Materials and Methods: Pfu polymerase gene sequence, derived from Pyrocuccus furiosus (Pfu) genomic DNA, was cloned and overexpressed in E. coli BL21 (DE3) pLysS. Upon overexpression, bacterial lysate containing the Pfu DNA polymerase was heated at 94°C for 5 minutes. Pfu DNA polymerase having high thermal stability was retained while the other bacterial proteins were denatured. The resulting thermo stable Pfu DNA polymerase was separated from the other debris of the denatured proteins by simple centrifugation.
Results: The enzymatic activity of the resulting Pfu DNA polymerase was estimated by comparing with the commercial Pfu DNA Polymerases. An estimated 50000 units of functional Pfu DNA polymerase was produced from a 400 ml culture.
Conclusion: The in-house produced Pfu DNA Polymerase could be used for routine amplification that requires high-fidelity such as cloning and DNA sequencing.
Materials and Methods: The whole plant of C. roseus was extracted using methanol extraction method. Phytochemical qualitative screening was carried out for C. roseus extract according to standard procedures used to test for the presence of alkaloid, saponin, terpenoid and steroid. Cytotoxicity was assessed using 3-(4,5-dimethylthiazol-2,5-diphenyltetrazolium bromide (MTT) assay. Plaque reduction assays were carried out to evaluate the antiviral activity of C. roseus extract against herpes simplex virus type 1 (HSV-1). These include post-treatment, pre-treatment and virucidal assays.
Results: C. roseus extract contain secondary metabolites such as alkaloid, saponin and terpenoid but does not contain steroid. Cytotoxicity screening against Vero cells using MTT assay showed that the CC50 values for crude extract of C. roseus was 0.5 mg/mL. The extract prepared from C. roseus possesses phytochemical compound that was non-cytotoxic to the cell with potential antiviral activity. Plaque reduction assays against herpes simplex virus type 1 (HSV-1) showed that the selective indices (SI = CC50 / EC50) of C. roseus extract in post-treatment, pre-treatment and virucidal assays were 36, 20 and 4.7 respectively. The results revealed that the extract prepared from C. roseus possesses phytochemical compound that was non-cytotoxic to the cell with potential antiviral activity.
Conclusion: This study showed that C. roseus extract has promising potential to be explored as anti-HSV-1 agent regardless of the mode of treatment.
Materials and Methods: Sensitivity of two biofilm-producing bacteria (P. mirabilis UCa4 and P. mirabilis UCe1) to bacteriocin, was investigated in planktonic and biofilm states by cell viability and crystal violet assay, respectively. Scanning electron microscopy (SEM) was also performed to determine the effect of bacteriocin on the morphology of the cells associated with biofilm.
Results: It was found that bacteriocin possessed bactericidal activity to biofilm-forming isolates in the planktonic state. However, bacteriocin interferes with the formation of biofilms and disrupts established biofilms. Bacteriocin reduced biofilm formation in the isolates of P. mirabilis UCa4 and P. mirabilis UCe1 with SMIC50 of 32 and 128 μg/mL, desirable SMIC50 of bacteriocin for biofilm disruption were 128 and 256 μg/mL, respectively. The SEM results indicated that bacteriocin affected the cell morphology of biofilm-associated cells.
Conclusion: The present findings indicated that bacteriocin from Bacillus sp. Sh10 has bactericidal properties against biofilm-forming isolates of P. mirabilis UCa4 and P. mirabilis UCe1 and has the ability to inhibit the formation of biofilm and disrupt established biofilm.
Materials and Methods: Isolated strains were identified by Gram staining, catalase assay, and 3 molecular identification methods; namely, (GTG) 5-PCR fingerprinting, ARDRA, and 16S rDNA gene sequencing.
Results: A total of 19 LAB strains belonging to 4 genera (Lactococcus, Leuconostoc, Lactobacillus and Enterococcus) were identified.
Conclusion: The experiments revealed that L. plantarum 15HN, L. lactis subsp. cremoris 44L and E. mundtii 50H strains, which were isolated from shiraz, cheese and shiraz, respectively, displayed a desirable tolerance to low pH and high bile salts, favorable anti-pathogen activity, and acceptable antibiotic susceptibility; hence, they could be considered as novel probiotic candidates and applied in the food industry.
MATERIALS AND METHODS: The antibacterial activity of MH, SH and TH against S. aureus was investigated by agar well diffusion, Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC), time-kill curve, microtiter plate and RT-qPCR analysis.
RESULTS: Agar inhibition assay showed that MH possess highest total antibacterial activity against S. aureus with an inhibition zone 25.1 mm compared with that of SH (22.2 mm) and TH (21.3 mm). The findings showed that when compared to SH and TH (MIC: 25% and MBC: 50%), MH honey had the lowest MIC (12.5%) and MBC (25%). After S. aureus was exposed to MH, SH, and TH, there was a decrease in colony-forming unit as seen by the time-kill curve. The lowest concentration 20% of MH, SH and TH was significantly found to inhibit S. aureus biofilm. The RT-qPCR results revealed that all the selected genes in S. aureus were downregulated in gene expression following exposure to each of the tested honeys. Comparing the total antibacterial, antibiofilm, and antivirulence activities of all the tested honeys, MH demonstrated the greatest levels of these properties.
CONCLUSION: According to this study, various types of each evaluated honey have the capacity to effectively suppress and modify the virulence of S. aureus via a variety of molecular targets.
MATERIALS AND METHODS: Activity was assessed using broth microdilution, time kill viability, microtiter plate, scanning electron microscope (SEM) and Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR).
RESULTS: The susceptibility tests revealed promising antibacterial activities of all honeys against both bacteria. The MICs of JH, KMH, GH, and AH ranged from 20% to 25% compared to MH (12.5%) against both bacteria. The MBCs of JH, KMH, GH, and AH ranged from 20% to 50% compared to MH (20%) against both bacteria. Treatment of both bacteria with 2× MIC (Minimum inhibitory concentration) of MH, JH, KMH, GH, and AH for 9 hours resulted in reduction in colony-forming unit (CFU/ml). SEM images showed that the morphological changes, cell destruction, cell lysis and biofilm disruption in both bacteria after exposure to all honeys. RT-qPCR analysis revealed that the expression of all genes in both bacteria were downregulated following treatment with all honeys. Among the all-tested honeys, MH showed the highest total antibacterial and antivirulence activities.
CONCLUSION: Our results indicate that all honeys activity included inhibition of both bacteria due to a decrease in expression of essential genes associated with both bacteria, suggesting that all honeys could potentially be used as an alternative therapeutic agent against certain microorganisms particularly against P. aeruginosa and S. pyogenes.
MATERIALS AND METHODS: The effect of TH on both bacteria was investigated using MIC, MBC, growth curve, time-kill curve, scanning electron microscopy (SEM) and RT-qPCR.
RESULTS: The MIC of TH against P. aeruginosa and S. pyogenes was 18.5% (w/v) and 13% (w/v) respectively and MBC was 25% (w/v) for both bacteria. Spectrophotometric readings of at least 90% inhibition yielded MIC90 values of TH, 18.5% (w/v) and 15% (w/v) for P. aeruginosa and S. pyogenes respectively. A time-kill curve demonstrated a bactericidal with a 4-log reduction estimated within 8 hours. Using SEM, loss of structural integrity and marked changes in cell shape were observed. RT-qPCR analysis showed that TH reduced the pattern of gene expression in both bacteria, with a trend toward reduced expression of the virulence genes of interest.
CONCLUSION: This study suggests that TH could potentially be used as an alternative therapeutic agent for microbial infection particularly against these two organisms.
MATERIALS AND METHODS: Crude methanol extract from P. betel powdered leaves was partitioned between chloroform and water. The fractions were tested against A. flavus UPMC 89, a strong aflatoxin producing strain. Inhibition of mycelial growth and aflatoxin biosynthesis were tested by disk diffusion and macrodillution techniques, respectively. The presence of aflatoxin was determined by thin-layer chromatography (TLC) and fluorescence spectroscopy techniques using AFB1 standard. The chloroform soluble compounds were identified using HPLC-Tandem mass spectrometry technique.
RESULTS: The results, evaluated by measuring the mycelial growth and quantification of aflatoxin B1(AFLB1) production in broth medium revealed that chloroform soluble compounds extract from P. betle dried leaves was able to block the aflatoxin biosynthesis pathway at concentration of 500μg/ml without a significant effect on mycelium growth. In analyzing of this effective fractions using HPLC-MS(2) with ESI ionization technique, 11 phenolic compounds were identified.
CONCLUSION: The results showed that the certain phenolic compounds are able to decline the aflatoxin production in A. flavus with no significant effect on the fungus mycelia growth. The result also suggested P. betle could be used as potential antitoxin product.
MATERIALS AND METHODS: Thirty-seven NiV genomes of human samples from Malaysia, Bangladesh, and India were subjected to phylogeny and metagenomic analysis to decipher the genome variability using MEGA11 software and the meta-CATS web server. Using the Single-Likelihood Ancestor Counting method, the synonymous and nonsynonymous mutations among NiV genes were identified. Further, the nonsynonymous variations were used to identify mutations in all the NiV proteins.
RESULTS: The NiV isolates were categorized into NiV-M, NiV-B, and NiV-I clades based on phylogenetic analysis. Metagenomic analysis revealed 1636 variations in the noncoding and coding regions of the genomes of the three clades of NiV. Further analysis of nonsynonymous mutations showed the phosphoprotein to be highly mutating, whereas the matrix protein was stable.
CONCLUSION: Deciphering the mutation pattern using a comparative genomics approach for human isolates provided valuable insight into the stability of NiV proteins which can be further used for understanding variations in host-pathogen interaction and developing effective therapeutic measures.
MATERIALS AND METHODS: Three groups were formed, consisting of twelve male Sprague Dawley rats: a control group designated as 0-day, and experimental groups labeled as 7-days and 14-days. Periodontitis induced by concurrent infection with sterile wire 0.2 mm insertion and E. faecalis inoculation is performed into the gingival sulcus located between the maxillary right 1st and 2nd molar teeth area. After euthanasia, tissue samples around the maxillary gingiva, maxillary jaw samples, kidney and heart tissues were obtained for quantitative Real-Time PCR assay and histopathological analysis.
RESULTS: Results showed at 7-days, there was an upregulation of E. faecalis gene expression in the gingiva, heart, and kidney samples as well as infiltration of the inflammatory cells at 7-days post induction, which consequently decreased at 14-days.
CONCLUSION: Thus, the study suggests dissemination of E. faecalis from gingival tissue to the heart, kidney which could be probable link between periodontal disease, heart, and kidney disease.