characterization of antifungal metabolite from serendipitiously isolated bacterial species

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“Characterization of Antifungal Metabolite From

Serendipitously Isolated Bacterial Strain”

Desai Dhananjay, Hajare Sampada, Rokade Snehal.

Post Graduate Students Department Of Microbiology

New Arts, Commerce And Science College Ahemadnagar

[email protected]

Guided by: Mr. Kukreja G. P. Head

Dept. of Microbiology

mailto:[email protected]

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INTRODUCTION

IFLs have emerged as an important cause of morbidity and mortality in immune-compromised patients.

Fungi are pathogenic for plants, animals, human, and other economically important fungi.

Challenge of planning searching of new bioactive compounds.

New bioactive molecule from natural sources Nature is host of amazing variety of clinically and economically important

products.

Bio prospecting

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INTRODUCTION

History of bioactive metabolite screening taxonomic characterization of microbial niche accelerate the rate at which

novel producer of bioactive metabolites relatedness to known strains of microorganisms.

63% of all tiny molecule of drugs launched between 1981 - 2006 Effective chemical entity must be identified, researched effective

manufacturing process require to commercialization of natural drug screening.

Antifungal drug: Unmet needs and challenges Treatment of invasive fungal infections is quite limited Finding a treatment that will kill the fungus and not harm our own cells more

difficult

Antifungal agent: Scenario of clinical practices Fluoropyrimidine analogs, Polyenes, Azoles, and Echinocandins

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INTRODUCTIONContribution of serendipity in scientific investigations

• finding one thing while looking for something else.• powerful interaction of chance with prepared mind.

Breakthrough in research by serendipity • 1880- Lithium in mood disorders• 1943- LSD-25• 1928- Discovery of Penicillin• Sildenafil- in Angina Pectoris

Johann Wolfgang Goethe wrote: “Discovery needs luck, invention, intellect—none can do without the other.”

Distribution of Drug Dis-covery

Non serendipitious 75.8%

Clinical derivatives 10.2%

Laboratory deriv-atives 8.1%

Clinic 3.7%

Laboratory 2.2%

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INTRODUCTIONAntifungal drug: a novel approach Antifungal-resistant has provided motivation for novel bioactive compound discovery Cloning and genetic engineering offer alternative approaches

The present investigation aimed to extract, purify and characterize the antifungal metabolite from serendipitously isolated bacterial species.

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MATERIALS AND METHODS Isolation of Bacterium• from a contaminated YPD agar plate on to which it was streaked from casein

agar plate where it was showing a pronounced proteolytic activity

Screening for antimicrobial activity• Fungi A.niger, A.flavus, F. oxysporum, Alternaria sp., bacteria s. typhi, B.subtilis

a)Agar cup diffusion method B)Dual culture technique

Methods for identification and characterization of the isolate

• Morphological characterization Gram staining and motility (hanging drop technique)

• Biochemical characterization Catalase test, Oxidase test, IMViC test, Sugar fermentation test, Starch

hydrolysis, Cellulose degrading test.

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MATERIALS AND METHODS Antibiotic Susceptibility Test (by using antibiotic poly disc method )• Gentamicin, Cefotaxime, Ciprofloxacin, Ceftriaxone, Ceftazidime ,

Ampicillin/sulbactam , Sparfloxacin , Cefadroxil , Lomefloxacin

Genotypic identification Partial (859BP) 16 S rRNA gene sequensing was done

Bioinformatics analysis• The DNA sequences were analyzed using online BLASTn• find out evolutionary relationship of bacteria. Were used to generate

phylogenetic tree was constructed by using MEGA 5

7

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MATERIALS AND METHODS

Submission of sequence to NCBI database • sequence obtained from 16S rRNA gene sequencing was submitted to

NCBI database

Extraction and purification of antifungal metabolites• Detection of appearance of protein- Ninhydrin test used

• Ammonium sulfate precipitation of bioactive metabolite ammonium sulfate use for 30%, 45%, 60%. Saturation Pellet was collected

and antifungal activity of pellet was checked against test fungi A.niger, A.flavus.

• Effect of pH on antifungal activity pH ranged 5, 7, 9.

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Dialysis• Pellet obtained from ammonium sulfate precipitation was partially purified by

using membrane dialysis technique (HiMEDIA LA393- 1MT)

Qualitative detection of protein (Dialyzed fraction)• By Ninhydrin method in which BSA as a standard and distilled water as blank

Quantitative estimation of protein (Dialyzed fraction)• By Folin - Lowry method in which BSA as a standard and solution without protein

stock used as a control.

Spectrophotometric analysis of antifungal metabolite• UV- Visible region (200-800 nm) in Multiskan® Spectrum (Thermo Electron Corp.)

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Fourier Transform Infrared Spectroscopy (FTIR) analysis of antifungal metabolites

• The FTIR spectrum of dialyzed fraction was recorded on SHIMADZU A 21374801518

Micro scale Optical Density Assay (MODA) for quantitative assay of antifungal activity (Dialyzed fraction)

• Antifungal potential of dialyzed fraction (concentration ranged 10-100%) was investigated against A. niger, A. flavus, F. oxysporum, Alternaria sp. By using 96 well plate

• Incubated at 370C for five days • Turbidity recorded everyday from day of inoculation at 550 nm

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Results Isolation of bioactive metabolite producing bacterium

Screening for antimicrobial activity of cell free broth

casein agar plate with

zone of proteolysis

Serendipitously observed antifungal activity on YPD agar plate

S. typhi B. subtilis A. niger A. flavus

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Results Screening for antifungal activity

Morphological IdentificationA. niger A. flavus

Isolated colonies of the bacterium on NA

Colony characteristics of the isolate on Nutrient agar

plate after 24 hrs incubation at 370C.

where bacterial isolate was grown on PDA for three days and then followed by inoculation of test fungi, Clear zone of inhibition were observed.

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Colony characteristics of

isolate

Size Pinpoint (< 1mm)

Shape Circular

Colour Offwhite

Margin Erose

Opacity Opaque

Consistancy Sticky

Elevation Flat

Gram

character

Gram negative rods

Motility Non motile

Capsule

staining

Capsule producing

Catalase and Oxidase tests

Test Observation Result

Catalase Fine bubbles arising

from the colony

after addition of

H2O2

+ve

Oxidase No colour change

on contact with

reagent on filter

paper

-ve

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IMViC test

Test Observation Result

Indole Test No development of pink

coloured ring on the

surface of tryptone broth

after addition of Kovac’s

reagent

-ve

Methyl Red

Test

No development of red

colour of GP broth after

addition of Methyl red

indicator

-ve

Voges

Proskauer Test

Development of red

colour of GP broth after

addition of VP I and VP

II reagent

+ve

Citrate

Utilization

Test

No colour change of

Simmon’s citrate slant

from green to blue

-ve

IMViC test

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Sugar fermentation

Test Acid Gas

Glucose ++ ++

Lactose - -

Maltose - -

Mannitol - -

Arabinose - -

Xylose - -

Galactose - -

Raffinose - -

Biochemical test interpretationsTest Observation Result

Cellulose Degradation TestZone of inhibition was observed

on CMC agar plate followed by

addition of 1% congo red

solution and decolorizing it by

NaCl solution

+ve

Starch Hydrolysis Zone of decolorization was

observed on starch agar plate on

addition of Gram’s iodine

+ve

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Antibiotic susceptibility test

Antibiotic susceptibility test

Interpretations of antibiotic susceptibility test

Antibiotic Zone of inhibition

(mm)

Interpretation

Amikacin (30mcg) 22 mm Intermediate

Lomefloxacin

(10mcg)

33 mm Sensitive

Cefadroxil (30mcg) 25 mm Sensitive

Sparfloxacin (30mcg) 30 mm Sensitive

Ampicillin/sulbactam

(10/10mcg)

30 mm Sensitive

Ceftazidime (30mcg) 30 mm Sensitive

Ceftriaxone (30mcg) 25 mm Sensitive

Ciprofloxacin (5mcg) 35 mm Sensitive

Cefotaxime (30mcg) 25 mm Sensitive

Gentamicin (10mcg) 25 mm Sensitive

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Molecular identification of isolate using 16srRNA gene sequence

NR 028986.1| Pseudomonas poae

NR 102514.1| Pseudomonas poae

NR 028987.1| Pseudomonas trivialis

NR 042392.1| Pseudomonas simiae

NR 025586.1| Pseudomonas antarctica

NR 024911.1| Pseudomonas rhodesiae

NR 117821.1| Pseudomonas marginalis

NR 029051.1| Pseudomonas salomonii

NR 113647.1| Pseudomonas fluorescens



NR 024928.1| Pseudomonas gessardii

NR 113600.1| Pseudomonas azotoformans

NR 025164.1| Pseudomonas costantinii

NR 042199.1| Pseudomonas lurida

B1

NR 114595.1| Pseudomonas tolaasii





84

79

67

62

2534

33

21

24

37

15

38

76

23

2

. On the basis of the position of sequence of the given bacterial samples

in the phylogenetic tree, B1 showed closest similarity to

Pseudomonas tolaasii.

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Antifungal potential of ammonium sulphate fraction against A. niger and A. flavus

Zone of inhibition (mm)

Concentration A.niger A.flavus

30% 17mm 11 mm

45% - -

60% 09 mm 08 mm

Effect of pH on antifungal activity

Zone of inhibition (mm)

pH A.niger A.flavus

5 - -

7 14mm 11mm

9 08mm 07mm

A. niger A. flavus A. niger A. flavus

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Spectrophotometric analysis of antifungal metabolite- (Dialyzed fraction)

Maximum absorbance of antifungal metabolite was observed at 280 nm.

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Fourier Transform Infrared Spectroscopy (FTIR) analysis of bioactive metabolites- (Dialyzed fraction)

Functional group of antifungal

metabolite (Dialyzed fraction)

Sr. No

Wave number (cm-1)

Functional group

1. 3332 N-H (stretching)

2. 1643 Amide carbonyl(stretching)

3. 3520 O-H (Stretching)

4. 2900.94-3028 C-H (Stretching)

5. 1080 C-N (Stretching)

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Blank

Positive

Control

10%20%

30%40%

50%60%

70%80%

90%100%

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Activity of test compound against A. niger

Day 1stDay 2ndDay 3rdDay 4thDay 5th

Concentration of test compound in %

Abs

orba

nce

at 5

50 n

m

Blank

Positive

Control

10%20%

30%40%

50%60%

70%80%

90%100%

0

0.5

1

1.5

2

2.5

Activity of test compound agaist A. flavus

Day 1stDay 2nd Day 3rdDay 4thDay 5th


Abs

orba

nce

at 5

50 n

m

Blank

Positive

Control

10%20%

30%40%

50%60%

70%80%

90%100%

00.20.40.60.8

11.21.4

Activity of test compound against F. oxysporum


Concentration of test compoundin %

Abs

orba

nce

at 5

50 n

m

Blank

Positive

Control

10%20%

30%40%

50%60%

70%80%

90%100%

00.20.40.60.8

11.21.41.61.8 Activity of test compound against Alternaria spp.



Abs

orba

nce

at 5

50 n

m

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only three classes of molecules are currently used in clinical practice and only one new class of antifungal drugs has been developed in the last 30 years

For many of the most common invasive fungal infections, the better tolerated azoles and Echinocandins have emerged as first-line agents

The spurred interest for the detection, extraction, purification and characterization of antifungal metabolite from the bacterial isolate which could a potential candidate for biotechnological or medicinal use.

change in concentration of metabolite does not result in significant change in activity while change period of incubation may show change in antifungal activity of metabolite

859 bp of 16S rRNA gene sequence was submitted to NCBI, Accession code; KU533778.

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Serendipity does not produce bioactive metabolites but serendipity plays crucial role in searching bioactive metabolites of bacterial origin

serendipitously this showed antifungal activity and the face of investigation turns to find out bioactive antifungal metabolite

16S rRNA (partial) gene sequencing was done and it is found to be a novel strain of Pseudomonas tolaasii.

concluded that the concentration of antifungal metabolite does not affect the fungal growth but the days of incubation play significant role in inhibition of test fungal sp.

The functional group of bioactive metabolite was assessed by FTIR of crude extract which apparently matches with reported extracellular lipodepsipeptide of P. tolaasii.

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FUTURE PROSPECT

1. Cytotoxic activity of antifungal metabolite against human cell line has to be studied.

2. Purification of active molecule which will important for antifungal activity.

3. To determine molecular weight of active molecule by using LC-MS 4. Gene responsible for production of antifungal activity can be isolated and study their genetic mapping and location 5. Use recombinant DN technology for excess production of antifungal metabolites.

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Coleman, J. J., Ghosh, s., Okoli, I. and Mylonakis. E. (2011) Antifungal Activity of Microbial Secondary Metabolites. Plos one. 6, 1-9.

Delany, I., Michelle, M., Fenton, A., Bardin, S., Aarons, S. and Gara, F. O. (2000) Regulation of production of the antifungal metabolite 2,4-diacetylphloroglucinol in Pseudomonas fluorescens F113: genetic analysis of phlF as a transcriptional repressor. Microbiol. 146, 537-546.

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Alvarez, S., P., Manuel, M. L., D. M and Oscar, P. K. (2016) Bacteriocins of lactic acid bacteria: extending the family. Appl Microbiol Biotechnol. 100, 2939–2951.

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http://www.ncbi.nlm.nih.gov/Taxonomy/tax.htm/

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PRESENTATION AND PUBLICATIONAbstract accepted titled “Serendipity: A novel way to investigate antifungal metabolite from bacterial origin”in 2nd European Microbiology Conference to be held on July 1415, 2016 in Cologne, Germany

Poster presentation entitled “Antimicrobial Screening of Secondary Metabolites from Serendipitously Isolated Bacterial Sp.”In National Conference on Recent Analytical Techniques in Quality Control of Food, Beverages & Nature Products. Held at Shri. S. H. College, Devgad, Sindhudurg. Dec. 18th – 19th, 2015. Poster presentation entitled “Antimicrobial Screening of Secondary Metabolites from Serendipitously Isolated Bacterial Sp.”In Avishkar University level Research Competition 2015, Pune. Dec.11th, 2015. Shortlisted, Poster presentation entitled “Antimicrobial Screening of Secondary Metabolites from Serendipitously Isolated Bacterial Sp.” In Avishkar Research Competition 2015, Ahmednagar. Oct. 9th, 2015

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SUGESSIONS ALWAYS WELCOME

characterization of antifungal metabolite from serendipitiously isolated bacterial species

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