METHODS: Comprehensive searches of the NCBI database were performed to identify published peer-reviewed articles and genomes of E. faecalis ST476. Each genome was analysed for resistome, virulome, OptrA variant and optrA genetic contexts. A phylogenetic comparison of ST476 genomes with publicly available genomes of other STs was also performed.
RESULTS: Sixty-six E. faecalis ST476 isolates from 15 countries (China, Japan, South Korea, Austria, Denmark, Spain, Czech Republic, Colombia, Tunisia, Italy, Malaysia, Belgium, Germany, United Arab Emirates and Switzerland) mainly of human and animal origin were identified. Thirty available ST476 genomes compared with genomes of 591 STs indicated a progressive radiation of E. faecalis STs starting from ST21. The closest ancestral node for ST476 was ST1238. Thirty E. faecalis ST476 genomes exhibited 3-916 SNP differences. Several antimicrobial resistance and virulence genes were conserved among the ST476 genomes. The optrA genetic context exhibited a high degree of or complete identity to the chromosomal transposon Tn6674. Only three isolates displayed an optrA-carrying plasmid with complete or partial Tn6674. The WT OptrA protein was most widespread in the ST476 lineage.
CONCLUSIONS: Linezolid-resistant optrA-carrying E. faecalis of the clonal lineage ST476 is globally distributed in human, animal and environmental settings. The presence of such an emerging clone can be of great concern for public health. Thus, a One Health approach is needed to counteract the spread and the evolution of this enterococcal clonal lineage.
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.
Materials and methods: Seventy-five enterococci isolates recovered from different clinical sources were re-identified by subculturing on selective medium, Gram staining, biochemical profiling (API 20 Strep), and 16s rRNA sequencing. Antimicrobial susceptibility testing (AST) was performed using Kirby-Bauer disc diffusion, E-test, and broth microdilution methods. PCR amplification was used to detect the presence of aminoglycoside modifying enzyme (AME) genes [aac(6')-Ie-aph(2")-Ia, aph(2")-Ib, aph(2")-Ic, aph(2")-Id, aph(3')-IIIa]. Descriptive data analysis was used to analyze the antibiotic susceptibility profiles and the distribution of HLAR genes.
Results: The majority of the isolates recovered from the clinical samples are E. faecalis (66.7%), with the highest recovery from the pus. The prevalence of HLGR (51%) is higher when compared to HLSR (45-49%). Analysis of the resistance genes showed that bifunctional genes aac(6')-Ie-aph(2")-Ia and aph(3')-IIIa contributed to the HLAR E. faecalis and E. faecium. The other AME genes [aph(2")-Ib, aph(2")-Ic, aph(2")-Id] were not detected in this study.
Conclusion: This study provides the first prevalence data on HLAR and the distribution of the AME genes among E. faecalis and E. faecium isolates from Malaysia. These highlight the need for continued antibiotic surveillance to minimize its emergence and further dissemination.
Materials and Methods: Extracted human teeth were biomechanically prepared, vertically sectioned, placed in the tissue culture wells exposing the root canal surface to E. faecalis to form a biofilm. At the end of the third week, all groups were treated for 15 min with the test solutions and the control. The results were analyzed both quantitatively and qualitatively.
Results: Statistical analysis was performed by using one-way analysis of variance and compared by the Mann-Whitney test using the Statistical Package for the Social Sciences (SPSS) software, version 20.0. The qualitative assay with the 3-week biofilm on the canal portion showed complete inhibition of bacterial growth for NaOCl, whereas samples treated with herbal solutions showed significant reduction of bacterial growth compared to control group, which showed 139.9 × 109 CFU/mL among the experimental herbal solutions groups. P. amarus has shown maximum bacterial count followed by C. longa and T. indica.
Conclusion: NaOCl 5% showed maximum antibacterial activity against 3-week biofilm on tooth substrate. T. indica, P. amarus, and C. longa showed statistically significant antibacterial activity against 3-week biofilm. The use of herbal alternatives might prove to be advantageous considering the several undesirable characteristics of NaOCl.
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.
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.