Enterococcus durans strain (failed endodontic treatment). Study Design: The direct contact test was used to study the antimicrobial activity. Bacterial suspensions were exposed for one hour to EC40, Cervitec (CE) and Cervitec Plus (CEP) varnishes. "Eradication" was defined as 100% bacterial kill. The formation of enterococci biofilms was tested on the surface of the varnishes after 24 hours of incubation and expressed as percentage of biofilm reduction. Results: EC40 eradicated all strains except E. faecalis ATCC 29212, where 98.78% kill was achieved. CE and CEP showed antimicrobial activity against all the strains, but most clearly against E. durans and E. faecalis 072. EC40 completely inhibited the formation of biofilm of E. faecalis ATCC 29212, E. faecalis 072 and E. durans. CE and CEP led to over 92% of biofilm reduction, except in the case of E. faecalis U-1765 on CEP (76.42%). Conclusion: The three varnishes studied were seen to be effective in killing the tested strains of enterococci and in inhibiting the formation of biofilm, the best results being observed with EC40.

Capsule Locus Polymorphism among Distinct Lineages of Enterococcus faecalis Isolated from Canals of Root-filled Teeth with Periapical Lesions

Introduction: Although Enterococcus faecalis is a member of the normal microbiota, it is also a major cause of nosocomial infections. Some strains of E. faecalis produce capsule, which contributes to pathogenesis through evasion of host defenses, and its production is dependent on the capsule (cps) operon polymorphism. This study investigated cps locus polymorphism in distinct lineages of E. faecalis isolated from canals of root-filled teeth with periapical lesions. Methods: Twenty-two E. faecalis isolates were evaluated regarding the cps operon polymorphism and genetic diversity. The 3 known CPS types were determined by polymerase chain reaction. This information was correlated with multilocus sequence typing data, which were used to define genetic lineages. Results: cpsA and cpsB were the only detected genes within the cps operon in 62.5% of E. faecalis strains (14/22), indicative of genotype CPS 1, which lacks capsule expression. The essential genes in the cps operon for capsule production were detected in the remaining strains, whereas 3 belonged to genotype CPS 5 and 5 strains to genotype CPS 2. A total of 14 sequence types (STs) were resolved in 22 E. faecalis isolates. Comparison with the E. faecalis international multilocus sequence typing database revealed that 9 STs were previously found, and that the 5 STs were novel. Conclusions: Certain E. faecalis genotypes from canals of root-filled teeth with periapical lesions belong to lineages associated with capsule expression and production of multiple virulence factors, which might account for their increased pathogenic potential. (J Endod 2012;38:58-61)

In vitro enterococcus faecalis biofilm formation on five adhesive systems

Objective: To determine the E. faecalis biofilm formation on the surface of five adhesive systems (AS) and its relationship with roughness. Study Design: The formation of E. faecalis biofilms was tested on the surface of four dual-cure AS: AdheSE DC, Clearfil DC Bond, Futurabond DC and Excite DSC and one light-cure antimicrobial AS, Clearfil Protect Bond, after 24 hours of incubation, using the MBEC high-throughput device. Results: E. faecalis biofilms grew on all the adhesives. The least growth of biofilm was on Excite DSC, Clearfil Protect Bond, and the control. Futurabond DC resulted in the greatest roughness and biofilm amount. There was a close relationship between the quantity of biofilm and roughness, except for Clearfil Protect Bond, which showed little biofilm but high roughness. Conclusion: None of the tested AS prevented E. faecalis biofilm formation, although the least quantity was found on the surface of Clearfil Protect Bond.

Comparative analysis of Enterococcus Faecalis biofilm formation on different substrates

Introduction: The aim of this study was to compare Enterococcus faecalis biofilm formation on different substrates. Methods: Cell culture plates containing growth medium and E. faecalis (ATCC 29212) were used to grow biofilm on bovine dentin, gutta-percha, hydroxyapatite, or bovine bone. Substrates were incubated at 37 C for 14 or 21 days, and the medium was changed every 48 hours. After the growth induction periods, specimens (n = 5 per group and per induction period) were stained by using Live/Dead, and the images were analyzed under a confocal microscope. The total biovolume (mm3), live bacteria biovolume (mm3), and substrate coverage (%) were quantified by using the BioImage_L software. Results obtained were analyzed by nonparametric tests (P = .05). Results: Biofilm formation was observed in all groups. Gutta-percha had the lowest total biovolume at 14 days (P < .05) and hydroxyapatite the highest at 21 days (P < .05). No significant difference was observed in green biovolume at 14 days. At 21 days, however, hydroxyapatite had the highest volume (P < .05). The percentages of coverage were similar among all substrates at 21 days (P > .05), but at 14 days, bovine bone presented the highest coverage (P < .05). Conclusions: E. faecalis was capable of forming biofilm on all substrates during both growth periods; hydroxyapatite presented the highest rates of biofilm formation. The type of substrate influenced the biofilm characteristics, according to the parameters evaluated

Analysis of genetic lineages and their correlation with virulence genes in enterococcus faecalis clinical isolates from root canal and systemic infections

Introduction: Enterococcus faecalis is a member of the mammalian gastrointestinal microbiota but has been considered a leading cause of hospital-acquired infections. In the oral cavity, it is commonly detected from root canals of teeth with failed endodontic treatment. However, little is known about the virulence and genetic relatedness among E. faecalis isolates from different clinical sources. This study compared the presence of enterococcal virulence factors among root canal strains and clinical isolates from hospitalized patients to identify virulent clusters of E. faecalis. Methods: Multilocus sequence typing analysis was used to determine genetic lineages of 40 E. faecalis clinical isolates from different sources. Virulence clusters were determined by evaluating capsule (cps) locus polymorphisms, pathogenicity island gene content, and antibiotic resistance genes by polymerase chain reaction. Results: The clinical isolates from hospitalized patients formed a phylogenetically separate group and were mostly grouped in the clonal complex 2, which is a known virulent cluster of E. faecalis that has caused infection outbreaks globally. The clonal complex 2 group comprised capsule-producing strains harboring multiple antibiotic resistance and pathogenicity island genes. On the other hand, the endodontic isolates were more diverse and harbored few virulence and antibiotic resistance genes. In particular, although more closely related to isolates from hospitalized patients, capsuleproducing E. faecalis strains from root canals did not carry more virulence/antibiotic genes than other endodontic isolates. Conclusions: E. faecalis isolates from endodontic infections have a genetic and virulence profile different from pathogenic clusters of hospitalized patients’ isolates, which is most likely due to niche specialization conferred mainly by variable regions in the genome.

Effect of photodynamic therapy with different formulations of methylene blue in teeth contaminated by Enterococcus faecalis

The aim of this study was to compare the disinfection of dentine using photodynamic therapy with methylene blue in different formulations. Thirty bovine teeth roots were autoclaved and incubated with a suspension of Enterococcus faecalis. The specimen were randomly divided into three groups: G1, the roots were filled with 10 mM methylene blue dissolved in water; G2, the roots were filled with 10 mM methylene blue dissolved in a mixture of glycerol: ethanol: water; G3, roots filled with 100 mM methylene blue dissolved in water. The groups were irradiated with a 660 nm diode laser with an output power of 100 mW for 4 min, energy density of 850 J/cm2 and after this procedure, the sensitizer was removed and microbial samples were collected from within the root canals. The samples were plated on mEnterococcus to count the colony-forming units (CFU/mL). The means were: Group 1=513×103, Group 2=1431×103 and Group 3=2.96×103. The statistical analysis detected higher disinfection achieved by G3 when compared with groups G1 and G2, and no significant difference between the groups G1 and G2 (P>0.05). The increase of the concentration of methylene blue dye achieved higher disinfection in photodynamic therapy.

Quantitative detection of Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa in human oral epithelial cells from subjects with periodontitis and periodontal health

Epithelial cells in oral cavities can be considered reservoirs for a variety of bacterial species. A polymicrobial intracellular flora associated with periodontal disease has been demonstrated in buccal cells. Important aetiological agents of systemic and nosocomial infections have been detected in the microbiota of subgingival biofilm, especially in individuals with periodontal disease. However, non-oral pathogens internalized in oral epithelial cells and their relationship with periodontal status are poorly understood. The purpose of this study was to detect opportunistic species within buccal and gingival crevice epithelial cells collected from subjects with periodontitis or individuals with good periodontal health, and to associate their prevalence with periodontal clinical status. Quantitative detection of total bacteria and Staphylococcus aureus, Pseudomonas aeruginosa and Enterococcus faecalis in oral epithelial cells was determined by quantitative real-time PCR using universal and species-specific primer sets. Intracellular bacteria were visualized by confocal microscopy and fluorescence in situ hybridization. Overall, 33 % of cell samples from patients with periodontitis contained at least one opportunistic species, compared with 15 % of samples from healthy individuals. E. faecalis was the most prevalent species found in oral epithelial cells (detected in 20.6 % of patients with periodontitis, P = 0.03 versus healthy individuals) and was detected only in cells from patients with periodontitis. Quantitative real-time PCR showed that high levels of P. aeruginosa and S. aureus were present in both the periodontitis and healthy groups. However, the proportion of these species was significantly higher in epithelial cells of subjects with periodontitis compared with healthy individuals (P = 0.016 for P. aeruginosa and P = 0.047 for S. aureus). Although E. faecalis and P. aeruginosa were detected in 57 % and 50 % of patients, respectively, with probing depth and clinical attachment level ≥6 mm, no correlation was found with age, sex, bleeding on probing or the presence of supragingival biofilm. The prevalence of these pathogens in epithelial cells is correlated with the state of periodontal disease.

Enterococcus faecium isolated from Lombo, a Portuguese traditional meat product: characterisation of antibacterial compounds and factors affecting bacteriocin production

Strain ST211CH, identified as a strain of Enterococcus faecium, isolated from Lombo produced a bacteriocin that inhibited the growth of Enterococcus spp., Listeria spp., Klebsiella spp., Lactobacillus spp., Pseudomonas spp., Staphylococcus spp. and Streptococcus spp. The mode of action of the bacteriocin named as bacteriocin ST211Ch was bactericidal against Enterococcus faecalis ATCC19443. As determined by Tricine-SDS-PAGE, the approximate molecular mass of the bacteriocin was 8.0 kDa. Loss in antimicrobial activity was recorded after treatment with proteolytic enzymes. Maximum activity of bacteriocin ST211Ch was measured in broth cultures of E. faecium strain ST211Ch after 24 h; thereafter, the activity was reduced. Bacteriocin ST211Ch remained active after exposure to various temperatures and pHs, as well as to Triton X-100, Tween-80, Tween-20, sodium dodecyl sulfate, NaCl, urea and EDTA. Effect of media components on production of bacteriocin ST211Ch was also studied. On the basis of PCR reactions targeting different bacteriocin genes, i.e. enterocins, curvacins and sakacins, no evidences for the presence of these genes in the total DNA of E. faecium strain ST211Ch was obtained. The bacterium most probably produced a bacteriocin different from those mentioned above. Based on the antimicrobial spectrum, stability and mode of action of bacteriocin ST211CH, E. faecium strain ST211Ch might be considered as a potential candidate with beneficial properties for use in biopreservation to control food spoilage bacteria.

Application of bacteriocinogenic Enterococcus mundtii CRL35 and Enterococcus faecium ST88Ch in the control of Listeria monocytogenes in fresh Minas cheese

Several strains of Enterococcus spp. are capable of producing bacteriocins with antimicrobial activity against important bacterial pathogens in dairy products. In this study, the bacteriocins produced by two Enterococcus strains (Enterococcus mundtii CRL35 and Enterococcus faecium ST88Ch), isolated from cheeses, were characterized and tested for their capability to control growth of Listeria monocytogenes 426 in experimentally contaminated fresh Minas cheese during refrigerated storage. Both strains were active against a variety of pathogenic and non-pathogenic microorganisms and bacteriocin absorption to various L. monocytogenes, Enterococcus faecalis ATCC 19443 and Lactobacillus sakei ATCC 15521 varied according to the strain and the testing conditions (pH, temperature, presence of salts and surfactants). Growth of L. monocytogenes 426 was inhibited in cheeses containing E. mundtii CRL35 up to 12 days at 8 degrees C, evidencing a bacteriostatic effect. E. faecium ST88Ch was less effective, as the bacteriostatic affect occurred only after 6 days at 8 degrees C. In cheeses containing nisin (12.5 mg/kg), less than one log reduction was observed. This research underlines the potential application of E. mundtii CRL35 in the control of L. monocytogenes in Minas cheese. (c) 2012 Elsevier Ltd. All rights reserved.

A preliminary study of the antibacterial potential of cetylpyridinium chloride in root canals infected by E. faecalis

The aim of this preliminary study was to verify the antibacterial potential of cetylpyridinium chloride (CPC) in root canals infected by Enterococcus faecalis. Forty human maxillary anterior teeth were prepared and inoculated with E. faecalis for 60 days. The teeth were randomly assigned to the following groups: 1: Root canal preparation (RCP) + 0.1% CPC with positive-pressure irrigation (PPI, Conventional, NaviTip®); 2: RCP + 0.2% CPC PPI; 3: RCP + 2.5% NaOCl PPI; 4: RCP + 2.5% NaOCl with negative-pressure irrigation system (NPI, EndoVac®); 5: Positive control; and 6: Negative control. Four teeth of each experimental group were evaluated by culture and 4 by scanning electron microscopy (SEM). In all teeth, the root canals were dried and filled with 17% EDTA (pH 7.2) for 3 min for smear layer removal. Samples from the infected root canals were collected and immersed in 7 mL of Letheen Broth (LB), followed by incubation at 37°C for 48 h. Bacterial growth was analyzed by turbidity of culture medium and then observed with a UV spectrophotometer. The irrigating solutions were further evaluated for antimicrobial effect by an agar diffusion test.The statistical data were treated by means, standard deviation, Kruskal-Wallis test and analysis of variance. Significance level was set at 5%. The results showed the presence of E. faecalis after root canal sanitization. The number of bacteria decreased after the use of CPC. In the agar diffusion test, CPC induced large microbial inhibition zones, similar to 2% chlorhexidine and large than 2.5% NaOCl. In conclusion, cetylpyridinium chloride showed antibacterial potential in endodontic infection with E. faecalis.

Complexation of 2-acetylpyridine- and 2-benzoylpyridine-derived hydrazones to copper(II) as an effective strategy for antimicrobial activity improvement

Complexes [Cu(2AcPh)Cl]center dot 2H(2)O (1), [Cu(2AcpClPh)Cl]center dot 2H(2)O (2), [Cu(2AcpNO(2)Ph)Cl] (3), [Cu(2BzPh)Cl] (4). [Cu(2BzpClPh)Cl] (5) and [Cu(2BzpNO(2)Ph)Cl] (6) were obtained with 2-acetylpyridine-phenylhydrazone (H2AcPh), 2-acetylpyridine-para-chloro-phenylhydrazone (H2AcpClPh), 2-acetylpyridine-para-nitro-phenylhydrazone (H2AcpNO(2)Ph), 2-benzoylpyridine-phenylhydrazone (H2BzPh), 2-benzoylpyridine-para-chloro-phenylhydrazone (H2BzpClPh) and 2-benzoylpyridine-para-nitro-phenylhydrazone (H2BzpNO(2)Ph). The hydrazones showed poor antibacterial effect against Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa but demonstrated significant antifungal activity against Candida albicans. Upon coordination to copper(II) the antibacterial and antifungal activities appreciably increased. H2AcpClPh, H2BzpClPh and their copper(II) complexes (2) and (5), respectively, were as active as fluconazole against C. albicans. (C) 2012 Elsevier Ltd. All rights reserved.

Evaluation of Radiopacity, pH, Release of Calcium Ions, and Flow of a Bioceramic Root Canal Sealer

Introduction: The aim of the present study was to evaluate the physicochemical properties of a bioceramic root canal sealer, Endosequence BC Sealer. Radiopacity, pH, release of calcium ions (Ca2+), and flow were analyzed, and the results were compared with AH Plus cement. Methods: Radiopacity and flow were evaluated according to ISO 6876/2001 standards. For the radiopacity analysis, metallic rings with 10-mm diameter and 1-mm thickness were filled with cements. The radiopacity value was determined according to radiographic density (mm Al). The flow test was performed with 0.05 mL of cement placed on a glass plate. A 120-g weight was carefully placed over the cement. The largest and smallest diameters of the disks formed were measured by using a digital caliper. The release of Ca2+ and pH were measured at periods of 3, 24, 72, 168, and 240 hours with spectrophotometer and pH meter, respectively. Data were analyzed by analysis of variance and Tukey test (P < .05). Results: The bioceramic endodontic cement showed radiopacity (3.84 mm Al) significantly lower than that of AH Plus (6.90 mm Al). The pH analysis showed that Endosequence BC Sealer showed pH and release of Ca2+ greater than those of AH Plus (P < .05) during the experimental periods. The flow test revealed that BC Sealer and AH Plus presented flow of 26.96 mm and 21.17 mm, respectively (P < .05). Conclusions: Endosequence BC Sealer showed radiopacity and flow according to ISO 6876/2001 recommendations. The other physicochemical properties analyzed demonstrated favorable values for a root canal sealer. (J Endod 2012;38:842-845)

Effect of low-dose gaseous ozone on pathogenic bacteria

Background: Treatment of chronically infected wounds is a challenge, and bacterial environmental contamination is a growing issue in infection control. Ozone may have a role in these situations. The objective of this study was to determine whether a low dose of gaseous ozone/oxygen mixture eliminates pathogenic bacteria cultivated in Petri dishes. Methods: A pilot study with 6 bacterial strains was made using different concentrations of ozone in an ozone-oxygen mixture to determine a minimally effective dose that completely eliminated bacterial growth. The small and apparently bactericidal gaseous dose of 20 mu g/mL ozone/oxygen (1: 99) mixture, applied for 5min under atmospheric pressure was selected. In the 2nd phase, eight bacterial strains with well characterized resistance patterns were evaluated in vitro using agar-blood in adapted Petri dishes (10(5) bacteria/dish). The cultures were divided into 3 groups: 1-ozone-oxygen gaseous mixture containing 20 mu g of O-3/mL for 5 min; 2- 100% oxygen for 5 min; 3- baseline: no gas was used. Results: The selected ozone dose was applied to the following eight strains: Escherichia coli, oxacillin-resistant Staphylococcus aureus, oxacillin-susceptible Staphylococcus aureus, vancomycin-resistant Enterococcus faecalis, extended-spectrum beta-lactamase-producing Klebsiella pneumoniae, carbapenem-resistant Acinetobacter baumannii, Acinetobacter baumannii susceptible only to carbapenems, and Pseudomonas aeruginosa susceptible to imipenem and meropenem. All isolates were completely inhibited by the ozone-oxygen mixture while growth occurred in the other 2 groups. Conclusion: A single topical application by nebulization of a low ozone dose completely inhibited the growth of all potentially pathogenic bacterial strains with known resistance to antimicrobial agents.

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.