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gothic wear shop Pongamia pinnata and relation to the virulence factors of multi

Antimicrobial activity of Aerva lanata, Boerhavia diffusa, Cassia tora, Pongamia pinnata and relation to the virulence factors of multi-drug resistant Escherichia coli and Pseudomonas aeruginosa

Article Summary: The studies was targeted at testing the efficacy of methanolic extract of medicinal plants for their antimicrobial activity and analyze the influence of plant extract on the virulent factors on microorganisms. The plants that were taken for study include Aerva lanata, Boerhavia diffusa, Cassia tora and Pongamia pinnata. P. pinnata showed better antimicrobial activity in comparison with others based on the area of inhibition. Studies were carried out to investigate the plasmid curing ability of the extract of P. pinnata. The extract was good at curing plasmids of both the strains taken for st

In spite of the supply of effective drugs, the battle against infectious diseases is certainly not over Clark, 1996. Furthermore the cause a lot of infections and deaths, specifically in the developing countries WHO, 1993, but the emergence and spread of antimicrobial resistance is now threatening to undermine our ability to treat infections and save lives Okeke et al., 2005.

Vegetation is uses as medicine for hundreds of years. India is really a rich supply of medicinal plants. Theyre widely used in ancient systems of drugs. Its reported that, 2 or 3 antibiotics that are launched each year are based on microorganisms Cowan, 1999. After a downturn for the reason that pace in recent decades, the pace is again quickening as scientists realize the effective lifespan of any antibiotic is restricted Schultes, 1978. New medicines have to be discovered with traditional, empirical and molecular approaches. Plants have limited less ability to synthesize secondary metabolites of which 12000 have been isolated, several estimated to become less than 10% from the total Harborne, 1998.

The research was targeted at testing the efficacy of methanolic extract of medicinal plants for their antimicrobial activity and focus the influence of plant extract about the virulent factors on microorganisms. The plants were selected based on the traditional knowledge that has accumulated previously. The antimicrobial activity was tested against bacterial pathogens that were resistant to various antibiotics Warrier et al., 1997.

Organisms and growth conditions

Microorganisms were from culture collections of Institute of biological sciences, Chennai. The organisms which were taken for study were Proteus mirabilis, Proteus vulgaris, Escherichia coli, Klebsiella pnemoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhi, Salmonella paratyphi A and B, and Bacillus subtilis. The fungal strains taken for study were Candidiasis and Candida tropicalis. Organisms were maintained in Muller Hinton agar. Overnight cultures were prepared by inoculating approximately 2 ml MHB with 2-3 colonies of each organism. Broths were incubated overnight at 37and#61616;C. The leaves were washed, shade dried and grinded. The dry powder of leaves 0.5 kg was initially

soaked at room temperature in methanol every day and night. The extract was filtered using Whatmann No 1 filter paper. It was repeated for two more days and other alike extract were cooled together and condensed at 40and#61616;C under reduced pressure using Buchi R-153 Rota vapour Vukovic et al., 2007.

This method was adopted to test the efficacy of numerous antibiotics against the organisms taken for study. Muller Hinton agar was prepared based on the manufacturers instructions and was poured on plates at a uniform thickness of four mm Pfaller et al., 2004. Using a cotton swab, an excellent lawn of cells was made at first glance from the agar. The discs containing the antibiotics were arranged on the surface from the inoculated plates. The plates were incubated at 37and#61616;C Gaudreau and Gilbert, 1997.

Muller Hinton agar was prepared based on the manufacturers instructions and was poured on plates in a uniform thickness of 4 mm. utilizing a cotton wool ball, a fine lawn of cells is made at first glance of the agar. A sterile cork borer was used to chop wells on 6 mm in diameter. To every well test solutions of measured amount of extract of various concentrations were added aseptically. The plates were incubated at 37and#61616;C and inhibition zones were measured Holder and Boyce, 1994. The percentage of inhibition was calculated as

Percentage of inhibition =___I____ and#61620; 100

To obtain the MIC of extract for the organisms, agar dilution method proposed by NCCLS was adopted. Twelve different concentrations of methanolic extract supplemented with the medium were prepared beginning with a preliminary concentration of 0.5-1024 g/ml of media. The organisms were streaked on agar surface and their growth was recorded. In line with the observations, the MIC was calculated Hanson and Martin, 1978.

Isolation of plasmid DNA and outer membrane protein

The antiplasmid effects of methanolic extract of P. pinnata was studied on strains of P. aeruginosa and E. coli containing plasmids. It was analyzed by supplementing the extract towards the medium at a power of 3 g/ml. The isolated plasmid was run on agarose gel electrophoresis and also the outcome was documented.

Studies were completed to analyze the effect of methanolic extract of P. pinnata on the expression of OMPs. The OMPs isolated were operate on SDS-PAGE and also the outcome was documented Hannah et al., 1992.

Estimation of superoxide dismutase and catalase

Experiments were carried out to analyze the influence of methanolic extract of P. pinnata on Superoxide dismutase and catalase activity of multidrug resistant P. aeruginosa and E. coli Misra and Fridovich, 1972; Beer and Siezer, 1974. The spectrophotometric readings were noted and the quantity of Superoxide dismutase and catalase was expressed in IU/mg of protein.

The antimicrobial susceptibility of numerous human pathogenic microorganisms against various classes of antibiotics includes Gentamycin G, Amikacin Ak, Erythromycin E, Amoxicillin Am, Tetracycline T, Chloramphenicol C, Cefpodoxime Cep, Cotrimazole Cc, Ciprofloxacin Cf, Vancomycin Va, Imipenum I. The results reveal that, the organisms which were taken for study were resistant against various classes of antibiotics Table 1; Fig. 1.

The methanolic extract was used after complete drying to ensure that any traces of solvent that is present may be removed. The methanolic extract was dissolved in calculated quantity of DMSO to organize three different concentrations 1 g of extract/l of DMSO, 2 g of extract/l of DMSO, 3 g of extract/l of DMSO. 50 l of the extract was put into each well Table 2; Fig. 2.

The minimal inhibitory power of the extract towards various organisms, the agar dilution process of calculating MIC/MLC was performed. Twelve different concentrations of methanolic extract supplemented with the medium of power of 0.5 g of extract/ml approx. of media to 1024 g of extract/ml of media proves the variations one of the 12 organisms were streaked on all of the 12 plates prepared with various dilutions. The development of the organism Table 3. Antimicrobial activity of A. Lanata, B. diffusa, C. tora, P. Pinnata were experimented and compared Table 4. In line with the observations, the MIC and MLC of extract of P. pinnata was calculated and tabulated Table 5.

Few changes in OMP profiles were observed using the strains which were grown with the extracts. E. coli and P. aeruginosa, there is complete inhibition within the expression of some membrane proteins Fig. 3. Studies were carried out to judge SOD and catalase activity in P. aeruginosa and E. coli under stress created by the extract of P. pinnata. The results demonstrated that the stress that was created by the methanolic extract of P. P. aeruginosa and E. coli.

The antiplasmid effects of methanolic extract of P. pinnata was studied on strains of P. aeruginosa and E. coli containing plasmids. Development of the strains containing plasmids was induced by adding antibiotic to the medium. Plasmid profiles of P. aeruginosa and E. coli shows that both strains were highly resistant. It was observed that, all the strains which were isolated from P. aeruginosa were above 23 kb in dimensions. The extract was effective in curing plasmids of both the strains taken for study at a power of 3 g/l. The mechanism of plasmid curing of methanolic extract of P. pinnata is unknown Fig. 4.

Bacillus subtilis, Salmonella typhi, Salmonella paratyphi A and B were highly susceptible to the amoxicillin. Klebsiella pnemoniae and Proteus vulgaris were resistant to Gentamycin. Bacillus subtilis, Klebsiella pnemoniae, Salmonella typhi were highly prone to Tetracycline. P. aeruginosa was highly prone to Cotrimazole. Bacillus subtilits and Staphylococcus were the only organisms which were susceptible against vancomycin. All of the organisms which were taken for study were prone to imipenum, the highest grade of antibiotic being used. Variation in resistant against antibiotics were observed in this research.

A. lanata showed considerable activity against Proteus mirabilis 43.33%, Salmonella paratyphi B 41.11%, Bacillus subtilis 37.78% and Candida tropicalis 36.67%. B. diffusa showed good antimicrobial activity against Bacillus subtilis 32.22%, Salmonella paratyphi B 38.89%, Candida tropicalis 36.37 and Proteus mirabilis 34.44%. C. tora did not show any activity against Staphylococcus aureus, P. aeruginosa, E. coli and Candida albicans. Of all the four plants which were taken for study, P. pinnata showed excellent activity against most of the pathogens.

The current study to evaluate the plasmid curing ability of extract of P. pinnata showed great results. The extract was effective in curing plasmids of both strains taken for study in a power of 3 g/l. The mechanism of plasmid curing of methanolic extract of P. pinnata is unknown.

Superoxide dismutase and catalase can contribute to the virulence of numerous human pathogenic bacteria through being able to neutralize toxic amounts of reactive oxygen species generated through the host. The results obtained in the study claim that the extract brings down the level of SOD and catalase in both the organisms and therefore lowers the virulence As a result it might be proposed the organism grown after administration from the extract was less virulent. The results also shows that SOD and catalase levels in P. aeruginosa, and E. coli influences virulence.

All of the four plant extracts tested within the study showed good antimicrobial activity against various bacterial pathogens. The methanolic extract of P. pinnata could bring down the virulence of both the strains taken for study.






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