Isolation and characterization of Anti- mycobacterials from Actinomycetes R. Ajay Kumar & Sabu Thomas Rajiv Gandhi Centre for Biotechnology Trivandrum OSDD/HCP0001/12FYP/ /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/ /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 Sites Sirumalai Karandhamalai Berizam Manakudy Thoothukudy Ponmudi Kovalam Manalpuram Kallaar Depth: ~10cm ~50gms soil Sterile plastic bags 1 gm serially diluted > Starch Casein Nitrate Agar + Nystatin (50µg/ml) + Nalidixic acid (100µg/ml) > 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 - sporulation B.Soft Agar stabs stored at 4°C C.Glycerol stocks at -80°C 400 X Microscopy

Medium-scale culturing Growth medium : Starch Casein Medium (50ml) Clumping, maximum biomass yield in days. Could not track 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 micron filter) Lyophilization 10mg/ml in waterREMA

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  g/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 31 E.coli B 20 S.aureus C 31 B.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 (1 st 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 mins mins3.585 minutes mins A C B D

Wayne’s model to simulate Latency in M. tuberculosis