Isolation and characterization of Antimycobacterials 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
• Duration
: January 22, 2013
: 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
• Date of joining of the JRF
: Balaji M
: April 15, 2013
Equipment procured :
Fermentor (Eppendorf)
Orbital shaker (Eppendorf)
-20°C freezer (Vestfrost)
Equipment yet to arrive :
37°C incubator (KEMI)
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) >
incubation @ RT, 4-10 days
Summary of isolation
•
•
•
•
•
•
Number of field trips made
Number of locations
Number of soil samples collected
Number of actinos isolated in this project
Number of isolates lost (unable to revive)
Number of viable isolates (currently)
:5
:9
: 54
: 290
: 84
: 206
Chalky colonies
with aerial
mycelium
400 X
Microscopy
Maintenance :
A.ISP-2 medium - sporulation
B.Soft Agar stabs stored at
4°C
C.Glycerol stocks at -80°C
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
Maximum growth: ~6 days
(1.3 cms, 5 gms)
Screening for Antimicrobial Activity
Against M. tuberculosis – by REMA
Against other bacteria – by cross streak method
Preparation of Culture filtrate
Filtration
(Whatman
#3 filter
paper + 0.2
micron filter)
Centrifugation
(6000 rpm, RT,
20mins)
REMA
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. .
• 100mg/ml (in DMSO)
• M. tuberculosis H37Rv
REMA: + Control (Rif, 1.0 mg/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
Partial & specific inhibition
Specific inhibition
# of isolates
M.smegmatis A
E.coli
B
S.aureus
C
B.subtilis
D
Pan inhibition
31
20
31
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 (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).
Some Interesting observations
Quorum sensing?
Antibiotic?
Thank you
5.392 mins
5.275 mins
sample prev iew
sample prev iew
0.08
0.05
0.04
0.04
0.03
0.03
A
0.02
0.02
0.01
0.01
0.00
0.00
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
0.08
Detector A (254nm)
P 50-224 nm
P 50 -224nm.dat
0.07
0.07
0.06
0.06
0.05
0.05
0.04
0.04
0.03
0.03
0.02
0.02
0.01
0.01
Volts
0.05
Volts
0.06
Volts
0.06
Volts
Detector A (254nm)
P 12-254
P12- 254
0.00
0.00
16
1
2
3
4
5
6
7
Minutes
8
9
10
11
12
13
14
Minutes
sample prev iew
sample prev iew
Detector A (254nm)
Strept 8-9-10 254
streptomycin 254 nm.dat
0.008
0.006
0.006
0.008
Detector A (254nm)
Actinomycin 254 nm
actinomycin 254 nm
0.007
0.007
0.006
0.006
0.005
0.005
0.004
0.004
0.003
0.003
0.002
0.002
0.001
0.001
0.000
0.000
-0.001
-0.001
0.003
0.003
0.002
0.002
Volts
0.004
Volts
0.004
0.001
Volts
0.005
Volts
0.005
C
B
0.001
0.000
D
0.000
1
2
3
4
5
6
7
8
9
Minutes
4.442 mins
10
11
12
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Minutes
3.585 minutes
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