In vitro regeneration and ploidy level analysis of Eulophia ochreata Lindl.

Various parameters including explant-type, medium compositions, use of phytohormones and additives were optimized for direct and indirect regeneration of E. ochreata, a medicinal orchid under threat. Protocorm-like-bodies (PLBs) proved to be the best explants for shoot initiation, proliferation and callus induction. Murashige and Skoog’s (MS) medium containing 2.5 mg L-1 6-benzylaminopurine (BAP), 1.0 mg L-1 kinetin (Kin) and additives (adenine sulfate, arginine, citric acid, 30 mg L-1 each and 50 mg L-1 ascorbic acid) was optimal for shoot multiplication (12.1 shoots and 7.1 PLBs per explant with synchronized growth), which also produced callus. Shoot number was further increased with three successive subcultures on same media and ~40 shoots per explant were achieved after 3 cycles of 30 days each. Additives and casein hydrolysate (CH) showed advantageous effects on indirect shoot regeneration via protocorm-derived callus. Optimum indirect regeneration was achieved on MS containing additives, 500 mg L-1 CH, 2.5 mg L-1 BAP and 1.0 mg L-1 Kin with 30 PLBs and 6 shoots per callus mass (~5 mm size). The shoots were rooted (70% frequency) on one by fourth-MS medium containing 2.0 mg L-1 indole-3-butyric acid, 200 mg L-1 activated charcoal and additives. The rooted plantlets were hardened and transferred to greenhouse with 63% survival rate. Flow-cytometry based DNA content analysis revealed that the ploidy levels were maintained in in vitro regenerated plants. This is the first report for in vitro plant regeneration in E. ochreata.

Antibiotic resistance reversal of multiple drug resistant bacteria using Piper longum fruit extract.

Present investigation was aimed to identify natural products of plant-origin as novel antibiotic resistance reversal agents. Aqueous and methanol extracts of Piper longum (fruits) were tested against multiple drug resistant (MDR) clinical isolates of Enterococcus faecalis, Staphylococcus aureus, Salmonella typhi, Shigella sonnei, as well as reference-plasmid-harboring strains of Escherichia coli (RP4) and Bacillus subtilis (pUB110). The crude methanol extract showed significant antibacterial activity with a minimal inhibitory concentration of 400 μg/mL against Bacillus subtilis (harboring pUB110 plasmid). Methanol extract could reverse the antibiotic resistance in clinical isolates of Shigella sonnei, with a curing efficiency of 42%. In comparison with methanol extract, aqueous extract showed antibiotic resistance reversal efficiency against wider range of clinical isolates. Aqueous extract showed strong antibiotic resistance reversal activities against R-plasmid harboring strains of clinical origin- Enterococcus faecalis, Staphylococcus aureus, Salmonella typhi with curing efficiencies of 64%, 50% and 32% respectively. This antibiotic resistance reversal may be attributed to the elimination of R-plasmids as the multiple antibiotic resistance genes are usually located on R-plasmids. Active biomolecules from P. longum may prove to be a source to develop MDR reversal agents of natural origin to contain the development and spread of plasmid borne multiple antibiotic resistance.

Antibacterial & antiplasmid activities of Helicteres isora L.

Background & objectives: The multiple drug resistance (MDR) is a serious health problem and major challenge to the global drug discovery programmes. Most of the genetic determinants that confer resistance to antibiotics are located on R-plasmids in bacteria. The present investigation was undertaken to investigate the ability of organic extract of the fruits of Helicteres isora to cure R-plasmids from certain clinical isolates. Methods: Active fractions demonstrating antibacterial and antiplasmid activities were isolated from the acetone extracts of shade dried fruits of H. isora by bioassay guided fractionation. Minimal inhibitory concentration (MIC) of antibiotics and organic extracts was determined by agar dilution method. Plasmid curing activity of organic fractions was determined by evaluating the ability of bacterial colonies (pre treated with organic fraction for 18 h) to grow in the presence of antibiotics. The physical loss of plasmid DNA in the cured derivatives was further confirmed by agarose gel electrophoresis. Results: The active fraction did not inhibit the growth of either the clinical isolates or the strains harbouring reference plasmids even at a concentration of 400 μg/ml. However, the same fraction could cure plasmids from Enterococcus faecalis, Escherichia coli, Bacillus cereus and E. coli (RP4) at curing efficiencies of 14, 26, 22 and 2 per cent respectively. The active fraction mediated plasmid curing resulted in the subsequent loss of antibiotic resistance encoded in the plasmids as revealed by antibiotic resistance profile of cured strains. The physical loss of plasmid was also confirmed by agarose gel electrophoresis. Interpretation & conclusions: The active fraction of acetone extract of H. isora fruits cured R-plasmids from Gram-positive and Gram-negative clinical isolates as well as reference strains. Such plasmid loss reversed the multiple antibiotic resistance in cured derivatives making them sensitive to low concentrations of antibiotics. Acetone fractions of H. isora may be a source to develop antiplasmid agents of natural origin to contain the development and spread of plasmid borne multiple antibiotic resistance.