LIST OF FIGURES

Figure

Title

Page

Paradigm of the Study

7 2 3 4 Gram-stained Pseudomonas aeruginosa Staphylococcus aureus Methodological Framework of the Study 17 22 36

LIST OF TABLES

Table
1

Title The Difference between Manuka Honey and Regular Honey Reference value for the zone of inhibtion of Gentamycin (+ control for Pseudomonas aeruginosa), Penicillin (+ control for Staphylococcus aureus) and Honey Zones of Inhibition of the Different Concentration of Honey against (Reference Strain) Staphylococcus aureus in Mueller-Hinton Broth Zones of Inhibition of the Different Concentrations of Honey against Staphylococcus aureus (Reference Strain) in Normal Saline Solution Zones of Inhibition of the Different Concentration of Honey against Pseudominas aeruginosa (Reference Strain) in Mueller- Hinton Broth Zones of Inhibition of the Different Concentration of Honey against Pseudominas aeruginosa (Reference Strain) in Normal Saline Solution Zones of Inhibition of the Different Concentration of Honey against Staphylococcus aureus (Clinical Isolate) in Mueller- Hinton Broth Zones of Inhibition of the Different Concentration of Honey against Staphylococcus aureus (Clinical Isolate) in Normal Saline Solution

Page 14 27

2.1

37

2.2

38

2.3

38

2.4

39

2.5

39

2.6

40

ACRONYMS USED IN THE STUDY

CFU DNA LPS LOS MHB MIC MHA MRSA mm NaCl W/v

Colony Formation Unit Deoxyribonucleic acid Lipopolysaccharides Lipooligosaccharide Mueller Hinton Broth Minimum Inhibitory Concentration Mueller Hinton Agar Methicillin Resistant Staphylococcus Aureus millimeter Sodium Chloride Weight over Volume

TABLE OF CONTENTS

Page Number

TITLE PAGE APPROVAL SHEET ACKNOWLEDGEMENT ABSTRACT TABLE OF CONTENTS LIST OF TABLE LIST OF FIGURES LIST OF PLATES

i ii iii iv v vi vii viii

Chapter

I. THE PROBLEM AND ITS SETTING

1.1 Introduction

1.2 Statement of the Problem

1.3 Null Hypotheses 1.4 Significance of the study 1.5 Theoretical Framework

4 4 5

1.6 Conceptual Framework 1.7 Scope and Delimitation 1.8 Definition of Terms

6 7 8

II. Review of Related Literature and Studies 2.1 Honey 2.2 Formation 2.3 Manuka Honey 2.4 Antibacterial Properties of Honey 2.5 The History of Medicinal Use of Honey 2.6 Apis negrocincta 2.7 Pseudomonas aeruginosa 2.7.1 Disease Association 2.7.2 Signs and Symptoms 2.8 Staphylococcus aureus 2.8.1 Disease Association 2.8.2 Duration of illness 2.5.4Transmission 2.9 Penicillin 2.9.1 Mode of Action 2.10 Gentamycin 2.10.1 Mode of Action 2.11 Related Studies 27 27 14 16 16 17 19 20 21 22 24 24 25 25 9 11

III. METHODOLOGY 3.1 Research Design 3.2 Research Locale 3.3 Research Procedure 31 31 32

IV. PRESENTATIONS, ANALYSIS and INTERPRETATION OF DATA V. SUMMARY, FINDINGS and RECOMMENDATIONS 5.1 General Summary of the Study 5.1 Summary of Findings 5.2 Conclusion 5.3 Recommendations REFERENCES APPENDICES CURRICUM VITAE

37 43 43 44 45 46 47 51

LIST OF PLATES

PLATES

TITLE

PAGE

A B C D

Preparation of Broth and Media Sample Honey Susceptibility Testing Negative Result of Antimicrobial Susceptibility Testing on Staphylococcus aureus

49 49 50 51

Negative Result of Antimicrobial Susceptibility Testing on Pseudomonas aeruginosa

51

Negative Result of Antimicrobial Susceptibility Testing on Pseudomonas aeruginosa (Reference Strain)

52

Negative Result of Antimicrobial Susceptibility Testing on Staphylococcus aureus (Reference Strain)

52

ACKNOWLEDGEMENT

We would like to express our heartfelt gratitude to the following persons who have shared the time, knowledge, and expertise throughout the conduct of this research: To our family, for their constant moral and financial support; raised us, supported us, taught us, and loved us. To them we dedicate this thesis To our research professor and panel members Dr. Adorico M. Aya-ay, Mr. Ace Ronald Sarabia, RMT, Mrs. Judee N. Nogodola, MS Bio, for their encouragement, insightful comments, and for equipping us with knowledge and guided us in the course of our study; To our adviser Ms. Avee Joy B. Dayaganon RMT, MS for, sharing her time, knowledge, and for motivating us to pursue this study; To the UIC laboratory headed by Sister Ma. Matilde Omandac RVM for, allowing the researchers to use the laboratory. To Mrs. Florence Alzate, RMT, MS for, imparting her knowledge and skill during the experiment and for her noteworthy advises in the making of the manuscript; To Mr. Rigil Kent Batistil, Richard Catulong and Shiela Mae Dauz who assisted us in performing the experiment especially in the preparation of materials needed. To Mr. Ponciano Almonte, who helped us and supported in this study, To the Almighty God who is our source of wisdom, without Him none of these would have been possible.

To those who have contributed their effort, time, knowledge, skill and showed simple acts of kindness that determined the success of this study, thank you very much.

M.B.II L.L.A.C M.A.G D.C.P

ABTRACT

TITLE RESEARCHERS

Antimicrobial Activity of Honey Against Staphylococcus aureus and Pseudomonas aeruginosa Breva, Mario II Crispo, Loigie Loraine Gayagas, Marwil Paderog, Danneth

TYPE ADVISER SUBJECT AREA CONFERRED DEGREE

Experimental Type Avee Joy Dayaganon, RMT, MS Medical Bacteriology Bachelor in Medical Laboratory Science

Ayurveda a group of Indian surgeon describes honey as the nectar of life and recommends its use in various ailments. The medicinal properties of honey have been known since ancient times but there was no recognition of its antibacterial properties, it was just known to be an effective remedy. This study was undertaken to extend existing knowledge about the effectiveness of the antibacterial properties of honey against Staphylococcus aureus and Pseudomonas aeruginosa. In this study two organisms were used, a Staphylococcus aureus and Pseudomonas aeruginosa, each bacterium was subjected for testing to different concentration (40%, 60%, 80%, and 100%) of same type of honey. Each sample has three replicates to test samples and examined under the supervision of our adviser. Honeys efficacy does not only depend on its high osmolality but it also depends on what type of organism has been healed. The antibacterial action of honey against

Staphylococcus aureus relies on the capacity of bacteria to ferment d-gluconic acid which is present in high concentration of honey. The result showed that honey is not effective antibacterial agent against

Staphylococcus aureus.

On the other hand antibacterial action of honey against

Pseudomonas aeruginosa does not depend on the fermentation process but it varies on the ability of Pseudomonas aeruginosa to produce a large number of extracellular protective and toxic substances. Some of these substances are slime glycolipoprotein, DNAse, and phospholipase that contribute to the pathogenicity of this opportunistic microorganism. The researchers highly recommend a more comprehensive study involving the determination of efficacy of honey as an antimicrobial agent.

CURRICULUM VITAE

Name: Mario B. Breva II Age: 19 Contact number: 09297142545

Personal Profile:

City Address: #5 Vermillion St. Spring Village Ma-a, Davao City Birth date: September 17, 1991 Nationality: Filipino Religion: Roman Catholic Civil status: Single Email Address: brevaii@yahoo.com Height: 55 Weight: 60 kg Sex: Male

Academic Background College: University of the Immaculate Conception (2008-Present) Secondary: University of the Immaculate Conception (2004-2008) Elementary: University of the Immaculate Conception (1998-2004)

CURRICULUM VITAE

Name: Loigie Loraine A. Crispo Age: 20 Contact number: 09072595624

Personal Profile: Provincial Address: Townsite Parang, Maguindanao City Address; General Luna St. Davao City Birth date: July 4, 1991 Nationality: Filipino Religion: Roman Catholic Civil status: Single Email Address: Gie_raine04@yahoo.com Height: 51 Weight: 50 kg Sex: Female

Academic Background College: University of the Immaculate Conception (2008present) Secondary: Notre Dame of Parang (2003-2007) Elementary: Notre Dame of Parang (19972003)

CURRICULUM VITAE

Name: Danneth Paderog Age: 24 Contact number: 09077960006

Personal profile: Provincial Address: Andap New Bataan Comval Province City Address: Buhangin Davao City Birth date: October 13,1986 Nationality: Filipino Religion: Roman Catholic Civil status: Single Email Address: danneth_padirog@yahoo.com Height: 54 Weight: 47 kg Sex: Female

Academic Background College: University of the Immaculate Conception Secondary: New Bataan National High School Elementary: Cabinuangan Central Elemantary School

CURRICULUM VITAE

Name: Marwil A. Gayagas Age: 20 years old Contact number: 09261271787

Personal Profile:

Provincial Address: Matalam North, Cotabato City Address: Fr. Selga St. Davao City Birth date: May 31, 1991 Nationality: Filipino Religion: Roman Catholic Civil status: Single Email Address: marwil_thepink@yahoo.com Height: 57 Weight: 62 kg Sex: Male

Academic Background College: University of Immaculate Conception (2008-Present) Secondary: Matalam National High School (2004-2008) Elementary: Linao Central Elementary School (1998-2004)

APPROVAL SHEET

This thesis entitled The Antimicrobial Activity of Honey against Staphylococcus aureus and Pseudomonas aeruginosa submitted by Mr. Mario B. Breva II, Ms. Loigie Loraine A. Crispo, Mr. Marwil A. Gayagas, and Ms. Danneth C. Paderog in partial fulfillment of the requirements for the degree of Bachelor of Science in Medical Laboratory has been examined and is recommended for oral examination, acceptance and approval.

Avee Joy B. Dayaganon, RMT, MS Adviser

PANEL OF EXAMINERS

Adorico M. Aya-ay, Ph.D. Chairman Judee N. Nogodula, MS Bio Member Ace Ronald C. Sarabia, RMT Member

Accepted and approved in partial fulfillment of the requirements for the degree of Master of Science in Medical Laboratory.

Avee Joy B. Dayaganon, RMT, MS Dean of Medical Laboratory Science Program

APPENDICES

Appendix A Reagents and Media

A. 1 Preparation of Mueller Hinton Agar Thirty four (34) grams of medium was dissolved in one liter demineralized water by heating in a boiling water bath and autoclave for 15 minutes at 121C (Forbes et al, 1998). A. 2 Preparation of Mueller Hinton Broth Twenty one (21) grams of Mueller Hinton Broth in 1 liter distilled water was mixed and heated with frequent agitation and boiled for 1 minute, then sterilized to 121 C for 15 minutes (Forbes et al, 1998). A. 3 Preparation of 0.5 McFarland Standard 0.5 mL of 0.048 M BaCl2 (1.175% w/v BaCl2 H2O) was mixed with 99.5 mL 0.3% N H2SO4 (1%v/v), 5 mL and distributed to sterile screw cap tubes, thightly sealed, and stored in dark place at room temperature (Forbes et al, 1998)..

Results using Reference Strain

Concentratio n of Honey 20% 40% 60% 80% 100% Concentratio n of Honey 20% 40% 60% 80% 100% Concentratio n of Honey 20% 40% 60% 80% 100% Concentratio n of Honey 20% 40% 60% 80% 100% Staphylococc us aureus in MHB 6 mm 6 6 6 6 mm mm mm mm Interpretatio n Resistant Resistant Resistant Resistant Resistant Interpretatio n Resistant Resistant Resistant Resistant Resistant Interpretatio n Resistant Resistant Resistant Resistant Resistant Interpretatio n Resistant Resistant Resistant Resistant Resistant Penicillin ( + control) 29 mm 31 mm 29.5 32 mm 32.2 mm Penicillin ( + control) 31 mm 32.6 mm 29.6 mm 29 mm 31 mm Gentamycin ( + control) 15 mm 15.5 mm 17 mm 16 mm 15.7 mm Gentamycin ( + control) 16 mm 17.5 mm 17 mm 15 mm 16.5 mm Interpretatio n Susceptible Susceptible Susceptible Susceptible Susceptible Interpretatio n Susceptible Susceptible Susceptible Susceptible Susceptible Interpretatio n Susceptible Susceptible Susceptible Susceptible Susceptible Interpretatio n Susceptible Susceptible Susceptible Susceptible Susceptible

Staphylococc us aureus in NSS 6 mm 6 6 6 6 mm mm mm mm

Pseudomona s aeruginosa in MHB 6 mm 6 6 6 6 mm mm mm mm

Pseudomona s aeruginosa in NSS 6 mm 6 6 6 6 mm mm mm mm

Results Using Clinical Isolates

Concentratio n of Honey 20% 40% 60% 80% 100%

Staphylococc us aureus in MHB 6 mm 6 6 6 6 mm mm mm mm

Interpretatio n Resistant Resistant Resistant Resistant Resistant

Penicillin ( + control) 31 mm 32.6 mm 29.6 mm 29 mm 31 mm

Interpretatio n Susceptible Susceptible Susceptible Susceptible Susceptible

Concentratio n of Honey 20% 40% 60% 80% 100%

Staphylococc us aureus in NSS 6 mm 6 6 6 6 mm mm mm mm

Interpretatio n Resistant Resistant Resistant Resistant Resistant

Penicillin ( + control) 31 mm 32.6 mm 29.6 mm 29 mm 31 mm

Interpretatio n Susceptible Susceptible Susceptible Susceptible Susceptible

Approved by: Ms. Avee Joy B. Dayaganon, RMT, MS Program Dean/Thesis Adviser

APPENDIX B

A. Preparation of Reagents and Media

B.

Sample Honey

C. Susceptibility Testing

D.

Negative Result of Antimicrobial Susceptibility Testing on Staphylococcus aureus

DI.

E. Negative Result of Antimicrobial Susceptibility Testing on Pseudomonas aeruginosa

F. Negative Result of Antimicrobial Susceptibility Testing on Pseudomonas aeruginosa (Reference Strain)

G. Negative Result of Antimicrobial Susceptibility Testing on Staphylococccus aureus (Reference Strain)