IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF BACTERIA HAVING ANTIMICROBIAL AND ANTIBIOFILM ACTIVITY

Objective: The aim of the current study was to isolate and identify the bacteriocinogenic strain exhibiting broad range antimicrobial activity and antibiofilm activity from soil of animal farms.Methods: In the current study, bacterial strains were isolated from soil of twelve different regions of animal farm all over India and screened for antimicrobial activity against Staphylococcus epidermidis, Micrococcus luteus, Pseudomonas fluorescence and Escherichia coli. Antibiofilm ability of these selected strains was checked on preformed biofilm of S. epidermidis and in addition biofilm disruption potential was also determined. The potent bacterial strain was identified at molecular level by 16S ribosomal DNA (rDNA) sequencing.Results: 30 out of 231 strains isolated from soil were selected on the basis of antibacterial activity against S. epidermidis. One potential candidate (GAS 101) exhibited ≥99% inhibition against S. epidermidis, M. luteus, P. fluorescence and E. coli and also showed antibiofilm activity. GAS 101 16S rDNA sequencing data identified it as Bacillus subtilis. The sequence of B. subtilis was submitted to genbank under accession no. KJ564301.Conclusion: B. subtilis GAS 101 isolated from soil of animal farm showed the antibacterial activity against all indicator organisms and also displayed antibiofilm activity against preformed biofilm and inhibited biofilm formation of S. epidermidis

ANTIBACTERIAL AND ANTIBIOFILM ACTIVITIES OF TRANS-CINNAMALDEHYDE NANOEMULSION AGAINST ESCHERICHIA COLI

Objective: Trans-cinnamaldehyde (TC) has shown antimicrobial activity against various microorganisms, but its direct use has some disadvantages such as skin irritation, low bioavailability, and low solubility. The objective of the present work was to develop the oil-in-water nanoemulsions (NEs) of TC to enhance its antimicrobial activity against Escherichia coli. Methods: The NEs of TC were prepared using triton x-100 and isopropyl alcohol as surfactant and cosurfactant. The developed NE was studied for size, zeta potential, and stability. NEs were evaluated for antimicrobial and antibiofilm activity against E. coli as indicator organism. NEs possible mode of action on E. coli was assessed by scanning electron microscopy (SEM). Results: Stable NEs of TC exhibited a particle size of 210 nm and were able to inhibit the growth of planktonic as well as biofilm cultures of E. coli at 67 μg/ml. The ruthenium red staining indicated the inhibition of glycoprotein layer formation in extracellular matrix after treating with NE. TC-NE exhibited substantial decrease in E. coli growth as well as its viability at its minimum inhibitory concentration as determined by 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The mode of action of cinnamaldehyde through β-galactosidase assay on E. coli ML-35p strain indicated that it altered the bacterial cell membrane permeability. SEM results showed the presence of holes on the cell wall of the E. coli in the presence of TC-NE. Conclusions: TC-NEs exhibited enhanced antimicrobial activity and were effective against E. coli biofilm. They also exhibited microbicidal activity and altered E. coli membrane permeability

Elucidating the Interacting Domains of Chandipura

The nucleocapsid (N) protein of Chandipura virus (CHPV) plays a crucial role in viral life cycle, besides being an important structural component of the virion through proper organization of its interactions with other viral proteins. In a recent study, the authors had mapped the associations among CHPV proteins and shown that N protein interacts with four of the viral proteins: N, phosphoprotein (P), matrix protein (M), and glycoprotein (G). The present study aimed to distinguish the regions of CHPV N protein responsible for its interactions with other viral proteins. In this direction, we have generated the structure of CHPV N protein by homology modeling using SWISS-MODEL workspace and Accelrys Discovery Studio client 2.55 and mapped the domains of N protein using PiSQRD. The interactions of N protein fragments with other proteins were determined by ZDOCK rigid-body docking method and validated by yeast two-hybrid and ELISA. The study revealed a unique binding site, comprising of amino acids 1–30 at the N terminus of the nucleocapsid protein (N1) that is instrumental in its interactions with N, P, M, and G proteins. It was also observed that N2 associates with N and G proteins while N3 interacts with N, P, and M proteins

Development and Characterization of Polyphenon 60 and Caffeine Microemulsion for Enhanced Antibacterial Activity

Green tea catechins and caffeine have exhibited antibacterial activity; however, their use is limited by lack of stability and effective delivery systems. Polyphenon 60 (P60) and caffeine were encapsulated in a single microemulsion (ME) formulation with an objective to lower the minimum inhibitory concentrations (MICs) of the individual agents against selected pathogens (S. epidermidis and E. coli). Combination of two natural compounds would advocate two different mechanisms on the bacterial growth thereby providing for better antibacterial activity. Thermodynamically stable ME was developed and characterized with an average particle size of 17.58 nm, further confirmed by TEM analysis. Antibacterial studies included chequerboard microdilution assay to determine the MIC and fractional inhibitory concentration (FIC) of both the natural compounds individually and in combination. MIC and FIC results indicated that the combination of the above two natural compounds was proficient in lowering the MICs of individual agents. Results of DPPH assay indicated that ME system preserved the long term antioxidative potential of P60 and caffeine. The cytotoxicity of the optimized formulation on Vero cell line by MTT assay was found to be nontoxic to mammalian cells

SMALL SCALE EXPRESSION, SOLUBILIZATION AND CHARACTERIZATION OF CHIKUNGUNYA VIRUS STRUCTURAL PROTEINS

  Objective: The severity and spread of Chikungunya fever in absence of effective antiviral therapy presents a serious public health threat. The present investigation aims to generate soluble and purified viral structural proteins that can be utilized to facilitate generation of reagents for development of both diagnostic and therapeutic measures.Methods: Bacterial expression system was used for optimization of expression and solubilization of structural proteins of CHIKV (Capsid, 6K and envelope proteins 1-3 [E1, E2 and E3]) as fusions with large (GST) and small (His and Strep) tags on a single platform. Affinity chromatography was used for small scale purification of viral proteins.Result: The effect of different tags, inducer concentrations, temperatures and duration of induction on solubilization of proteins has been optimized and small scale purification of all the structural proteins has been attempted. Utility of these solubilized proteins has been shown by analyzing the interaction of E2 with all the structural proteins using pull down assay.Conclusion: Small scale purification of all five structural proteins and ectodomains of envelope proteins E1 and E2 has been standardized. The data and reagents generated can be utilized for large scale purification and studying CHIKV biology.Keywords: Chikungunya virus, Protein expression, Solubilization, Purification

Development of Nanoemulsion Based Gel Loaded with Phytoconstituents for the Treatment of Urinary Tract Infection and in Vivo Biodistribution Studies

Purpose: A nanoemulsion based gel containing Polyphenon 60 (P60) and cranberry (CRB) has been developed to deliver via intravaginal route for the treatment of urinary tract infection. Methods: Polyphenon 60 and cranberry were loaded in a single nanoemulsion gel (NBG) by ultra-sonication method and characterized for particle size, rheological properties, in vitro release and growth curve analysis. P60+CRB NBG were radiolabelled using technetium pertechnetate (99mTc) to perform in vivo pharmacokinetic studies in animals. Results: The finalized NE had a droplet size of 58±1 nm. In vitro release of 90.92 ± 0.6% in 8 hr for P60 and 99.39 ± 0.5% in 6 hr for CRB was observed in simulated vaginal fluid. Growth curve of E. coli indicated the inhibitory action of nanoemulsion based gel at the fifth hour of inoculation. Gamma scintigraphy studies on female Sprague-Dawley rats showed transport of nanoemulsion based gel from the vaginal cavity into the systemic circulation. Further, biodistribution studies with radiolabelled P60+CRB NBG showed significant higher uptake of radiolabelled actives by kidney (3.20±0.16) and urinary bladder (3.64±0.29), when administered intravaginally. Conclusion: The findings suggested 99mTc-P60+CRB NBG can potentially be transported through vaginal cavity and reach the target organs and showed effective distribution in organs affected in urinary tract infectio