In vitro antibacterial activity of Quercus Infectoria gall extracts against multidrug resistant bacteria

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by

MASRAH BINTI MALIK

Dissertation submitted in partial fulfillment of the requirements forthe Degree of Bachelor ofHealth Sciences (Biomedicine)

2013

INVITROANTIBACTERIAL ACTIVITY OF Quercus mfectoria GALLEXTRACTS AGAINST MULTIDRUGRESISTANT BACTERIA

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CERTIFICATE

This is to certify that the dissertation entitled “Zn vitro antibacterial activity of Quercus infectoria gall extracts against multidrug resistant bacteria” is the bona fide record of research work done by Miss Masrah Binti Malik during the period from July 2012 till June 2013 undermysupervision.

Supervisor,

Dr. Wan Nor Amilah bt Wan Abdul Wahab Lecturer

School of HealthSciences Health Campus

Universiti SainsMalaysia

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ACKNOWLEDGEMENT

In the nameofAllah, theMostGracious and the Most Merciful

Alhamdulillah, all praises to Allah for His blessings as He gave me strength, courage and health to conduct this research and complete my dissertation well. Special appreciation goes to my supervisor who let me experience the microbiology research works beyond the textbooks. Dr. Wan Nor Amilah. A million thanks for all of her guidance, constant encouragement and moral support, patience and critical appraisal at every stage of this project. Her invaluable help of constructive comments and recommendations throughout my research and dissertation works have contributed to the successofthisproject.

I would also like to express my deepest gratitude and acknowledgement to Dr.

See Too Wei Chun, the Coordinator of final year student research project for always being understanding and toleranceto us in completing ourproject.

My acknowledgement also goes to the laboratory staffs of Department of Microbiology and Parasitology, School ofMedical Sciences especially Pn. Rosliza, Pn.

Roziawati, Pn. Nur Fasihah and Pn. Amanina, laboratory staffs of School of Health Sciences especially Pn. Wan Razlin and Cik Kurunisa and all the postgraduatestudents especially Lukman, Zaharaini and Nurzila for sharing their immense knowledge and co­ operations that they gave me during my research works.

Sincere thanks to all my friends especially Fatimah, Amira and Atiqah for their kindness and moral support during my research works and throughout completing this dissertation. Thanksforthe friendship and memories.

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Last but not least, my deepest gratitude goes to my beloved parents, Mr. Malik BinAli and Mrs. Hjh. Hajirah Bt. Kadir and also to my siblings for their endless love, prayers and encouragement. To those who indirectly contributed in my project, your kindnessmeans a lot to me. Thankyou ven- much.

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TABLE OF CONTENTS

Page CERTIFICATE ii

iii ACKNOWLEDGEMENTS

v TABLEOF CONTENTS

vii LIST OFTABLES

viii LISTOF FIGURES

ix LIST OF PLATES

x LISTOF ABBREVIATIONS

xi

ABSTRAK

xiii ABSTRACT

1.0INTRODUCTION 1-4

1.1 Background of study 1 1.2 Problem statement 2 1.3 Rationaleofstudy 3

1.4 Objectives of study 3 2.0 LITERATURE REVIEW 5-14

2.1 Quercus infectoria 5

2.2 Medicinalplants extraction 6

2.3 Antibacterial activity ofQuercus infectoria galls extracts 7 2.4 MDR bacteria and mechanism of resistance 8 2.5 Emergence of MDR bacterial infections 10

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2.6 Antimicrobial susceptibilitytesting (AST) 13

15-20 3.0 METHODOLOGY

3.1 Study design 15

3.2 Equipment and instrument 15 3.3 Material 16

3.4 Plant materials andpreparation of crudeextracts 17 3.5 Bacterial strains 18

3.6 Screening of antibacterial activity 18 3.7Determinationof MIC and MBC 19 3.8 Statistical analysis 20

21-39 4.0 RESULTS

4.1 Disc Diffusion Test 21

4.2 Minimum Inhibitory Concentration (MIC) 34 4.3 Minimum Bactericidal Concentration (MBC) 38

4.4 Summary of MIC and MBC 39

5.0DISCUSSION 40

6.0 CONCLUSION 46

REFERENCES 47

APPENDICES 57

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LIST OF TABLES

Page Table 3.1: Equipment and instrument used 15

1

Table 3.2: Materialsused 16 2

3 21

23 4

5 25

6 27

7 29

34 8

9 Table 4.7: Determination of MBC values of Q. infectoria galls 38 extractsagainst MDR bacteria

10 39

Table 4.2: The mean of inhibition zones diameter of different concentrations of ethanol extracts from galls of Q. infectoria against MDR bacteria

Table 4.5: Inhibitionzonesdiameter of 5 mg/disc (250 mg/ml) extracts from galls ofQ. infectoria against MDR bacteria

Table 4.3: Inhibition zones diameter of I mg/disc (50 mg/ml) extracts from galls ofQ. infectoria against MDR bacteria

Table 4.4: Inhibition zones diameter of 2 mg/disc (100mg/ml) extracts fromgalls of Q. infectoriaagainstMDR bacteria

Table 4.8: Summary of MIC and MBC values of Q. infectoria galls extracts againstMDRbacteria

Table 4.1: The mean of inhibition zones diameter of different concentrations of aqueous extracts from galls ofQ. infectoria against MDR bacteria

Table 4.6: Determinationof MIC values ofQ. infectoria galls extracts againstMDR bacteria

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LTST OF FIGURES

Page

Figure2.1; Gallsof Q. infectoria 5

1

Figure 2.2: Part ofQ. infectoriaplant 6 2

3 22

4 24

5 26

6 28

7 30

Figure 4.1: The mean of inhibition zones diameter of different concentrations of Q. infectoria aqueous galls extracts against MDR bacteria

Figure 4.2: The mean of inhibition zones diameter of different concentrations of Q. infectoria ethanol galls extracts against MDR bacteria

Figure 4.5: Comparison of the mean of inhibition zones diameter of between 5 mg/disc (250 mg/ml) aqueous and ethanol extracts from galls of Q. infectoria against MDR bacteria

Figure 4.4: Comparison of the mean of inhibition zones diameter between 2 mg/disc (100 mg/ml) aqueous and ethanol extracts from galls of Q. infectoriaagainst MDR bacteria

Figure 4.3: Comparison of the mean of inhibition zones diameter between 1 mg/disc (50 mg/ml)aqueous and ethanol extracts from galls of Q. infectoriaagainstMDR bacteria

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LIST OF PLATES

Page Plate 3.1’. Determination ofMIC value

1 20

Plate 3.2: Determination of MBCvalue

2 20

3 31

4 32

5 35

6 35

7 36

8 36

9 37

Plate 4.1: Disc Diffusion test of 1 mg/disc (50 mg/ml) and 2 mg/disc (100 mg/ml)extracts from galls ofQ. infectoria against MDR bacteria

Plate 4.3: Determination of MIC values of Q. infectoria galls extracts against MRSA

Plate 4.7: Determination ofMIC values of Q. infectoria galls extracts against ESBL K. pneumoniae

Plate 4.2: Disc Diffusion test of5 mg/disc (250 mg/ml) extracts from galls of Q. infectoriaagainstMDR bacteria

Plate 4.6: Determination of MIC values of Q. infectoria galls extracts against ESBL E. coli

Plate 4.5: Determination ofMIC values ofQ. infectoria galls extracts againstMDR Acinetobacter sp.

Plate 4.4: Determination of MIC values ofQ. infectoriagalls extracts against MRCoNS

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LIST OF ABBREVIATIONS

AST- Antibiotic susceptibility testing

CDC - Centre forDisease Controland Prevention CLSI- Clinicaland Laboratory Standards Institute DMSO-Dimethyl sulfoxide

ESBL - Extended spectrum beta lactamase

HA-MRSA -Hospital acquired- methicillin resistant Staphylococcus aureus MBC — Minimum bactericidal concentration

MDR - Muliidrug resistant MHA— Mueller Hinton agar MHB - Mueller Hinton broth

MIC - Minimum inhibitoryconcentration

MRCoNS - Methicillin resistant coagulase negative Staphylococcus MRSA - Methicillin resistant Staphylococcus aureus

CA-MRSA- Community acquired - methicillinresistant Staphylococcus aureus

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ABSTRAK

Kajian mengenai aktiviti antimikrob daripada tumbuh-tumbuhan telah lama dijalankan untuk mengenalpasti potensi ubat yang selamat digunakan pada masa hadapan bagi mengurangkan kesan rintangan mikroorganisma yang tidak diingini.

Kajian ini dijalankan untuk menilai aktiviti antibakteria ekstrak biji manjakani (Q.

infectoria) terhadap isolat klinikal bakteria rintangan pelbagai dadah (MDR) melalui

pencairan mikro bersiri bergandapada kepekatan antara 5.00 mg/ml - 0.01 mg/ml dan kepekatan bakterisidal minimum (MBC). Tiga kepekatan yang berbeza (1, 2 dan 5 mg/disk) daripada ekstrak akuas dan etanol biji manjakani tclah digunakan untuk perbandingan kepekatan disk optimum semasa ujian saringan dengan lima isolat klinikal bakteria MDR iaitu MRSA, MRCoNS, MDR Acinetobacter sp., ESBL E. coli danESBL K. pneumoniae. Kami mcndapati bahawa. diameterpercncatan zon 5 mg/disk adalah lebih besar daripada diameterzonperencatan 1 mg/disk ekstrak akuas terhadap MRSA, MRCoNS dan MDR Acinetobacter sp. Begitu juga, terdapat perbezaan yang signifikan dari diameter zon diperhatikan antara2 mg/disc dan 5 mg/disc ekstrakakuas terhadap ketiga-tiga jenis isolat. Untuk ekstrak etanol, diameter zon perencatan adalah lebihbesar diperhatikan dengan 5 mg/disk terhadap MRSA dan MDR Acinetobacter sp.

berbanding dengan 1 mg/disk. MRCoNS adalah bakteria yang paling menunjukkan kesan perencatan tertinggi diikuti oleh MRSA. Kedua-dua ekstrak menunjukkan kesan perencatan yang lemah terhadap MDR Acinetobactersp. manakala tiada zon perencatan

perencatan yang signifikan terhadap MRSA dan pada 5 mg/disc; terdapat perbezaan yang ketara dalam saiz zon antara kedua-duaekstrak juga diperhatikan dalam MRCoNS (p <0.05). Nilai MIC dan MBC daripada ekstrak adalah dari 0.08 mg/ml - 2.50 mg/ml.

dirckodkan untuk isolat ESBLs. Kesemua tiga kepekatan ekstrak menunjukkan saiz zon kaedah penentuan kepekatan perencatan minimum (MIC) menggunakan teknik

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Nilai untuk MIC ckstrak akuas dan ctanol tcrhadap MRSA masing-masing adalah 0.08 mg/ml dan 0.16 mg/ml manakala nilai MIC untuk kcdua-dua ckstrak tcrhadap MRCoNS adalah sama (0.08 mg/ml). Nilai MBC ekstrak akuas terhadap MRSA dan MRCoNS adalah lebih tinggi daripada nilai MIC manakala nilai MBC ekstrak etanol terhadap MRSA dan MRCoNS adalah sama dengan nilai MIC. Nilai MBC kedua-dua ekstrak terhadap semua bakteria Gram negatif adalah sama dengannilai MICiaitu MDR Acinetobacter sp. (0.63 mg/ml), ESBL E. coli (2.50 mg/ml) dan ESBL K. pneumoniae (1.25 mg/ml). Biji manjakani adalah sumber yang berpotensi baik sebagai ejen antimikrob kerana keberkesanan dalam aktiviti antibakteria terhadap bakteria rintangan pelbagai dadah.

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ABSTRACT

Antimicrobial activities of plants have long been evaluated for their potential

microorganisms. The study was conducted to evaluate the antibacterial activity of Quercus infectoria gall extracts against multidrug resistance (MDR) bacterial clinical isolatesby determination ofminimum inhibitory concentration (MIC) usingthe twofold serial microdilution technique at concentration ranging from 5.00 mg/ml to 0.01 mg/ml and minimum bactericidal concentration (MBC) values. Three different concentrations (1, 2 and 5 mg/disc) of aqueousand ethanol extracts of Q. infectoria galls were used for comparison of optimum disc concentration during screening test with five MDR bacterial clinical isolates namely MRSA, MRCoNS, MDR Acinetobacter sp., ESBL E.

coli and ESBL K. pneumoniae. We found that, the inhibition zones diameter of 5 mg/disc were significantly larger than inhibition zones diameter of 1 mg/disc aqueous extract against MRSA, MRCoNS and MDRAcinetobacter sp. Similarly, the significant difference ofinhibitory zone diameter was observed between 2 mg/disc and 5 mg/disc of aqueousextract against all three isolate strains. For ethanol extract, larger inhibition zones diameter was observed with 5 mg/disc diffusion plate of MRSA and MDR Acinetobacter sp. as compared to 1 mg/disc diffusion plate. Among all tested bacteria, MRCoNS was the most susceptible followed by MRSA during screening. Both extracts

inhibition zone observed for ESBLs isolates. All of the three concentrations of extracts showed inhibition zone size which was significantly different against MRSA and at 5 mg/disc; there was a significant difference in the zone sizes between both extracts was also observed in MRCoNS (p< 0.05). The MIC and MBC values ofthe extracts ranged from 0.08 mg/ml to 2.5 mg/ml. The MIC values for aqueous and ethanol extracts showed weak inhibitory effects against MDR Acinetobacter sp. while there was no safe remedies in the future to minimize the unwanted resistance effects of

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against MRSA were 0.08 mg/ml and 0.16 mg/ml respectively whereas the MIC value for both extracts against MRCoNS were the same (0.08 mg/ml). The MBC values of aqueous extracts against MRSA and MRCoNS were above their MIC values whereas

the MBC values of ethanol extracts were the same with the MIC values against MRSA and MRCoNS. MBC values ofboth extracts against all Gram negative bacteria tested were the same with their MIC values; MDR Acinetobacter sp. (0.63 mg/ml), ESBL E.

coli (2.50 mg/ml) and ESBL K. pneumoniae (1.25 mg/ml). The Q. infectoria gall extracts may be considered as apotentially good source of antimicrobial agents due to theeffectiveness in their invitro antibacterial activityagainst MDRbacteria.

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