Microbiological profiles in clinical and subclinical cases of mastitis in milking Jafarabadi buffalo.

Mastitis is one of the important diseases affecting the dairy industry across the globe. Identification of bacterial pathogens associated with mastitis becomes essential in order to understand the etiology of disease which in turn will help to new strategies to control it. Microbial diversity analysis using pyrosequencing is widely studied for mastitis pathogens in dairy cows. However it is unexplored in case of buffalo. In the present study 16SrDNA gene pyrosequencing was used to characterize microbiota associated with clinical and subclinical mastitis in 28 Jafarabadi buffalo. The obtained sequencing data were analyzed by Quantitative Insights into Microbial Ecology (QIIME) and statistical analysis was done using Paleontological Statistics (PAST). Pyrosequencing produced 47.3 million base pairs reads. Phylogenetic profiles using ribosomal database revealed differences in abundance of Staphylococcus (25.95%, 10.09% and 0.03%), Enterococcus (10.80%, 8.72% and 0.36%), Escherichia (8.88%, 0.38% and 0.00%), Streptococcus (3.97%, 0.42% and 0.00%), Lactococcus (3.73%, 23.96% and 0.01%), and Ralstonia (0.54%, 12.72% and 0.00%), genera in clinical, subclinical and healthy samples, respectively. Different microbial profiles in clinical and subclinical mastitis in buffalo suggest the composition of bacteria in the milk is more diverse and complex hence single therapeutic regimes cannot be applied.

Role of ligninolytic enzymes of white rot fungi (Pleurotus spp.) grown with azo dyes.

BACKGROUND: Total three Pleurotus species (P. ostreatus, P. sapidus, P. florida) was compared for ligninolytic enzyme production grown with Coralene Golden Yellow, Coralene Navy Blue and Coralene Dark Red azo dyes in liquid medium under shaking condition. RESULTS: The biodegradation competency varied from species to species and it was found that P. ostreatus, P. sapidus and P. florida to 20 ppm dye concentration shows 88, 92 and 98 % decolorization, respectively for all three dyes. Production pattern of laccase, manganese dependent peroxidase and lignin peroxidase were studied during the growth of the organisms for 10 days. Laccase was found to be the major extracellular ligninolytic enzyme produced by fungus with negligible detection of lignin peroxidases. In all concentration of three dye studied, maximum laccase activity was observed on day 8, for 20 mg/l of dye laccase specific activity was 1-1.58 U/mg in P. ostreatus, 0.5-0.78 U/mg in P. sapidus and 1-1.92 U/mg in P. florida. Different factors (dye concentration, pH, protein and sugar estimation) influencing the ability of Pleurotus species to degrade dyes is documented and degradation was attributed to microbial action irrespective of pH change. HPTLC analysis of samples indicated degradation of dyes into intermediate products. CONCLUSION: Level of ligninolytic enzymes is playing a major role in degradation of dye, which is dependent on time of incubation and species of fungi.

Mitigation of acrylamide by l-asparaginase from Bacillus subtilis KDPS1 and analysis of degradation products by HPLC and HPTLC.

The use of bacterial l-asparaginase (LA) is one of the alternative approaches for acrylamide reduction in food stuffs as it catalyzes the conversion of l-asparagine to l-aspartic acid and ammonia. In present investigation, purification of extracellular LA from isolate of Bacillus subtilis sp. strain KDPS-1 was carried out by solid state fermentation process. The effects of solid substrates, initial moisture content, moistening agents, temperature, and incubation time on LA production was studied, and the highest asparaginase activity (47 IU/ml) was achieved in the medium having orange peel as substrate. The enzyme was purified to homogeneity by diethylaminoethyl (DEAE) cellulose ion exchange chromatography; with 84.89 % yield and 12.11 fold purity. LA showed stimulant activity against ß-mercaptoethanol and was greatly inhibited by Zn(2+) and Hg(2+) metal ions. Reduction of acrylamide in fried potatoes was detected by high performance liquid chromatography, which showed clear degradation of acrylamide by height and area (%) in the chromatograms of standard sample to that of the test sample. Hydrolysates analysis by high performance thin layer chromatography confirmed the test sample to be LA.

A novel alkaline keratinase from Bacillus subtilis DP1 with potential utility in cosmetic formulation.

The Bacillus subtilis DP1 was isolated from poultry farm soil at Anand district, India. The highest enzyme production (379.65U/ml) was obtained at pH 10.0, a temperature of 37°C and a growth period of 72h. The extracellular keratinase was purified by gel filtration chromatography with 27.98 purification fold. Purity was also confirmed by High-Performance Liquid Chromatography (HPLC) analysis, where a major peak having retention time of 2.5min was obtained on C18 column using photo diode array detector. Purified keratinase was stable in a broad range of pH (8-12) and temperature (20-50°C) with optimum at pH 10.0 and 37°C. The metallic ions, Ca(2+) and Mg(2+) enhance keratinase activity. Secondary structure from Circular Dichroism (CD) spectra implies that purified keratinase is largely ß-pleated sheet rich protein. For preparation of dehairing cream formulation, compatibility studies of excipients were carried out. Fourier transform infrared spectroscopy (FTIR) spectra of sodium stearate, calcium carbonate and sodium lauryl sulphate shows no reactivity of functional groups and hence mixture was compatible for formulation of keratinase dehairing cream. Prepared biological depilatory was able to remove hair more efficiently compared to marketed formulations.

Functional expression and purification of Anabaena PCC 7120 XisA protein.

Anabaena PCC 7120 xisA gene product mediates the site-specific excision of 11,278 bp nifD element in heterocysts formed under nitrogen starvation conditions. Although XisA protein possesses both site-specific recombinase and endonuclease activities, till date neither xisA transcript nor XisA protein has been detected. Gene encoding XisA protein was isolated from plasmid pMX25 and overexpressed in Escherichia coli BL21 DE3 yielding 7.7 mg enzyme per L of growth culture in soluble fraction. His-tagged XisA was purified using Ni-NTA affinity chromatography with 95% recovery. The purified XisA showed a single band on SDS-PAGE with molecular mass of 52 kDa. Identity of XisA was confirmed by MALDI-TOF analysis and functionality of enzyme was confirmed using restriction digestion. A PCR based method was developed to monitor excision by XisA, which displayed near 100% activity in E. coli within 1 h at 37 (°)C on LB under static condition.

Medicinal and antimicrobial role of the oyster culinary-medicinal mushroom Pleurotus ostreatus (higher Basidiomycetes) cultivated on banana agrowastes in India.

Oyster mushrooms, species of the genus Pleurotus, are recognized for producing secondary metabolites with important medicinal properties. Investigations were carried out to evaluate the antioxidative and antimicrobial properties of the edible mushroom Pleurotus ostreatus (MTCC142) extracts cultivated on banana agrowastes. Ethanolic extracts showed antimicrobial activities against gram-positive and gram-negative bacteria, and their in vitro antifungal activities against all fungi tested revealed a promising role. Qualitative phytochemical analysis of Pleurotus grown on yeast dextrose broth and banana agrowaste confirmed the presence of steroids, cardiac glycosides, terpenoids, and alkaloids, whereas ethanolic extract after 40 days exhibited a phenol concentration of 521.67 µg/mL in banana waste compared to 155 µg/mL in yeast dextrose broth. The minimum inhibitory concentration of ethanolic extracts ranged from 19.74 to 56.84 mg/mL and 35.53 to 102.31 mg/mL in solid-state and submerged grown mycelium extracts, respectively, after 40 days. Moreover, banana agrowaste could be a significant economic source for the production of the oyster mushroom P. ostreatus. The nutritive, medicinal, and antimicrobial properties of P. ostreatus can be used to develop a new nutraceutical formulation; it can also be used as an additive to routine and fast food.

Isolation and partial purification of erythromycin from alkaliphilic Streptomyces werraensis isolated from Rajkot, India.

An alkaliphilic actinomycete, BCI-1, was isolated from soil samples collected from Saurashtra University campus, Gujarat. Isolated strain was identified as Streptomyces werraensis based on morphological, biochemical and phylogenetic analysis. Maximum antibiotic production was obtained in media containing sucrose 2%, Yeast extract 1.5%, and NaCl 2.5% at pH 9.0 for 7 days at 30 °C. Maximum inhibitory compound was produced at pH 9 and at 30 °C. FTIR revealed imine, amine, alkane (C[bond, double bond]C) of aromatic ring and p-di substituted benzene, whereas HPLC analysis of partially purified compound and library search confirmed 95% peaks matches with erythromycin. Chloroform extracted isolated compound showed MIC values 1 µg/ml against Bacillus subtilis, ≤0.5 µg/ml against Staphylococcus aureus, ≤0.5 µg/ml against Escherichia coli and 2.0 µg/ml against Serretia GSD2 sp., which is more effective in comparison to ehtylacetate and methanol extracted compounds. The study holds significance as only few alkaliphilic actinomycetes have been explored for their antimicrobial potential.

Human MECP2 gene at Q28 arm of X chromosome as a suitable target for monitoring PCR inhibition in a nested, multiplexed HIV-1 DNA detection protocol.

Human MECP2 gene located at q28 arm of X chromosome was identified as target for thermal co-amplification with HIV-1 proviral DNA of infected individuals. The selected MECP2 gene-specific primers functioned at a wide range of annealing temperature, extension time and exhibited no significant interaction with pathogen specific primers. A 466 bp PCR amplicon originating from human MECP2 gene was found to be diagnostic for inhibition-free PCR reaction when co-amplified with the HIV-1 target gene in a multiplexed, nested PCR reaction. The 5' end of the MECP2 primers were engineered to position an EcoRI restriction endonuclease site to facilitate rapid cloning in various DNA vector molecules at the corresponding EcoRI sites. Cell mass of Escherichia coli (XL1Blue) harboring the recombinant plasmid when added to pleural fluid of HIV-1 infected individuals co-infected with Mycobacterium tuberculosis, generated the diagnostic 466 bp MECP2 PCR amplicon as well as the 194 bp PCR amplicon of target gene from M. tuberculosis. The experiment underlined potential of the region spanning nucleotide position 4118099 to 4118552 of human MECP2 gene (NCBI accession number NT_011726.13) as a reliable target for multiplex PCR to accommodate a wide range of thermal cycling and multiplex reaction conditions. In both cases of this study, electrophoresis-based separation of the 466 bp MECP2 fragment and the 232 bp and 194 bp HIV-1 and M. tuberculosis fragments respectively was distinct and unambiguous. The potential of this human MECP2 gene available from human genome or recombinant plasmid as a potent target to monitor PCR inhibition for a range of different PCR reactions is discussed.

Excision of Anabaena PCC 7120 nifD element in Escherichia coli: Growth kinetics and RecA regulated xisA expression and DNA rearrangement.

Anabaena PCC 7120 nifHDK operon is interrupted by an 11 kb DNA element which is excised during the development of heterocysts by Excisase A, encoded by the xisA gene residing on the element. The excision is a site-specific recombination event that occurs at the 11 base pair direct repeats flanking the element. Earlier work showed the excision of the 11 kb element in Escherichia coli at a frequency 0.3%. We report here the excision of this element at 1.1% and 1.98% in E. coli DH5alpha, and 1.9% and 10.9% in E. coli JM 101 when grown on Luria broth and minimal media, respectively. Excision of nifD element in isogenic recA(-) (RK1) and recA+ (RK2) E. coli JM101 P1 transductants, showed similar results to that of E. coli JM101 and DH5alpha, respectively. A plasmid pMX32, carrying a xisA defective 11kb element, showed no excision in E. coli RK2 strain. In contrast to Anabaena PCC 7120, excision of nifD element did not increase in E. coli DH5alpha grown in iron-deficient conditions. A PxisA::lacZ transcriptional fusion, used to detect the expression of elusive xisA gene, showed maximal beta-galactosidase activity in the stationary phase. The results suggest that the excision event in E. coli may involve additional factors, such as RecA and that the physiological status can influence the excision of nifD element.