Isolation and characterization of antimicrobial peptides from Sin Croaker (Jhonius Dussumieri)

1. ANTIMICROBIAL PEPTIDES FROM
CROAKER FISH, Johnius Dussumieri.
BY: AJIT ANTONY (OST-2015-26-02)
EXTERNAL GUIDE: DR. ROSAMMA PHILIP,
PROFESSOR AND HEAD,
DEPARTMENT OF MARINE BIOLOGY, MICROBIOLOGY AND BIOCHEMISTRY,
COCHIN UNIVERSITY OF SCIENCE AND TECHNOLOGY.
INTERNAL GUIDE: DR. N RAJENDRAN,
ADJUNCT PROFESSOR AND ACADEMIC COORDINATOR
DEPARTMENT OF MARINE MICROBIOLOGY AND MARINE DRUGS
KERALA UNIVERSITY OF FISHERIES AND OCEAN STUDIES.

2. 2
OBJECTIVE

3. ANTIMICROBIAL
PEPTIDES
3

4. ORGANISM
USED
Classification
Kingdom : Animalia
Phylum : Chordata
Sub-Phylum : Vertebrata
Super-class : Gnathostomata
Class : Actinopterygii
Order : Perciformes
Family : Sciaenidae
Genus : Johnius
Species : dussumieri

5. 5
MATERIALS AND METHODS:
1 2 3 4 5

6. 6 6
1
The peptide was extracted by modified
acetic acid-acetone precipitation method.

7. Antimicrobial activity was tested against the
above mentioned bacterial strains.
2
7
GRAM POSITIVE STRAIN GRAM NEGATIVE STRAIN
Bacillus cereus Pseudomonas aeruginosa
Staphylococcus aureus Edwardsiella tarda
Aeromonas hydrophila
Escherichia coli
Vibrio harveyi
Vibrio parahaemolyticus
Vibrio alginolyticus
Vibrio cholera
Vibrio proteolyticus
Vibrio vulnificus
Crude peptide sample was reconstituted in sterile
MilliQ and loaded on 6mm discs
Using a micropipette with sterile tip, with each disc
containing 4 mg of the peptide
These impregnates were placed on plates seeded with
microbial strains
Kept at 4oC for 30 minutes
Incubated overnight at 37oC
Zone of inhibition and the activity was recorded

8. The peptide sample was then subjected to solid
phase extraction using Sep- Pak C-18 Cartridges
(Waters, USA).
The cartridge was conditioned with methanol and
equilibrated with 0.1% Tri Fluoro Acetic Acid (TFA).
The sample was loaded on to the cartridge,
followed by a wash with 0.1% TFA.
The analyte trapped in the cartridges were eluted
with 6 ml each of 5%, 40%, 80% and 100%
Acetonitrile in 0.1% TFA.
Fractions were labelled as 5%, 40% and 80% and
subjected to lyophilisation.
3
8

9. 4
9

10. 10
• Liquid growth inhibition assay was employed to test the antimicrobial activity of the active
fractions purified by the cation exchange chromatography. Different bacterial suspensions were
prepared for the assay and it was carried out in a 96 well microtitre plate.
• Mid-logarithmic phase bacteria were diluted to 104CFU/ml in 50 mM HEPES (4-(2-
hydroxyethyl)-1-piperazine ethane sulfonic acid) buffer pH 6.8. 10µl FPLC fractions and 10µl of
the diluted bacterial suspension were loaded in to the microtiter well and incubated at room
temperature for two hours.
• HEPES buffer served as the Blank, served as negative control and served as positive control. A
set of negative and positive controls were maintained for the assay. The negative control
comprised of 10µl of bacterial suspension and 10µl of 50Mm Tris-HCl.
• 10µl of bacterial suspension along with 10µl of different concentrations of two fold serially
diluted antibiotic Ampicillin ( Vibrio alginolyticus ) and Chloramphenicol ( Staphylococcus
aureus and Bacillus cereus)served as positive control. After incubation for 2 hours, at room
temperature, 80µl of MH (Mueller-Hinton) broth was added to each well.
• Absorbance was measure using a microplate reader (Tecan, USA) at a reference wave length of
600 after incubation at 370C for another 16 hours with shaking at 100 rpm.
5

11. 13
RESULTS
0
5
10
15
20
25
30
16
12
10
16
12
11
12
15
10
8
20
26
Zone of inhibition (mm)

12. 14
RESULTS
Conductivity
Elution of sample
At 280 nm
% of Buffer

13. 15
RESULTS
Conductivity
Elution of sample
At 280 nm
% of Buffer

14. 16
RESULTS
Conductivity
Elution of sample
At 280 nm
% of Buffer

15. Inhibition % = 100 – Growth Percentage
Growth % = (OD of Test/ OD of Control) X 100
17
RESULTS
Antimicrobial activity of 5% FPLC fraction
% Inhibition
Sl.
No.
Fraction Staphylococcus
aureus
Bacillus cereus Vibrio alginolyticus
1 Jd5- 1 0.00 77.69 94.75
2 Jd5- 2 61.17 0.00 0.00
3 Jd5- 3 44.96 0.00 15.36
4 Jd5-4 18.25 41.97 98.05
5 Jd5-5 40.98 97.41 66.85
6 Positive control
70
95.4 99.63
ANTIMICROBIAL ASSAY: Liquid
Growth Inhibition Assay

16. 18
RESULTS
0
10
20
30
40
50
60
70
80
90
100
Jd5- 1 Jd5- 2 Jd5- 3 Jd5-4 Jd5-5
% of Inhibition
Staphylococcus aureus Bacillus cereus Vibrio alginolyticus
Antimicrobial activity of 5% FPLC fractions

17. 19
RESULTS
% Inhibition
Sl.
No.
Fraction Staphylococcus
aureus
Bacillus cereus Vibrio
alginolyticus
1 Jd 40-1 56.58 96.23 100
2 Jd 40-2 55.72 0 78.52
3 Jd 40-3 23.54 50 93.82
4 Jd 40-4 0.00 91.25 87.99
5 Jd 40-5 43.60 59.02 93.32
6 Positive
control
70 95.4 99.63

18. 20
RESULTS
Antimicrobial activity of 40% FPLC fractions from Johnius dussumieri
0
10
20
30
40
50
60
70
80
90
100
Jd 40-1 Jd 40-2 Jd 40-3 Jd 40-4 Jd 40-5
% of Inhibition
Staphylococcus aureus Bacillus cereus Vibrio alginolyticus

19. 21
RESULTS
% Inhibition
Sl.
No.
Fraction Staphylococcus
aureus
Bacillus cereus Vibrio
alginolyticus
1 Jd80- 1
68.82 0.00 0.00
2 Jd80- 2
66.77 0.00 0.00
3 Positive 49 95.4 99.63
0
10
20
30
40
50
60
70
Jd80- 1 Jd80- 2
% of Inhibition
Staphylococ cus aureus Bacillus cereus Vibrio alginolyticus

20. CONCLUSION
22

22. REFERENCES
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• Antony, S. P., Afsal, V. V., & Philip, R. (2013). Antimicrobial peptides from the indian white shrimp, Fenneropenaeus Indicus: Isolation
and In Vitro activity profile. In New Developments in Blue Biotechnology and Environmental Pollution Assessment (pp. 111–118).
• Chaithanya, E. R., Philip, R., Sathyan, N., & Anil Kumar, P. R. (2013). Molecular Characterization and Phylogenetic Analysis of a Histone-
Derived Antimicrobial Peptide Teleostin from the Marine Teleost Fishes, Tachysurus jella and Cynoglossus semifasciatus. ISRN
Molecular Biology, 2013, 1–7.
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https://doi.org/10.1016/j.peptides.2010.11.005
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peptide Himanturin from round whip ray Himantura pastinacoides and its phylogenetic significance. Results in immunology, 2, 120-
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