Isolation and characterization of Anti-mycobacterials from Actinomycetes

R. Ajay Kumar & Sabu Thomas

Rajiv Gandhi Centre for Biotechnology Trivandrum 695014

OSDD/HCP0001/12FYP/2012-123/Fin/2412

Objectives of the proposal:

• Isolation of actinomycetes from different ecosystems.

• Screening of CF against M.tuberculosis H37Rv

• Purification of inhibitory principles from CF.

• Characterization and identification of potent molecules.

• Identification of potent actinomycetes.

• Creation of a Repository of actinomycetes.

OSDD/HCP0001/12FYP/2012-123/Fin/2412

• Date of sanctioning : January 22, 2013• Duration : 3 years

• Total amount sanctioned : 31,80,000/-• Amount sanctioned for the first year : 22,10,400/-• Amount sanctioned for equipment : 16,75,000/-

• Name of the JRF : Balaji M• Date of joining of the JRF : April 15, 2013

Fermentor (Eppendorf)

Orbital shaker (Eppendorf)

-20°C freezer (Vestfrost)

Equipment yet to arrive :

37°C incubator (KEMI)

Equipment procured :

Sample collection

SitesSirumalaiKarandhamalaiBerizamManakudyThoothukudyPonmudiKovalamManalpuramKallaar

Depth: ~10cm ~50gms soilSterile plastic bags

1 gm serially diluted > Starch Casein Nitrate Agar + Nystatin (50µg/ml) + Nalidixic acid (100µg/ml) > incubation @ RT, 4-10 days

Summary of isolation

• Number of field trips made : 5• Number of locations : 9• Number of soil samples collected : 54• Number of actinos isolated in this project : 290• Number of isolates lost (unable to revive) : 84• Number of viable isolates (currently) : 206

Chalky colonies with aerial mycelium

400 X

Maintenance :

A.ISP-2 medium - sporulationB.Soft Agar stabs stored at 4°CC.Glycerol stocks at -80°C

400 X

Microscopy

Medium-scale culturing

• Growth medium : Starch Casein Medium (50ml)

Clumping, maximum biomass yield in 11-14 days. Could not track growth.

@RT, 200 RPM

Solution: Introduction of 2 glass marbles (1.3 cms, 5 gms)

Maximum growth: ~6 days

Screening for Antimicrobial Activity

Against M. tuberculosis – by REMA Against other bacteria – by cross streak method

Preparation of Culture filtrate

Centrifugation(6000 rpm, RT, 20mins)

Filtration (Whatman

#3 filter paper + 0.2

micron filter)

Lyophilization

10mg/ml in water REMA

Bacteria Killed(Sensitive)

Bacteria Not Killed(Resistant)

REMA - Principle Viable bacterium

incubation

12

+ Drug/CF

M.tb + CF/L (5.0 & 2.5mg/ml) in 96 well plates - 7 days

20µl of resazurin (0.02% in water, w/v) on day 7

Color change was observed on day 8.

REMA – test against M.tuberculosis H37Rv

• CF extracted with MeOH. .

• 100g/ml (in DMSO)

• M. tuberculosis H37Rv

REMA: + Control (Rif, 1.0 g/ml). Neg control – no inhibitor. MC- Medium alone.

Example of REMA

21 CFs tested – No activity

2/12 inhibitary at 2.5mg/ml

Results of REMA

Inhibitory activity against other bacteria

M. smegmatis E. coli S. aureus B. subtilis

# of isolates M.smegmatis A 31E.coli B 20S.aureus C 31B.subtilis D 31

No inhibition Partial & specific inhibition

Pan inhibition

Specific inhibition

• The isolates grown in Nutrient broth (3-4 days). • ~ 100mg of mycelia frozen in liquid N2, ground with

mortar & pestle.

• DNA extracted with phenol-chloroform.

• PCR with Universal primers

• 1500bp PCR product sequenced.

S. variabilis (4) S. albofaciens (2) S. violaceorectus (1) S. cinnamomeus (1)

Identification by 16S rRNA gene Sequencing

• Unable to isolate during rainy season (or when the soil is extremely wet). Absence of spores? Failure of vegetative mycelia to grow? Could not isolate from sea water.

• Colonies that were obtained on selective media (1st isolation) were subsequently unrevivable (>30%).

• The activity that was exhibited in the Cross streak method was not reproducible following culturing in liquid medium. CF did not inhibit the test strains (agar well diffusion method).

Hurdles

Antibiotic?

Some Interesting observations

Quorum sensing?

Thank you

HPLC profiles. Solvent MeOH. Absorbance 254nm.

A. P12; B. P50; C. Streptomycin SO4; D. Actinomycin-D.

5.275 mins

4.442 mins 3.585 minutes

5.392 mins

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0.008Detector A (254nm)Actinomycin 254 nmactinomycin 254 nm

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Detector A (254nm)Strept 8-9-10 254streptomycin 254 nm.dat

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Wayne’s model to simulate Latency in M. tuberculosis