METHODS: Enterococcus faecalis, Streptococcus sanguinis, Fusobacterium nucleatum, Porphyromonas gingivalis and Prevotella intermedia were suspended as follows: Iso-osmotic group 0.9% NaCl; Hypo-osmotic group "ultrapure water"; Hyper-osmotic group 9% NaCl solution for 120 hours before exposure to 0.0001% NaOCl for 10 minutes. Quantitative analyses of viable cells were performed at 0 and 120 hours and after exposure to NaOCl to obtain colony forming units (CFU/mL). A linear mixed-effects model was used to find the association between mean CFU/mL (logarithmic transformation) and the interaction of solution Group and Time (P<0.001).
RESULTS: F. nucleatum, P. gingivalis and P. intermedia did not survive after 24 hours in any of the solutions and were excluded from further testing. For S. sanguinis there were significant differences at each time interval, when holding solution group constant. After 120 hours, the Hyper-osmotic group presented with the highest CFU/mL and was significantly different to the Iso-osmotic group (P<0.001). For E. Faecalis, there was a significant difference for each pairwise comparison of time (P<0.001) in mean CFU/mL between 0 hours and 120 hours for the Iso-osmotic and Hyper-osmotic groups. At 120 hours, no significant differences were found between the three groups. Significant differences were also found between 0 hours and Post-NaOCl administration, and between 120 hours and Post-NaOCl administration for all three groups (P<0.001). Exposure to NaOCl after hypo-osmotic stress was associated with significantly less CFU/mL for S. sanguinis compared to hyperosmosis and iso-osmosis (P<0.001) and for E. Faecalis only compared to hyperosmosis (P<0.001).
CONCLUSION: S. sanguinis and E. faecalis were able to withstand osmotic stress for 120 hours. Hypo-osmotic stress before contact with NaOCl was associated with lower viable bacterial numbers, when compared to the other media for the above species. Hyper-osmotic stress was associated with higher viable bacterial numbers after NaOCl exposure for E. faecalis.
MATERIALS AND METHODS: An estimated 120 human root dentin disks were prepared, sterilized, and inoculated with E. faecalis strain (ATCC 29212) to develop a 3-weeks-old biofilm. The dentin discs were exposed to group I-control group: 5.25% sodium hypochlorite (NaOCl) (n = 20); group II-1% ALX + 5.25% NaOCl (n = 40); group III-1% alexidine (ALX) (n = 40) (Sigma-Aldrich, Mumbai, India); group IV-negative control: saline (n = 20). After exposure, the dentin disks were stained with the fluorescent live/dead dye and evaluated with a confocal scanning electron microscope to calculate the proportion of dead cells. Statistical analysis was done using the Kruskal-Wallis and Mann-Whitney U test (p < 0.05).
RESULTS: The maximum proportion of dead cells were seen in the groups treated with the combination of 1% ALX + 5.25% NaOCl (94.89%) and in the control group 5.25% NaOCl (93.14%). The proportion of dead cells presented in the 1% ALX group (51.79%) and negative control group saline (15.10%) were comparatively less.
CONCLUSION: The antibacterial efficiency of a combination of 1% ALX and 5.25% NaOCl was more effective when compared with 1% ALX alone.
CLINICAL SIGNIFICANCE: Alexidine at 1% could be used as an alternative endodontic irrigant to chlorhexidine, as alexidine does not form any toxic precipitates with sodium hypochlorite. The disinfection regimen comprising a combination of 1% ALX and 5.25% NaOCl is effective in eliminating E. faecalis biofilms.
METHODS: Root canal preparation was performed using stainless steel K-files™ and F4 size protaper with irrigation protocols of 6% NaOCl + 2% CHX; 3.5% QIS; 2% QIS and sterile saline. Biofilms were prepared using E. faecalis adjusted and allowed to grow for 3 days, treated with irrigants, and allowed to grow for 7 days. AFM was performed and surface free energy calculated. MC3T3 cells were infected with endo irrigant treated E. faecalis biofilms. Raman spectroscopy of biofilms were performed after bacterial re-growth on root dentine and exposed to different irrigation protocols and collagen fibers analysed collagen fibers using TEM. Antimicrobial potency against E. faecalis biofilms and cytoxicity against 3T3 NIH cells were also. Resin penetration and MitoTracker green were also evaluated for sealer penetration and mitochondrial viability. Data were analysed using One-way ANOVA, principal component analysis and post-hoc Fisher's least-significant difference.
RESULTS: Elastic moduli were maintained amongst control (5.5 ± 0.9) and 3.5% QIS (4.4 ± 1.1) specimens with surface free energy higher in QIS specimens. MC3T3 cells showed reduced viability in 6%NaOCl+2%CHX specimens compared to QIS specimens. DNA/purine were expressed in increased intensities in control and 6% NaOCl + 2% CHX specimens with bands around 480-490 cm-1 reduced in QIS specimens. 3.5% QIS specimens showed intact collagen fibrillar network and predominantly dead bacterial cells in confocal microscopy. 3.5% QIS irrigant formed a thin crust-type surface layer with cytoplasmic extensions of 3T3NIH spread over root dentine. Experiments confirmed MitoTracker accumulation in 3.5% treated cells.
SIGNIFICANCE: Novel QIS root canal irrigant achieved optimum antimicrobial protection inside the root canals facilitating a toxic effect against the Enterococcus faecalis biofilm. Root dentine substrates exhibited optimum mechanical properties and there was viability of fibroblastic mitochondria.
METHODS: Seven optrA-carrying E. faecalis obtained from chicken faeces (n=3, August 2017) and retail chicken meat (n=4, August 2017) in Tunisia were analysed. Antimicrobial susceptibility was determined by disc diffusion, broth microdilution and Etest against 13 antibiotics, linezolid and tedizolid, respectively (EUCAST/CLSI). optrA stability (∼600 bacterial generations), transfer (filter mating) and location (S1-PFGE/hybridization) were characterized. WGS (Illumina-HiSeq) was done for four representatives that were analysed through in silico and genomic mapping tools.
RESULTS: Four MDR clones carrying different virulence genes were identified in chicken faeces (ST476) and retail meat (the same ST476 clone plus ST21 and ST859) samples. MICs of linezolid and tedizolid were stably maintained at 8 and 1-2 mg/L, respectively. optrA was located in the same transferable chromosomal Tn6674-like element in ST476 and ST21 clones, similar to isolates from pigs in Malaysia and humans in China. ST859 carried a non-conjugative plasmid of ∼40 kb with an impB-fexA-optrA segment, similar to plasmids from pigs and humans in China.
CONCLUSIONS: The same chromosomal and transferable Tn6674-like element was identified in different E. faecalis clones from humans and animals. The finding of retail meat contaminated with the same linezolid-resistant E. faecalis strain obtained from a food-producing animal highlights the potential role of the food chain in the worrisome dissemination of optrA that can be stably maintained without selective pressure over generations.
METHODS: Root canal was prepared using stainless steel K-files™ and ProTaper™ and subjected to manual and ultrasonic irrigation using 6% NaOCl+2% CHX, 6% NaOCl+2% QAS and saline as control. For confocal-microscopy, Raman spectroscopy and SEM analysis before and after treatment, Enterococcus faecalis cultured for 7 days. Raman spectroscopy analysis was done across cut section of gutta percha/sealer-dentine to detect resin infiltration. Indentation of mechanical properties was evaluated using a Berkovich indenter. The contact angle of irrigants and surface free energy were evaluated. Mineralization nodules were detected through Alazarin red after 14 days.
RESULTS: Control biofilms showed dense green colonies. Majority of E. faecalis bacteria were present in biofilm fluoresced red in NaOCl+2% QAS group. There was reduction of 484cm-1 Raman band and its intensity reached lowest with NaOCl+2% QAS. There was an increase in 1350-1420cm-1 intensity in the NaOCl+2% CHX groups. Gradual decrease in 1639cm-1 and 1609cm-1 Raman signal ratios were seen in the resin-depth region of 17μm>, 14.1μm> and 13.2μm for NaOCl+2% QAS, NaOCl+2% CHX and control groups respectively. All obturated groups showed an intact sealer/dentine interface with a few notable differences. 0.771 and 83.5% creep indentation distance for NaOCl+2% QAS ultrasonic groups were observed. Highest proportion of polar component was significantly found in the NaOCl+2% QAS groups which was significantly higher as compared to other groups. Mineralized nodules were increased in NaOCl+2% QAS.
SIGNIFICANCE: Favorable antimicrobial and endodontic profile of the NaOCl+2% QAS solution might suggest clinical use for it for more predictable reduction of intracanal bacteria.
METHOD: Bacterial cell viability was performed by using microplate AlamarBlue assay. Atomic force microscopy was used to determine morphological changes in the surface of bacterial cells. Cytotoxicity and phytotoxicity were determined by brine shrimp lethality and Lemna minor bioassay. Caco-2 (colorectal adenocarcinoma) cell line was used for the evaluation of the anticancer effects.
RESULT: Among the fractions tested, ethyl acetate (EA) fraction was found to be active with minimum inhibitory concentration (MIC) of 750 μg/mL against E. faecalis, but other fractions were found to be insensitive to bacterial growth. Microscopically, the EA fraction-treated bacteria showed highly damaged cells with their cytoplasmic content scattered all over. The LC50 value of the EA fraction against brine shrimp was more than 1000 μg/mL showing the nontoxic nature of this fraction. Chloroform (CH), EA, and methanol (MOH) fractions of C. excavata were highly herbicidal at the concentration of 1000 μg/mL. EA inhibited Caco-2 cell line with an IC50 of 20 μg/mL.
CONCLUSIONS: This study is the first to reveal anti-E. faecalis property of EA fraction of C. excavata leaves, natural herbicidal, and anticancer agents thus highlight the potential compound present in its leaf which needs to be isolated and tested against multidrug-resistant E. faecalis.