Effects of the probiotics Lactococcus lacttis (MTCC-440) on Salmonella enteric serovar Typhi in co-culture study.

Lactococcus lactis (L. lactis)a probiotics microorganism having wide range of benefits on human health, and also protects the body from pathogenic microorganism. This study was conducted to determine the co-culture effect with the probiotic strain L. lactis (MTCC440) on Salmonella enterica serovar Typhi (S.Typhi). The existing problem was to determine the individual growth of both strains during co-culture. Growth kinetics study was performed and observed for 28 h and used to determine specific growth rate of S. Typhi under co-culture study. In growth kinetics study maximum specific growth rate (µ) of S. Typhi under monoculture and co-culture study was achieved 0.695 h-1 and 0.35 h-1 respectively. The maximum cell mass of L. lactis and S. Typhi was obtained 0.15 g/L and 0.18 g/L respectively. In co-culture study, L. lactis was found effective for the inhibition of 73% growth of S. Typhi due to lactic acid production.

Expression, purification and evaluation of recombinant lipoprotein of Salmonella typhi as a vaccine candidate.

Lipoprotein has been reported as a vaccine candidate against many pathogenic bacteria, it plays direct role as a virulence-associated function. Here the approach is toward the expression of recombinant lipoprotein of Salmonella typhi in prokaryotic host and its evaluation as a vaccine candidate. Lipoprotein gene (lp1) was cloned in pET32a expression vector in addition to Bam HI and Hind III restriction sites, and BL21(pLysS) was used as prokaryotic expression host for transformation. Lipoprotein induction was performed by IPTG and 55 kDa (31 kDa of Gene +24 kDa of vector additional protein with His-tag) was analyzed by 12% SDS-PAGE. The recombinant lipoprotein was purified by Ni-NTA affinity chromatography due to the addition of 6X His-tag in recombinant lipoprotein. Western blot analysis using anti-His tag polyclonal antibodies confirmed the specificity of recombinant lipoprotein. Immunogenicity and protection study of recombinant lipoprotein against S. Typhi was performed in BALB/c mice. Adjuvants IFA and alum salts were used to enhance the immune response. ELISA results proved that biologically active truncated recombinant lipoprotein (31 kDa) is a suitable immunogen. Alum salts used as adjuvant was effective for long-lasting immunity.

A label-free genosensor for BRCA1 related sequence based on impedance spectroscopy.

A simple approach based on electrochemical impedance spectroscopy of a DNA hybridization sensor for breast cancer is reported by immobilizing a BRCA1 related 19 mer oligonucleotides ssDNA sequence on a glassy carbon electrode modified with a poly 5-carboxyindole conducting polymer. Differential pulse voltammetry, UV-vis spectrophotometry, FT-IR and impedance spectroscopy are performed for the study of electropolymerization of 5-carboxyindole, chemical immobilization of ssDNA and the hybridization event at the electrode surface. A significant change is observed in the impedance spectra before and after hybridization of ssDNA with the target probe under optimum conditions. The above described technique is found to be efficient enough to detect a single base mismatch in a target DNA sequence. All the experiments are performed in Tris-HCl buffer 20 mM (pH 6.5). A detection limit of 1 × 10(-17) M with a linear range from 1 × 10(-8) to 1 × 10(-18) is obtained after hybridization for the optimum time period. The proposed method is simple, free from any redox indicator/marker and possesses high sensitivity and selectivity. This technique is successfully demonstrated for detection of a single base mutation in a BRCA1 related sequence.

Bioconversion of silver salt into silver nanoparticles using different microorganisms.

Abstract: Recently, silver nanoparticles have generated enough interest due to their immense usage. Until now chemical synthesis has been a fast method to produce nanoparticles, but the release of environmental pollutants has raised caution. So a more subtle biochemical approach is in research. Three microorganisms, Aspergillus flavus, Phoma exigua and Bacillus megaterium, were selected to reduce silver nitrate to silver nanoparticles in aqueous form. All three microorganisms showed varying reducing capacity, of which Phoma exigua showed the highest, when their biomass was incubated in silver nitrate for 48 hours. The nanoparticles showed all the optical and physical properties, which were analyzed by measuring the surface plasmon resonance and TEM. It was also observed that the fungal species have varied growth in silver nitrate and can be used to produce silver nanoparticles, directly incubating the inocula of fungus in the media containing silver nitrate. On comparing all the characteristics and results produced, Aspergillus flavus was found to be the most effective microorganism, which can convert the silver nitrate to silver nanoparticles in aqueous condition. The bacterial species showed no growth with incubation with silver nitrate.

Biosynthesis of cephalosporin-C acylase enzyme: optimal media design, purification, and characterization.

7-aminocephalosporanic acid (7-ACA) is the key intermediate for the synthesis of semisynthetic cephalosporin antibiotics and enzyme cephalosporin-C acylase (CPC acylase) plays an important role in the conversion of cephalosporin-C to 7-ACA. With an aim to increase the yield of 7-ACA production by Micrococcus luteus, a stepwise strategy, statistical medium was applied for optimizing the medium composition for the production of CPC acylase. Purified enzyme was found to be of 80 kDa. The optimum pH and temperature for the production of 7-ACA were 7.6 and 340C, respectively. The Km and Vmax were estimated to be 9.43 mg/mL and 7.65 U/mL, respectively.

Fast-growing, non-infectious and intracellularly surviving drug-resistant Mycobacterium aurum: a model for high-throughput antituberculosis drug screening.

OBJECTIVES: Enoyl acyl-carrier-protein reductase (InhA), the primary endogenous target for isoniazid and ethionamide, is crucial to type-II fatty acid biosynthesis (FAS-II). The objectives of this study were first to generate InhA mutants of Mycobacterium aurum, secondly to characterize InhA-mediated isoniazid and ethionamide resistance mechanisms across those mutants and finally to investigate the interaction of InhA with enzymes in the FAS-II pathway in M. aurum. METHODS: Spontaneous mutants were generated by isoniazid overdose and limited broth dilution, while for genetically modified mutants sense-antisense DNA technology was used. Southern hybridization and immunoprecipitation were both used to identify the InhA homologue in M. aurum. The latter method was further used to compare the level of InhA expression in M. aurum with that in corresponding mutants. Isoniazid/ethionamide susceptibility modulation was examined in vitro and ex vivo using a resazurin assay as well as by cfu counting. In addition, circular dichroism and the bacterial two-hybrid system were exploited to investigate the interaction of InhA with other enzymes of the FAS-II pathway. RESULTS: A Mycobacterium tuberculosis InhA homologue was detected in M. aurum. Susceptibility to isoniazid/ethionamide was significantly altered in genetically modified mutants and simultaneously InhA was overexpressed in both spontaneous and genetically modified mutants. InhA interacts with other FAS-II enzymes of M. aurum in vivo. CONCLUSION: Close resemblance of isoniazid/ethionamide action on InhA between M. tuberculosis and M. aurum further supports the use of fast-growing and intracellularly surviving drug-resistant M. aurum to substitute for highly virulent, extremely slow-growing M. tuberculosis strains in the early stage of antituberculosis inhibitor screening.

Oligodynamic Boons of Daptomycin and Noble Metal Nanoparticles Packaged in an Anti-MRSA Topical Gel Formulation.

BACKGROUND: Daptomycin is a popular anti-MRSA antibiotic, especially for surgical wound infections. The side-effects of Daptomycin dosage through intravenous administration have prompted the experimental use of topical Daptomycin. Also, combinatorial drug therapy involving noble metal nanoparticles and conventional antibiotics have proved beneficial in the past. The synergistic oligodynamic effect of Daptomycin with nanoparticles for topical application was attempted for the first time in this work. OBJECTIVES: The present study was focused on topical gel formulation containing Daptomycin combined with mycogenic gold, silver and bimetallic gold and silver nanoparticles and evaluation of their synergistic antibacterial effect against an MRSA strain. METHODS: An efficient approach for fungal growth was discussed wherein the biomass was cultivated under non-limiting conditions, followed by the addition of gold salt, silver salt and bimetallic (Gold and silver) solution. The metal salt reduction efficacy was evaluated using Cyclic Voltammetry. Formation of nanoparticles was observed by visual color changes and confirmed by UV-visible characteristic peaks. The mycosynthesized metallic and bimetallic nanoparticles were characterized by various advanced analytical methods. Further, Daptomycin was combined with nanoparticles in a topical gel formulation. The properties of the topical gels were evaluated and their antimicrobial activity was investigated against an MRSA strain associated with burn infections though disc diffusion method. RESULTS: Formation of nanoparticles was observed by visual color changes and confirmed by UVvisible characteristic peaks. XRD spectra revealed the crystalline nature of nanoparticles whereas TEM confirmed the presence of spherical nanoparticles. The bio fabricated nanoparticles were characterized using ICP-MS, XRD and TEM. The UV-Visible spectrum of the gold, silver, bimetallic nanoparticles showed a characteristic peak at 550 nm, 450 nm, and 480 nm, respectively. ICP-MS of the residual salt concentration depicted more than 75% bioconversion of metal salt to metal nanoparticles. TEM showed the formation of uniform, spherical monometallic nanoparticles. XRD results were in sync with the dynamic light scattering experiments which determined that the gold, silver, bimetallic nanoparticles ranged between 10-20 nm, 5-30 nm, and 20-40 nm respectively and were crystalline in nature with the face centered cubic symmetry. Topical gels combining Daptomycin and nanoparticles were formulated and characterized. The in-vitro drug release studies indicated controlled release of antibiotic from bimetallic nanoparticles and Daptomycin combination in topical gel formulation. The MIC values reduced for the combinatorial drug and the average synergistic antimicrobial effect was 37% and the increase in efficacy of Daptomycin due to the synergistic effect with bimetallic nanoparticles was 43%. CONCLUSION: Topical gels were formulated using the biologically synthesized gold, silver and bimetallic gold-silver nanoparticles and modern-day antibiotic Daptomycin to combat burn infections. The topical gel formulations showed enhanced antimicrobial activity against methicillin-resistant Staphylococcus aureus at lower MIC values as compared to individual nanoparticle or antibiotic. The best results were obtained with bimetallic nanoparticles in topical gel formulation as it assisted in controlled drug release up to 94.6% and improved antimicrobial effect i.e. 43%.

Improved production of Daptomycin in an airlift bioreactor by morphologically modified and immobilized cells of Streptomyces roseosporus.

The increased threat of drug resistance has challenged the existence of several conventional and non-conventional antibiotics in the recent times. Daptomycin is a novel cyclic-lipopeptide antibiotic produced by Streptomyces roseosporus that has progressed as a significant anti-MRSA (methicillin-resistant Staphylococcus aureus) antibiotic. But, the economic practicality of this highly valued secondary metabolite is deterred by its poor production and tedious processing methodology. The present study aims at strategic improvement of Daptomycin production through morphological variations of S. roseosporus cells. Free cells, pelletized cells and immobilized cells on ultra porous refractory brick and silk sachets were investigated for the production of Daptomycin in a lab-scale 2.0 l air-lift bioreactor. The effect(s) of nitrogen source, inoculum size and oxygen stress were analyzed for pellet formation of S. roseosporus. Interestingly, free cells produced 750 mg/l of Daptomycin in a single batch. But, the three phase broth viscosity increased due to vigorous growth of free cells which hampered the oxygen transfer rate. The cell degeneration over the time deterred pellet reusability. 1430 mg/l Daptomycin was produced using pellets for 2 batches. On the contrary, mechanical stability, buoyancy and reusability of refractory bricks and silk sachets were beneficial. Daptomycin production was recorded for 6-8 batches. Immobilized cells on refractory bricks and silk sachets led to 4895 mg/l and 3623 mg/l Daptomycin production respectively. Cell immobilization improved the three phase broth rheology and hence, the hydrodynamics within the reactor. Therefore, whole-cell immobilization could contribute to the ameliorated production of this life-saving drug.

Mycofabrication of gold nanoparticles and evaluation of their antioxidant activities.

Gold nanoparticles have found prominence in pharmaceutical applications due to their unique physical properties as well as their inert nature. Mycosynthesis of noble metal nanoparticles is less stringent and eco-friendly. In this paper, we have reported the economically-viable synthesis of gold nanoparticles, mediated by five different fungal strains Aspergillus flavus NCIM650, Phoma exigua NCIM1237, Aspergillus niger NCIM 616, Aspergillus niger NCIM 1025 and Trichoderma reesei NCIM 1186. An efficient approach for fungal growth was discussed wherein the biomass was cultivated in non-limiting conditions, followed by addition of gold salt solution. Cyclic Voltammetry studies were conducted to show the varying reducing capacities of these strains. The surface plasmon peaks for gold nanoparticles produced by Aspergillus flavus NCIM650, Phoma exigua NCIM1237, Aspergillus niger NCIM 616, Aspergillus niger NCIM 1025 and Trichoderma reesei NCIM 1186 were recorded as 536nm, 543nm, 542nm, 560nm, 537nm respectively. Based on the cyclic voltammetry studies and UV-Visible spectroscopy, transmission electron microscopy (TEM) analysis was done. Among the five strains, gold nanoparticles fabricated by Aspergillus niger NCIM 616 gave quite promising results. The antioxidant activities were evaluated using DPPH quenching assay and hydrogen peroxide assay.

Development of probiotic candidate in combination with essential oils from medicinal plant and their effect on enteric pathogens: a review.

Medicinal plants and probiotics both have very high potential in terms of their antimicrobial activity against antibiotic-resistant enteric pathogens. The probiotics being enteric microorganism do not have any parasitic effect on human beings. They have been an integral part of daily food for centuries. They have been shown to have health beneficiary properties. The probiotics retard the growth of the microorganisms, while essential oil kills them. Combining the effect of medicinal plant extract and probiotics may be a new approach due to their complementary antimicrobial effects and practically no side effects. The synergistic effect of the essential oil and probiotics will be necessarily higher than using them alone as health product.

Middle ear of human and pig: a comparison of structures and mechanics.

INTRODUCTION: Animal studies are an essential method to evaluate implants for the middle ear prior to clinical studies. New Zealand rabbits, guinea pigs and chinchillas are, among other small mammals, well established animal models, but their auditory system is significantly smaller compared to human. Hence, the suitability of the domestic pig (sus scrofa domesticus) as a new animal model for research on the middle ear (ME), that would match the human in size, was investigated. METHODS: Thirty halved pig heads were obtained from the butcher and each middle ear was dissected. Using a digital light microscope, several anatomical magnitudes were determined for 24 specimens, namely the planar projected area of the tympanic membrane (TM), in relation to the stapes footplate as well as the dimensions and weight of the ossicles, in order to determine the effective lever ratios. Using normal and micro computed tomography (CT), six porcine temporal bones were scanned and the geometric data obtained were transferred into a finite element model (FEM) simulation of the porcine middle ear. The transfer function was determined and compared to those from humans determined by measurements and simulations, respectively. RESULTS: The anatomy investigated presented itself as highly comparable to that of the human. Differing from literature, no fourth ossicle could be found. The porcine dimensions and lever ratios determined match the humans far better than those of all established animal models. The obtained transfer function was congruent to the human one. This underlines the suitability of the pig as an animal model for middle ear implants.

Trace analysis of cefotaxime at carbon paste electrode modified with novel Schiff base Zn(II) complex.

Cefotaxime a third generation cephalosporin drug estimation in nanomolar concentration range is demonstrated for the first time in aqueous and human blood samples using novel Schiff base octahedral Zn(II) complex. The cefotaxime electrochemistry is studied over graphite paste and Zn(II) complex modified graphite paste capillary electrodes in H(2)SO(4) (pH 2.3) using cyclic voltammetry and differential pulse voltammetry. Cefotaxime enrichment is observed over Zn(II) complex modified graphite paste electrode probably due to interaction of functional groups of cefotaxime with Zn(II) complex. Possible interactions between metal complex and cefotaxime drug is examined by UV-vis and electrochemical quartz crystal microbalance (EQCM) techniques and further supported by voltammetric analysis. Differential pulse voltammetry (DPV) with modified electrode is applied for the determination of cefotaxime in acidified aqueous and blood samples. Cefotaxime estimation is successfully demonstrated in the range of 1-500 nM for aqueous samples and 0.1-100 microM in human blood samples. Reproducibility, accuracy and repeatability of the method are checked by triplicate reading for large number of samples. The variation in the measurements is obtained less than 10% without any interference of electrolyte or blood constituents.

Cephalosporins determination with a novel microbial biosensor based on permeabilized Pseudomonas aeruginosa whole cells.

A new potentiometric microbial biosensor based on Pseudomonas aeruginosa was developed in this study for detecting the cephalosporin group of antibiotics. Preliminary results with the biosensor indicated that P. aeruginosa cells, when treated with lysozyme, showed more efficiency in detecting cephalosporin C in a wide concentration range of 0.1-11 mM with high sensitivity compared to the normal cells. Optimization of the three important biosensor design parameters permeabilized cell contents, quantities of gelatin, and glutaraldehyde resulted in high performance of the biosensor. The optimized values of the above parameters were cell contents 2.5 mg/cm(2), gelatin 8.5 mg/cm(2), and 0.25% glutaraldehyde. The assay conditions, namely phosphate buffer pH, ionic strength, and temperature, were optimized for best performance of the biosensor. The specificity test of the biosensor towards detecting different beta-lactam antibiotics showed good response only for the cephalosporins. The operational and storage stability in detecting cephalosporin C indicated very good potential of the biosensor in detecting cephalosporins with high accuracy.

Microbial production of 7-amino-cephalosporanic acid and new generation cephalosporins (cephalothin) by different processing strategies.

The development of beta-lactam antibiotics has been a continuous battle of the design of new compounds to withstand inactivation by the ever-increasing diversity of beta-lactamases. Semisynthetic cephalosporins like cephalothin were synthesized from 7-amino-cephalosporanic acid (7-ACA), and thiophene-2-acetic acid using cephalosporin-C acylase enzyme was studied. The production of cephalosporin-C acylase by Pseudomonas diminuta was used and the growth kinetics studied. The optimum condition of enzyme activity was determined by using response surface methodology. A 2(3) full-factorial composite design was employed for experimental design and the result analyzed. The pH value and temperature for optimum activity were 6.5 and 32 degrees C, respectively. The structural analog compound similar to the side-chain of semisynthetic cephalosporins, e.g., thiophene-2-acetic acid, was added. HPLC data analysis indicate that the concentration of cephalothin was 1.6 mg/mL.

Continuous production of cephalosporin-C by immobilized microbial cells using symbiotic mode in a packed bed bioreactor.

Cephalosporins are usually produced semisynthetically from Cephalosporin-C, which is exclusively produced by Cephalosporium acremonium. Free cell studies for the production of Cephalosporin-C had some limitation such as pulpy growth of fungus causing an appreciable rise in the broth viscosity affecting the transfer of oxygen and other nutrients into the cells. High cell concentrations cannot be maintained because of wash out phenomenon at high dilution rates. The whole cell immobilization technique is a potentially important process for Cephalosporin-C biosynthesis, where increase cell densities were maintained and broth-handling problems were reduced. Cephalosporin-C fermentation is a highly aerobic process. The symbiotic relationship of Cephalosporium acremonium and Chlorella pyrenoidosa has been used to increase oxygen transfer rate to the fungi by co-immobilizing it with algae. Co-immobilization of whole cells of fungus and algae were carried out in different immobilizing agents and the systems were coated with polyacrylamide resin of pharmaceutical grade to overcome the problems of leakage. The operational stability of immobilized systems in a packed bed reactor was also studied.

Influence of medium constituents on the biosynthesis of cephalosporin-C

Acremonium chrysogenum NCIM 1069 was used for the biosynthesis of cephalosporin-C (CPC) in batch mode of cultivation. The effect of different medium constituents for better yield of CPC was thoroughly investigated. From the results of the fermentation, it was found that ammonium sulphate as inorganic nitrogen source and methionine at the concentration of 0.4 percent are most suitable for higher yield of antibiotic. The variation in the C/N ratio on the biosynthesis of CPC showed that a C/N ratio of 8.0 is most suitable for maximum production of CPC