Nano-microbial based technology employing polyvalent phage conjugate: A next generation weapon for antimicrobial resistance lurking behind wastewater.

Worldwide, due to a dearth of innovative interventions, new forms of antimicrobial resistance (AMR) are being discovered every day in clinical and environmental settings. Therefore, it is necessary to remove these contaminants directly or indirectly from the environment. Nanomicrobial-based technology employing nanomaterials with microbes is a new paradigm that finds a place in the antimicrobial crisis. Microbial entities such as phages can be used to treat antimicrobial resistance, but phage resistance is challenging and limits its applicability. Similarly, nanotechnology will not be able to selectively remove resistant strains from the environment individually. Therefore, we employ nanomicrobial-based technology that aims to fill these gaps. In the present study, polyvalent phages were isolated from wastewater with an easy-to-use modified multi-host sequential approach, characterized and conjugated with magnetite (Fe3O4) nanoparticles with the modified formulation to form nanomicrobial conjugates (NMCs). These NMCs were subjected to characterization and in vitro antibacterial studies. The results indicated a significant polyvalency of phages in the order of Caudovirales. Transmission electron microscopy (TEM) analysis of Fe3O4 nanoparticles formed by the co-precipitation method showed a particle size of 30 ± 5 nm and the selected area electron diffraction (SAED) pattern indicates a single-phase crystalline structure. To form NMCs, isolated phages (105 PFU/mL) were immobilized onto Fe3O4 nanoparticles. Further, surface modification of Fe3O4 nanoparticles enables the covalent association of phages. Biosurfactant-functionalized Fe3O4 nanoparticles (FNMCs) were found to have higher phage loading capacity, with a significant value of p < 0.0127 and a zeta potential of -22.2 mV. TEM studies and in vitro biofilm assay showed that NMCs exhibit promising antibacterial activity against various resistant bacterial strains. Pilot studies showed that NMCs can selectively eliminate up to 98.3% of AMR in wastewater. Thus, these findings indicate a synergistic effect of both phage and nanomaterial and this technology is expected to be a new lead in wastewater management.

Antiviral potential of some novel structural analogs of standard drugs repurposed for the treatment of COVID-19.

SARS-CoV-2 pandemic has claimed millions of lives across the world. As of June 2020, there is no FDA approved antiviral therapy to eradicate this dreadful virus. In this study, drug re-purposing and computational approaches were employed to identify high affinity inhibitors of SARS-CoV-2 Main protease (3CLpro), Papain-like protease (PLpro) and the receptor domain of Spike protein. Molecular docking on 40 derivatives of standard drugs (Remdesivir, Lopinavir and Theophylline) led to the identification of R10, R2 and L9 as potential inhibitors of 3CLpro, PLpro and Spike protein, respectively. The binding affinity of R10, R2 and L9 towards 3CLpro, PLpro and Spike protein were 4.03 × 106, 3.72 × 104 and 1.31 × 104M-1, respectively. These inhibitors interact with the active site or catalytic amino acid residues of 3CLpro, PLpro and Spike protein. We also examined the stability and dynamic behavior of protein-inhibitor complex by employing molecular dynamics simulation. RMSDs, RMSFs and variation in secondary structure of target proteins alone or in complex with their respective inhibitors were used to ascertain the integrity of proteins' structure during simulation. Moreover, physicochemical and ADMET properties of R10, R2 and L9 along with Remdesivir, Lopinavir and Theophylline were determined. In vitro and In vivo studies are needed to further validate the potential of these derivatives before they can be developed into potential drug molecules.Communicated by Ramaswamy H. Sarma.

Synergistic combination of natural bioadhesive bael fruit gum and chitosan/nano-hydroxyapatite: A ternary bioactive nanohybrid for bone tissue engineering.

In this work, we have explored the polysaccharide nature of bael fruit gum (BFG) motivated from the current findings about the substantial role of the polysaccharides in bone tissue engineering. The nanocomposite scaffold (CSH-BFG) was prepared by blending BFG, nano-hydroxyapatite (n-HA) and chitosan (CS) by co-precipitation approach and compared with n-HA and CS binary system (CSH). The analysis of different properties was carried out by SEM, TEM, FTIR, XRD and mechanical testing. The CSH-BFG scaffolds revealed a rough morphology and uniform distribution of particles along with strong chemical interactions among different components compared to the CSH scaffold. The incorporation of BFG in the scaffold resulted in significant increase of the compressive strength, compressive modulus, protein adsorption, biodegradation and swelling behaviour. The ternary system exhibited superior antibacterial activity against different bacterial pathogens compared to the binary system. The in vitro biomineralization ability was elucidated from the formation of thick apatite layer complementing the result of ARS study in the CSH-BFG nanocomposite. Our findings also revealed that BFG reinforced CSH nanocomposite exhibited enhanced cell adhesion and proliferation, osteogenic differentiation along with phenomenal cytocompatibility. Overall, our results signified that the fabricated CSH-BFG nanocomposite carries enormous potential to be applied in the bone remodelling procedures.

Resol based chitosan/nano-hydroxyapatite nanoensemble for effective bone tissue engineering.

It is the first report where different amounts of resol resin (RS) were incorporated with chitosan-hydroxyapatite (CHA) to develop a triconstituent nanoensemble CHA-RS(0.5,1,2), via simple co-precipitation method. The results of SEM, TEM, TGA and mechanical analysis revealed irregular interconnected rough morphology with homogenous distribution of needle shaped particles having average size ranging between 12 and 19nm, possessing higher thermal stability and mechanical strength, respectively relative to CHA (binary) nanocomposite. The CHA-1RS nanocomposite showed enhanced protein adsorption and ALP activity with excellent apatite formation ability compared to CHA-RS(0.5,2) and CHA nanocomposites. Thus, CHA-1RS nanocomposite was selectively tested as bare implant in the repair of critical-size calvarium defect (8mm) in albino rat. The histopathological and radiological investigations indicated that CHA-1RS prompted the bone regeneration ability as early as 2 weeks postimplantation demonstrating remarkably faster healing of calvarial defect relative to Cerabone. These findings have placed CHA-1RS on the pedestal to be employed as a potential alternative biomaterial for bone tissue engineering.

Evaluation of anti-methicillin-resistant Staphylococcus aureus (MRSA) activity and synergy of some bioactive plant extracts.

Anti-methicillin-resistant Staphylococcus aureus (MRSA) activity of ethanolic extracts of four medicinal plants namely Acorus calamus (rhizome) Hemidesmus indicus (stem), Holarrhena antidysenterica (bark), and Plumbago zeylanica (root), were detected with inhibition zone size ranged from 11 to 44 mm and minimum inhibitory concentration (MIC) varied from 0.32 to 3.25 mg/mL. Further, ethyl acetate, acetone and methanol fractions of above plants demonstrated antibacterial activity. The potency of these fractions based on zone of inhibition and MIC value was relatively higher in P. zeylanica (ethylacetate fraction), followed by acetone fractions of H. indicus, A. calamus, and H. antidysenterica. Time kill assay with most promising fractions of these plant extracts, demonstrated concentration-dependent killing of MRSA within 9-12 h of incubation. Interestingly, synergistic interaction among alcoholic extracts and some fractions of above four plants was evident against MRSA. Further, synergistic interaction of these extracts was detected with one or more antibiotics tested (tetracycline, chloramphenicol, ciprofloxacin, cefuroxime and ceftidizime). The findings also validate the traditional uses of above plants against infectious diseases. Phytochemical studies demonstrated flavonoids and phenols as major active constituents. Further investigations are needed to characterize the active principle and its interaction mechanism with antibiotics.

Toxicity, stability and pharmacokinetics of amphotericin B in immunomodulator tuftsin-bearing liposomes in a murine model.

OBJECTIVES: In the present study we evaluated the pharmacokinetics and toxicity of amphotericin B in immunomodulator tuftsin-loaded liposomes in a murine model. METHODS: Stability of amphotericin B liposomes was tested by incubating one volume of liposomal formulations of amphotericin B with nine volumes of serum. The pharmacokinetics of amphotericin B in Candida albicans-infected mice treated with conventional and tuftsin-loaded amphotericin B liposomes was evaluated over a period of 24 h. In vitro toxicity of amphotericin B deoxycholate, as well as amphotericin B liposomes, was tested by incubation with human erythrocytes for 1 h at 37 degrees C. To assess amphotericin B-induced in vivo toxicity, BALB/c mice were injected with three doses of amphotericin B deoxycholate, as well as amphotericin B liposomal formulations on days 1, 2 and 3 post C. albicans infection. Blood from treated mice was taken by retro-orbital puncture to test renal function parameters such as serum creatinine and urea. RESULTS: In vitro stability studies revealed that tuftsin-bearing amphotericin B liposomes released only 11% of the total liposomal amphotericin B in the serum, while it was found to be 19% from identical tuftsin-free amphotericin B liposomes. Both tuftsin-loaded as well as tuftsin-free liposomal formulations of amphotericin B induced approximately 20% haemolysis of erythrocytes at a dose of 40 mg/L, while the same amount of drug in amphotericin B deoxycholate caused 100% lysis of the erythrocytes. Pharmacokinetic studies revealed that subsequent to administration of various formulations of amphotericin B, there was 32 mg/L amphotericin B in the systemic circulation of mice treated with tuftsin-bearing amphotericin B liposomes, while it was 25 mg/L for amphotericin B liposomes, 4 h post drug administration. In vivo toxicity studies demonstrated that the amphotericin B deoxycholate formulation induced elevations in serum creatinine (approximately 300% of control) and blood urea (approximately 380% of control) values, while these values were substantially less (blood urea approximately 150% of control and serum creatinine approximately 210% of control) in the animals treated with the tuftsin-loaded amphotericin B liposomal formulation. Further, the administration of amphotericin B deoxycholate (1 mg/kg) in BALB/c mice at a dose of 1 mg/kg body weight led to the accumulation of 18.6 +/- 5.25 g/kg (of amphotericin B) in kidneys. On the other hand, administration of liposomal amphotericin B and tuftsin-bearing liposomal amphotericin B at a dose of 5 mg/kg body weight resulted in accumulation of 8.8 +/- 2.0 and 4.0 +/- 1.6 g/kg of amphotericin B, respectively, in the kidneys of treated animals. CONCLUSIONS: Co-administration of immunomodulator tuftsin along with liposomal formulations of amphotericin B successfully minimizes toxicity, as well as other side effects of the drug. Interestingly, tuftsin also increased the stability of liposomal amphotericin B. Superior efficacy, reliable safety and favourable pharmacodynamics of tuftsin-loaded amphotericin B liposomes suggest their potential therapeutic value in the management of fungal infections.

Effect of certain bioactive plant extracts on clinical isolates of beta-lactamase producing methicillin resistant Staphylococcus aureus.

Ethanolic extracts and some fractions from 10 Indian medicinal plants, known for antibacterial activity, were investigated for their ability to inhibit clinical isolates of beta-lactamase producing methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus (MSSA). Synergistic interaction of plant extracts with certain antibiotics was also evaluated. The MRSA test strains were found to be multi-drug resistant and also exhibited high level of resistance to common beta-lactam antibiotics. These strains produced beta-lactamases, which hydrolyze one or other beta-lactam antibiotics, tested. The extract of the plants from Camellia sinensis (leaves), Delonix regia (flowers), Holarrhena antidysenterica (bark), Lawsonia inermis (leaves), Punica granatum (rind), Terminalia chebula (fruits) and Terminalia belerica (fruits) showed a broad-spectrum of antibacterial activity with an inhibition zone size of 11 mm to 27 mm, against all the test bacteria. The extracts from the leaves of Ocimum sanctum showed better activity against the three MRSA strains. On the other hand, extracts from Allium sativum (bulb) and Citrus sinensis (rind) exhibited little or no activity, against MRSA strains. The antibacterial potency of crude extracts was determined in terms of minimum inhibitory concentration (MIC) by the tube dilution method. MIC values, of the plant extracts, ranged from 1.3 to 8.2 mg/ml, against the test bacteria. Further, the extracts from Punica granatum and Delonix regia were fractionated in benzene, acetone and methanol. Antibacterial activity was observed in acetone as well as in the methanol fractions. In vitro synergistic interaction of crude extracts from Camellia sinensis, Lawsonia inermis, Punica granatum, Terminalia chebula and Terminalia belerica was detected with tetracycline. Moreover, the extract from Camellia sinensis also showed synergism with ampicillin.TLC of the above extracts revealed the presence of major phytocompounds, like alkaloids, glycosides, flavonoids, phenols and saponins. TLC-bioautography indicated phenols and flavonoids as major active compounds.