Materials and methods: Root canal exudates of 30 patients were tested against MTA Fillapex (Angelus), Metapex (BioMed), zinc oxide eugenol (Deepak Enterprise), Endomethasone (Septodont), Endoflas FS (Sanlor Laboratories), MTA (Angelus) (positive control), and glycerine (negative control). Children with failed endodontic cases were included in the study. Tube dilution and agar diffusion methods were used to check the antifungal efficacy of the root canal sealers. In tube dilution method, 24-well culture plates containing freshly mixed material along with Candida albicans were used. Wells containing MTA (Angelus) along with Sabouraud dextrose agar and Candida albicans served as positive control while glycerine along with Sabouraud dextrose agar and Candida albicans served as negative control. All plates were incubated at 37°C for 24 hours. Growth of the fungi was monitored after 24 hours by the presence of the turbidity. The samples were recultured to test the experimental material using agar well diffusion method, and the Petri plates were incubated for 24 hours and 72 hours. Zone of inhibition was measured after respective time period. Paired t test was used for the data analysis.
Results: It was seen in tube dilution method Endomethasone showed least turbidity while maximum was shown by Metapex; similar results were seen in case of agar well diffusion method in which largest zone of inhibition was shown by Endomethasone while smallest was by Metapex.
Conclusion: It was concluded that Endomethasone showed maximum efficacy against Candida albicans as compared to Metapex.
How to cite this article: Singh S, Srivastava B, Gupta K, et al. Comparative Evaluation of Antifungal Efficacy of Five Root Canal Sealers against Clinical Isolates of Candida albicans: A Microbiological Study. Int J Clin Pediatr Dent 2020;13(2):119-123.
METHODS: The enzyme was purified in two steps using affinity and size exclusion chromatography. Enzyme assays were performed using the malachite green assay and enzymatic product was identified using gas chromatography-mass spectrometry (GC-MS) analysis. Sequence analysis of PmSTS was performed using multiple sequence alignment (MSA) against plant sesquiterpene synthase sequences. The homology model of PmSTS was generated using I-TASSER server.
RESULTS: Our findings suggest that the recombinant PmSTS is mainly expressed as inclusion bodies and soluble aggregate in the E. coli protein expression system. However, the addition of 15% (v/v) glycerol to the protein purification buffer and the removal of N-terminal 24 amino acids of PmSTS helped to produce homogenous recombinant protein. Enzyme assay showed that recombinant PmSTS is active and specific to the C15 substrate FPP. The optimal temperature and pH for the recombinant PmSTS are 30 °C and pH 8.0, respectively. The GC-MS analysis further showed that PmSTS produces β-sesquiphellandrene as a major product and β-farnesene as a minor product. MSA analysis revealed that PmSTS adopts a modified conserved metal binding motif (NSE/DTE motif). Structural analysis suggests that PmSTS may binds to its substrate similarly to other plant sesquiterpene synthases.
DISCUSSION: The study has revealed that homogenous PmSTS protein can be obtained with the addition of glycerol in the protein buffer. The N-terminal truncation dramatically improved the homogeneity of PmSTS during protein purification, suggesting that the disordered N-terminal region may have caused the formation of soluble aggregate. We further show that the removal of the N-terminus disordered region of PmSTS does not affect the product specificity. The optimal temperature, optimal pH, Km and kcat values of PmSTS suggests that PmSTS shares similar enzyme characteristics with other plant sesquiterpene synthases. The discovery of an altered conserved metal binding motif in PmSTS through MSA analysis shows that the NSE/DTE motif commonly found in terpene synthases is able to accommodate certain level of plasticity to accept variant amino acids. Finally, the homology structure of PmSTS that allows good fitting of substrate analog into the catalytic active site suggests that PmSTS may adopt a sesquiterpene biosynthesis mechanism similar to other plant sesquiterpene synthases.