Screening of 40 Plant extracts for antimicrobial activity against Mycobacterium tuberculosis

Richin John Koshy

Supervisor: Maria Lerm

Overview

• Background

•Hypothesis/Aim

• Materials and methods

• Results

• Conclusion

Epidemiology of MDR (TB)

• http://wwwnc.cdc.gov/travel/yellowbook/2010/

http://wwwnc.cdc.gov/travel/yellowbook/2010/chapter-5/tuberculosis.htm

Morphology

• Mycobacteria are small rod shaped aerobic bacilli

• Mycobacteria are present in the environment, soil and water

• Mycobacteria have high concentration of lipids and wax in the cell wall making them resistant to standard staining techniques

• Mtb grow quite slowly and colonies do not appear on solid media up to 2.5 -5 week

• Media which are used in growth of mycobacteria are selective media, semisynthetic media and broth media

www.lookfordiagnosis.com

Pulmonary TB

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niaid.nih.gov

Treatment of TB

Drug Development

• Screening of Chinese herbal medicine led to introduction of artemisinin from the leaves of Artemisia annua which is used worldwide for treatment against malaria

• At present, there are eight to ten drugs with therapies lasting to 18-24 months of which there are only four drugs which are actually developed to treat TB

• Shifting the screening strategy from a single enzyme targets to a whole bacterial cell level have been showing successful results as it has a holistic approach and only gives hits that can enter the bacterium through the cell wall

Hypothesis

• Whole cell screening method is a more effective way than molecular/chemical approaches to identify new, anti-mycobacterial drug candidates/lead compounds

• Traditionally used plants are a valuable source for the discovery of modern drugs

Aim

To screen 40 plant extracts prepared from plants used traditionally to treat tuberculosis and other infections in Sudan for possible antimicrobial activity against Mtb

Materials and Methods

Mycobacteria culture in medium for 2-3 weeks followed by

inoculation in fresh broth for 1 week

Preparation of plant extracts along

with DMSO to ranges of

treatments

Analysis of bacterial growth by

luminometry with treatment of plant

extracts and controls

Confirming inhibition of growth as observed in luminometry by CFU

plating to rule out inhibition by luciferase

pH Assay

Confirming screening of plant extracts showing inhibition

Student paired t test was done assuming Gaussian distribution which show statistically insignificant hits

Detecting changes in mycobacterial growth by CFU plating

Determination of pH

A 22 6.3-6.4

A 27 6.3-6.4

A 28 6.3-6.4

A 29 6.3-6.4

A 30 6.3-6.4

Conclusions

• High-throughput screening using luciferase determines mycobacterial growth giving conclusive results

• 40 extracts of selected medicinal plants were analysed for antimycobacterial activities which yielded 5 extracts to have distinct antimycobacterial properties

• Further studies were carried out in order to measure colonies in the presence of selected medicinal extracts showing inhibition of bacterial growth, thereby ruling out inhibition due to luciferase

• Inhibition of growth by these extracts was observed in both systems giving inference about their antituberculous activity which appears to be meaningful

Acknowledgement

• First I will like to give my greatest appreciation to my supervisor Maria Lerm for her direction, encouragement and support throughout the period of my project. I am happy that I had the opportunity to work under her supervision and ever thankful for all that she taught me

• I also express my gratitude to Johanna Raffetseder, Marie Larsson, ElsjePienaar and Daniel Eklund for introducing me to most of the methods iused in this project and more and also for always being there to answer numerous questions I had.

• Finally but not the least, I will like to say a big thanks to all the staff of the department

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