Isolation and characterization of Anti mycobacterials from Actinomycetes OSDD/HCP0001/12FYP/2012-123/Fin/2412 R. Ajay Kumar & Sabu Thomas Rajiv Gandhi Centre for Biotechnology Trivandrum 695014

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

Equipment procured : Fermentor (Eppendorf) Orbital shaker (Eppendorf) -20 ° C freezer (Vestfrost) Equipment yet to arrive : 37 ° C incubator (KEMI)

Sites Sirumalai Karandhamalai Berizam Manakudy Thoothukudy Ponmudi Kovalam Manalpuram Kallaar

Sample collection

Depth: ~10cm ~50gms soil Sterile 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

Maintenance : A.ISP-2 medium - sporulation B.Soft Agar stabs stored at 4

°

C C.Glycerol stocks at -80

°

C 400 X

Microscopy

400 X

Medium-scale culturing

• Growth medium : Starch Casein Medium (50ml) @RT, 200 RPM Clumping, maximum biomass yield in 11-14 days.

Could not track growth.

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) REMA Filtration (Whatman #3 filter paper + 0.2 micron filter)

Lyophilization

10mg/ml in water

REMA - Principle Viable bacterium

incubation + Drug/CF

Bacteria Killed (Sensitive) Bacteria Not Killed (Resistant)

12

REMA – test against M.tuberculosis H37Rv

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.

Example of REMA • CF extracted with MeOH. .

• 100 m g/ml (in DMSO) •

M. tuberculosis

H37Rv

REMA: + Control (Rif, 1.0

m

g/ml). Neg control – no inhibitor. MC- Medium alone.

Results of REMA

21 CFs tested – No activity 2/12 inhibitary at 2.5mg/ml

Inhibitory activity against other bacteria

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

No inhibition Pan inhibition Partial & specific inhibition Specific inhibition # of isolates

M.smegmatis

E.coli

S.aureus

B.subtilis

A 31 B 20 C 31 D 31

Identification by 16S rRNA gene Sequencing

• 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)

Hurdles

• 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).

Some Interesting observations Quorum sensing?

Antibiotic?

Thank you

5.275 mins

sample prev iew

Detector A (254nm) P 12-254 P12- 254

0.06

0.05

0.04

0.03

0.02

A

0.01

0.00

0.006

1 2 3 4 5 6 7 8 9 Minutes sample prev iew 10 11 12 13 14 15 16

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

0.01

0.00

0.02

0.006

0.05

0.04

0.03

0.06

0.005

0.005

0.004

0.003

0.004

0.003

C

0.001

0.002

0.000

1 2 3 4 5 6 7 8 Minutes

4.442 mins

9 10 11 12 0.000

0.001

0.002

5.392 mins

sample prev iew 0.08

0.07

Detector A (254nm) P 50-224 nm P 50 -224nm.dat

0.06

0.05

0.04

0.08

0.07

0.06

0.05

0.04

0.03

0.03

0.02

0.01

0.00

1 2 3 4 5 9 10 11 6 7 Minutes sample prev iew 8 0.002

0.001

0.000

-0.001

0.008

0.007

0.006

0.005

0.004

0.003

Detector A (254nm) Actinomycin 254 nm actinomycin 254 nm

1 2 3 4 5 6 7 8 9 10 11 Minutes

3.585 minutes

12 12 13 14 0.00

0.02

0.01

B

0.008

0.007

0.006

0.005

0.004

0.003

0.002

13 14 15 -0.001

0.001

0.000

D

HPLC profiles. Solvent MeOH. Absorbance 254nm.

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

Wayne ’ s model to simulate Latency in

M. tuberculosis