UNIVERSITI PUTRA MALAYSIA

ISOLATION AND CHRACTERIZATION OF PYOCYANINE AND 1- PHENAZINOLE FROM PSEUDOMONAS AERUGINOSA

(AN ENDOPHYTE)

MAJID ESHAGID

FSMB 1998 3

ISOLATION AND CHRACTERIZATION OF PYOCYANINE AND 1-PHENAZINOLE FROM PSEUDOMONAS AERUGINOSA (AN ENDOPHYTE)

By

MAJID ESHAGID

Thesis Submitted in Fulfillment of the Requirement for the degree of Master of Science

In the Faculty of Food Science and Biotechnology Universiti Putra Malaysia

March 1998

Dedicated to ...... ,

Memory of my father

ACKNOWLEDGMENTS

I would like to express my most sincere gratitude and deep appreciation �o the

Chairman of my supervisory committee, Assoc. Prof. Dr. Abdul Manaf Ali for his

invaluable contribution, generous offering of time and careful supervision.

I am indebted to the member of my supervisory committee, Dr Zaiton Hassan,

Prof. Dr. Mohamed Ismail Abdul Karim and Dr. Khozirah Shaari for their

encouragement, constructive suggestion, guidance and helping for analysis of data.

Next, I would like to extend heartfelt thanks to Prof. Dr. F. Tomita, Prof. Dr. K.

Kawazu and Assoc. Prof. Dr. Mawardi Rahman especially for their valuable comments

on isolation of endophytes and chemistry. I am sincerely grateful to Puan Mimi (UKM),

Puan Hayzan (MARDI), Encik Zainol and Encik Wahab (FSAS, UPM) for their

technical guidance.

Furthermore, I would like to acknowledge UPM for supporting the research and

providing the Graduate Assistantship by the Malaysian government through IRP A

(Intensification of Research Priority Areas), grant no. 51113. I wish to express my deep

appreciation to all the staff of the MKT for offering me their hospitality.

I also wish to thank all my fellow graduate students and my friends for their

hospitality and help. Special acknowledgment is due to Mr. Imran Wiart and Mr. Nasir

Tsafe Umar for providing valuable inputs during my study.

m

I would like to record my sincere appreciation to all the final year undergraduate

students who worked in the MKT during the course of my study for their friendship that

made my study very pleasant and memorable.

Words are not enough to express my heartfelt feelings to my mother, sister and

brothers for their encouragement and support. lowe a depth of gratitude to them which,

can never be repaid

IV

TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 111

LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vlll

LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. IX

LIST OF PLATES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Xl

LIST OF ABBREVIATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. Xll

ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. Xlll

ABSTRAK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XVI

CHAPTER

I INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . '" . . . . . . . . . . , . . . . . . 1

IT LITERATUREREVIEW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

The History of Antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . 5 The Chemical Structure of Main Antibiotics . . . . . . '" . . . . . . . . . . . . . . . . . 8 Phenazine Antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Sources of Antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . 1 6 Antibiotics from Bacteria . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . 1 7 Endophyte . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 9

ill SCREENING, ISOLATION AND CHARACTERIZATION OF ANTIMICROBIAL PRODUCER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . 23

Isolation of Endophytic Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Plant Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Maintenance of the Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Target Microbes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Preparation of Target Microbes for Antimicrobial Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Cultivation of Bacterial Isolates for Antimicrobial Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Diagnostic Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . 28 Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . .. . . . . 33 API-20NE Test Strips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 Identification of Bacteria Through Fatty Acid Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

v

Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1

IV ISOLATION AND IDENTIFICATION OF BIOACTIVE COMPOUNDS FROM AN ENDOPHYTIC BACTERIA AND EVALUATION OF ITS POTENTIAL ANTIMICROBIAL ACTMTY AND PRODUCTMTY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 50 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

Paper Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 52 Chloroform Extraction . . . ' " . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ' " . . . . . 53 Analytical Thin Layer Chromatography . . . . . . . . . . . . . . . . . . . . . . . . .. . 54 Silica Gel Column Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Preparative Thin Layer Chromatography (PTLC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 56 Recrystalization of Orange Compound . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 Structural Elucidation by Spectroscopic Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 57

Evaluation of the Potential Antimicrobial Activity of Bioactive Compounds and the Ability of P. aeruginosa to Produce Pyocyanine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

Quantitative Bioassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 Evaluation of Pyocyanine Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 59 Extraction and Purification of Antimicrobial Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 Structure Determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 Antimicrobial Activities . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 1 Pyocyanine Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 7 1

V STUDIES ON THE CYTOTOXICITY OF PYOCYANINE .

AND I -PHENAZINOLE AGAINST THE HUMAN AND ANIMAL CELL LINES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . '" . . . . . . . . . 76

Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 Assay Media . . . . . , . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . 76 Cultivation of He La and 3T3 Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 Cultivation of CEM-SS Cell Line . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 77 Cultivation of Sfl) Insect Cell Line . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 78 MTT -Microculture Tetrazolium Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

VI GENERAL DISCUSSION AND CONCLUSION . . . . . . . . . . . . . . . . . . . . . . 86 General Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 86

VI

Conclusion . . .. . . . .. . ..... . . . . . . ... . . . ... . . . . .. . .... . . . . .. . . . . . . . . . . . . . . . . . . . . ... 90

BmLIOGRAPHY . . . . . . . . . . . .. .... . . . . . . . . .... . . .. ... . . . . . ...... . . . . . . . .. .. .. . . . . . . . . . . . . 9 1

APPENDIX A . ... . . . . . . . . . . . . . .. . . . . . . . . . ... . . . . . . . . . . ... . . . . . . . .. .... . . . . . ... . . . . . . . .. . ... 1 0 1

APPENDIX B . . . ... . . . . ... . ....... . .. .. .. . .... . .. . . . . . . .... . . . . . . . .. .. .. . . . . . . . .. . .. ... . . . . 108

APPENDIX C . . . .... . . . . . . . . . . .... . . . . . . . ... . . . . ... . . . . . . . . . . . ........ . . . . . . . . . . . .. .... .... 1 09

VITA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 1 1 0

VII

LIST OF TABLES

Table Page

2.1 Key to the main antibiotic families (Berdy, 1 974) . . , . .. . . . . .. . . . . . . . . . '" . .. . . . . . . . . . . . . . ,. . . . . .. . . . . . . . . . . . . . .. 9

2.2 Collected data on the antibacterial effect of pyocyanine on the selected organisms (Waksman and Woodruff, 1942) . . . . . . . . . .. . . . . . . , . . . . . , . . . . . 1 5

2.3 Commercially produced antibiotics from bacteria

3 . 1 The antimicrobial activity of the endophytic bacteria against target microbes

3.2 The antimicrobial activity of

1 8

3 9

isolate 32. 1 against an array of pathogens . , . .. .. , .. . .. , . ... . . , .. . .. . .. .. 40

3.3 Different characteristics of the isolate ... ... ... ... ............ ..... .... . .. 47

3 .4 Fatty acid composition of the UPM strain . . .. . . . . .. . . . ,. . . . . .. . . .. . . . ... 49

4.1 Two different media for production of pyocyanine by P. aeruginosa . . . . . . . . .... . . . .. . .... . . .. . .. 58

4.2 Solvent systems used for analytical thin layer chromatography

4.3 Comparison of l3C NMR of I-phenazinole from the literature and experiment

4.4 Comparison ofUV-VIS (nm) of

6 1

69

pyocyanine from the literature and experiment . . . . . . . . . . . . . . . . . . . . . . . . . 70

4.5 The MIC values (Ilgldisk) of pyocyanine and I-phenazinole against target microorganisms

5. 1 Mean of inhibitory concentration at 50% cells reduction (ICso, Ilglml) of the pyocyanine

72

and I-phenazinole against the cell lines . . . . . . . . . . . . . . . . . . . . . . . .. . ... . . . . 8 1

VIII

LIST OF FIGURES

Figure Page

2. 1 Structure and numbering of the phenazine nucleus 1 1

2.2 Structure of pyocyanine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3

2.3 Structure ofHemipyocyanine (l-phenazinole) . . , . . . . . . . . . . . . . . . . . . . . . . . . . 16

3 . 1 Steps for isolation of endophytic bacteria

3 .2 Sample processing for whole-cell fatty acid analysis

24

of bacteria (from Miller and Berger, 1985) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

3 .3 Comparison between antimicrobial activity of the isolate and positive control against target microbes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

3 .4 A fatty acid methyl ester chromatogram of the UPM strain

4. 1 Detection ofbioactive compounds produced by

49

Pseudomonas aeruginosa by biological means . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

4.2 Procedure for isolation and purification ofbioactive compounds produced by Pseudomonas aeruginosa . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . 63

4.3 UV-VIS of l-phenazinole . . . . . . . . . . . . . . . . . . . . . . . . . . , . .. . . . . . . . . . . . . . . . . . . . . . 65

4.4 IR of I-phenazinole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

4.5 GeMS of I-phenazinole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . 66

4.6 1 H NMR of I-phenazinole . . . . . . . . . . . . . . . . . .. . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 67

4.7 l3C NMR of I-phenazinole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

4.8 GeMS of pyocyanine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .. . . . . . . . . . . . . . . . . . . . . 69

4.9 UV-VIS of pyocyanine . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

IX

4.10 The MIe values of the Supernatant, chloroform extract and pure compounds against Bacillus subtilis (B28) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

4.11 Comparing the yield of pyocyanine by MacDonald medium (a) and Hellinger medium (b) . . . ... ... ... ... ... .... 73

x

LIST OF PLATES

Plate Page

3.1 Photograph of confluent growth of the bacterium on the Cetrimide agar . . . ... ...... ...... ... ... ... ...... ..... 45

3.2 Photograph of the test results on API-20NE test strips for identification of the isolate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 45

3.3 A scanning electron micrograph of the isolate showing flagellum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

3.4 A scanning electron micrograph of the isolate showing colonies of bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

4.1 Bulk extraction of antimicrobial compounds from supernatant using chloroform as solvent . . . . . . . . . . . . . . . . . . . . . .. . . . . . . 61

4.2 Separation of bioactive compounds by silica gel column chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . '" ... ... ... 62

4.3 PTLC of bioactive compounds (note the well separated orange and blue bands)

5.1 Cytotoxic effect of pyocyanine on HeLa cells (a, treated cells and b, control) and CEM-SS cells (c, treated cells and d, control)

5.2 Cytotoxic effect of pyocyanine on 3T3 cells (a, treated cells and b, control) and Sf9

62

82

insect cells (c, treated cells and d, control) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

5.3 Cytotoxic effect of I-phenazinole on HeLa cells (a, treated cells and b, control) and EMC-SS cells (c, treated cells and d, control) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 84

5.4 Cytotoxic effect of I-phenazinole on 3T3 cells (a, treated cells and b, control) and Sf9 insect cells (c, treated cells and d, control) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

XI

ATCC

CDCl3

DIP

DMSO

EDTA

EI

PBS

FCS

GCMS

ICso

IR

LC/MS

MIC

MIS

MLD

MRS A

NCI

NMR. NRRL

PC

PTLC

SI

TLC

UV

VIS

LIST OF ABBREVIATIONS

American Type Culture Collection

Deuterized chloroform

Direct insert probe

Dimethyl sulphoxide

Ethylenediamine tetraacetic acid

Electron impact

Fetal bovine serum

Fetal calf serum

Gas chromatography mass spectrometry

Inhibitory Concentration at 50010 cells reduction

Infrared

Thermospray liquid chromatography/mass spectrometry

Minimum inhibitory concentration

Microbial Identification System

Minimum lethal dose

Methicillin Resistant Staphylococcus auerus

National Cancer Institute

Nuclear magnetic resonance

Northern Regional Research Laboratories

Paper chromatography

Preparative thin layer chromatography

Similarity index

Thin layer chromatography

Ultraviolet

Visible

XII

Abstract of the thesis presented to the Senate ofUniversiti Putra Malaysia in fulfillment of the requirement for the

Degree of Master of Science

ISOLATION AND CHRACTERIZATION OF PYOCYANINE AND 1-PHENAZINOLE FROM PSEUDOMONAS AEROGINOSA (AN ENDOPHYTE)

By

MAJID ESHAGID

March 1998

Chairman: Assoc. Prof. Dr. Abdul Manaf Ali

Faculty: Food Science and Biotechnology

Endophytic bacteria are organisms that colonize internal plant tissues. These

organisms are believed to be useful for pharmacological screening and agricultural

programs for biological control of plant pests and diseases. This project was designed to

screen endophytic bacteria to be used for pharmaceutical purpose.

Bacterial endophytes were isolated from the twigs of 50 different healthy tropical

rain forest plants collected at Kemensal Hill, Ulu Kelang, Selangor. After preparing the

pure culture of the bacteria, the isolates were subjected to bioassay using disk diffUsion

method against Bacillus cereus NRRL 1447 (Gram-positive bacteria), Pseudomonas

aeruginosa ATCC 60690 (Gram-negative bacteria), Bacillus subtilis B28 and B29

[mutant (deficient in DNA repair) and wild type], Candida lipolytica ATCC 2075

XIII

(yeast), Sacchromyces /ipolytica ATCC 16617 (yeast) and Aspergillus ochraceous

ATCC 398 (fungi). Out of the 79 bacterial endophytes that were isolated from 50

tropical plants, 12 of them showed antimicrobial activity. One of the isolate (no. 32. 1)

that possesses the best inhibition activity against target microorganisms was selected for

further study. By employing biochemical tests, miniaturized multi test system (API

20NE kit) and whole cellular fatty acid profiles the isolate was identified as

Pseudomonas aeruginosa UPM strain.

Judicious combinations of chromatographic techniques were adopted in

purifying the active compounds from the fermentation media. As a result, two bioactive

compounds were purified. The structure of these two bioactive compounds were

elucidated by means of spectroscopic techniques including ultraviolet spectroscopy

(UV), infrared spectroscopy (IR), nuclear magnetic resonance spectroscopy (IH and

13C-NMR), mass spectroscopy (MS), also by comparison with the literature and they

were identified as pyocyanine and I-phenazinole.

Minimum inhibitory concentration of pyocyanine against P. aeruginosa, B.

subtilis (B29) and methicillin resistant Staphylococcus auerus was 4.6875 �gldisk, while

against B. subtilis (B28) and B. cereus was 9.375 Jlgldisk. I-Phenazinole had the MIC

value of 37.5 Jlgldisk against P. aeruginosa, C. lipolytica, A. ochraceous. The MIC

value of this compound was 75 and 150 Jlgldisk against B. cereus, B. subtilis (B28 and

B29), S. lipolytica and MRSA, respectively. Therefore, pyocyanine revealed

antibacterial activity whereas, I-phenazinole was active against all target microbes. The

XIV

antimicrobial activity of I-phenazinole was less than pyocyanine. The cytotoxicity of the

compounds were tested against the HeLa cells (Human cervical adenocarcinoma), 3T3

(Mouse fibroblast), T-Lymphoblastic Leukemic cells (CEM-SS) and Sf9 insect cells. 1 -

Phenazinole was found to be more toxic than pyocyanine against the tested cell lines,

except for CEM-SS. Cytotoxic activity of the compounds against 3T3 cell line (non­

cancerous mouse fibroblast) was lower than against the tumor cell lines. Thus, the

compounds revealed fairly selective cytotoxic activity against tumor cell lines. The

compounds showed cytotoxic effect on Sf9 insect cells and therefore could be used for

biological control.

xv

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia bagi memenuhi syarat ijazah Master Sains

PENULENAN DAN PENCIRIAN "PYOCYANINE" DAN" I-PHENAZINOLE" DAR! PSEUDOMONAS AERUGINOSA (ENDOFIT)

Oleh

MAJID ESHAGID

Mac 1998

Pengerusi : Prof. Madya Dr. Abdul Manaf Ali

Fakulti: Sains Makanan dan Bioteknologi

Bakteria endofitik adalah organisma yang dapat hidup di dalam tisu tumbuhan.

Organisma-organisma ini dipercayai berguna untuk penyaringan farmakologi serta

rancangan pertanian disebabkan oleh fungsinya sebagai kawalan biologi untuk haiwan

perosak tumbuhan dan penyakit. Projek ini dirancang untuk menyaring bakteria

endofitik bagi tujuan farmaseutikal.

Bakteria endofitik diisolatkan daripada ranting 50 batang pokok yang berlainan

dari hutan hujan tropika di Bukit Kemensal, Ulu Kelang, Selangor. Selepas kultur tulen

bakteria dilakukan, isolat tersebut telah diuji dengan bioasai kaedah penyerapan calera

terhadap Bacillus cereus NRRL 1447 (bakteria Gram-positif), Pseudomonas aeruJ:,inosa

XVI

ATCC 60690 (bakteria Gram-negatif), Bacillus subtilis B28 dan B29 [mutan( tidak

berupaya membaiki DNA) dan jenis liar], Candida lipolytica ATCC 2075 (yis),

Sacchromyces /ipo/ytica ATCC 16617 (yis) dan Aspergillus ochraceous ATCC 398

(fungi). Daripada 79 bakteria endofit yang diasingkan, 12 bakteria telah menunjukkan

aktiviti antimikrob. Salah satu isolat bemombor 32. 1 yang menunjukkan sifat aktiviti

perencatan terbaik terhadap mikroorganisma sasaran telah dipilih untuk ujian lanjut.

Dengan penggunaan ujian biokimia, sistem ujian "multi miniaturized"(API 20NE kit)

serta keseluruhan profil sel asid lemak, isolat tersebut telah dikenalpasti sebagai

Pseudomonas aeruginosa strain UPM.

Sebatian-sebatian aktif telah ditulenkan daripada media fermentasi menggunakan

pelbagai teknik kromatografi. Dua sebatian bioaktif telah ditulen. Kedua-dua struktur

sebatian aktif ini ditentukan dengan pelbagai teknik spektroskopik termasuk

spektroskopi ultra ungu (UV), spektroskopi infra-merah (IR), spektroskopi nuklear

magnetik resonan (lH dan 13C_NMR), spektroskopi jisim (MS) dan perbandingan nilai di

dalam rujukan dan dikenalpasti sebagai pyocyanine dan I-phenazinole.

Kepekatan minima bagi perencatan untuk pyocyanine terhadap P. aeruginosa, B.

subtilis (B29) dan Staphylococcus aureus lentang metisilin (MRSA) adalah 4.6875

J.1g1disk, manakala bagi B. subtilis (B28) dan B. cereus adalah 9.375 J.1g1disk. 1 -

Phenazinole mencatatkan nilai MIC sebanyak 37 .5 J.1g1disk ke alas P. aeruginosa, C.

lipo/ytica, A. ochraceous, manakala nilai MIC sebanyak 75 J.1g1disk terhadap B. cereus,

B. subtilis (B28 dan B29), S. lipolytica dan 1 50 J.1g1disk terhadap MRSA. Dengan

XVII

demikian, pyocyanine menunjukkan spektrum aktiviti antibakteria yang luas dan

sebaliknya I-phenazinole adalah aktif terhadap semua mikrob sasaran. I-Phenazinole

menunjukkan aktiviti antimikrob yang kurang berbanding dengan pyocyanine.

Kesitosikan sebatian-sebatian tulen telah diuji terhadap sel HeLa (Human cervical

adenocarcinoma), sel fibroblas mencit-3T3 (titisan bukan kanser), sel leukemia T­

limfoblastik (CEM-SS) dan sel serangga Sf'). Kesitosikan I-phenazinole adalah lebih

tinggi berbanding dengan pyocyanine terhadap sel yang diuji kecuali CEM-SS. Aktiviti

sitotoksik sebatian-sebatian tersebut terhadap sel 3T3 adalah kurang daripada sel kanser.

Jadi, sebatian-sebatian tersebut telah menunjukkan pemilihan aktiviti sitotoksik terhadap

sel pertumbuhan. Sebatian-sebatian tersebut juga menunjukkan kesan sitotoksik ke atas

sel serangga SF9 dan justeru itu, mungkin dapat digunakan sebagai kawalan biologi.

XVIII

CHAPTER I

INTRODUCTION

Antibiotics are chemical substances produced by metabolism of living

organisms which have inhibitory activity against microorganisms and some other

animal cells, e.g., tumor cells, or viruses. The principle drugs used in the treatment of

infectious diseases fall into three categories: antibiotics, sulfonamides, and

chemotherapeutics (Murray et al., 1995). Collectively, they may be referred to as

antimicrobics. Antibiotics can be either broad-spectrum antibiotics, active against

many organisms, or narrow-spectrum antibiotics, active against only a restricted

range of organisms.

During World War n, the demand for chemotherapeutic agents to treat

wound infections led to the development of a production process for penicillin and

the beginning of the era of antibiotic research. This continues to be the most

important area of industrial microbiology today. Intensive screening programs in all

industrial countries continue to increase the number of described antibiotics: 5 1 3

antibiotics were known in 1961, 407 in 1972, 7650 in 1 985, and currently about

8000. In addition, about 3000 antibiotically active substances have been detected in

lichens, algae, higher animals, and plants. Each year, about 300 new antibiotically

active materials are detected; of which 30-35% are secondary components from

fermentation with known antibiotics. Approximately 1 0,000 different antibiotics

have been characterized, but only 123 are currently produced by fermentation (perry

2

and Staley, 1997). In addition, more than 50 antibiotics are produced as

semisynthetic compounds, and three antibiotics namely, chloramphenicol,

phosphonomycin, and pyrrolnitrin, are produced completely synthetically (Crueger

and Crueger 1990). Worldwide antibiotic production is over 100 000 tons per year

and estimated gross sales for 1980 were US$4.2 billion. The annual gross sales in the

United State alone are US$lbillion, with cephalosporin in leading position, followed

by ampicillin and the tetracycline. Feed-additive antibiotics are believed to have a

world market of US$lOO million annually. Before 1960, about 5% of the newly

isolated antibiotics were therapeutically useful. In the following years new antibiotics

were discovered at an approximately constant rate, but the percentage of the new

antibiotics which actually came on the market decreased from 2.6% in 1961-1965 to

1% in 1966-1971(Crueger and Crueger 1990). This is primarily because of severe

cost increase in development and clinical testing, thus those manufacturers produce

only those compounds that clearly show promising therapeutic progress. About ten

years are likely to elapse before an agent can be marketed, at an average cost of

US$10,000,000 to US$20,000,000 (Greengrass, 1997).

Most antibiotics are produced by bacteria, actinomycetes and fungi but

compounds may be obtained from many other living organisms such as plants,

lichens, algae and higher animal organisms (Berdy, 1985). Antibiotic producers can

be isolated from various natural sources such as soil, water, plants and animals.

Microorganisms from plants (endophytes) are one of the sources that scarcely have

been screened for pharmacological purposes but they have a good potential for

production of antibiotics (Dreyfuss, 1987).

3

Considering the very large number of known compounds, it may seem

questionable whether the search for new antibiotics should continue. The reasons for

continued research are:

• In many cases the properties of natural antibiotics are not optimal for therapeutic

application. The following improvements are needed: greater activity with

uncharged or diminished toxicity, decreased side effects, broader antimicrobial

range, greater selectivity against certain pathogens, improved pharmacological

properties.

• Suitable antibiotics are not available in many fields of human medicine or in

nonmedical areas. Numerous tests have been made of new and semisynthetic

substances, but no significant breakthroughs have been made.

• Since the beginning of chemotherapy, the number of resistant strains has

increased. Multiple- and cross-resistance can occur, i.e., if resistance develops to

one antibiotic it may simultaneously develop to others having the same mode of

operation or uptake mechanism. Careless use of antibiotics has been responsible

for much increase in resistance, but even with careful use in chemotherapy,

resistance still develops, albeit at a slower rate, Currently, the only alternative for

overcoming the resistance problem is the discovery of new antibiotics.

Improved antibiotics can be obtained by modifying known compounds using

either chemical or genetic means (mutasynthesis, protoplast fusion, and recombinant

DNA technology). However, antibiotics with entirely new basic structures can be

expected only from screening, especially by the use of new test procedures and by

research on new groups of microorganisms.

4

Based on the perspectives presented, the followings were the objectives of this

study.

• To isolate bacterial endophytes from tropical rain forest plants.

• To screen the isolates for antimicrobial activity.

• To select and identify one isolate with a broad-spectrum of antimicrobial activity.

• To purify and characterize the antimicrobial metabolites of the isolate.

CAHPTER II

LITRERATURE REVIEW

The History of Antibiotics

The pre-scientific era of antibiotics has its roots in folk medicine many years

before Pasteur, Tyndall and others. They recognized the antagonism of

microorganisms in the last third of 19th century (Florey et al., 1949). The Mayans

used a fungus known as cuxum for treatment of ulcers and intestinal infections.

Florey and co-workers (1949) found several references in the medical literature of

the 19th century on the use of microorganisms for therapeutic purposes. For example,

Mosse, in 1852, published his experiences in healing wounds with yeast.

The scientific era in the history of antibiotic can be divided into three periods.

The first period up to the early 1940s is characterized by the discovery of the first

known antibiotics, mycophenolic acid, and later by the discovery, isolation and

therapeutic use of penicillin. Florey and co-workers (1949) reported that the first

antibiotic isolated in the 1896 by Gosio was from mouldy maize. From such mouldy

maize, Gosio isolated a fungus, which he named Penicillium glaucvum, but it

probably is a strain of P. brevi-copactum. After the cultivation of this fungus, Gosio

was able to isolate a small amount of a crystalline substance from the cultivation

medium. He found that this substance was phenolic and tested its antibacterial

effects. The substance inhibited the growth of Bacillus anthracis.

6

In 1913, Alberg and Black isolated a phenolic substance from a Penicillium

of the same kind of culture. According to its color reaction, they believed that the

substance was identical with that of Gosio and named it mycophenolic acid. In 1928,

Fleming discovered an antibacterial agent, produced by a Penicillium contaminating

an agar plate culture of staphylococci. During observations on staphylococcal

colonies, he noticed that some of them, growing in the neighborhood of the

contaminating fungus, became apparently lysed. He picked the contaminating

penicillium up, grew it on the surface of nutrient broth and found that an antibacterial

substance was secreted into the medium. This broth was found to contain penicillin.

The systematic screening of actinomycetes and discoveries of the

fundamental types of antibiotics characterized the second period. In the 1940s,

systematic screening programs began in the search for actinomycetes capable of

producing antibiotics. The first screening surveys made in W aksman' s laboratory and

elsewhere, established that nearly 50% of all cultures isolated from soil (mostly

Streptomyces species) were active mainly against Gram-negative bacteria. Apart

from actinomycetin, actinomycin was the first antibiotic isolated from a culture of an

actinomycete (Waksman and Woodruff, 1941). Closely related substances were later

isolated throughout the world and tens of actinomycins are known at present.

Another antibiotic, proactinomycin was later separated into several components.

Streptothricin was isolated in the same year (Waksman and Woodruff, 1942). In

September 1943, the work of Waksman's group culminated in the isolation of

streptomycin from Streptomyces griseus (Schatz et al., 1944). Streptomycin started a

great number of studies on the production of antibiotics by actinomycetes. In 1945,

research on Streptomyces aureojaciens produced an antibiotic, aureomycin (known