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Republic of Iraq Ministry of Higher Education and Scientific Research Baghdad University College of Science

Study the Effect of Antibiotics and Propolis on Pathogenicity of The Methicillin

Resistant Staphylococcus aureus (MRSA)

A Thesis Submitted to the College of Science /University of

Baghdad in Partial Fulfillment of the Requirements for the Degree of Master of Science in

Biology/Microbiology

By

Elaf Basim Nori AL-Sheikh B.Sc. in Biology / University of Baghdad /College of Science

(2011)

Supervised by:

Assist.prof.Dr.Hanaa Saleem Yossef

Mahram /1435 November /2013

G هل ا الرحمن

S الرحيل

يؤتي الحكمة من يشاء ومن يؤت [الحكمة فقد أوتي خيرا كثيرا وما

]يذكر إال أولو األلباب

هل ا العظيل

) ۲٦۹(سورة البقرة / اآلية

Supervisor Certification

I certify that this thesis was prepared under my supervision at the

Department of Biology / College of Science / University of Baghdad, as

a partial requirement for the degree of Master of Science in

Microbiology.

Signature: Asst.prof. Dr.Hanaa S. Yossef Supervisor Biology Department College of Science University of Baghdad Date: In view of the available recommendation, I forward this thesis for debate by the examining committee.

Signature: prof. Dr.Sabah N.Alwachi Head of Biology Department College of Science University of Baghdad Date:

Committee Certification

We, the examining committee, certify that we have read this thesis

and examined the student in its contents and that in our opinion; it is

adequate for awarding Degree of Master of Science in Microbiology.

Dr. Rajwa H.Essa

Chairman (Assistant Professor) / /2013

Dr. Sameer Abdul Ameer Abid Ali Alash Dr. Rana Saadi Aboud

Member (Lecturer) Member (Assistant Professor) / /2013 / /2013

Dr. Hanaa Saleem yossef

Supervisor (Assistant professor) / /2011

Approved by The Council of The College of Science, University of

Baghdad.

Prof.Dr. Saleh M. Ali

(Professor) Dean of College of Science University of Baghdad / /2013

declaration

This is to certify that the Desertation entitled:

“Study the Effect of Antibiotics and Propolis on Pathogenicity of The Methicillin Resistant Staphylococcus aureus (MRSA)”.

Submitted by:

elaf basim nori al-sheikh

Department of: Biology

College of: Science

has been linguistically corrected and its language in its

present form is acceptable.

dr. nazar aziz auda

Department of Biology,

College of Science, University of Baghdad

2013

Dedication

To

I introduce my work with respect.

@Elaf

Whom who gave me their endless love,

Whom gave me their patience and support,

Who lighten my way to achieve this work,

Acknowledgements

Thanks for Al-mighty God for his generosity and mercy. I would like to sincerely

thank and express of my great appreciation, heart felt gratitude and thankful to my

supervisor “Dr.Hanaa saleem ” for his scientific guidance, recommendation, advice,

encouragement and support, also I am grateful for his patience, generosity and

kindness during the all period of this research.

I would like to express of my profound thanks to the all staff members Department of

Biology, for their kindness, help and encouragement.

I express of my special thanks to the all members and staff in the Bacteriology Lab.

of AL-Kindy Teaching Hospitals, for their help in the collection of clinical

specimens and help in the identification of isolates.

Also I would like to express of deeply thanks and grateful to my friend “Dr. Thura

awad ” for her helpful, generosity and kindness.

My special, great and deepest love, thankful and grateful go to the most persons that

I love, whom without their patience, understanding, support and most of all love, the

completion of this work would not have been possible, “my lovely Family”.

Finally, I would like to thank everyone that helps me in some way or another during

my research work.

@Elaf

summary

This study included collection of 100 swab specimens from

patients in AL-Kindy Teaching Hospital and teaching laboratories of

Medical City Hospitals in Baghdad during the period from August to

December 2012 , these swab specimens differed in their sources which

included 19 nasal swab, 16 wound swab, 27 burn swab, 7 pus swab, 15

sputum swab, 10 corneal swab and 6 urine swab . Only 38 (38%) isolates

were identified as Staphylococcus aureus, 38 isolates of them (82.6%)

were coagulase-positive (COPS), while 8 isolates(17.3%) were coagulase

negative (CONS), from total 46 isolates of Staphylococci.

The distribution of Methicillin resistance among Staphylococcus

spp isolates was investigated by disc diffusion method. In this study, 21

isolates (55.26%) out of 38 (38%) isolates were identified as

Staphylococcus showed to be resistant to the Methicillin while 17 isolates

(44.73%) were sensitive. The highest rate of Methicillin resistance

Staphylococci were obtained from wound and pus swabs.

The results obtained from antimicrobial sensitivity test showed that

MRSA isolates were resistant to many other antimicrobial agents in

addition to the Methicillin (Multi-drug Resistant).

Antimicrobial activities of different antimicrobial agents include

(crude Ethanolic Extract Propolis (EEP)), Lysostaphin, Ciprofloxacin and

Vancomycin) were tested singly and in combination against selected

isolates (MRSA S3) by using agar disc diffusion assay. Results revealed

that S. aureus (MRSA- S3) was more sensitive to 2 µg/ml concentration

of EEP than other antimicrobial agents used in this study.

However, antibacterial activity of the other three reagents were

significantly less than propolis, where MRSA isolate showed resistance

for their low concentrations. In comparing the pharmacodynamics of

lysostaphin, vancomycin and Ciprofloxacilin against selected isolate

(MRSA -S3) there was no significant differences with (P>0.05) between

those drugs at the diameter of inhibitions zone which were (11, 11and

10mm) respectively, the effected concentration of them were

(5.625µg/ml,16µg/ml and 3%).

The results showed a synergistic effect of this combination on the

selected isolate (MRSA- S3), as well as, same results obtained when

combined propolis with ciprofloxacin. The bacterial growth was

inhibited with elevation of inhibition zone to (11, 13mm) respectively,

while selected isolate (MRSA-S3) showed ahigh resistance against the

combination of vancomycin with (propolis and lysostaphin ) ,so they

effects was antagonistic to S. aureus.

The ability to produce slime layer by MRSA isolates was also

investigated and the results showed that all isolates of MRSA have

produced a slime layer when tested by tube method, but the amounts of

adherent materials were differ among the isolates. However, the results

by Congo red agar method showed that 57% of MRSA isolates produced

strong slime layer and 43% of MRSA revealed negative result. Similarly

the ability of MRSA to produce biofilm by tissue culture plate (TCP) was

investigated and the results indicated that MRSA isolates showed strong

ability to form abiofilm , and the OD value of biofilm formation ranged

between (0.262 - 0.311nm). moreover the OD value of biofilm formation

significantly increased after addition of 1% glucose to the media.

The Antimicrobial activity of propolis and lysostaphin on the

biofilm formed by MRSA isolates were investigated and the results

showed that propolis at MIC (2µg/ml) were significantly inhibited the

biofilm formation by MRSA S3 isolates when used alone, the optical

density significantly (P<0.05) decreased to (0.027nm) in comparison with

control group (0.3875nm).

Statistical analysis showed a slightly effect of lysostaphin under

therapeutic concentration (5.625µg/ml ) on biofilm formation ability of

(MRSA- S3) optical density was reduced to (0.312 nm) in compared to

control group (0.389nm). On the other hand, the results showed that the

synergistic combination of propolis with (lysostaphin, ciprofloxacin) at

therapeutic concentration were inhibited the biofilm formation by

selected isolates.

Histo-pathological effect of different concentrations of propolis,

lysostaphin and ciprofloxacin on the keratitis treatment induced in the

rabbit eyes was studied. Organs of rabbits (eyes) showed different

pathological changes in a corneas treated with 2µg/ml propolis alone

were significantly less inflamed than untreated MRSA S3–infected

corneas. The corneal epithelium and endothelium looked like an

uninfected cornea, clinical signs of keratitis in the experimental rabbit

were completely disappeared with score 0: they had normal and healthy

appearance compared with negative control group (uninfected eyes).

Corneas treated with lysostaphin were highly edematous in

comparison to uninfected eyes. Histopathological changes showed highly

neutrophils infiltrations with highly damaged endothelial layer, corneas

treated with lysostaphin had similar appearance to untreated corneas.

On the other hand, when combined propolis with lysostaphin and

propolis with ciprofloxacin results induced amoderate recovering in the

infected eyes, the pathological changes included moderate corneal

ulceration, conjunctively edema, and minor accumulation of fibrin in the

anterior chamber were the primary changes noted in those treated groups,

than untreated corneas. While when compared the current results with a

healthy cornea, it was found that the infected group still inflamed after

treated with reagents combinations, also there’s significant (P<0.05)

difference in comparable to un treated eyes.

List of contents Subject Page No. Summary I List of Contents 12 List of Figures 12 List of Tables ۱۲ List of Abbreviations 12

Chapter One : Introduction and Literature Review

Series Subject Page No.

1 Introduction & Literature Review 1

1-1 Introduction 1 1-2 Literature Review 4 1-2-1 Staphylococcus 4 1-2-2 Methicillin Resistant Staphylococci 4 1-2-3 Mechanism of Methicillin Resistance 6 1-2-4 Pathogenesis of Methicillin Resistance Staphylococci 8

1-2-5 Virulence factors 9 1-2-6-A Therapy of Methicillin-Resistant Infection 11

1-2-6-B Mechanism of Resistance Staphylococci to

Antimicrobial agents (Multi-drug Resistant

Staphylococci)

13

1-2-7 Biofilm and Slime layer and their role in the Pathogenesis

14

1-2-8 Mechanism of Boifilm Formation 16 1-2-9 Keratitis 17 1-2-10 Lysostaphin 19 1-2-11 Propolis 20

Chapter Two: Materials and Methods


2 Materials and Methods 23

2-1 Materials 23

2-1-1 Laboratory Equipments and Apparatus 23 2-1-2 Chemicals and biological materials 24 2-1-2-3-1 Antibiotic discs 26 2-1-2-4-2 Antibiotic Powders 26 2-1-3 Culture Media 27 2-1-3-1 Ready-made media 27 2-2 Methods 27 2-2-1 Culture Media preparation 27

2-2-1-1 Ready to use culture media 27 2-2-1-2 Laboratory Prepared Media 27 2-2-1-2-A Acetoin production media 27

2-2-1-2-B Blood Agar 28 2-2-1-2-C Tributyrin Agar 28 2-2-1-2-D Skim-Milk Agar 29 2-2-1-2-E Urea Agar medium 29 2-2-1-2-F Nutrient Gelatin medium 30 2-2-1-2-G Congo-red Agar 30 2-2-2 Stains, Reagents and Solution 31 2-2-2-1 Gram Stain Kit 31 2-2-2-2 Catalase reagent 31

2-2-2-3 Oxidase reagent 31 2-2-2-4 Nitrate test reagent 31 2-2-2-5 Acetoin production test reagent 32

2-2-2-6 Safranin stain solution 32

2-2-2-7 McFarland standard solution 32 2-2-2-8 Phenol red 33 2-2-3 Staphylococcus aureus isolation and identification 33 2-2-3-1 Specimen's collection 33 2-2-3-2

Staphylococcus aureus isolation 33

2-2-3-3 Identification of Staphylococcus aureus 34 2-2-3-3-A Microscopic examination 34 2-2-3-3-B Cultural characteristics 34

2-2-3-3-B-I Growth on mannitol salt agar 34

2-2-3-3-B- II detection of hemolysis on human blood agar 34

2-2-3-3-C Biochemical tests 34

2-2-3-3-C-1 Catalase test 34

2-2-3-3-C-2 Oxidase test

34

2-2-3-3-C-3 Coagulase test 35

2-2-3-3-C-4 Acetoin production test 35

2-2-3-3-C-5 DNase production test 35

2-2-3-3-C-6 Mannitol fermentation 35

2-2-3-3-C-7 Gelatin liquefaction 36

2-2-3-3-C-8 Protease production 36

2-2-3-3-C-9 Nitrate reduction test 36 2-2-3-3-C-10 Urease test 36

2-2-3-3-C-11 Autoanalyzer staph system(vitak- 2 Compact)

37

2-2-3-3-C-12 Antibiotic susceptibility test 37

2-2-3-4 Preservation of bacterial isolates 39

2-2-4 Effect of (lysostaphin and propolis) singly and in

combination with antibiotics, against the multi-drugs

resistant Staphylococcus aureus

40

2-2-4- 1 Prepareation of stoch solutions 40

2-2-4- 1 –A Prepareation of stoch solutions( singly

concentrations of antimicrobial)

40

2-2-4-1-A-1 Prepareation of lysostaphin stoch solution 40

2-2-4- 1-A-2 Prepareation of vancomycin stock solution 40

2-2-4- 1-A-3 Prepareation of ciprofloxacin stock solution 40

2-2-4- 1-A-4 Propolis 41

2-2-4- I- A-4-1 Propolis collection 41

2-2-4-1-A-4-2 Ethanolic Extract of Propolis (EEP)

41

2-2-4-1-A-4-3 High-performance liquid chromatography analysis

of propolis(HPLC)

41

2-2-4-1-A-4-4 Detection of propolis structure by using Fourier transform infrared (FTIR)

43

2-2-4-1-B Preparation of stoch solutions (combination


43

2-2-4-1-B-1 The combination of lysostaphin and vancomycin 43

2-2-4-1-B-2 The combination of propolis and ciprofloxacin 44

2-2-4-1-B-3 The combination of propolis and lysostaphin 44

2-2-4-1-B-4 The combination of propolis and vancomycin 44

2-2-4-2 procedure for detection of the antimicrobial activity 45

2-2-4-2-A disc diffusion method with some modifications 45

2-2-4-2-B Minimum inhibitory concentration (MIC) 46

2-2-5 Detection of the bacterial ability to produce slime

layer and Biofilm formation

46

2-2-5-1 Congo-red agar method 46

2-2-5-2 Tube Method 47

2-2-5-3 Tissue culture plate method (TCP) 47

2-2-6 Effect of Lysostaphin, propolis and antibiotics

singly and in combination on MRSA biofilm

49

2-2-7 Antimicrobial activity in Treatment of Experimental

Staphylococcal Keratitis

50

2-2-7-1 Bacteria 50

2-2-7-2 Induction of experimental S. aureus keratitis 50

2-2-7-3 Treatment 51

2-2-7-4 Determination of the number of viable bacteria 51

2-2-7-5 Histopathological evaluation 52

2-2-8 Statistical analysis. 52

Chapter Three: Results and Discussion


3 Results and Discussion 53 3-1 Isolation and Identification of Staphylococcus aureus 53 3-1-1 Cultural Characteristics 53 3-1-2 Microscopically Characteristics 53 3-1-3 Biochemical Tests 54 3-2 Detection of Methicillin Resistance Staphylococci 56 3-2-1 Antibiotic susceptibility of Methicillin Resistance

Staphylococci 60

3-2-2 Antimicrobial activity of propolis, lysostaphin, ciprofloxacin and vancomycin

63

3-2-2-1 Antimicrobial activity of lysostaphin. 63 3-2-2-2 Antimicrobial activity of propolis 64

3-2-2-2-A Physical Properties of Iraqi Propolis 67 3-2-2-2-B Identification of chemical compounds in propolis by

using Fourier transform infrared (FTIR and HPLC).

68

3-2-2-2-B- I FTIR analysis of propolis 68 3-2-2-2-B- II HPLC analysis of propolis 69 3-2-2-3 Antimicrobial activity of vancomycin 70

3-2-2-4 Antimicrobial activity of ciprofloxacin 71

3-2-3 Antimicrobial activity of antibiotics and propolis

combinations

73

3-2-4 Detection of Slime layer and Biofilm formation by Methicillin Resistance Staphylococci

78

3-2-4-A Congo-red agar method (CRA) 78

3-2-4-B Tube Method 79

3-2-4-C Biofilm assay by Tissue Culture Plates Method (TCP) 80

3-2-5 Activity of propolis, lysostaphin and other antibiotics as a single and combination to reduced S.aureus (MRSA) biofilm formation

83

3-2-6 Experimental study on laboratory animals 88

3-2-6-1 Ocular Response to Infection 88

3-2-6-2 Histopathological examination 97

Conclusions and Recommendations

Series Subject Page No. Conclusions 104 Recommendations 105

References and Appendix

Series Subject Page No. References 106 Appendix 142

List of Figures


1-1 Mechanism of Antibiotics Resistance 7 1-2 Structure of S. aureus 11

3-1 The percentage of S.aureus isolated from collected

specimens.

53

3-2 Detection of Methicillin-Resistance Staphylococci by

antibiotic disc diffusion method using Methicillin disc

(ME 5μg/disc) ,A: Detection of Methicillin Resistance S.

aureus (MRSA), B: Detection of Methicillin sensitive S.

aureus (MSSA)

57

3-3 The Percentages Methicillin resistance S. aureus (MRSA) to the Methicillin sensitive (MSSA) S. aureus.

58

3-4 Antibiotic susceptibility of Methicillin resistance S. aureus.

60

3-5 Effect of lysostaphin at concentration (5.625 (µg/ml) on S.aureus isolates.

64

3-6 Inhibitory effect of Ethanolic extracted propolis , A:(EEP (4µg/ml)) ,B: (EEP (2µg/ml )) on S.aureus(S3) isolates by using (disc) method.

65

3-7 Effect of different concentration of EEP (2 µg/ml for

S10,S15,S3andS43) and (4 µg/ml for S57 )on S.aureus

isolates.

66

3-8 Effect of vancomycin on S.aureus isolates. 71 3-9 Effect of combination lysostaphin – propolis , A:OD of

control,B:OD after treated with lysostaphin(5.62 µg/ml)+propolis(2 µg/ml).

74

3-10 Slime layer production on the Congo red agar (CRA). A:

Negative production of slime layer by S. auerus (MRSA)

on CRA, B: Strong slime layer production by S. auerus

(MRSA) on the CRA

79

3-11 Slime layer production at the bottom of glass tube

(Christensen et al., 1982; Freeman et al., 1989), A:

strong positive, B: moderate positive , C:weak,

D:control.

80

3-12-A Biofilm formation on tissue culture plate using BHI as a 82

medium,A:control, B: Tissue culture plate(BHI).

3-12-B Biofilm formation on tissue culture plate using BHIglu

as a medium,A:control , B: Tissue culture plate

(BHIglu).

82

3-13 Percentage of biofilm formation using BHI & BHIglu . 82

3-14 A: normal eyes without infection consider as negative control B: untreated eyes that infected with MRSA S3, and consider as positive control.

89

3-15

A: eyes that received propolis with at concentration (2 µg/ml), B: normal eyes.

90

3-16 Eye treated with lysostaphin(5.62µg/ml) plus propolis(2 µg/ml).

90

3-17 Eye treated with combination ciprofloxacin(3%) and

propolis (2 µg/ml).

91

3-18 Eye treated with lysostaphin (5.62µg/ml) no significant

difference when compared with untreated group.

92

3-19 Section of eye (ciliary body) (control negative ) showing

normal structure of ciliary body(CB)&ciliary

process(CP),(400X), (H&E).

97

3-20 Section of eye (retina layer)(control positive) showing

degeneration(D) &damage of the retina layers ,rod(R)

,nuclei of rod(NR),nuclei of bipolar (NP),Muller fibers

(M) ,(400X), (H&E).

98

3-21 Section of eye ball (ciliary body&sclera) treated with

propolis at concentration (2µg/ml) look like normal

appearance of ciliary proceses(CP) &sclera(S),ciliary

muscle (CM),(400X), (H&E).

99

3-22 Section of eye (retina layer) treated wih lysostaphin at

concentration (5.625 µg/ml) showing still there was

degeneration(D) &damage of retina layers after

treatment , outer plexiform (OP) ,cones (C) , ganglion

cells (G),(200X), (H&E).

100

3-23 Section of eye (cornea and ciliary process layer layer

showing mild inflammatory cells(IN) infiltrate of ciliary

101

process(CP) and oedema(O) after treated with propolis

(2µg/ml) and lysostaphin(5.625 µg/ml) ,(400X), (H&E).

3-24 Section of eye (cornea) layer showing mild

inflammatory cells(IN) infiltrate ,odema (O),stroma (S),

after treated with propolis (2µg/ml )and

ciprofloxacin(3%) ,(400X), (H&E).

101

List of Tables


2-1 Apparatus and Equipments. 23 2-2 Chemical and biological materials. 24 2-3 Antibiotic discs used in the study. 26 2-4 Antibiotic Powders used in this study. 26 2-5 Culture media used in the study. 27 2-6 zone diameter interpretation standards 39 2-7 Combination of lysostaphin and vancomycin. 43 2-8 Combination of propolis and ciprofloxacin . 44 2-9 Combination of propolis and lysostaphin . 44 2-10 Combination of propolis and vancomycin. 44 2-11 Classification of bacterial adherence by tissue

culture plate method. 49

3-1 The biochemical tests and their results for S. aureus.

54

3-2 Number of isolates and percentage of S.aureus. 56

3-3 The number of MRSA isolates from according to

the sources of collection.

59

3-4 Effect of different concentration of ciprofloxacin on

isolates using disc method in inhibition zone (mm).

72

3-5 Effect of lysostaphin and EEP on isolates using

disc method.

73

3-6 Effect of propolis and ciprofloxacin on S.aureus

isolates.

77

3-7 Inhibition of biofilm –forming capacity of the selected isolates at different EEP concentration.

83

3-8 Inhibition of biofilm –forming capacity of the

selected isolates at different lysostaphin

concentrations.

85

3-9 Inhibition of biofilm –forming capacity of the selected isolates at different EEP and lysosotaphin

86

concentrations.

3-10 Inhibition of biofilm –forming capacity of the selected isolates at different EEP and Cipro. Concentrations.

87

3-11 Number of bacterial isolates from coronea. 92

List of Abbreviations

Abbreviation Meaning Agr accessory gene regulator BHIglu Brain heart infusion Broth (TSB) with 1% glucose. CA-MRSA Community-Acquired Methicillin-Resistant Staphylococcus

aureus CFU Colony forming unite

Clf Clumping factors CONS Coagulase-Negative Staphylococci COPS Coagulase-Positive Staphylococci CRA Congo red agar method DNase Deoxyribo nuclease EEP Ethanolic extracts of propolis FDA Food and drug administration FnBP Fibronectin Binding Proteins FTIR Fourier transform infrared HA-MRSA Healthcare-Associated Methicillin-Resistant Staphylococcus

aureus HPLC performance liquid chromatography-High ica operon intercellular adhesion operon MIC Minimum Inhibitory Concentration Min Minute MR-CONS Methicillin-Resistant Coagulase-Negative Staphylococci MR-COPS Methicillin-Resistant Coagulase-Positive Staphylococci MRSA Methicillin Resistance Staphylococcus aureus MRSE Methicillin-Resistance Staphylococcus epidermidis MSCRAMMs Microbial surface components recognizing adhesive matrix

molecules MSSA Methicillin-Sensitive Staphylococcus aureus NCCLS National committee for clinical standards Nm Nanometer PBP2a Penicillin-Binding Protein 2a PBPs Penicillin Binding Proteins PBS Phosphate buffer saline PIA polysaccharide intercellular adhesion PVL Panton-Valentine leukocidin SCCmec Staphylococcal Cassette Chromosome SSTI Skin and soft tissue infection

TCP Tissue Culture Plates method TSST-1 toxic shock syndrome toxin 1 TSS Toxic shock syndrome VRE Vancomycin-Resistant Enterococcus VISA Vancomycin Intermediately Sensitive S. Aureus VRSA Vancomycin-Resistant S. aureus

Chapter One

Introduction&Literature

review

1 Introducion &Literature review

1-1 Introducion

The Staphylococci, Gram positive bacteria, which tend to grow in

clumps, hence their descriptive name (Todar, 2004). Staphylococci are

nonmotiled, non-spore forming and usually non-capsulated. They grow

over a wide range of temperatures (10-42 ºC) with an optimum growing

temperature of 37ºC .They are normally aerobic but are also facultatively

anaerobic (Sangvik et al., 2011).

S. aureus is a common species of this genus and is easily identified

by its ability to produce coagulase and hence clot human plasma (Witte et

al., 2006). Staphylococcal infection can affect many sites and organs of

the human body. Invasion of the skin cause impetigo, cellulitis. In the

lungs abscesses and pneumonia are the result. Infection of the heart leads

to endocarditis. Meningitis and abscess formation can be the result of

infection to the central nervous system as well as, Keratitis can be the

result of eye infection (Rahman, 2011).

Microbial keratitis is defined as a stromal infiltrate associated with

an overlying epithelial defect with or without an anterior chamber

reaction (Keay et al., 2006). It is potentially blinding and is a major

cause of ocular morbidity if appropriate treatment is not initiated

promptly. It is widely accepted that the spectrum of microorganisms

responsible for corneal ulceration varies in all geographic regions (Ly et

al., 2006).

S. aureus , partially methicillin resistant S. aureus (MRSA), are

the most common bacterial pathogens associated with an ulcerative

keratitis. Infections caused by S. aureus which accounts for up to 31% of

cases of keratitis with the majority of ocular surface infections (Bharathi

et al., 2007). Staphylococcus keratitis can result in irreversible corneal

scarring, leading to the loss of visual acuity. Tissue damage during

Staphylococcus keratitis results from the action of bacterial products on

ocular tissues and from the host inflammatory response to infection

(Ikonne and Odozor, 2009).

The prevalence of methicillin resistant S. aureus (MRSA) is

responsible for bacterial keratitis form further complicate the therapy of

these infections, where this bacterium showing resistance to different

antibiotics, Since 2002, the first three vancomycin-resistant MRSA

strains have been cultured in the United States (Kaye et al., 2010), as well

as, new class of antibiotics has been instrumental in changing

management strategies for the treatment of corneal infections.

Nonetheless, emerging patterns of resistance even to these new classes of

antimicrobial agents (Koetsie, 2011; Sueke et al., 2013).

S. aureus has many mechanisms have been linked to increased its

virulence and pathogenicity, one of them ability to biofilm formation

mediate adhesion of bacteria to host cells, also to abiotic surfaces

(Krismer and Peschel, 2011). Biofilm is a conglomeration of microbial

cells in a sessile multicellular community, (Bryers, 2008), which

represent the protective factor for bacteria, were embedded in a self-

produced, hydrated matrix of polysaccharide, teichoic acid and protein

(Stewart & Costerton, 2001; Longauerova, 2006). Biofilm effects

chronic infection by providing innate protection to micro-organisms from

opsonophagocytosis and antibiotic agents (Von et al., 2005 ).

Antibiotic treatment of patients harboring a biofilm will lead to

temporary suppression of infective symptoms, by killing free-floating

bacteria shed from the biofilm population (Novick, 2006).

However, it does not kill the sessile bacteria within the biofilm

which are afforded between 1000 and 1500 times the antibiotic resistance

of their planktonic counterparts and so, when the antimicrobial

chemotherapy is stopped, the biofilm acts as a nidus for recurrence of the

infection (Thomas, 2008).

All those events have stimulated the continuing quest for an agent

that provides rapid and complete microbicidal activity with minimal toxic

effects and susceptibility to mechanisms of microbial resistance. The

problem of emerging antimicrobial resistance and the need to find more

effective antimicrobial agents stimulated us to initiate investigation of

potent antimicrobial agent as a tool for the management of ocular

infection. Therefore , this study aimed to

1- Investigating the effect of lysostaphin singly and in combination

with vancomycin, against the multi-drugs resistant Staphylococcus

aureus .

2- Detection the effect of Iraqi propolis singly and in combination

with other antibiotics on the multi-drugs resistant Staphylococcus

aureus with lysostaphin and compare it effect.

3- Investigating the effect of lysostaphin on biofilm of the multi-drugs resistant Staphylococcus aureus .

4- Studying the effect of Iraqi propolis on biofilm of the multi-drugs resistant S. aureus and compare it with lysostaphin effect.

5- In-vivo study to evaluate the efficacy of Lysostaphin, Iraqi propolis singly and in combination against the multi-drugs resistant S. aureus (MRSA) Keratitis induced in rabbit using histological sections.

1-2 Literature Review

1-2-1Staphylococcus

Staphylococci is a Gram-positive bacteria, with diameters of 0.5 –

1.5 μm. The designation of the genus Staphylococcus is derived from its

characteristically form of growth. (in Greek: staphyle) (Makins, 1993 and

Todar, 2004), its characterized by individual cocci, which divide in more

than one plane to form grape-like clusters (Harris et al., 2002). The

staphylococci are non-motile, non-spore forming, facultative anaerobes

that grow by aerobic respiration or by fermentation. Although more than

30 species of Staphylococcus are described (Götz et al., 2007). S. aureus

and S. epidermidis appear to be important in their interactions with

humans (Doktorgrades et al., 2008).

Staphylococcus aureus is the most virulent and the best known

member of the Staphylococcus genus. Susceptible to lysis by lysostaphin

but not lysozyme. Their cell walls contain peptidoglycan (mucopeptide)

and teichoic acid, are important cell-adherence factors. Their

peptidoglycan chains are linked by pentaglycine bridges (attacked by

lysostaphin); while micrococci do not have glycine residues in their

peptide bridges ( are resistant to lysostaphin), and neither do they possess

teichoic acids (Holt et al.,1994; Collee et al.,1996).

1-2 -2 Methicillin Resistant Staphylococci

Staphylococcus aureus is a leading cause of hospital- and

community-associated infections. (Loffler and Macdougall, 2007), in

1941, all strains of staphylococcal bacteria were susceptible to penicillin,

while at three years later, one strain of S.aureus appeared resistant to

penicillin (Lim and, Strynadka, 2002). Today, especially in hospitals,

there are strains of S.aureus bacteria that are resistant to, not just one,

but nearly all known antibiotics.

In the 1950s, penicillinase-producing strains of S. aureus were so

common, lead to that the Penicillin was becoming useless against

Staphylococcal infections (Chambers, 1988; Livermore,2006). The

introduction of Methicillin (A semisynthetic, narrow spectrum β-lactam

antibiotic of the Penicillin class) into clinical practice in 1959 and 1960

solved this problem, for a time (Chambers, 1988; Enright et al., 2002).

At the beginning of 1961 there were reports from the United Kingdom of

S. aureus isolates that had acquired resistance to Methicillin (Methicillin-

resistant S.aureus, MRSA), MRSA isolates were soon recovered from

other European countries, later from Japan, Australia, and the United

States (Enright et al., 2002).

Methicillin-resistant Staphylococcus aureus (MRSA) had emerged

as an important cause of skin and soft tissue infections (SSTI). The

understanding of the molecular epidemiology and virulence of MRSA

continues to expand (Ellis et al., 2009). Infections caused by methicillin-

or oxacillin- resistant S. aureus (MRSA) are mainly nosocomial and are

increasingly reported from many countries worldwide (Tiemersma et al.,

2004) .

The seriousness of this problem has been compounded by the fact

that these organisms are frequently resistant to most of the commonly

used antimicrobial agents, including the Aminoglycosides, Macrolides,

Chloramphenicol, and Tetracycline. Although initially susceptible to the

Fluoroquinolones, MRSA strains have rapidly developed widespread

resistance to this class of antimicrobial agent. In addition, in accordance

with the National Committee for Clinical Laboratory Standards (NCCLS,

2003) MRSA strains should be considered to be resistant to all

Cephalosporin’s and other β-lactams antibiotics (Seguin et al., 1999).

Several techniques used to detect MRSA epidemiology, recently,

the most important one is Molecular typing techniques have been used

with increasing frequency in studies of the epidemiology of MRSA and

also for a better understanding of the evolutionary relationships among

MRSA clones (Olivera and Lencastre, 2002).

1-2-3 Mechanisms of Methicillin Resistance(MRSA)

Staphylococci have two primary mechanisms for resistance to

lactam antibiotics: the expression of an enzyme (the PC1 β-lactamase)

which cleaves the beta-lactam ring structure, confers resistance to

penicillin but not to semi-synthetic penicillin such as methicillin,

oxacillin, or cloxacillin (Brooks et al., 2007). Resistance to methicillin

was termed “intrinsic” because it was not due to destruction of the

antibiotic by β-lactamase (Chambers, 1997), and the acquisition of a gene

encoding a modified penicillin-binding protein (PBP), known as PBP 2a,

found in MRSA and coagulase-negative staphylococci. PBP 2a was

intrinsically resistant to inhibition by β-lactams antibiotics (Fuda et al.,

2005).

In gram-positive bacteria the cell wall, a key structural component,

comprises the outer most layer of the cell, whereas in gram-negative

bacteria the cell wall lies underneath an additional layer known as the

outer membrane. At the molecular level, the cell wall is a meshwork of

glycan (polysaccharide) chains interconnected by peptide cross-links,

known as peptidoglycan (Thumanu et al., 2006). Biosynthesis of the

peptidoglycan was accomplished by the membrane-bound enzymes

known as penicillin-binding proteins (PBPs) (Sauvage et al., 2008). Each

bacterium contains several different types of PBPs that not only act as

enzyme catalysts for peptidoglycan synthesis during cell growth but also

function in cell septation and, in some species, sporulation. PBPs are

localized to the extracellular surface of the cytoplasmic membrane via a

membrane anchor (Goffin and Ghuysen, 2002). These biosynthetic

proteins may catalyze both a glycosyltransferase activity (for the

elongation of glycan strands) and a transpeptidase activity (for the

interconnection of the glycan strands by peptide crosslinking) (Van

Heijenoort et al., 2001).

Figure (1-1): Mechanism of Antibiotics Resistance (Todar,2009)

β-Lactam antibiotics irreversibly acylate the catalytic serine within

the transpeptidase active site of the biosynthetic PBPs and hence inhibit

peptide cross-linking, the loss of this catalytic activity impairs the ability

of the bacterium to control the integrity of its cell wall. (Llarrull et al.,

2009). The basis for β-lactam resistance by PBP 2a-containing MRSA

strains was the intrinsically lower reactivity of its transpeptidase domain

to β-lactam acylation (Zapun et al., 2008). Presence of the mecA gene

defines MRSA; the mecA gene, which codes for the penicillin binding

protein PBP2a, confers virtually complete resistance to all β-lactam

antibiotics including the semisynthetic penicillin (Weese et al.,2005).

PBP2a had a very low affinity for β-lactam antibiotics, and is

thought to aid cell wall assembly when the normal PBPs are inactivated

(Duquette and Nuttall, 2004).

1-2-4 Pathogenesis of Methicillin Resistance Staphylococci

Staphylococcus aureus is a major threat to human health and well-

being the world over (Stevens, 2007). The pathogenicity of S. aureus was

came from the expression of an arsenal of virulence factors, which can

lead to acute diseases such as, superficial skin lesions, or to more serious

infections, such as pneumonia, mastitis, urinary tract infections,

osteomyelitis, endocarditis, and even sepsis. In very rare cases, S. aureus

causes meningitis (Rahman, 2011), in addition to acute disease, S. aureus

can cause chronic infections, many of which are mediated by the ability

of this pathogen to adhere to medical devices and form biofilm (O’Neill

et al., 2007).

Staphylococcus aureus bacteremia is associated with serious

complications including endocarditis in 30% to 40% of cases bacteremia

(Snyder et al., 2010), greater importance is that patients with MRSA

bacteremia had higher rates of morbidity and mortality than patients with

infection caused by Methicillin-sensitive strains (Brunsvold and

Napolitano, 2007; Snyder et al., 2010). Over the past 40 years, MRSA

infections have become endemic in most U.S. hospitals and hospitals

worldwide (Fridkin et al., 2005). The major mechanism of patient-to-

patient spread of resistant microorganisms is on the hands, equipment or

apparel of healthcare workers (Safdar et al., 2007).

The majority of infections caused by community-acquired MRSA

(CA-MRSA) are skin and soft tissue infections (Brasel and Weigelt,

2007). Respiratory infections caused by MRSA are especially challenging

(Lam and Wunderink, 2007). Pneumonia is the second-most common

clinical manifestation of CA-MRSA (Brasel and Weigelt, 2007).

The epidemiology of MRSA has changed radically since 1999; in

particular, true community-acquired MRSA (CA-MRSA) infections have

been reported in patients with no clear risk factors, these CA-MRSA

clones predominantly infect young and previously healthy patients and

have now spread throughout the world (Durand et al., 2006). CA-MRSA

infections have been reported in North America, Europe, Australia, New

Zealand, and France (Vandenesch et al., 2003; Dauwalder et al., 2008).

Control spread of MRSA in the healthcare setting and in the

community is clearly essential. Understanding the natural history and

epidemiology of MRSA colonization and infection is fundamental to

devise effective strategies for prevention and control (Lee, 2003; Safdar

et al., 2007).

1-2- 5 Virulence factors

Colonization of host skin or mucosal surfaces and involves

bacterial attachment to host cells often via components of the

extracellular matrix (Dziewanowska etal ,2000). Here, numerous surface

proteins of S. aureus that belong to the microbial surface components

recognizing adhesive matrix molecules protein family promote the

attachment to the host tissue, e.g. the clumping factors A(Clf A), the

fibronectin-binding proteins (FnBP)A and B (Patti etal ,1994). On the

other hand, the organism produces molecules that decrease body immune

responces, lead to inhibit the effectiveness of complement mediated,

antibody-mediated opsonophagocytosis and block effectors of host

immune cell killing, such as reactive oxygen species and antimicrobial

peptides. Ultimately, the organism expresses specific factors that damage

host cells and degrade components of the extracellular matrix,

contributing to persistence and facilitate spread within normally sterile

sites of the host (Doktorgrades et al., 2008).

Virulence factors contribute to S. aureus pathogenesis include,

pore-forming toxins (alpha-hemolysin), Panton-Valentine

leukocidin(PVL), superantigens (Enterotoxin ,Toxic shock syndrome

toxin-1, and exfoliative toxin), phagocytosis inhibitors (protein A and

Polysaccharide capsule ). immune evasion molecules (chemotaxis

inhibitory protein, and staphylokinase, bone sialoprotein binding protein,

extracellular adherence protein, coagulase, Lipase and others (Brooks et

al., 2007).

Figure(1-2). Structure of S. aureus. Panel A shows the surface and secreted proteins.

The synthesis of many of these proteins is dependent on the growth phase, as shown

by the graph, and is controlled by regulatory genes such as agr. Panels B and C show

cross sections of the cell envelope. Many of the surface proteins have a structural

organization similar to that of clumping factor (Clf) , including repeated segments of

amino acids (Panel C). TSST-1 denotes toxic shock syndrome toxin 1 (Patti et

al.,1994).

1-2-6-A Therapy of Methicillin-Resistant Infection

Methicillin-resistant Staphylococcus aureus (MRSA) is a

multidrug resistant organism. Although “Methicillin-resistance” actually

means β-lactam antibiotic resistance, there are several other antibiotics

that are usually ineffective against MRSA, especially healthcare-

associated MRSA (HA-MRSA), such as Aminoglycosides and

Fluoroquinolones.

There were four Food and Drug Administration (FDA)-approved

antibiotics for the treatment of MRSA infections: Vancomycin, Linezolid,

Daptomycin, and Tigecycline. Vancomycin was a glycopeptide

antimicrobial, inhibits ribonucleic acid and cell wall synthesis, it had

lethal membrane effects(Chambers, 1988 ). Vancomycin was the drug of

choice for treatment of infections caused by Methicillin-resistant

Staphylococci S. aureus (Chambers, 1997). In addtion, Daptomycin was

a lipopeptide antibiotic active only against gram-positive organisms. Its

activity attrebuted to bacterial cell membranes binding ability in a

concentration-dependent and calcium-requiring process, causing rapid

depolarization lead to rapid bacterial cell death without cell lysis (Wise

etal.,2001; Silverman et al,2003).

Additionally Tigecycline was the first clinically available drug in a

new class of antibiotics called the glycylcyclines. Tigecycline targeted the

bacterial ribosome so its considered a bacteriostatic agent (Gales

etal.,2008).these two antibiotics( Daptomycin, Tigecycline ) is have

proven activity against MRSA infections .Linezolid was the first of a new

antibiotic class, the oxazolidinones. It was developed in the 1990s as a

response to the rising incidence of MRSA and the increased use and

emerging resistance to Vancomycin. Its mechanism of action involved

selectively binding to the bacterial 50S ribosomal subunit, where it

inhibited the formation of a functional initiation complex (Kutscha-

Lissberg et al., 2003; Reed, 2007).

Teicoplanin and Oritavancin are similar to Vancomycin. Both of

them were glycopeptides active against gram-positive cocci including

Methicillin-susceptible and-resistant strains of Staphylococci (Chambers,

1997; Reed, 2007). Ceftobiprole was the first Cephalosporin to have anti-

MRSA activity in addition to its broad-spectrum coverage (Von Eiff et

al., 2005).

1-2-6-B Mechanism of Resistance Staphylococci to antimicrobial

agents (Multi-drug Resistant Staphylococci)

Until the mid-1990s the majority of MRSA isolates exhibited a

multiresistance phenotype (Strommenger et al., 2003), such organisms

are also frequently resistant to most of the commonly used antimicrobial

agents, including (Aminoglycosides, Macrolides, Lincosamides,

Chloramphenicol, Tetracycline, and Fluoroquinolones or combinations of

these antibiotics) (Lee, 2003; Safdar et al., 2007) . In addition, Methicillin

resistant Staphylococci are resistant to all Penicillins, Carbapenems

(Chaieb et al., 2007), as well as, should be considered to be resistant to all

Cephalosporins, and other β-lactams (such as Ampicillin-sulbactam,

Amoxicillin-clavulanic acid, Ticarcillin-clavulanic acid, Piperacillin-

tazobactam) (Lee, 2003).

With the emergence of MRSA, Vancomycin became more heavily

used as it was the primary agent employed for MRSA infections .Over

use of Vancomycin had led to the emergence of Vancomycin resistance.

Vancomycin-resistant enterococcus (VRE) began to emerge in the late

1980s, after that, Vancomycin intermediately sensitive S. aureus (VISA)

was emerged in the late 1990s, while Vancomycin-resistant S. aureus

(VRSA) began to emerge in the early 2000s (Reed, 2007). The first

highly-Vancomycin-resistant strain was isolated in 2002, this strain was

shown to carry a plasmid which contains, among other resistance genes,

the vanA gene plus several additional genes required for Vancomycin

resistance ( Sievert et al, 2002). Proteins encoded by these genes were

responsible for lowering the cell wall affinity for Vancomycin (Sibbald et

al., 2006). On the other hand, Aminoglycoside resistance related to the

aacA-aphD gene, coding for a bifunctional enzyme which conferring

cross-resistance to clinically used Aminoglycosides such as Gentamicin,

Tobramycin, Kanamycin, and, when overexpressed, Amikacin

(Strommenger et al., 2003).

As well as, resistance to Erythromycin in Staphylococci is usually

associating with resistance to other Macrolides , there are three genes

(ermA, ermB, and ermC) encoding methylases have been found in

Staphylococci (Zmantar etal., 2010). Another mechanism of inducible

resistance to Erythromycin was conferred by the msrA gene, which

encoded an ATP-dependent efflux pump (Chaieb et al., 2007). In

addition,Tetracycline resistance in S. aureus is either based on

modification of the ribosome encoded by the widely disseminated tetM

gene or mediated by tetK encoded efflux pump (Schmitz et al., 2001).

1-2-7 Biofilm and Slime layer and their role in the Pathogenesis

A biofilm can be defined as a sessile community, surface-

associated microorganism, characterized by cells that are irreversibly

attached to a living or nonliving substratum to form a multilayered cell

clusters that embedded in a matrix of extracellular polysaccharide (slime),

that they have produced, which facilitates the adherence of these

microorganisms to the surfaces, protect them from host immune system

and antimicrobial therapy (Jabra-Rizk et al., 2006; Mathur et al., 2006).

Microbial biofilms represent an important determinant of human

chronic infections (TuQuoc et al., 2007). Staphylococci are the most

common bacterial pathogens causing foreign-body infections and medical

device-associated infections. The ability of biofilm formation on

biomaterials was the major contributing factor to such infections (Lim et

al., 2004). Biofilm enhance chronic infection by providing innate

protection to micro-organisms from opsonophagocytosis and antibiotic

agents (Stewart & Costerton, 2001).

Antibiotic treatment of patients harbouring a biofilm will lead to

temporary suppression of infective symptoms, by killing free-floating

bacteria shed from the biofilm population (Stewart and Costerton, 2001).

However, it does not kill the sessile bacteria within the biofilm which are

afforded between 1000 and 1500 times the antibiotic resistance of their

planktonic counterparts and so, when the antimicrobial chemotherapy is

stopped, the biofilm acts as a nidus for recurrence of the infection (Wu et

al., 2003).

The chemical compositions of slime layer consists of two

polysaccharide fractions. Polysaccharide I (>80%) is a linear homoglycan

of at least 130 residues of β-1,6-linked N-acetylglucosamine, 15–20% of

which are deacetylated and therefore positively charged. Polysaccharide

II (<20%) is structurally related to polysaccharide I, but has a lower

content of non-N-acetylated D-glucosaminyl residues contains phosphate

and ester-linked succinate, rendering it anionic. The structure of this

polysaccharide is unique and, according to its function in intercellular

aggregation, it was referred to as polysaccharide intercellular adhesin

(PIA) or polymeric N-acetyl-glucosamine (PNAG) (Gӧtz, 2002). PIA

mediates the contact of the bacterial cells with each other, resulting in the

accumulation of a multilayered biofilm (Rachid et al., 2000).

Both S. aureus and S. epidermidis rank among the clinically most

significant gram-positive bacterial pathogens that form biofilm layer was

responsible for 50 to 70% of intravenous-catheter-related infections

(Cotter et al., 2009). The Methicillin-resistant S. aureus (MRSA) biofilms

are present in more than 65% of all bacterial infections. This includes

both device-related infections and chronic non-device-related infections

(Del Pozo et al., 2009).

1-2-8 Mechanism of Boifilm Formation

Over the last decade interest in Staphylococcal biofilm

mechanisms has also intensified, arising initially from the importance of

this phenotype as a virulence determinant in S. epidermidis (McCann etal

,2008 ). However, S. aureus is also an adept biofilm former, which

enhanced its already considerable virulence capacity (O’Neill et al.,

2008).

The structured life style of bacterial biofilm communities involves

a coordinated sequence of events and a multistep process, characterized

by attachment of the cells to a surface by physicochemical interactions

(early adherence), which is followed by growth-dependent intercellular

accumulation of bacteria in multilayered cell clusters (intercellular

adhesion), glycocalyx formation, maturation of the biofilm, and finally

escape of the bacteria from the biofilm (dissemination) (TuQuoc et al.,

2007; Seidl et al., 2008).

Multiple factors have been found to influence bacterial attachment,

including bacterial surface proteins, physicochemical interactions,

hydrophobic interactions, presence of host proteins, and polysaccharides

like the capsular polysaccharide adhesin, the autolysin AtlE, and other

Staphylococcal surface proteins (Knobloch et al., 2004; TuQuoc et al.,

2007). After initial attachment, the accumulation step in biofilm

formation depends on the production of PIA (Lim et al., 2004), because

it mediates cell-to-cell adhesion of proliferating cells (Knobloch et al.,

2004).

Synthesis of PIA requires enzymes encoded by the intercellular

adhesion (ica) operon. The intercellular adhesion operon (ica operon) is

composed of the icaR (regulatory) gene and icaADBC (biosynthesis)

genes (Dobinsky et al., 2003; Cotter et al., 2009). PIA production was

not always correlated with biofilm formation. However, recently it has

become clear that there were at least two mechanisms of biofilm

development in S. aureus (TuQuoc et al., 2007). One mechanism requires

the production of an extracellular polysaccharide, termed polysaccharide

intercellular adhesin (PIA), the ica gene cluster is required for the

production of PIA, and this locus is essential for biofilm formation in

PIA-producing strains (O’Neill et al., 2008 ).

Later studies determined that mutations in the ica locus in

multiple S. aureus strains did not impair biofilm capacity, revealing a

second, ica-independent mechanism of biofilm formation (O’Neill et al.,

2008). Examination of Methicillin-resistant S. aureus (MRSA) strains

indicated that these isolates predominantly form the ica-independent

biofilms (O’Neill et al., 2008; Lauderdale et al., 2009).

The PIA-independent ways includes: Teichoic acids, surface-

exposed charged polymers, which constituted an important part of the S.

aureus cell wall (Seidl et al., 2008). Its function in primary adherence as

a component of the biofilm matrix as well, and the autolysin Atl

(Lauderdale et al., 2009).

1-2-9 Keratitis

Keratitis, it’s a disease of the cornea (infectious keratitis’ and

"ulcerative keratitis), can result from direct infection with viruses,

bacteria, fungi, yeast, and amoebae or from immune-related

complications such as the sterile keratitis associated with Lyme disease.

Bacterial keratitis was a sight-threatening process.A particular feature of

bacterial keratitis is a rapid progression; corneal destruction may be

complete in 24-48 hours with some of the more virulent bacteria. Corneal

ulceration, stromal abscess formation, surrounding corneal edema and

anterior segment inflammation are characteristic of this disease(Ly et

al.,2007)

Bacterial keratitis can occur in a variety of mammals and can be

caused by multitudes of bacterial species such as Pseudomonas

aeruginosa, Staphylococcus aureus, Streptococcus pneumonia, and

Serratia species. This disease characterized by pain, redness,

inflammation, and opacity. The common risk factors for infectious

keratitis include ocular trauma, contact lens wear, recent ocular surgery,

preexisting ocular surface disease, dry eyes, lid deformity, corneal

sensational impairment, chronic use of topical steroids, and systemic

immunosuppression (Bharathi et al .,2009; Stapleton et al. ,2012).

The pathogen Staphylococcus aureus is a leading cause of

bacterial keratitis can result in irreversible corneal scarring, leading to the

loss of visual acuity(Dajes etal.,2002b)

Multidrug-resistant strains of S. aureus further complicate the

therapy of these infections (Moreira and Daum, 1995). Tissue damage

during Staphylococcus keratitis results from the action of bacterial

products on ocular tissues and from the host inflammatory response to

infection(Chusid and Davis ,1979; Callegan et al., 1994).

In the rabbit keratitis model, strains producing α toxin have been

shown to cause extensive tissue damage and ocular inflammation.

Purified α toxin injected into the rabbit cornea in nanogram quantities

caused corneal epithelial erosions, marked edema, and ocular

inflammation (Moreau et al., 1997). β Toxin has been shown to induce

edema in rabbit eyes during keratitis and when purified toxin is injected

into the cornea. δ Toxin has not been specifically analyzed as a virulence

factor for keratitis. However, strains producing δ toxin, but not other

hemolysins, produce minimal corneal virulence,suggesting that δ toxin is

not an important virulence factor in keratitis (Callaghan et al., 1997).

Proteins other than alpha-toxin could contribute to ocular

virulence during S. aureus keratitis. The expression of multiple proteins

potentially involved in virulence was controlled by the accessory gene

regulator (Agr) system. Mutants defective in Agr demonstrate reduced

expression of some proteins normally induced in stationary phase (e.g.,

beta-toxin) and do not express many other such proteins, including

several hemolytic toxins and enzymes. (Kornblum et al.,1990). Agr-

defective mutants produce increased quantities of coagulase and the cell

wall-associated proteins, including protein A, clumping factor,

fibronectin binding protein (Foster and McDevitt, 1994).

1-2-10 Lysostaphin

Lysostaphin is an antimicrobial agent belonging to a major class

of

antimicrobial peptides and proteins known as the bacteriocins ,Class III

bacteriocins include large peptides which are generally heat-labile. This

class of bacteriocins was further subdivided by Heng and co-workers into

two distinct groups: (i) the bacteriolytic enzymes (or bacteriolysins) and

(ii) the non-lytic antimicrobial proteins (Heng etal., 2007).

Staphylococci have been shown to produce bacteriolysins, from

which lysostaphin is considered to be the prototype. Lysostaphin, an

extracellular enzyme secreted by strains of S. simulans biovar

staphylolyticus (Schindler and Schuhardt,1964; Maria etal.,2011) was

probably the first staphylococcin (bacteriocin produced by staphylococci)

discovered. Its bactericidal activity against staphylococci relies on its

capability of cleaving the peptidoglycan present in the bacterial cell

walls. Lysostaphin is a monomeric zinc-containing metallo-enzyme of

246 amino acids. It has a molecular mass of ~27 kDa, a pH optimum of

7.5 (Trayer & Buckley,1970; Maria etal.,2011).

Mechanism of action lysostaphin came from its ability to cleave

the Staphylococcal cell wall between the third and fourth glycine of the

pentaglycine cross-bridges (Grüdling and Schneewind, 2006). This

enzymatic cleavage leads to destabilization of the staphylococcal cell

wall, loss of osmotic equilibrium and rapid lysis of the staphylococci,

often within seconds (Zygmunt & Tavormina,1972; John etal., 2008) .

Lysostaphin was highly effective against both rapidly

dividing S. aureus as well as quiescent cells (Harrison&Crop,1964).

Current research demonstrated the important role of lysostaphin in lysis

Staphylococci biofilms, leading to disrupted the sessile cells as well as the

extracellular biofilm matrix (Wu etal ,2003).

The cloning of the lysostaphin gene from Staphylococcus

simulans into expression systems like E. coli, as well as new purification

procedures led to produce of highly purified recombinant lysostaphin

with consistently high specific activity (Mierau etal.,2005). The

availability of recombinant lysostaphin, in the face of a vanishing list of

effective anti-Staphylococcal agents, has generated renewed interest and

research into lysostaphin as a potential anti-Staphylococcal agent ( Maria

etal.,2011 ).

1-2-11 Propolis

propolis is a resinous substance collected by worker bees (Apis

mellifera) from the bark of trees and leaves of plants. The term

"propolis" is derived from the Greek, πρω, pro, meaning "for" or "in

defence of", and πολιζ, polis, "the community or city", and thus has the

meaning of "for the defense of the community" (Salatino et al., 2005).

This bee salivary and enzymatic secretions-enriched material is

used by bees to cover hive walls to ensure a hospital-clean environment.

As a natural honeybee hive product, propolis extracts have been used

both internally and externally for thousands of years as a healing agent in

traditional medicine. Propolis shows a complex chemical compositions

responsible for its biological properties- such as antibacterial, antiviral,

antifungal, among other activities, have attracted the researchers' interest

(Simone-Finstrom and Spivak, 2010).

The biological activity of propolis may vary according to different

plant sources and chemical composition may differ depending on the

geographic location (Trusheva et al.,2006), It had strong characteristic

smell and taste (Abu Fares et al., 2008). It was lipophilic, yellow-brown

to dark brown or sometimes greenish in color (Münstedt and Zygmunt,

2001; Sorkun et al., 2001). when it is cold, It was hard, but becomes soft

and very sticky when it is warm (Salatino et al., 2005).

In laboratory tests, studies have shown broad spectrum

antimicrobial activity of various propolis extracts. Synergism with certain

antibiotics has been demonstrated (Naher et al .,2011). Depending upon

its composition, propolis may show powerful local antibiotic and

antifungal properties. Many authors have demonstrated propolis

antibacterial activity against Enterococcus spp, Escherichia coli, and

Staphylococcus aureus. Reports have pointed out propolis efficient

activity against Gram-positive bacteria and limited action against Gram-

negative bacteria (Park etal.,2005).

Different researchers Kosalec et al.,(2004); Trusheva et al.,(2006)

have been reported that propolis antibacterial activity was attributed to a

number of phenolic compounds, mainly flavonoids, phenolic acids and

their esters and some prenylated coumaric acids were isolated from

propolis in several countries (Katircio and Nazime,(2006). Although

numerous researchers have been reported the biological activities of

propolis collected worldwide, information about Iraqi propolis are still

absent.

Over 300 compounds have been isolated from propolis, the main

components are resins and balsams, which contain flavonoids and

phenolic acids or their esters; highly variable wax contents; volatile oils;

pollen and impurities (Almedia and Menezes, 2002; Kumar et al., 2009).

The active compounds are flavanoids (flavones, flavanols, flavanones, and

flavononoles) and phenolic compounds (phenolic acids and their esters).

However, it should be pointed out that most of studies carried out have

not been aimed at determining a complete chemical composition, but have

merely determined some of the components of interest, particularly the

flavonoids (Xu et al., 2009; Mello et al., 2010).

Propolis contains some enzymes; for instance succinic

dehydrogenase, glucose-6-phosphatase, adenosine triphosphatase and acid

phosphatase (Tikhonov and Mamontova, 1987), and small quantities of

terpens, tannins, traces of secretions from the salivary glands of bees and

possible contaminants (Almedia and Menezes, 2002). Also contains some

minerals such as magnesium, calcium, iodine, potassium, sodium, copper,

zinc, manganese and iron, in addition to vitamins like B1, B2, B6, C and

E, and a number of fatty acids (Naher et al., 2011; Marcio et al., 2012).

Chapter Two Materials & Methods

2 Materials &Methods

2-1 Materials

2-1-1 Laboratory Equipment and Apparatus

The equipments and apparatus used throughout the present study were

shown in table 2-1:

Table 2-1: Apparatus and Equipments

Company / Origin Apparatus Fanem (Brazil) Autoclave

Janetzki (Germany) Centrifuge BioTek (USA) Micro ELISA Auto Reader

Dragon-med( spain) Micropipetter Kottermann (Germany) Distillator

Shimadzu (8300) (Japan) Fourier transform infrared (FTIR) Ishtar (Iraq) Freezer

Shimadzu (Japan) High performance liquid Chromatography (HPLC)

Labinco (Netherland) Hot plate Magnetic Stirrer Nüve (Turkey) Incubator

Olympus (Japan) Light Microscope Gema medical S.L. (Spain) Membrane filter unite

Dragon-med (Spain) Micropipette Memmert (Germany) Drying Oven

Milwaukee (Italy) pH meter

BioTek (USA) Plastic tissue culture plate(96-well flat bottom)

Ishtar (Iraq) Refrigerator Sartorius (Germany) Sensitive balance

Gallenkamp (England) Shaker Incubator Varian (Australia) UV-visible Spectrophotometer Biomerieux(USA) Vitak-2 Compact

Fanem (Brazil) Vortex Memmert (Germany) Water bath

2-1-2 Chemicals and biological materials

Table 2-2: Chemical and biological materials

ID Materials Company / Origin

1 Acetic acid Fluck / Switzerland

2 Alpha naphthol BDH/ England

3 Autoanalyzer Vitak-2/USA

4 Barium chloride (BaCl2) BDH/ England

5 Bovine serum albumin (BSA) BDH/ England

6 Bromophenol blue Himedia / India

7 Calcium chloride (CaCl2) BDH / England

8 Chloroform SD-fine / India

9 Congo red stain BDH/ England

10 Eosin Sigma Aldrich

11 Ethyl alcohol (ethanol) GCC / England

12 Formalen Analar, SDFCL (Mumbai)

13 Gelatin powder GCC / England

14 Glucose BDH / England

15 Gram stain kit Syrbio / Syria

16 Hematoxylin Sigma Aldrich

17 Human blood Al-Kadimia blood bank

18 Hydrogen chloride Riedel-Dehan / Germany

19 ketamine hydrochloride Euro Pharmacy/ IndiaMart

India

20 Lysostaphin Bio neer (Japan)

21 Meat extract BDH/ England

22 N,N,-Dimethyl-1-naphthylamine BDH/ England

23 Normal saline solution US(Biological(USA)

24 Peptone BDH/ England

25 Propolis Local commercial

26 Phenol red BDH / England

27 Phosphate buffer saline Himedia/ India

28 Potassium hydroxide(KOH) Fisons Scientific Apparatus LTD./ England

29 Safranin stain BDH/ England

30 Skim milk powder Himedia/India

31 Sodium Chloride GCC / England

32 Sucrose BDH / England

33 Sulfanilic acid Redial-dehaeny/ Germany

34 Sulphuric acid (H2SO4) GCC/ England

35 Tributyrin SAfc / India

36 Tryptone BDH / England

37 Urea extra pure powder SD-fine / India

38 Xylazine BDH /England

39 Xylose BDH /England

40 Yeast extract Himadia / India

41 Zinc powder BDH/ England

2-1-3 Antibiotic

2-1-3-1 Antibiotic discs

Antibiotics discs are listed in table 2-3:

Table 2-3: Antibiotic discs

Company(Origin) Concentration

(µg) Company(Origin)

ID

Himedia / India 5 Methicillin (ME) 1

Himedia / India 30 Tetracyclin (TE) 2

Himedia / India 30 Vancomycin (VA) 3

Himedia / India 5 Ciprofloxacin (CIP) 4

Himedia / India 5 Rifampin (RA) 5

Himedia / India 2 Clidamycin (CD) 6

Himedia / India 15 Clarithromycin (CLR) 7

Himedia / India 10U Penicillin(p) 8

2-1-3-2 Antibiotic Powders

Table 2-4: Antibiotic Powders

Manufacture Company Antibiotics ID

Julphar/ UAE Vancomycin vial 1

United/Jordan Ciprofloxacin tablet 2

2-1-4 Culture Media

2-1-4-1 Ready culture media

All culture media used for isolation and identification of bacteria

throughout this study are listed in table 2-5.

Table 2-5: Culture media

ID Medium Company (origin)

1 Blood agar base, Brain heart infusion

(BHI) agar, BHI broth, , Mueller Hinton

agar, Mannitol salt agar, Nutrient agar,

Nutrient broth, Urea agar base, Mueller-

Hinton broth,Nitrate reduction broth

Trypton Soya broth

Hi-media (India)

2 DNase agar Oxoid (England)

2-2 Methods

2-2-1 Culture Media preparation

2-2-1-1 Ready to use culture media:

All media listed in table 2-5 were prepared according to the

manufacturer company, pH was adjusted by 0.1N NaOH or 0.1N HCl,

then sterilized by autoclaving (121 °C, 15 minute and 15 pound/Inch2).

The prepared media were distributed into sterile tubes or Petri dishes.

2-2-1-2 Laboratory Prepared Media

Acetoin production media (Collee et al.,1996) 2-2-1-2-A-

The medium was composed of the following materials:

Tryptone-------------------10g

Meat extract---------------3g

Yeast extract--------------1g

Glucose---------------------20g

All these materials were dissolved in 900ml of distilled water,

after adjusteding the pH to 7.2, the volume was completed to 1000ml

then distribute52d into tubes in 5ml for each and sterilized by autoclave.

This medium was used to detect the ability of bacteria to fermented

glucose and acetoin production.

B-Blood Agar (Benson, 2002 ) 2-2-1-2-

This medium was prepared according to the instruction of

manufacturer company, sterilized by autoclave, then after cooled to 50ºC,

5ml of sterile defibrinated human blood was added to each 100ml of the

medium, mixing well then poured in a sterile Petri-dish. This medium is

suitable for the cultivation and isolation of bacteria and for the detection

of haemolytic activity and the kind of haemolysis.

2-2-1-2-C- Tributyrin Agar (Sirisha et al., 2010)

This medium was prepared by dissolving:

Pepton ---------------------------------5g

Yeast extract--------------------------3g

Calcium Chloride (CaCl2)----------1g

Tributyrin---------------------------10ml

Agar------------------------------------20g

In one liter of D.W., then pH was adjusted to 8.0, boiled for 1

minute autoclaved, cooled to 45°C and poured into sterile Petri-dishes.

This medium was used for detecting ability of bactreia to produce lipase

enzyme.

2-2-1-2-D- Skim-Milk Agar (Collee et al., 1996)

This medium consists of:

Nutrient agar, sterile --------------------------------87.5ml

Skimmed milk, sterile ------------------------------12.5ml

The Nutrient agar was prepared according to the instruction of

manufacturer company, sterilized by autoclaved , cooled to 50ºC.The

skim milk was prepared according to the instruction of manufacturer

company, sterilized by autoclaved, cooled to 40-45ºC. 12.5ml of sterile

skimmed milk was added for each 87.5ml of sterile nutrient agar, mixed

well then poured in a sterile Petri-dish. This medium was used for

detecting the ability of bacteria to produce Protease enzyme.

2-2-1-2-E-Urea Agar medium (Collee et al., 1996)

This medium consists of:

Urea base agar (Christensen's medium)------------------ 95ml

Urea solution -------------------------------------------------- 5ml

The Christensen's medium was prepared according to the

instruction of manufacturer company, the pH was adjusted to 6.8-6.9 and

sterilized by autoclaved, cooled to about 50ºC, then 5ml of (40% urea

solution) was filtering throw Millipore filter unit (0.2 µm) was aseptically

added to each 95ml of Christensen's medium, mixed well and dispensed

into a sterile tubes (slant). This medium was used for detecting the ability

of bacteria to produce urease enzyme.

2-2-1-2-F- Nutrient Gelatin medium (Harley and Prescott, 2002)

The medium was composed of

Peptone------------------------------------5g

Meat extract-------------------------------3g

Gelatin-----------------------------------120g

All these materials were dissolved in 900ml of distilled

water, after adjustment of pH to 6.8 the volume was completed to the

1000ml then sterilized by autoclaved ,this medium was used to

demonstrate the gelatin liquefaction by bacteria.

2-2-1-2-G-Congo-red Agar (Freeman et al.,1989)

The medium was composed of:

Brain-heart infusion broth-----------------------------37g

Sucrose---------------------------------------------------50g

Congo-red stain-----------------------------------------0.8g

Agar-agar------------------------------------------------10g

All these materials were dissolving in 900ml of distilled water with

the exception of Congo red stain and autoclaved. The Congo red stain

was dissolved in 100ml of distilled water and autoclaved . Separately

from the other medium constituents. Then added when the agar had

cooled to 55ºC and poured in sterile Petri dishes. This medium was used

for detecting bacterial ability to produce slime layer.

Stains, Reagents and Solution 2-2-2

Gram Stain Kit 2-2-2-1

Gram Stain Kit consists of:

Violet stain solution / Lugo Iodin solution/ Alcohol Acetone solution/

safranin.

2-2-2-2 Catalase reagent (Harley and Prescott, 2002)

This reagent was prepared as 3% of hydrogen peroxide (H2O2).

2-2-2- 3 Oxidase reagent (Vandepitte et al., 2003)

This reagent was prepared by dissolved of 1g of N,N,N,N,-tetra methyl-

P-phenylene-diamine dihydrochloride in 100ml of distilled water. Stored

in dark bottle and used immediately.

2-2-2-4 Nitrate test reagent (Harley and Prescott, 2002)

This reagent was consisted of:

1. Solution (A): prepared by dissolving 8g of sulfanilic acid in 1 litter of

5N acetic acid (1 part glacial acetic acid to 2.5 parts distilled water).

2. Solution (B): prepared by dissolved 6ml of N,N,-dimethyl-1-

naphthylamine in 1 litter of 5N acetic acid.

3. Zinc powder.

2-2-2-5 Acetoin production test reagent (Barritt’s reagent) (Collee et

al., 1996)

This reagent was amixture two solutions:

1. Solution (A): prepared by dissolving 5g of alpha-naphthol in 100ml

of absolute ethanol.

2. Solution (B): prepared by dissolved 40g of potassium hydroxide in

100ml of distilled water.

2-2-2-6 Safranin stain solution

This solution was prepared by dissolving 0.1g of safranin stain in

100ml of distilled water .

2-2-2-7 McFarland standard solution (Collee et al., 1996; Benson,

2001)

McFarland’s standard solution 0.5 is the turbidity standard

solution which is the most widely used method of inoculum preparation

or standardization, this solution has aspecific optical density to provide a

turbidity comparable to that of bacterial suspension containing 1.5×108

CFU/ml.

This solution was prepared as the following:

Solution (A): 1g of barium chloride (BaCl2) was dissolved in 100ml of

distilled water.

Solution (B): 1ml of concentrated sulphuric acid (H2SO4) was added and

the volume was completed to 100ml by distilled water.

0.5ml of solution (A) was added to 99.5ml of solution (B) and stored in

dark bottle until used.

2-2-2-8 Phenol red (Forbes et al., 2007)

This indicator was prepared by dissolving 0.5g phenol red, 1g

pepton and 0.5g Sodium Chloride in 100ml of D.W., then the pH was

adjusted to 7.4.

2-2-3 Staphylococcus aureus isolation and identification

Specimen's collection 2-2-3-1

From August to December 2012 One hundred specimens were

collected from patients in AL-Kindy Teaching Hospital and Medical City

Teaching Hospitals (Baghdad /Iraq). The specimens were included 19

nasal swab,16 wound swab, 27 burn swab,10 corneal scraping swab , 7

pus, 15 sputum and 6 urine culture.

2-2-3-2 Staphylococcus aureus isolation

The collected specimens were inoculated on the blood agar which

prepared as mentioned in (2-1-3-1),and incubated at 37ºC for 24 hours.

The isolates were examined for their shape, size, colour, pigments, and

haemolytic activity. Then transferred and streaked on Mannitol salt agar

(2-1-3-1) which considered as selective and differential medium for the

isolation, purification and identification of Staphylococci, and for

detecting the ability of each isolate to ferment mannitol. All plates were

incubated at 37ºC for 24 hours then a single pure isolated colony was

transferred to Nutrient agar medium (2-1-3-1) to be kept and to carry out

other biochemical tests to confirm the identification of isolates.

2-2-3-3 Identification of Staphylococcus aureus

2-2-3-3-A-Microscopic examination (Benson, 2002):

These isolates were stained by Gram stain to detect their response

to stain, shapes and their arrangement.

Cultural characteristics: B- 2-2-3-3-

The colonies were grown on blood agar and tested for their shape,

size, color and blood heamolysis.

2-2-3-3-B-I-Growth on mannitol salt agar (Kloos and Bannerman,

1995)

The plates were streaked with a pure colony of tested bacteria, then

incubated at 37°C for 24 hours. This medium was used for selective

isolation and cultivation of bacteria from specimens.

2-2-3-3-B-II-detection of hemolysis on human blood agar

Human blood agar was inoculated with an overnight bacterial

culture and incubated at 37°C for 24 hours. A clear zone around the

colonies was indicated abeta hemolysis behavior.

C- Biochemical tests: 2-2-3-3-

2-2-3-3-C-1-Catalase test (Brooks et al., 2007)

Single isolated colony of bacteria was removed from culture plate

by wooden applicator stick and placed on a glass slide then 1-2 drops of

3% H2O2 were mixed with the cells on the slide. The appearance of gas

bubbles indicates a positive test.

2-2-3-3-C-2-Oxidase test (Brooks et al., 2007)

Single isolated bacterial colony was placed on a piece of filter

paper by a wooden applicator stick, then 2-3 drops of oxidase reagent (2-

2-2-3) were added to the filter paper. Change in color to dark purple

within 20-30 seconds indicates a positive test.

2-2-3-3-C-3-Coagulase test (Harley and Prescott, 2002)

A 0.5ml of thick 18-24hrs bacterial broth culture was added to

0.5ml of citrated plasma (Plasma was obtained from human blood by

adding 1.25ml of sodium citrate for each 5ml of blood and then

centrifuged; the upper layer is the plasma ) in a tube, then incubated at

37°C and examined every 30 minutes for 4hrs. The clot formation

indicated a positive result, while the negative tubes re-examined after

24hours.

2-2-3-3-C-4-Acetoin production test (Collee et al., 1996)

This test was used to detect the ability of bacteria to produce

acetoin. A small amount of colony growth of the tested bacteria was

inoculated into acetoin production medium (2-2-1-2-A), then incubated at

30 °C for 14 days. After incubation, 1ml of potassium hydroxide solution

and 3ml of α-nephthol were added to that medium, gently mixed, then left

at room temperature ( 26-28°C ) until the pink colour was observed after

1-2 hours (a positive result).

2-2-3-3-C-5- DNase production test (Collee et al., 1996)

DNase agar was heavily streaked by the activated bacteria then

incubated for 18–24hours at 37°C. Conversion of medium green color to

yellow color indicated the positive result.

2-2-3-3-C-6- Mannitol fermentation (Kloos and Bannerman, 1995)

Coagulase-positive Staphylococci will produce yellow colonies

with yellow zone on mannitol salt agar as a result of utilizing of mannitol

(as positive result); whereas other species of staphylococci will produce

small pink to red colonies with no color change to medium.

2-2-3-3-C-7- Gelatin liquefaction (Collee et al., 1996)

Tubes that contain nutrient gelatin agar medium (2-2-1-2-F) were

inoculated with small amount of colony growth and incubated at 37°C for

24 hours. In order to confirm the result, all tubes were placed in

refrigerator for 15 minutes. If the medium still liquid, then the result was

considered as a positive result.

2-2-3-3-C-8- Protease production (Collee et al., 1996)

Skim milk agar (2-2-1-2-D) was inoculated with a pure isolated

colony and incubated for 24 hours at 37°C. A positive result was

observed as clear zone around the colony. This medium was used to

detect ability of bacteria to produced protease enzyme.

2-2-3-3-C-9- Nitrate reduction test (Collee et al., 1996; MacFaddin,

2000):

The over night grown (2-3) bacterial colonies were transferred by

sterile cooling loop full into tubes containing 5ml of nitrate reduction

broth prepared as in (2-1-3-1) then incubated at 37C◦for 24hr, after that a

few drops of α-naphtol reagent and few drops of sulfonilic acid (prepared

as in2-2-2-4) were added to the bacterial growth appearance of red color

means a positive result. If the color don't appear that not mean a negative

result, the bacteria may have the ability to reduct the nitrate to free

nitrogen directly ,the test about this can be done by adding zinc powder

disappear of red color is an evidence to positive result ,while it's

appearance meaning a negative result.

2-2-3-3-C-10- Urease test :( Collee et al., 1996):

Using a sterile loop, tubes of urea medium (prepared as in step (2-

2-1-2-E) were inoculated with a colony of tested bacterium, and

incubated for 24-72 hours. Urease positive strains split off ammonia from

the urea which raises the pH and causes the phenol red (which is the pH

indicator) turn to red-pink.

2-2-3-3-C-11- Autoanalyzer staph system (vitak- 2 Compact)

Is an identification system for the genera Staphylococcus . This

test applied according to the supplied company instructions in AL-Kindy

Teaching Hospital as following:

1. Loop full of Fresh culture after 24hrs (single colony). Put in 3ml of

normal saline, shacked and measured by turbidity meter.

2. The gram positive card entered in the tube , and put it in the

casket place .

3. Interd the casket in the room number 1 of the vitek 2 compact to lode

bacterial suspension.

4. Moved the casket from room one to room two to begin reading( late for

6 hrs the results appeared on the computer screen as a tabes contained all

detailed of bacterial isolate.

12- Antibiotic susceptibility test (Benson, 2002; Morello et al., 2006)

This test performed by modified Kirby-Bauer method as the following:

1- From an overnight culture plate, 4-5 colonies of bacterial isolate

were picked up by sterilized inoculating loop and emulsified in 5ml

of sterile normal saline until the turbidity is approximately

equivalent to that of the McFarland No. 0.5 turbidity standard

which prepared as mentioned in (2- 2-2-7).A sterile swab

was dipped into the bacterial suspension, any excess fluid was

expressed against the side of the tube.

2- The surface of a Mueller-Hinton agar plate was inoculated by

bacterial isolate as follows: The whole surface of the plate was

streaked with the swab, then the plate was rotated through a 45º

angle and streaked the whole surface again; finally the plate was

rotated another 90º and streaked once more.

3- By a sterile forceps the antimicrobial disc was picked up and

placed on the surface of the inoculated plate. The disc was pressed

gently into full contact with the agar.

4- The step (3) was repeated to all antimicrobial discs under the test,

spaced evenly a way from each other.

5- The plates were incubated at 35ºC for 18-24 hours.

6- After incubation, the plates were examined for the presence of

inhibition zone of bacterial growth (clear rings) a round the

antimicrobial discs, if there was no inhibition zone the organism

was reported as resistant to the antimicrobial agent in that disc. If a

zone of inhibition surrounded the disc, the diameter of the zone

inhibition was measured (by millimetres) and compared their sizes

with values listed in a standard chart, (Table 2-6).

Table 2-6: Zone diameter interpretation standards (Benson, 2002;

Morello et al., 2006)

Antimicrobial Agents

Disc Potency

Diameter of Inhibition zone (mm)

Resistant Intermediate Susceptible

Penicillin 10U ≤28 - ≥29

Clarithromcin 15µg ≤13 14-17 ≥18

Ciprofloxacin 5µg ≤15 16-20 ≥21

Clindamycin 10µg ≤14 15-20 ≥21

Methicillin 5µg ≤9 10-13 ≥14

Rifampin 5µg ≤16 17-19 ≥20

Tetracycline 30µg ≤14 15-18 ≥19

Vancomycin 30µg _ _ ≥15

2-2-3-4 Preservation of bacterial isolates (Vandepitte et al., 2003)

Two methods were employed for the preservation of bacterial isolate:

Short time preservation (Harely and and Prescott, 2002) 1-

Single pure colony of bacterial isolate was streaked on the nutrient

agar culture plates and on the nutrient agar slants. Incubated at 37ºC for

24 hours, sealed well and stored at 4ºC in the refrigerator one month for

the plates and three months for the slants.

2- Long time preservation (Vandepitte et al., 2003)

A loopful of overnight growth pure culture was added to nutrient

broth and incubated at 37°C. After 18 hours glycerol was added to the

inoculums in final concentration reached 15-50% and stored at -20°C.

bacteria can stored for months or years in low temperature without

significant loss of viability.

2-2-4 Effect of (lysostaphin and propolis) singly and in combination

with antibiotics, against the multi-drugs resistant Staphylococcus

aureus

The effect of lysostaphin and propolis were tested on the multi-drugs

resistant Staphylococcus aureus All assays were accomplished in

triplicate.

2-2-4-1 Preparation of stock solutions (Guignardet etal.,2013)

2-2-4-1-A Preparation of stock solutions( singly concentrations of

antimicrobial)

1 - Preparation of lysostaphin stock solution

Different concentrations of lysostaphin were prepared by

dissolving 4.5mg of lysostaphin in 50 ml of strelized D.W.to prepare

90µg/ml as a stock solusion. Sterility was done by filtering through

Millipore filter unit (0.2 µm) then serial concentrations were prepared

(90,45,22.5,11.25 and 5.62 µg/ml ).

2- Preparation of vancomycin stock solution

Different concentrations of vancomycin were prepared by

dissolving 1.6mg of vancomycin in 100 ml of strelized D.W. To prepare

16µg/ml as a stock solusion .Sterility was done by filtering through

Millipore filter unit (0.2 µm) then serial concentrations were prepared

(16,8,4,2 and 1 µg/ml) .

3 - Preparation of ciprofloxacin stock solution

Different concentrations of ciprofloxacin stock were prepared by

dissolving 12g of ciprofloxacin in 100 ml of sterilized D.W.To prepare

12% as a stock solution. Sterility was done by filtering through Millipore

filter unit (0.2 µm) then serial concentrations were prepared

(12,6,3,1.5and 0.75 % ).

4- Propolis

1-Propolis collection

Propolis samples were obtained from Karbala on August 2012,

Through personal communication with bee keepers, all propolis samples

were collected by scrapping from frames and walls of the beehives. The

collected samples were kept desiccated in the dark place to their

processing according to Syamsudin et al., (2009).

2-Ethanolic Extract of Propolis (EEP)

Crude propolis was freezed and grounded in a mortar, then 1.6mg of

fine powder were extracted with a total 100 ml of 70% (v/v) ethanol, final

propolis suspension (16 µg/ml) was obtained, with shaking for 7 days at

room temperature (26-28°C). The mixture was centrifuged at 3000×g for

15 minute, and supernatant was collected and kept in dark at room

temperature until used as ethanol extracted propolis (EEP) (Mbawala et

al ., 2010). Sterility was done by filtering through Millipore filter unit(0.2

µm).This EEP was used as a stock solution to prepare (16, 8, 4, 2 and 1

µg/ml) concentration in order to determine MIC concentration of propolis

against Staphylococcus isolates.

3- High-performance liquid chromatography analysis of propolis

(HPLC) (Watson et al.,1986)

The propolis analysis was carried out in Ministry of Science and

Technology by liquid chromatography using Shimadzu 10AV-LC

equipped with binary pump model LC-10A Shimadzu. The eluted peaks

were monitored by UV-Vis 10A-SPD spectrophotometer.

The procedure was applied by using column type phenomenex C-18.3μm

particle size (50 × 4.6 mm I.D). The mobile phase was methanol:

deionized water (80:20 v/v). Separation of propolis was carried out under

optimum conditions, with flow rate at 1.2 ml/minute and UV detection set

at 280 nm.

Preparation of sample

Propolis( 0.5g) were dissolved in hexane: ethanol (40:60 v/v) mixture,

agitated in an ultrasonic bath for 20 minutes. Two fractions were

separated. The hexane fraction (lower layer) which contains the resin part

was weighed after drying. The methanol fraction (upper layer) was

analyzed by HPLC column as previously mentioned (2-2-4-1-A-4-3) for

the quantitative identification of active constituents. 50µg/ml of the

standards sequence was as follows:

1. α-pinenine

2. β-pinenine

3. Isoferulic acid

4. Cinammic acid

5. Chrysin

6. B-caryphyllene Medicarpin

7. Napthoquinone

8. Trans-verbenol

9. Poplins

10. Medicarpin

Concentration of the sample was calculated following this equation:

· Concentration of sample (μg/ml) = (Area of sample/Area of standard) × con. of standard (50µg/ml) ………………..(5)

4- Detection of propolis structure by using Fourier transform

infrared (FTIR) (Charyulu, et al., 2009)

Chemical structure of propolis was examined by using Fourier

Transform-Infrared spectrophotometer (FTIR). This was done at wave

length of 400- 4000 cm-1. This spectrum was used to determine the

functional chemical groups that are found in the propolis structure (� C-

H aliphatic bond range from 3000- 2800 cm-¹, � C-H aromatic bond

range from 2940-3000 cm -¹, � C�C aromatic bond range from 1560–

1420 cm -¹, � C=N bond range from 1680-1600 cm -¹,� O-H bond

range from 3300- 3000 cm -¹)

2-2-4-1-B Preparation of stock solutions (combination


1- The combination of lysostaphin and vancomycin in table (2-7)

Table 2-7: Combination of lysostaphin and vancomycin

Tube No.

Combination of lysostaphin and Vancomycin

Lysostaphin Vancomycin

Concentration ( µg/ml) Concentrtion ( µg/ml)

1 5.62 8

2 5.62 16

2- The combination of propolis and ciprofloxacin in table (2-8)

Table 2-8: Combination of propolis and ciprofloxacin

Tube No.

Combination of Propolis and Ciprofloxacin

Propolis Ciprofloxacin

Concentration ( µg/ml) Concentrtion %

1 2 3

2 4 3

3-The combination of propolis and lysostaphin in table(2-9)

Table 2-9: Combination of propolis and lysostaphin

Tube No.

combination of propolis and lysostaphin

Propolis Lysostaphin

Concentration ( µg/ml) Concentrtion( µg/ml)

1 2 5.62

2 4 5.62

4- The combination of propolis and vancomycin in table(2-10)

Table 2-10: Combination of propolis and vancomycin

Tube No. combination of propolis and Vancomycin

Propolis Vancomycin

Concentration ( µg/ml) Concentrtion( µg/ml)

1 2 8

2 4 16

2-2-4-2 Procedure for detection of the antimicrobial activity

2-2-4-2-A disc diffusion method (Bauer et al., 1966) with some

modifications:

1. From an overnight culture plate, 3-4 colonies of (S. aureus strains)

were picked up by a sterile inoculating loop and dispersal in 5 ml of

sterile normal saline until the turbidity is approximately equivalent

to that of the McFarland No.0.5 turbidity standard which prepared

as mentioned in ( 2-2-2-7 ).

2. A sterile cotton swab was dipped into the bacterial suspension; any

excess fluid was expressed against the side of the tube.

3. The surface of a Mueller-Hinton agar plate was inoculated by

bacterial isolate as the following; The whole surface of the plate

was streaked with swab, then the plate was rotated through a 45°

angle and the whole surface was streaked again; finally the plate

was rotated another 90° and streaked once more, left for drying at

room temperature few minutes.

4. Filter paper discs with diameter of 7mm were prepared, sterilized

and then impregnated into desired concentration of each the

antimicrobial concentration.

5. All discs were placed on the inoculated plate by a sterile forceps.

The plates were incubated at 37°C for 18-48 hours.

6. After incubation, the plates were examined for the presence of

inhibition zone (clear zone) around the discs, if there was no

inhibition zone the organism was reported as resistant to the

inhibitor agent in that disc if a zone of inhibition surrounded the

disc, the diameter of the zone of inhibition was measured in

millimeters.

2-2-4-2-B Minimum inhibitory concsntration (MIC)

MIC is the lowest concentration of antimicrobial agent that inhibit

the growth of a particular microorganism by broth dilution method. The

minimum inhibitory concentration (MIC) of the lysostaphin, propolis

single and in combination with other antibiotics was determined by using

two-fold dilution in nutrient broth at 37°C. After incubation period,

examined the tube for development of turbidity. If turbidity occurs, the

microorganisms are resistant to the antibiotic concentration and if no

turbidity, it considered as sensitive (Guignard et al., 2013).

2-2-5 Detection of the bacterial ability to produce slime layer and

Biofilm formation

The ability of Staphylococci to produce slime layer and biofilm

formation were tested by three methods; Tube method, Congo red agar

method, and Tissue culture plate method. The bacterial ability to produce

slime layer were applied on Twenty-one isolates that identified as S.

aureus (MRSA).

1- Congo-red agar method (Freeman et al., 1989; Mathur et al.,

2006)

The Congo-red agar medium which prepared as mentioned in (2-

2-1-2-G) was inoculated with a suspension of bacterial isolate that

turbidity is approximately equivalent to the McFarland No. 0.5 turbidity

standard. The plates were incubated aerobically for 24-48 hours at 37ºC.

A positive result was indicated by black colonies with a dry crystalline

consistency. Non- slime producing colonies usually remained pink. An

undetermin results was indicated by a darkening of the colonies but with

the absence of a dry crystalline colonial morphology.

Tube Method (Christensen et al., 1982; Freeman et al., 1989) 2-

This is a qualitative assessment of slime production a standard

glass culture tubes were used in this method as the following:

Tryptone soya broth (10ml) were inoculated with a suspension of

bacterial isolate that turbidity is approximately equivalent to the

McFarland No. 0.5 turbidity standard. The tubes were incubated

overnight (18-24) hours at 37ºC. The cultured tubes and the control tube

(without bacterial growth)were then emptied of their contents and stained

by adding 10ml of safranin stain solution (2-2-2-6). Each tube was then

gently rotated to ensure uniform staining of any adherent material on the

inner surface and the contents gently decanted. The tubes were then

placed upside down to drain. A positive result was indicated by the

presence of an adherent layer of stained material on the inner surface of

the tube or visible film lined the walls of the tube. Ring formation at the

liquid-air interface was not considered indicative of slime production.

3- Tissue culture plate method (TCP) (biofilm assay) (Christensen et

al., 1985; Lim et al., 2004; Mathur et al., 2006)

The tissue culture plate assay described by Christensen et al.

(1985) is the most widely used and was considered as a standard test for

the detection of biofilm formation. This method was applied on twenty –

one isolates of Methicillin resistance Staphylococci MRSA .These

isolates selected according to the multi-drug resistance pattern. The

influence of media composition on biofilm formation were also

investigated, therefore two media were used to evaluate biofilm

formation; brain heart infusion broth (BHI), and BHI with 1% glucose.

A suspension of bacterial isolate that equivalent to the McFarland

No. 0.5 turbidity standard were inoculated in BHI and incubated for 18

hours at 37ºC in stationary condition then diluted 1:100 with fresh BHI

and with BHI supplemented 1% glucose. Individual wells of sterile,

polystyrene, 96-well, flat-bottomed tissue culture plate were filled with

0.2ml aliquots of the diluted cultures and only broth served as control to

check sterility and non-specific binding of media.

The tissue culture plates were incubated for 24 hours at 37ºC. The

contents of each well were gently removed by tapping the plates, the

wells were washed three times with phosphate-buffered saline (pH 7.2) to

remove free-floating “planktonic” bacteria. Adherent organisms were

fixed by air drying and stained with 0.1% safranin. Excess stain was

rinsed off by distilled water or tap water and plates were kept for drying

and immediately de-stained with 95% ethanol. De-staining solution is

then transferred to a new well and the amount of the safranin extracted in

the de-staining solution is estimated with a Micro ELISA autoreader at

wavelength of 490nm. TCP method allows a quantitative measure of the

mass of biofilm cells.

The mean of (OD) value obtained from media control well was

deducted from all the test OD values. Classification (Table 2-11) based

on OD values obtained for individual strains of Staphylococcus spp. were

used for the purpose of data simplification and calculation.

Table 2-11: Classification of bacterial adherence by tissue culture plate

method (Christensen et al.,1985; Mathur et al.,2006)

Mean OD values Adherence Biofilm formation

< 0.120 Non Non/ Weak

0.120 – 0.240 Moderately Moderate

> 0.240 Strong High

2-2-6 Effect of Lysostaphin, propolis and antibiotics singly and in

combination on MRSA biofilm (Miranda-Novales et al., 2006)

Different concentrations of antibiotics ,lysostaphin and propolis

as mentioned in ( 2-2-4-1).used in this study to detect their effect on

biofilm formation depending on the method was applied by Miranda-

Novales et al., (2006) with simple modification as following steps:

1. A suspension of bacterial isolate that equivalent to the McFarland

No.0.5 were inoculated in tryptone soya broth and incubated for 6

hours at 37ºC.

2. The broth culture was diluted with the same volume of antibiotics

solution in combination by adding 1ml of the broth culture to each

tube that content antibiotic solution in combination to obtain the final

concentration as mentioned in ( 2-2-4-1).

3. bacterial-drug solution (0.2ml) from each tube was placed into the

aplastic tissue culture plate wells in triplicate for each tube, and

allowed to adhere up for additional 6 hours. The control was

performed in the absence of antibiotics as negative control.

After incubation, the contents of each well was removed and

washed three times with phosphate-buffer saline (pH 7.2) then the

adherent bacteria were fixed by air drying and stained with 0.1% safranin.

Excess stain was removed by washing with water. After drying de-stained

with 95% ethanol. De-staining solution is then transferred to a new well

and the amount of the safranin extracted in the de-staining solution is

estimated with a Micro ELISA autoreader at wavelength of 490nm and

compared with the negative control.

2-2-7 Antimicrobial activity in Treatment of Experimental Staphylococcal Keratitis (Irina etal.,2012)

1-Bacteria

MRSA strain S3 which used in the study was isolated from human

cornea. strain was propagated on Mueller-Hinton agar plates. Fresh

cultures were cultured for each experiment by inoculating bacteria onto

new plates and incubating the plates at 37°C for 18 h. then diluted with

normal saline solution to obtain bacterial suspension with 1.5 × 108

CFU/ml by adjusting to (0.5 Mc Farland standard).

2- Induction of experimental S. aureus keratitis ( Irina etal.,2012 )

Eighteen young healthy white rabbits (weight, 2.5 to 3.0

kg),brought from Bio-technology research center in AL-Nahrain

University were treated and maintained in plastic cages under optimum

condition of food, water, light and temperature..All rabbits were

anesthetized by intramuscular injection of xylazine (3 mg/kg of body

weight) and ketamine hydrochloride (35 mg/kg). Examination under a

stereo microscope, the central corneal epithelium (diameter, 7.5 mm) was

scraped with a needle. A total of 100 μl of a freshly prepared S. aureus

cell suspension injected intrastromally into the center of the cornea with a

30-gauge needle.

3- Treatment (Irina etal.,2012)

Following intrastromal injection of the bacterial cell suspension,

the rabbits were randomly divided into six treatment groups, group 1 was

treated topicaly with 2μg/ml the ethanolic extract of propolis drops;

group 2 received topical 5.62 μg /ml lysostaphins; group 3 was treated

with 3% ciprofloxacin drops along with 2μg/ml the ethanolic extract of

propolis drops, group 4 was treated with topical 2μg/ml the ethanolic

extract of propolis in combination with 5.62 μg/ml lysostaphin drop,

group 5, the control group (without infection), and group 6 was treated

with phosphate-buffered saline (PBS) drops. Two therapeutic regimens

were used. In the early-onset therapy schedule, infection was left to

develop undisturbed for 4 h prior to the initiation of therapy and topical

treatment was applied every 30 min during the first 5 hrs and then every

hour for four additional hours. In the late-onset therapy schedule,

infection was allowed to proceed for 10 hrs and topical treatment was

applied every 30 min for 5 h. One hour after the last drop application, all

rabbits with the MRSA-infected eyes were killed and their corneas

excised for bacterial quantification.

4- Determination of the number of viable bacteria (Irina etal.,2012)

For bacterial counting, uniform corneal were removed aseptically

from the rabbits eyes .The corneal were rinsed and homogenized in sterile

phosphate-buffered saline (3 ml/cornea). Aliquots of the corneal

homogenates were serially diluted in the same buffer, plated in triplicates

onto Muller-Hinton agar plates (100 μl per plate) and incubated for 24 h

at 37°C to determine the number of colony forming units (CFU) per

cornea . The lower limit of detection was one colony per plate, i.e., 30

CFU per cornea.

5- Histopathological evaluation

Histological sections were prepared according to Humason (1972)

.The infected eyes were fixed by 10% formalin, then washed by tap water

for several min., passing through a serial concentrations of alcohol (50%,

70%, 80%, 90% and 100%) for 2hrs in each concentration, then cleared

by xylol for 2hrs, saturated with paraffin at 60ºC for 3hrs, embedded in

pure paraffin; the blocks were then cut into sections with 5µm in

thickness by using Microtome.

These sections were held on glass slides using Myer’s albumin; they were

left at 37ºC to dry , Haematoxylin stain was applied for 5-10mins, washed

by tap water then with acidic alcohol then washed by tap water. After that

Eosin stain is used for 15-30seconds and then washed by distilled water.

Serial concentrations of alcohol (70%, 90%, and 100%) were then used

for 2mins in each concentration, cleared by xylol for 10mins; then

Canada balsam was used, covered by slide cover and examined by light

microscope.

2-2-8 Statistical analysis.

The Statistical Analysis System- SAS (2010) was used to effect of

treatments in study parameters. The LSD test the comparative between

means and Chi-square test to comparative between percentage in this

study.

Chapter Three Results & Discussion

3 Results and Discussion

3-1 Isolation and Identification of Staphylococcus aureus

3-1-1 Cultural Characteristics

one hundred specimens at first were cultured on a blood agar media,

isolates that suspected to be S. aureus isolates were more purified by

ABC streaking method. The isolates that appeared yellow colour,round,

convex, smooth, mucoid, raised on the surface of the medium, and

hemolysis surrounding colonies. Figure (3-1) showed only 38(38%)

isolates gave positive result on Mannitol Salt Agar these isolates can

tolerate the high concentrations of NaCl (7.5%) and ferment mannitol, an

acidic by product is formed that will cause the phenol red in the agar to

turn yellow( Appak, 2006).

Figure 3-1: The percentage of S.aureus isolated from collected specimens

3-1-2 Microscopically Characteristics

The 38 isolates were examined under light Microscope after

staining by Gram stain and the cells appeared as Gram-positive cocci

irregular clustered in large number (in grape- like irregular clusters).

38%

62%

S.aureus

other bacteria

3-1-3 Biochemical Tests

The results of S. aureus are summarized in table (3-1).

Table 3-1: The biochemical tests and their results for S. aureus

Test 38 Isolates

Gram stain 100% positive

Acetoin production 100% Positive

Catalase 100% Positive

Oxidase 100% Negative

Coagulase 100% Positive

DNase 100% Positive

Mannitol fermentation 100% positive

Gelatin liquefaction 100% Positive

Protease production 100% Positive

Nitrate reduction 100% Positive

Urease 100% Positive

Hemolysis behavior 86% β hemolysis

· Catalase test was performed and all 38 isolates gave positive results.

Hence staphylococci were differentiated from streptococci which

give negative results (Brooks et al., 2007).

· An oxidase test was performed to differentiate them from

Micrococcus which gave a positive oxidase test (Collee et al., 1996;

Mack, 2006).

Depending on tube coagulase test, present results showed 38 isolates

(82.6%) out of 46 isolates were able to produce coagulase enzyme

(coagulase-positive) (CoPS) that reacts with plasma substance called

"Coagulase-Reacting Factor" (CRF) to form a complex, which in turn

reacts with fibrinogen to form fibrin (the clot), while only 8 isolates

(17.3%) were coagulase-negative (CoNS). Coagulase testing is the single

most reliable method for identifying S. aureus. (Koneman et al., 1997;

Kateete et al., 2010).

· The fact that coagulase-positive bacteria are also able to hydrolyze

DNA makes the DNase test a reliable mean of confirming S.

aureus identification (Benson, 2001).The positive result was seen as

an opaque degradation zone around the DNase positive colonies

(Appak, 2006).

· When S. aureus allowed to grow on gelatin containing media

(gelatin liquefaction test), gelatin was liquefied by all isolates,

additionally, nitrate reduction test was performed for further

identification because the Staphylococcus species often reduce

nitrate to nitrite (Holt et al., 1994; MacFaddin, 2000). Also some

isolates showed the ability to produce acetoin and other did not. A

pink to red color appearing in the upper layer of the medium

indicate production acetoin from glucose (Roberson et al., 1992).

· Extracellular protease represents one of the most important groups

of hydrolytic enzyme. All isolates of S. aureus were able to produce

extracellular protease detected as clear zones of casein hydrolysis

around colonies in skim milk agar plate (Vazquez et al., 2008).

· In the present study, all S. aureus isolates produced urease. Urease

test is one the invasive test, consequently rising medium pH was

detected by phenol red indicator when ammonia produced by S.

aureus (Berry and Sagar, 2006).

· Staphylococcus aureus on human blood agar were yellow colonies.

staphylococcal isolates (100%) developed β hemolytic

behavior.Hemolytic proteins are extracellular protein and

commonly isolated from pathogenic bacteria, and β-hemolysins are

one of the important bacterial virulence factor. β-hemolysis caused

due to hemolysin production as clear zone along the streak on blood

agar plate with incubation period (Pandey et al., 2010).

· Finally the vitak- II Compact Staph system was used to confirm the

identification and typing of Staphylococcus aureus isolates which

previously identified by conventional biochemical tests, the result

from vitak- II Staph system were in agreement with those obtained

from biochemical identifications ,Appendix (no.1).

· The incidence of S. aureus varied among collected specimens (Table

3-2), depending on the source of isolation and type of clinical

specimens,volume of sample ,and according to diseses.

Table 3-2: Number of isolates and percentage of S.aureus

Sources of swab

Specimens

Number of

Specimens

%of S. aureus in each

specimen.

Sputum 15 5 (13.1%)

corneal swab 10 4 (10.5%)

Burn 27 13(34.2%)

Nasal swab 19 7 (18.4%)

Pus 7 2 (5.3%)

Urine 6 4 (10.5%)

Wounds 16 3 (7.9%)

Total 100 38 (38%)

3-2 Detection of Methicillin Resistance Staphylococci To investigate the distribution of Methicillin resistance

Staphylococci in the community and among the patients in the hospitals,

the antibiotic sensitivity test was applied to all 38 isolates that were

proved to belong to the genus Staphylococcus aureus, the test was

performed by using Methicillin antibiotic discs (5μg/disc) and applied the

antibiotic disc diffusion method (Figure 3-2).

Figure 3-2: Detection of Methicillin-Resistance Staphylococci by antibiotic disc

diffusion method using Methicillin disc (ME 5μg/disc) ,A: Detection of

Methicillin Resistance S. aureus (MRSA), B: Detection of Methicillin sensitive S.

aureus (MSSA)

From the collected clinical samples, 21(55.26%) out of 38 were

MRSA and the rest were MSSA 17(44.73%), (figure 3-3). Present results

were agree with peck et al., (2009) which showed that only (51.4%) of

isolates were methicillin resistant and (48.6%) were sensitive. Also,the

finding of AL-alem, (2008) showed that the ratio of MRSA strain was

(56%), while MSSA strains was (44%) in Turkish hospitals besides, the

prevalence of MRSA strain was (59%), and MSSA strain was (41%) in

Libyan hospitals.

B A

Resistance to methicillin may due to prevalence of mecA gene

among S. aureus isolates, which coding for penicillin- binding proteins

with very low affinity to β-lactam antibiotics including Methicillin

(Chambers, 1997; zetola et al., 2005).

On the other hand, current results did not agree with the results of a

local study by Al-Maliki, (2009) who showed that the percentage of

Methicillin resistance S. aureus (MRSA) to the Methicillin sensitive S.

aureus were (80.3% ,16.4%) respectively, while the intermediate

resistance to the Methicillin in the S. aureus was 3.3%. As well as, Al-

Hasani, (2011) reported that the ratio of Methicillin resistance S. aureus

(MRSA) was( 83.70% ). On the other hand, Al-Geobory, (2011)

showed that the ratio of Methicillin resistance S. aureus (MRSA) was(

90.90%). These observed differences may due to the variation in the

geographic area, sources of clinical specimens, genetic background and

the collection site of isolates.

Figure 3-3:The Percentages Methicillin resistance S. aureus (MRSA) to the

Methicillin sensitive (MSSA) S. aureus.

MRSA; 55%;

MSSA; 44%

MRSA

MSSA

the results showed that burn swabs obtained the highest rate of

Methicillin resistance Staphylococci (28.6) followed by nasal, wound,

sputum, cornea, pus, and urine in the rates of resistance 19%, 14.3%,

14.3% and 9.5% ,9.5% and 4.8% respectively (Table 3-3).

The wide spread of S. aureus in nasal, wound and sputum may due

to its presence as normal flora in human body, in addition to the truth that

the staphylococcus aureus bacterium was considering the particular

causative agent of hospital nosocomial infections.

Table 3-3: The number of MRSA isolates according to the sources of

collection

Type of swab

Specimens

No. of

samples

No. of

MRSA

Percent.

No. of

MSSA

Percent.

Sputum swab 15 3 14.3 2 11.8

cornral swab 10 2 9.5 2 11.8

Burn swab 27 6 28.6 7 41.2

Nasal swab 19 4 19 3 17.6

Pus swab 7 2 9.5 0 0

Urine swab 6 1 4.8 3 17.6

Wounds swab 16 3 14.3 0 0

Total 100 21 100 17 100

3-2-1 Antibiotic susceptibility of Methicillin Resistance Staphylococci

The pattern of antibiotics resistance of (MRSA) to penicillin,

Rifampin, tetracycline, Ciprofloxacin, Clindamycin, Clarithromycin and

Vancomycin was determined by using the disk diffusion method

according to the National Committee for Clinical Laboratory Standards

(now Clinical Laboratory Standards Institute) guidelines (NCCLS, 2003).

The results showed that MRSA isolates were resistant to Penicillin

(100%), Rifampin (33%), Tetracycline (52%), Ciprofloxacin (47%),

Clindamycin (28 %%). Clarithromycin (57%) and Vancomycin (7%)

(Figure 3-4).

Figure 3-4: Antibiotic susceptibility of Methicillin Resistance S.aureus(MRSA)

All isolates were (100%) resistant to penicillin, this result similar to

other local study by Zeidan, (2005), and Al-Jundiy, (2005) who observed

0%10%20%30%40%50%60%70%80%90%

100%

%Re

sist

ance

Antibiotics

high resistance levels to β-lactam antibiotics, the percentage of

penicillinG resistant in both studies were 90%, 90.5% respectively.

Similar result obtained by Brady et al., (2007) they observed that all

isolates were resistant to penicillin and other β-lactam antibiotics, other

study from neighboring country Iran done by Aghazadeh et al., (2009)

showed results in agreement with this study results. Resistance to

penicillinG and other β-lactam antibiotics may due to the presence of β-

lactamase enzymes that hydrolyze lactam ring and inactivate the

antibiotic (Llarrull et al., 2009).

Current results showed resistant of MRSA isolates to Vancomycin,

Ciprofloxacin ,Tetracycline and Clarithromycin at the percentages

(7%,47%,52%,57%) respectively. Similar result obtained by Al-Jumaily

et al., (2012) revealed that all isolates were resistant to Vancomycin,

Ciprofloxacin and Tetracycline (4.7%,45.8%,58.5%) respectively. while

Udo et al., (2006) work on MRSA isolated from burn patients at Kuwait

general hospital, reported that 96% of MRSA were resistant to

Gentamycin, Tetracycline, Erythromycin and Ciprofloxacin. There are

two mechanisms of Tetracycline resistance in Staphylococci, first, a

membrane protein mediates active efflux of the drug, and in the second a

cytoplasmic protein reduces the sensitivity of the ribosome to the drug.

The major gene in Staphylococci encoding active efflux tet(K) is usually

plasmid encoded and mediates resistance to Tetracycline and

Doxycycline but not Minocycline. Most clinical CoNS and S. aureus

seem to bear the tet(K) determinant (Archer and Climo, 1994;

Strommenger et al., 2003).

This results came in harmony with those obtained from previous

local study done by Zeidan, (2005) who showed that the rates of resistant

to Tetracycline, Rifampin and Vancomycin were (52%,35%,10%)

respectively. Furthermore, similar result obtained by Al-Jundiy, (2005)

was reported highly resistance to Tetracycline (67.6%) by (MRSA).

As well as, MRSA isolated in this study had vancomycin resistant,

however, the rates of resistance have remained low throughout the period

of study as well as worldwide (kim et al., 2004). The incidence of

infection due to vancomycin resistant organism has significantly

increased during the past decade. This is important because vancomycin

has been the drug of choice for the treatment of infections due to MRSA.

On the other hand, resistance of MRSA isolates to other antibiotics like

Clindamycin also investigated in this study, the results showed high

resistant of MRSA isolates reach to 28%. Similar result was obtained by

Al-Hasani, (2011) who reported high levels of Clindamycin resistance

(31.3%).

Generally, current results showed that Methicillin resistance

Staphylococci also resistant to other β-lactam antibiotics and

Cephalosporin but this feature was differ from one antibiotic to another

and this may due to the type of antibiotic and how much that used among

the patients in the community. In addition to that the resistance toward

any antibiotic was depended on the amount of β-lactamase enzyme or the

amount of (penicillin-binding protein) PBB 2a that produced by each

strain of bacteria. All these reasons could create variations in the rate of

resistance (Fuda et al., 2005 ).

As a result of the increasing percentage of infections caused by the

MRSA displaying intermediate sensitivity or resistance to vancomycin,

antibiotic therapy is becoming more difficult and often fails (Rayner &

Munckhof, 2005; Livermore, 2006). Hence, there is an urgent need for

the development of alternative therapeutics.

3-2-2 Antimicrobial activity of propolis, lysostaphin, ciprofloxacin

and vancomycin

3-2-2-1 Antimicrobial activity of Lysostaphin

On the other hand, current study was done to investigate the

antimicrobial activity of lysostaphin against selected isolates of

S.aureus (MRSA), the results of qualitative analysis have revealed that

lysostaphin had weakly effect against five isolates (S10,S15,S43and

S57) ,the inhabition zone was (9,9,8 and 9mm respectively)while S3 had

(11mm) when used lysostaphin at effected concentration (5.625 µg/ml) .

As well as by using quantitative method depending on the optical density

(OD at 620nm). statistical analysis confirm that there is no significant

effect of lysostaphin on the tested isolates,where the optical density after

treated with lysostaphin reduced ( from 0.391, 0.386, 0.389, 0.379,

0.381) to (0.352, 0.361, 0.326, 0.351, 0.349) Figure( 3-5). These results

came in harmony with Kusuma et al., (2007) demonstrated that the

development of lysostaphin resistance by two strains of (MRSA)

Consistent with the mutations found in previously reported lysostaphin-

resistant S. aureus variants, these two variants had mutations in their

femA genes, resulting in non functional FemA proteins and, thus,

monoglycine cross bridges in the peptidoglycan.

Kusuma and Kokai-Kun., (2005) determined the lysostaphin

susceptibility of various strains of S. aureus , 50% of isolates were

sensitive to lytic activity of lysostaphin, the MIC ranged between 0.005-

0.031 µg/ml for the strains examined with zones of inhibition of >13mm.

while lysostaphin-resistant S. aureus strains had no zone of inhibition.

Sabala et al., (2012) showed high sensitivity for lysostaphin by all

S.aureus isolates that the MIC was around 0.0015-0.003μg/ml, but

inhibition of bacterial growth was not observed even with 5 μg/ml. The

main difference between several studies and present study due to

Methicillin-resistant Staphylococcus aureus (MRSA) strains show strain-

to-strain variation in resistance level, in genetic background, and also in

the structure of the chromosomal cassette (SCCmec) that carries the

resistance gene mecA (Kim et al., 2011).

Figure 3-5: Effect of lysostaphin at concentration (5.625 (µg/ml) on S.aureus

isolates

3-2-2-2 Antimicrobial activity of propolis

The results of disc diffusion method of crude ethanolic extract of

propolis (EEP) showed that all S. aureus (MRSA) isolates were sensitive

to EEP. The S3 isolate was highly sensitive to EEP than other isolates at

concentrations ranged (16, 8, 4 and 2 µg/ml) with inhibition zones (29,27,

27and 23mm respectively )This results showed that the minimum

inhibitory concentration (MIC) was 2 µg/ml for S10, S15, S3 and S43,

with inhibition zones of (12, 10, 23 and 11mm) respectively, while S57

was the highest resistant among the selected isolates (MIC = 4 µg/ml)

with inhibition zone (11mm) in diameters Figure (3-6) .

0.29

0.3

0.31

0.32

0.33

0.34

0.35

0.36

0.37

0.38

0.39

0.4

S10 S15 S3 S43 S57

0.391 0.386 0.389

0.379 0.381

0.352 0.361

0.326

0.351 0.349

OD

620

nm

Isolates

OD:control

OD:after treated withlysostaphin

Figure 3-6: Inhibitory effect of Ethanolic extracted propolis , A:(EEP (4µg/ml))

,B: (EEP (2µg/ml )) on S.aureus(S3) isolates by using (disc) method

As well as, S3 (MRSA) isolate showed a highly microbial growth

inhibition (MIC=2 µg/ml) depending on the quantitative analysis, the

inhibitory effect was observed by mean absorbance values (OD at

620nm) of microbial cultures obtained in the presence of propolis was

lower than positive control (microbial growth), hence OD value was

decreased from 0.387nm to 0.077 Figure(3-7). These results were in

agreement with those of Helaly et al., (2011) noticed that the EEP had an

antimicrobial activity against Staphilococcus aureus, with 2500 μg/mL

MIC for (N31 and N25) isolates, while MIC was 1250 μg/mL for N6.

While Stan et al., (2013) showed strong inhibitory effect of propolis on

the growth of Staphylococcus aureus 13024. MIC value of propolis was

(0.1875 mg/mL).

A

B

Figure 3-7: Effect of different concentration of EEP (2 µg/ml for

S10,S15,S3andS43) and (4 µg/ml for S57 )on S.aureus isolates

Antimicrobial activities of crude extract of Al-Museiab propolis

(EEMP) tested by Hendi et al., (2011) at different concentration (10%,

20%, 30%), showed that S. aureus was higher sensitive to EEMP where

the effect of EEMP was elevated when the concentration increased to

20% and 30%. The zones inhibition of S. aureus were 28 mm and 30 mm

respectively.

The present results about S. aureus were in agreement with those

obtained by several authors who found that the inhibition zones obtained

by propolis from Mongolia, Albania, Egypt and Brazil were 24, 21.8,

24.3, and 21.8 mm respectively (Kujumgiev et al., 1999; Darwish et al.,

2010 ).

These results are comparable with results obtained by Prytzyk et

al. (2003) who found that the inhibition zone for Bulgarian propolis was

(20 mm) also with results obtained by Stepanovi et al., (2003) who found

out that the inhibition zone of propolis from different geographical areas

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

S10 S15 S3 S43 S57

0.284

0.234

0.387

0.311

0.233 0.231 0.215

0.071

0.283

0.218

OD

620

nm

Isolates

OD:control

OD:after treated withpropolis

of Serbia ranged from 18 - 23 mm. Also, results were in accordance with

previous reports (Fernandes et al., 2005; Gonsales et al., 2006).

Helaly et al., (2011) found that EEP showed different antibacterial

activity against different S. aureus isolates, the MIC values were (5000,

2500 and 1250µg/ml) for Staphylococcus isolates N4, N31, N25 and N6

respectively. Biological and pharmaceutical activity of propolis

contributed to the propolis contain active compound such as phenols,

flavonoids and alkaloids that possessing antibacterial activities, Ophori et

al.,(2010) who reported that the antimicrobial activity of propolis is as a

result of the high content of flavonoids. However, the variation might

reflect the difference in the composition of the propolis and type of

extracts with increased of concentration. Flavonoids were regarding

largest component of that the phenolic compound and it had

pharmaceutical and antimicrobial activities. Kumer et al., (2008)

explained that the concentration of flavonoids differs from sample to

other sample of propolis attributed to geographical area and concentration

of propolis extracts. Where Al-Zubady, (2009) reported that the

inhibition zones of S. aureus was 16 mm after treated with 150%

concentration of propolis collected from, Karbala.

3-2-2-2-A Physical Properties of Iraqi Propolis

physical properties of Iraqi propolis showed wide variation

especially in color between different samples of Iraqi propolis depending

on geographical origin and flora vegetation in that area. Propolis used in

this study had brownish yellow color, rigid waxy texture with midly

aromatic odor. This results came in agreement with Ali et al., (2012)

they showed that the iraqi propolis collected from different areas had a

broad range of varieties, it ranges between brownish yellow in Ba'qubah

propolis sample and dark brown in Al-Ramadi propolis sample depending

on flora vegetation that was mixed with different plants, and according to

geographical position.

Flavonoids and polyphenolic compounds form about 45-50% of

propolis composition in general and they had an important role in

propolis color (Burdock et al., 1998; Araujo et al., 2012). Whereas Silva

and co-workers (2008) reported chemical composition and botanical

origin of red propolis as a new type of Brazilian propolis, which

depended on secretions of plant species that were often mentioned as its

probable botanical source.

On the other hand, Texture of Iraqi propolis may be rigid or rigid

waxy depending on the amount of beeswax, and this extrusive

proportionate with beeswax that increased the softness of propolis from

rigid to rigid waxy or waxy, and in general, propolis contains about 30%

of wax, which affects the texture (Cohen et al., 2004). These results

approximating to Mot et al., (2010), their study was conducted on

Romanian propolis, and flora was complex or Meadow, high or low

content of mixture of deciduous forests.

Concerning Iraqi propolis odor, most of the samples were aromatic

resinous depends on flora vegetation, and types of chemical compounds

were essential and aromatic oils, which form 10% of propolis

composition (Szliszka et al., 2011).

3-2-2-2-B Identification of chemical compounds in propolis by using

Fourier transform infrared (FTIR and HPLC).

I - (FTIR) analysis of propolis

Identification of separated chemical compounds was indeed done

with FTIR Spectroscopic device. showed that the propolis sample

contain The (C–H) bands of aromatic compounds appear in the 3300–

2700cm−1 range, carbonyl group (C=O) gave rise to a strong absorption

in the region 1820–1660 cm-1, the peak was often the strongest in the

spectrum. Also (O–H) group found at 3300–2500 cm-1, and C=C band

was a weak absorption near 1650 cm-1. Aromatic ring as general gave

rise weak absorption in the region 1650–1450 cm-1. Aromatic bond υ C-

C in 1500-1550 cm-1 appendix(no.2, no.3).Some of these compounds

were similar to results of Ali and co-workers (2012), who identified ten

chemical compounds from Iraqi propolis for different regions of (AL-

Sulaymania, Erbil, Duhok, Nineveh, Kirkuk, Salah Al-din, Diyala and

Al-Anbar).

II - HPLC analysis of propolis

The analysis of propolis ethanol extract using HPLC revealed several

peaks had important bioactive-natural chemical compounds in Iraqi

propolis and these were flavanone, chrysin, galangin and phenolic acid

caffeic acid, naringenin, p-coumaric acid and pinocembrin, α-pinenine in

compared with standard peaks of compound appendix(No.4. No.5).

These compounds were similar to results of Naama and co-workers.,

(2010) that identified six chemical compounds from Iraqi propolis for

different regions of Baghdad area by TLC and HPLC, these compounds

were: chrysin, galangin and caffeic acid, naringenin, p-coumaric acid and

pinocembrin. As well as, some of these compounds were similar to

results of Al-Shamary, (2012) who identified several chemical

compounds from Iraqi propolis by HPLC, theses chemical compounds

included (α-pinenine, cinammic acid, isoferulic acid, poplins, medicarpin,

napthoquinone, trans-verbenol, chrysin and B-caryphyllene). While

Darwish and co-worker., (2010) investigated Jordanian propolis from

Amman city by column chromatography and identified three pure

phenolic compounds: pinobanksin-3-O-acetate, pinocemberin and

chrysin.

The determination of the chemical characteristics of EEP showed that

the phenolic compounds were mainly responsible for the anti-CoNS

activity of EEP collected from the Southeast of Brazil (Mantovani et al.,

2008).

3-2-2-3 Antimicrobial activity of Vancomycin

The sensitivity of S.aureus (MRSA) isolates to lysostaphin were

compared with vancomycin activity, current results showed that the

vancomycin had weak effect on all MRSA selected isolates at

concentration (8µg/ml) except MRSA S43 was more resistant to

vancomycin ,were the therapeutic effect was (16µg/ml). Inhibition zone

for S10, S15and S43 was (10mm), while it was (11mm) for S3 and S57.

However, the statistical analysis showed a slight change in the optical

density for these isolates after incubated with vancomycin when

measured at a wavelength of 620 nm. Optical density reduced (from

0.286, 0.235, 0.389, 0.298 and 0.235OD) to (0.253, 0.212, 0.354, 0.273

and 0.2018 OD) for bacterial suspensions without and with vancomycin

respectivly Figure( 3-8). Present results confirm previous result by Climo

et al., (2013) reported reduced suitability of MRSA at concentration

(8µg/ml ) to vancomycin.

Statistical analysis showed a slight effect of vancomycin on all

isolates in comparable with lysostaphin at effected concentration . It

inhibits early stages in cell wall peptidoglycan synthesis. In this study the

result of S. aureus susceptibility was disagreement with Placencia et al.,

(2009) reported that MIC ratio of vancomycin and lysostaphin trended

toward significance with p values of 0.07 and 0.1, respectively.

Vancomycin is the drug of choice for serious infections caused by S.

aureus strains that are resistant to beta-lactam antibiotics and for patients

who have potentially life-threatening allergy to the latter drugs. Recently,

several anecdotal reports have questioned the efficacy of vancomycin for

both MSSA and MRSA (Chang et al., 2003; Soriano et al., 2008).

However, In comparing the pharmacodynamics of vancomycin and

lysostaphin to the selected isolate (S3), we found no significant

differences between the two drugs at the diameter of inhibitions zone

which were (11mm). While Dajcs et al., (2000) revealed a significant

difference between lysostaphin and vancomycin activity against MRSA,

MICs were (0.0625µg/ ml and 1.2207µg/ml) respectively. Present result

proved a highly resistance of MRSA S3 isolate when compared to MRSA

isolated from Iraqi hospital (Babylon hospital) by Habeeb, (2011) which

was highly sensitive to vancomycin with 18mm inhibition zone. Finally,

overall, 67% of studies with vancomycin for the treatment of MRSA have

reported sterilization of vegetations for fewer than half of them (Climo et

al., 2013).

Figure 3-8: Effect of vancomycin on S.aureus isolates

3-2-2-4 Antimicrobial activity of ciprofloxacin

Regarding to ciprofloxacin, it revealed a weak effect against selected

S. aureus isolates, except S3 and S57. The effected concentration 3%

with inhibition zone (10mm) for MRSA S3, while it was 12% for MRSA

S57 with inhibition zone (10mm) Table( 3-4).

Table 3-4: Effect of different concentration of ciprofloxacin on

isolates using disc method in inhibition zone (mm)

Isolates

Inhibition zone (mm) at different

Concentration of Cipro.(%)

0.75 1.5 3 3 12

S10 0 0 0 0 0

S15 0 0 0 0 0

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

S10 S15 S3 S43 S57

0.286

0.235

0.389

0.298

0.235 0.253

0.212

0.354

0.273

0.2018

OD

620

nm

isolates

OD :Control

OD:after treated withvancomycin

S3 0 0 10 0 0

S43 0 0 0 0 0

S57 0 0 0 0 10

LSD value 3.500 *

* (P<0.05).

A study of Narita, (2008) reported that ciprofloxacin has been

used as a single agent to treat staphylococcal carriage or infection, it has

failed to eradicate the organism or resistance has developed in a

proportion of patients. In Taiwan, National data from 2000 (TSAR

program) has demonstrated 40% S. aureus (including MSSA and MRSA)

in vitro resistance to ciprofloxacin (Fukuda et al., 2002). While Dajcs et

al., (2004) found that MIC value of ciprofloxacin against S.aureus

(MRSA) was differ depending on the nature of individual isolate,

ciprofloxacin revealed activity at concentration (0.25µg/ml) against

MRSA301, while (16.88µg/ml) against MRSA 30155.

The quantitative test depending on the optical density reported that

S. aureus isolate S3 had a slight change in the OD value after incubated

with ciprofloxacin; it was reduced vegetation bacterial growth to (0.368)

compared with the control bacterial growth value (0.401).

The difference in the level of susceptibility to certain drugs could be

attributed to the frequency at which that individual drug has been used in

the hospital during the period of study. Furthermore, the antibiotic

resistance could be transferred from one bacterial strain to another by

transposable gene (Transposes), which could be transposed from the

chromosome to the proper plasmid (Mansouri et al., 2001).

In conclusion the antibacterial activity of propolis extracts were

more effective than antimicrobial agent due to the affinity of

antimicrobial agent and reaction with cell component and have specific

receptors on bacterial cell wall or specific carrier into the cell for stopped

the enzymes and coenzymes or interference with bioactivities such as

inhibition of protein and nucleic acid synthesis (Matthew et al., 2007).

3-2-3 Antimicrobial activity of Antibiotics and propolis combinations

The activities of propolis combination with antibiotics (vancomycin

and ciprofloxacin) on the bacterial isolates were examined in this study.

In addition this study considered the first time dealt with effect of

propolisA- lysostaphin combination on MRSA isolates.

Statistical analysis showed significant differences (P<0.05)

between effect of propolis and lysostaphin–propolis combination on

bacterial isolates but there was no significant differences between

lysostaphin and lysostaphin –propolis combination at level (P>0.05).

Propolis alone was completely active against all isolates since the

inhibition zones were ranged from (10-23mm) in diameter. Furthermore,

the optical density of bacterial culture were reduced significantly after

treated with propolis alone there were (0.23, 0.215, 0.077, 0.283 and

0.218nm) for (S10, S15,S3, S43,S57) respectively, but there’s no effect of

lysostaphin –propolis combination on the S.aureus isolates except

S3isolate showed slightly decreases in the OD value was reduced (from

0.389 to 0313nm) (figure 3-9).

Figure 3-9:Effect of combination lysostaphin - propolis A:OD of control, B:OD

after treated with lysostaphin(5.62 µg/ml)+propolis(2 µg/ml)

In contrast, lysostaphin was mostly inactive since the inhibition

zone was zero, except on the five isolates, the inhibition zone was range

(8-11mm)while it was reach to 12 mm in diameter after treated with

lysostaphin –propolis combination. Current results showed an

antagonistic effect of compensation toward propolis activation against

MRSA isolates (table 3-5).

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

S3

0.389

0.313

OD

620

nm

Treatment

A:OD of control

B:OD after treatment

Table 3-5: Effect of lysostaphin and EEP on isolates using disc

method

* (P<0.05), 0:no inhibition zone

Present results of vancomycin -propolis combination on the

bacterial isolates were; antagonistic words to S. aureus isolates. Statistical

analysis showed significant differences (P<0.05) between effect of

propolis, vancomycin and vancomycin-propolis combination on bacterial

isolates . vancomycin and propolis alone were completely active against

all isolates since the effected concentration were ranged from (8-

16µg/ml) and (2-4 µg/ml) respectively, the optical density of bacterial

cultures after incubated with effected concentration of vancomycin

weakly reduced ranged from (0.2018- 0.354nm). While propolis reduced

the OD values ranged (from 0.077 to 0.283nm) of bacterial cultures

significantly,while there’s no effect of vancomycin -propolis combination

on all selected isolates. These results agreed with Habeeb et al., (2011)

showed significant differences between effect of propolis and

vancomycin –propolis combination on bacterial isolates.

Same results obtained when combined of lysostaphin-

vancomycin togather, we’re not significantly better than the combination

of propolis with vancomycin, both regimen produced highly antagonism

Isolates Inhibition zone (mm)after treated with Lysostaphin (5.62µg/ml)+ EEP(2 µg/ml)

S 10 0

S15 0

S3 12

S43 0

S57 0

LSD value 3.554 *

in reducing S. aureus (MRSA) growth. Since the inhibition zones were

zero for all selected isolates. This result may due to isolates provide an

opportunity to gain insight into the range of adaptive strategies employed

by staphylococci to survive in the presence of glycopeptides (Vavra et al.,

2001), who obtained one of six clonally identical MRSA isolates (A–F)

from the blood of this patient who was receiving vancomycin therapy had

an MIC of vancomycin (10–12 mg/L) associated with an increased MIC

of the endopeptidase lysostaphin and slightly increased cell wall

thickness.

Among the isolates from dialysis patient, the MIC of vancomycin

changed from a susceptible to intermediate phenotype in the same

organism (isolate C) in which the MIC of lysostaphin increased. Thus,

lysostaphin resistance was the most distinctive change associated with the

vancomycin-resistant phenotype (Pfeltz et al., 2000; Deresinski, 2009).

In spite of Chuang et al., (2012) statement that vancomycin was

the most active agent against all MRSA isolates(100%) according to

antibiotic susceptibility test, but was less effective than in that report of

Blomquist,(2006); Freidlin et al., (2007). Although vancomycin retains

extremely high efficacy against MRSA, S. aureus with reduced

susceptibility to vancomycin was identified (Hawser et al., 2011). My

results proved several previous studies through reduced suitability to

vancomycin by MRSA isolates in this study. Since prior vancomycin use

is a risk factor for MRSA with reduced vancomycin susceptibility, and no

convincing evidence shows that routine vancomycin prophylaxis is

effective in elective cataract surgery (Gordon, 2001; Fridkin et al., 2003).

Moreover, the results of effectiveness ciprofloxacin-propolis

combination on the bacterial isolates were; synergistic to words all

MRSA isolates. Statistical analysis showed significant differences

between effect of propolis and ciprofloxacin –propolis combination

depending on inhibition zones were decreased to reach (10-13mm).

However, there were highly significant differences between ciprofloxacin

and ciprofloxacin–propolis combination at level (P<0.05), ciprofloxacin

was inactive against (S10,S15 and S43) the inhibition zones was zero,

while had slight activity against (S3 and S57) with 10mm inhibition

activity after 24hrs of incubation, while results showed significant

elevation of inhibition zones for all isolates were became

(10mm,10mm,13mm,11mm and11mm) for (S 10, S15, S3, S43 and S57)

respectively, after treated with ciprofloxacin–propolis combination Table

(3-6) . These results came in harmony with Oksu et al., (2005) asserted

that the synergism activities between ciprofloxacin and propolis on the S.

aureus, which found the combination of ciprofloxacin and propolis had

better effects than either agent alone. As well as, Helaly et al., (2011)

reported increase ciprofloxacin activity against S.aureus N3, where

inhibition zone elevated (from 5mm to 40mm) in diameters after

incubated with ciprofloxacin–propolis combination.

Table 3-6 :Effect of propolis and ciprofloxacin on S.aureus isolates

Isolates Concentration of . cipro (µg/ml) + propolis(µg/ml)

Inhibition zone (mm)

S10 3 + 4 10 S15 3 + 2 10 S3 3 + 2 13

S43 3 + 2 11 S57 3 + 4 11

LSD value --- 2.250 * * (P<0.05).

3-2-4 Detection of Slime layer and Biofilm formation by Methicillin

Resistance Staphylococci

Slime production has been reported in strains of all Staphylococcus

spp. associated with the infection of biomedical devices (Mathur et al.,

2006). The ability of Staphylococcus spp. to adhere and form

multilayered biofilms on host tissue and other surfaces is one of the

importance mechanisms by which they were able to persist in the diseases

(Jabra-Rizk et al., 2006). An association was observed between

multiresistance and biofilm production. The biofilm environments

seemed to increase genetic exchanges and hence may contributed to

multiresistance phenotypes (Araujo et al., 2006). From this point the

ability of Methicillin Resistance Staphylococci (MRSA) were tested to

produce slime layer and biofilm formation by three methods; (Tube

method, Congo red agar method, and Tissue culture plate method).

Additionally comparison between isolates and their ability to produce

slime layer and biofilm formation were evaluated.

A- Congo-red agar method (CRA)

This method was described by Freeman et al., (1989) to detect the

slime layer production by bacteria using a specially prepared solid

medium.

The results showed that 57% of MRSA isolates produced a strong slime

layer indicated by formation of black colonies with dry crystalline

consistency, while 43% of MRSA isolates were non-slime producer

indicated by formation of pink colonies with no change in the color of the

medium (figure 3-10).This result agreed with the study by Akiyama et al.,

(1997) who found that 50% of S. aureus isolates produced strong slime

layer on the Congo red agar and 40% was indeterminate producer and

only 10% showed negative result (pink colonies). Also, current study was

agreement with the study by Al-Hasani, (2011) showed 60% of CoPS

isolates were slime producer.

Figure 3-10 : Slime layer production on the Congo red agar (CRA). A: Negative

production of slime layer by S. auerus (MRSA) on CRA, B: Strong slime layer

production by S. auerus (MRSA) on the CRA.

B- Tube Method

The adherence ability of Methicillin Resistance S.aureus to

smooth surfaces (slime layer production) was achieved by method

described by Christensen et al., (1982). (figure 3-11).The results showed

that 100% of MRSA tested isolates were produced slime layer by this

method but the amount of adherent material was differ among the isolates

(ranged from weak to strong).

A B

Figure 3-11: Slime layer production at the bottom of glass tube (Christensen et

al., 1982; Freeman et al., 1989), A: strong positive, B: moderate positive ,

C:weak, D:control

C- Biofilm assay by Tissue Culture Plates Method (TCP)

This method that described by Christensen `et al. ,(1985) was

applied on isolates of Methicillin resistance Staphylococci selected

according to the multidrug resistance pattern. To evaluate the influence of

media composition (specially the influence of glucose). On the biofilm

production, the test was performed by using two types of media, the first

one was Brain heart infusion broth (BHI) and the second medium was

BHI supplemented with 1% glucose (BHI glu) appendix (No.6). First type

of media Brain heart infusion broth (BHI) in figure (3-12) showed that

the twenty –one isolates in the BHI gave the OD values ranged from

0.081 to 0.311. These values indicated weak biofilm formation and strong

adherence according to the classification of Christensen et al. ,(1985) and

Mathur et al. (2006).

Currently, the results showed that the use of second medium

(BHIglu) in figure (3-13) showed significantly increased bacterial growth,

A B C D

the OD values of twelve isolates were

(S10,S15,S3,S43,S57,S22,S35,S72,S86,S18,S11 and S37).

` However, the increasing in the OD value of (S79, S66, S91, S56,

S40, S35, S30, S26, S64 and S50) was considered non-significant.This

results agreed with previous studies reported that the presence of sugar

was played an important role in the stimulation of biofilm formation in

Staphylococcus spp .The impact of glucose in the induction of biofilm

formation in S. aureus and S. epidermidis also reflected by the fact that

most of the biofilm adherence assays included high concentration of

either glucose or sucrose (Dobinsky et al., 2003; Seidl et al., 2008) .

Fitzpatrick et al, (2005) demonstrated that biofilm formation was

increased four- to eight fold in all MRSA isolates when grown in brain

heart infusion (BHI) medium supplemented with glucose compared to

BHI alone . These observations suggested a strong dependence between

growth condition and biofilm formation in Staphylococci, so using of

various sugar sources was essential for biofilm formation (Mathur et al.,

2006).

O’Neill et al., (2007) demonstrated that under standard laboratory

conditions in BHI medium, only 8% of 114 MRSA and 18% of 98 MSSA

isolates were capable of biofilm development. These percentages

dramatically increased to 74% for MRSA and 84% for MSSA isolates

when grown on BHI supplemented with 1% glucose.

Figure 3-12:A: Biofilm formation on tissue culture plate using BHI as a

medium,A:control, B: Tissue culture plate(BHI)

Figure 3-12:B: Biofilm formation on tissue culture plate using BHIglu as a

medium,A:control , B: Tissue culture plate (BHIglu)

Figure 3-13: Percentage of biofilm formation using BHI & BHIglu

0

10

20

30

40

50

60

Strong Moderate Weak

23.8

33.3

42.8

23.8

57.1

19.0

%

BHIB before additionBHI glu. after addition

A

B

A

B

3-2-5 Activity of lysostaphin, propolis and other antibiotics as a

single and combination to reduced S.aureus (MRSA) biofilm

formation

The formation of biofilm, is difficult to eradicate by standard

antibiotic therapy. Thus, researchers are still looking for alternative

options to eliminate the biofilm-forming microorganisms (Budzyńska et

al., 2011; Kurlenda and Grinholc, 2012). The antibiofilm activity of Iraqi

propolis has not been yet studied therefore this study was done to

determine the antibacterial activity and especially antibiofilm formation

activity of this bee product against S.aureus (MRSA) .

Current results demonstrated highly inhibition in biofilm the mass

that formed by S.aureus (MRSA-S3) which was achieved in the

presence of propolis at concentrations (16, 8, 4,2 and 1 µg /ml), OD

values ranged (from -0.453 to 0.098nm). Statistical analysis confirmed

high activity of propolis was at MIC (2 µg/ml) which prevent MRSA S3

from biofilm, the optical density significantly decreased to (0.027nm) in

comparable with control group (0.3875nm) table(3-7). Present results

came in harmony with Stan et al., (2013) that revealed development S.

aureus biofilm the of inert substratum was inhibited by Romanian

propolis tincture at a concentration of (0.1875) mg/ml.

Table 3-7: Inhibition of biofilm –forming capacity of the selected

isolates at different EEP concentration

Isolate

Propolis (µg/ml)

S. aureus (S3)

16 8 4 2 1

OD of control cell 0.3875

OD of turbidty cell 0.0091 0.0038 0.0012 0.027 0.098

LSD value 0.233 * 0.178 * 0.181 * 0.153 * 0.084 *

* (P<0.05).

This results confirmed the high activity of Iraqi propolis used in

this study companing to that reported by Helaly et al., (2011) which

marked the ability of propolis to reduce biofilm forming mass by

S.aureus N25,N31, were used highly concentration ranged from (100 to

0.05mg/ml). The OD values ranged (from 0.1 to 0.55 nm) after 24hrs of

incubated period.

Other studies demonstrated MIC values for Staphylococcus sp.

higher than MIC value determined from present study: Schazzocchio et

al., (2006) recorded MIC values for 42 strains of Staphylococcus spp. 2.5

mg/mL, as well; Kouidhi et al. (2010) found that EEP possessed excellent

protective effects against biofilms activity of oral streptococci.

However, there is only a few published reports which study the

effects of propolis against biofilms-forming staphylococci or multidrug

resistant pathogens were investigated. It was found that ethanol extracts

of propolis can inhibit growth of the multidrug resistant bacteria, such as

methicillin-resistant S. aureus (MRSA), Enterococcus spp., and

Pseudomonas aeruginosa (AL-Waili et al.,2012).

The main components responsible for propolis biological activity

as antibiofilm agent are polyphenols and flavonoids (Sforcin & Bankova,

2011; Chaillou& Nazareno, 2009).

Budzyńska et al. (2011) studied 22 synthetic flavonoids of which

three 3-arylideneflavones, revealed efficient antimicrobial activity against

S. aureus. Interestingly, 3-arylideneflavone inhibited the initial adhesion

of bacteria to abiotic surfaces which resulted in blocking the biofilm

formation.

Moreover, this study investigated the ability of lysostaphin to

inhibit biofilm formation capacity the selected isolate (MRSA-S3). table

(3-8) showed weak decrease in the biofilm forming capacity of the tested

isolate S3 detected by lysostaphin alone. Statistical analysis showed

slightly effect of lysostaphin under Concentration (5.625µg/ml), optical

density was reduced to (0.312 nm) in comparable with control group

(0.389nm). While, previous study by Shah et al., (2004) showed

effective role of lysostaphin against biofilm formation as a coating for

catheters; Also, in a mouse model, lysostaphin has been used to eradicate

S. aureus biofilms from a catheterized jugular vein (Kokai-Kun et al.,

2009).


isolates at different lysostaphin concentrations

Isolate

Lysostaphin (µg/ml)

S. aureus 3

90 45 22.5 11.25 5.625

OD of control ( at 490nm)

0.389

OD after treatment(at 490nm )

0.183 l 0.214 0.263 0.264 0.312

LSD value 0.113 * 0.084 * 0.069 * 0.078 * 0.089 NS

* (P<0.05).

The difference between current and previous study may due to

lysostaphin –resistance of selected strains for the present study. Wu et al.,

(2003) showed that the disruption of S. aureus biofilms was specific for

lysostaphin-sensitive S. aureus, as biofilms of lysostaphin-resistant S.

aureus were not affected.

Studies the effect of propolis in combination with (lysostaphin,

ciprofloxacin) showed that Iraqi propolis had antibiofilm action, as well

as a synergistic effect with antibiotics acting on the proteins (Orsi et al.,

2012).These observations were further confirmed by other authors

(Fernandes et al., 2005), in addition Orsi et al., (2006) explained the

synergistic effects of propolis to diminish the resistance of the bacteria

cell wall to antibiotics, as suggested by Mirzoeva et al., (1997) promoting

beta-lactamics action on the peptideoglycan synthesis - an important

structure of bacteria cell wall (Silva, 2000) . Since, the statistical

analysis of current results, showed significant differences between effect

of propolis and combination of (lysostaphin –propolis )as antibiofilm

forming capacity of selected isolate S3, the OD value was decreased to

reach (0.289nm) in treated bacterial biofilm in comparable with control

group had (0.387nm). However, there were highly significant differences

between lysostaphin and lysostaphin–propolis combination at level

(P<0.05) (table 3-9).


isolates at different EEP and lysosotaphin concentrations

Isolate

Lysostaphin (µg/ml) + Propolis (µg/ml)

S. aureus 3

Lyso+ propolis 90+16

Lyso+ propolis

45+8

Lyso+ propolis 22.5+4




OD of control ( at490 nm)

0.387

OD after treatment(at490 nm )

0.067 0.165 0.266 0.274 0.289 0.381

LSD value 0.102 * 0.093 * 0.088 * 0.081 * 0.085* 0.079 NS

* (P<0.05).

Regarding ciprofloxacin combined with propolis, the presence of

combination decreased biofilm formation of S. aureus (MRSA S3). Such

decrease was prominent at 3% of ciprofloxacin with 2 µg/ml propolis.

Table (3-10).showed decrease in biofilm forming optical density from

(0.3879nm) to reach (0.238nm) after 24hrs of incubation period. This

result confirm a significant difference at antibiofilm formation capacity

between propolis and ciprofloxacin-propolis combination at level

(P<0.05).


isolates at different EEP and Cipro. concentrations

Isolate

Cipro % + Propolis (µg/ml)

S. aureus S3

Cipro+propolis 12+16

Cipro+propolis 6+8

Cipro+propolis 3+4

Cipro+propolis 3+2

Cipro+propolis 1. 5+2

Cipro+propolis 0.75+1

OD of control ( at 490 nm)

0.3879

OD after treatment(at 490 nm )

0.0021 0.097 0.131 0.238 0.382 0.389

LSD value 0.122 * 0.954 * 0.085 * 0.104 * 0.149 NS 0.034 NS

* (P<0.05).

The antimicrobial action of propolis is controversial and not

completely understood. The biological activity of propolis (EEP) may

vary according to its composition and seems to be multidirectional

(Speciale et al., 2006), involving several mechanisms such as the

disorganization of the cytoplasmatic membrane and the cell wall; partial

bacteriolysis; formation of pseudo multicellular colonies; and inhibition

of protein synthesis (Duran et al., 2006) It is assumed that the synergistic

effect of main components of propolis extracts like flavonoids (quercetin,

galangin, pinocembrin) and caffeic acid and/or cinnamic acid, probably

influence the microbial membrane or cell wall sites, resulting in

functional and structural effects (Dziedzic et al., 2013).

3-2-6 Experimental study on laboratory animals

Staphylococci are a leading cause of endophthalmitis (Pushker et

al., 2002) and bacterial keratitis, with the causative organism usually

thought to originate in the patient’s ocular surface, skin, or nasal cavity

(Oksuz et al., 2005). Despite the availability of numerous efficient

antibiotic agents, the management of S. aureus infections remains a

challenge due to the constantly increasing incidence of clinical isolates

resistant to antibiotics(Chuang et al., 2012). Antimicrobial resistance

results in increased illness, and health-care costs, highlighting the need

for novel antimicrobial agents.

Present study aimed to investigate role of (propolis, lysostaphin and

Double Combination of Some Antibiotics) in the treatment of Keratitis

caused by experimental local isolate methicillin resistant Staphylococcus

aureus (MRSA) in Rabbits.

3-2-6-1 Ocular Response to Infection

The ocular response to bacterial challenge and treatment was

examined depending on the scoring system: 0 = clear or normal cornea;

+1 = light to dense opacity at wound site; +2 = dense opacity fully

covering the pupil; +3 = dense opacity fully covering the anterior

segment; and +4 = corneal perforation or phthisis bulbi (i.e., shrunken

eye). All eyes were normal before infection, at 4 hours Post infection no

significant differences in disease scores 0 (P > 0.05) were observed

between the eyes of rabbits that were left untreated.

The doses of examined materials were selected depending on the

effected concentration calculated from the primary in vitro study. The

MIC value of propolis was determined as 2 µg/ml used for treated

group1.

Group 2: received lysostaphin at concentration (5.625 µg/ml). group: 3

included eyes that received propolis plus ciprofloxacin at concentration (2

µg/ml +3%), while group 4: eyes that received propolis plus lysostaphin

with concentration (2 µg/ml +5.625 µg/ml); group: 5 normal eyes without

infection consider as negative control ; `group:6 untreated eyes that

infected with MRSA S3, and consider as positive control.

Dense corneal opacity score 4: obscured anterior chamber details in

all four groups in addition to positive control group. Frank corneal ulcers,

mucous secretion and completely closed of eyes were observed in

untreated eyes (figure 3-14).

Figure 3-14: A: normal eyes without infection consider as negative control B:

untreated eyes that infected with MRSA S3, and consider as positive control

B A

After 5hrs of treatment the degree of inflammation (mucus

secretion, ulcerative morphology) was significantly lower in eyes treated

with propolis which had score2: when compared with other treated

groups and positive control group had score 4: 24hrs of treatment clinical

signs of keratitis in experimental rabbit were completely disappear for the

eyes receiving propolis alone with score 0: they had normal and healthy

appearance compared with negative control group (uninfected eyes)

(Figure 3-15).

Figure 3-15:A: eyes that received propolis at concentration (2 µg/ml )

B: normal eyes

On the other hand, eyes treated with lysostaphin plus propolis and

ciprofloxacin-propolis combination had significantly lower response to

both compensation, opacity score were 3: in comparable to negative

control group(figure 3-16)(figure 3-17).

Figure 3- 16: Eye treated with lysostaphin(5.62 µg/ml ) plus propolis(2 µg/ml )

Figure 3- 17: Eye treated with combination ciprofloxacin(3%) and propolis

(2µg/ml)

This results confirmed highly effectivity of Iraqi propolis than

Turkish propolis used in the previous study by Oksuz et al., (2005)

reported that the corneal opacity scores were significantly lower in eyes

that received propolis plus ciprofloxacin, the mean corneal opacity score

was: 1.25 than in those treated with propolis, the corneal opacity score

was 2.0 ; as well as, 3.625 in control eyes.

However, eyes receiving lysostaphin alone showed no changed in

the degree of inflammation, the mucous secretion and eyebrows closed

continually, with smaller size and shrunken eyes with opacity scores 4:

there’s no significant difference when compared with untreated group

(infected without treatment)(figure 3-18).

figure 3-18: Eye treated with lysostaphin (5.62µg/ml)no significant difference

when compared with untreated group

Current results demonstrated a highly effect of propolis on

S.aureus (MRSA) keratitis, when corneal dilution from group 1 plated on

the agar, no bacterial colonies grew on the plates after incubation for 24 h

at 37°C. The mean bacterial number was (0.0 cfu/ml), its equal to

negative control group (healthy eyes) has (0.0 cfu/ml) table(3-11), while

highly significant difference when results compared to positive control

group 5 (infected eyes without treatment).

Table 3-11:Number of bacterial isolates from eye

Isolate

CFU/ML

LSD value

Treatments

Controls

Staph. aureus (S 3)

Propolis 2 µg/ml

Lysostaphin 5.625 µg/ml

Prop+ lyso

2 µg/ml +5.625 µg/ml

Prop+ cipro

2 µg/ml +3%

+ control (infected)

-control (healthy)

---

0.0

7.31 4.28 3.94 7.42 0 2.418 *

* (P<0.05).

Total eradication of bacteria was observed in the treatment of

group1. The antibacterial activity of propolis against S. aureus (MRSA3)

was shown both in vivo and in vitro. Several studies have demonstrated

that propolis might act as a potent anti-inflammatory agent against both

acute and chronic inflammation (Massaro et al., 2011). The observed

anti-inflammatory effect of propolis could be attributed to its content of

flavonoids, phenolic acid and caffeic acid. Flavonoids were reported to

inhibit the activity of the enzymes involved in the conversion of

membrane polyunsaturated fatty acids, such as phospholipase A 2,

cyclooxygenase, and lipo oxygenase, to inhibit the release of the

lysosomal enzymes from rabbit polymorphonuclear leukocytes, and to

scavenge free radicals ( Araujo et al., 2012).

Propolis of Extracts were found to have an inhibitory effect on

enzyme dihydrofolate reductase similar to the well-known non-steroidal

anti-infl ammatory drugs (Oksuz et al., 2005). This property may explain

part of its anti inflamatory action. Probably the anti-inflammatory effect

of propolis and the decrease in bacterial count are thought to have played

role in reaching this a results.

As well as, Result from group2, were treated with lysostaphin

indicated no significant effect of lysostaphin against S.aureus (MRSA S3)

used in this study. The statistical analysis confirmed no significant

differences were observed in the mean bacterial number of treated group

in comparable with positive control group5 (infected eyes without

treatment) , the mean bacterial number was (7.31x106 cfu/ml and

7.42x106 cfu/ml) respectively.

This study differ with Dajcs et al., (2000) demonstrated the

effective role of lysostaphin at concentration (2.8 mg/ml) in reduced

bacterial number of S.aureus (MRSA) infected rabbit eyes; the

CFU/cornea was decreased to 0.85 compared to 6.59 CFU/cornea in the

untreated eyes.

On the other hand, present results came in harmony with Vavre et

al.; (2001) reported 33.33% of MRSA isolated from USA in 1999 was

initially associated with an increased MIC of the endopeptidase

lysostaphin and slightly increased cell wall thickness, finally became

highly resistance to lysostaphin, these isolates had diversity of change

documented in the cell walls of isolates associated with altered cross-

bridge structure. For example, substitution of one or more sites in the

pentaglycine cross-bridge by a non-glycine moiety, e.g. serine, with or

without acquisition of the lysostaphin resistance gene produced isolates

resistant to lysostaphin.Boyle-Vavra et al.,(2001) demonstrated that a

single genetic or biochemical event cannot account for all resistance

observed to date.

In addition, study of Wootton et al., (2005) reported 88.8% of

S.aureus (MRSA) was resistances to lysostaphin contribute to, or

predispose to the development of, a thickened cell wall and glycopeptide-

intermediate resistance. Lysostaphin resistance in S. aureus is associated

with the genes femA,B, the former responsible for increasing serine

content and decreasing glycine content of the peptidoglycan interpeptide

bridge and the latter for shortened glycine bridges (Komatsuzawa et al.,

2002; Scher et al., 2006).

Moreover, this results may due to activity of immune systems of

lab animals inhibited activity of antibacterial efficiency of lysostaphin on

MRSA by sera with high anti-lysostaphin antibody titers. Dajcs et al.,

(2002b) mention that low neutralization titers of the serum from

immunized animals may reflect a minimal number of antibody molecules

specific for epitopes within or adjacent to the catalytic site of lysostaphin.

However, the mean bacterial number from corneal cultures of

group3, treated with a combination of propolis and lysostaphin at

concentration (2µg/ml +5.625µg/ml ) demonstrated mild effect of this

combination against MRSA S3 keratitis with bacterial number was

(4.28x106cfu/ml).

There was a significant difference when compared to the positive

control group5 at level (P<0.05). As well as, the combination of propolis

and Ciprofloxacin caused a significant reduction of the mean bacterial

number (3.94 x106 cfu/ml) of MRSA S3, when used to treat rabbit eyes in

group4 after 24hrs of incubation at 37° C. No significant different

(P>0.05) between both group at the mean bacterial number of the S.

aureus (MRSA S3) colonies.

The mean bacterial number MRSA S3 isolated from non-treated

rabbit eyes (positive control group) was (7.42 x106 cfu/ml), the statistical

analysis confirm significant difference (P<0.05 ) at the mean bacterial

number between non treated (positive control) group5 and those in other

four treated groups.

Reported study of Dajcs et al., (2004) quantitatively compare

effectiveness of ciprofloxacin against S.aureus (MRSA) isolates in a

rabbit keratitis model, the levels of effectiveness of ciprofloxacin(0.3%)

aganist of S.aureus (MRSA301: MRS30155:MRSA60171).

Ciprofloxacin produced less significant reductions in the number of CFU

per cornea for MRSA301, while did not produce signifcant reduction in

the mean bacterial number CFU/ML of (MRSA30155; MRSA 60171)

compared to that of the untreated control group. From this results, can

conclude that the effect of ciprofloxacin depending on the resistance of

individual strain which differ from one to another.

Studies of ciprofloxacin resistance by Miller et al., (2008)

demonstrated that the therapeutic success or potency of an antibacterial

agent is a complex interelationship between drug and its ability to reach

the target site (pharmacokinetics), the microbial pathogen and

susceptibility to the selective drug (pharmacodynamics), and the

underlying immune status of the patient. The third partner in this

complex relationship is what both the drug and the pathogen do to the

patient. This interplay is described by the patient’s age, genetic

background, underlying disease and prior antimicrobial exposure.

Results of this study were more effectively than other previous

studies. Oksuz et al., (2005) showed there were significantly fewer

bacteria in eyes receiving propolis plus ciprofloxacin than in those treated

with ciprofloxacin or propolis or control eyes treated with PBS. The mean

number of bacteria in corneal cultures was: (42.875± 6.49 cfu/ml) in eyes

treated with propolis-ciprofloxacin combination. While the mean

bacterial number was (192.37 ± 46.97cfu/ml and 219.37 ±51.44 cfu/ml)

in group received ciprofloxacin and group treated with propolis

respectively.

From above results, we concluded a total eradication of bacteria

was observed in the group1: treated with propolis; followed by group3

and group4, while the less effective treatment in group2: they received

lysostaphin. Since, bacteria may be resistant to several antimicrobial

drugs, propolis may constitute alternative for treating this pathogen.

Although the properties of propolis have been the subject of several

investigations, it is difficult to compare the results of different studies,

due to the different composition and different methods used for the

evaluation of propolis antibacterial activities (Balestrin et al., 2005). This

results came in agreement with Onlen et al., (2007) found higher activity

of propolis in treated rabbit keratitis than other rabbit resaved

ciprofloxacin-propolis combination, ciprofloxacin and dexamethasone

were found to be statistically the same (P>0.05), According to the mean

bacterial counts and corneal opacity scores in comparable with positive

control group (infected without treatment).

3-2-6-2 Histopathological examination

An uninfected healthy cornea is shown in Figure (3-19) . As

expected, the multilayered corneal epithelium and endothelial monolayer

are intact. The only cells present in the stroma are keratocytes. The

anterior chamber is free of any cellular infiltrate. Twenty-four hours after

infection with S. aureus (MRSA S3), the untreated cornea Figure(3-20)

experienced substantially more pathologic changes to the eyes of rabbits.

Marked increases in corneal pathologic effects were observed at 15 and

25 hours Post infection and included chemosis, iritis, corneal epithelial

erosion, and opacity caused by fibrin in the anterior chamber and PMN

infiltration into the cornea. This results agreed with Karicherla and

Hobden, (2009) reported highly edematous, damaged endothelial layer.

The stromal layer is engorged with neutrophils, and the epithelium is

desquamating. The anterior chamber is full of proteinaceous exudate and

neutrophils in experimental rabbit had keratitis.

Figure 3-19: Section of eye (ciliary body) (control negative ) showing normal

structure of ciliary body(CB)&ciliary process(CP),(400X), (H&E).

CP

CB

R

NR

NP D

M

Figure 3-20: Section of eye (retina layer)(control positive) showing

degeneration(D) &damage of the retina layers ,rod(R) ,nuclei of rod(NR),nuclei

of bipolar (NP),Muller fibers (M) ,(400X), (H&E).

Corneas treated with propolis alone at concentration (2 µg/ml )figure

(3-21) were significantly less inflamed than untreated MRSA S3–infected

corneas. The corneal epithelium and endothelium looked like an

uninfected cornea figure (3-19), and few neutrophils were observed in

either the stroma or the anterior chamber. The histopathology of these

corneas were essentially the same as of uninfected corneas significantly

no inflammation, few neutrophils in the stroma and anterior chamber, and

relatively normal corneal epithelium and endothelium.

Figure 3-21: Section of eye ball (ciliary body&sclera) treated with propolis at

concentration (2µg/ml) look like normal appearance of ciliary proceses(CP)

&sclera(S),ciliary muscle (CM),(400X), (H&E).

CP

S

CM

However, corneas treated with lysostaphin only Figure ( 3-22) were

highly edematous in comparable to uninfected eyes. Histopathological

changes showed highly neutrophils infeltrations with higly damaged

endothelial layer, as well as, the anterior chamber are full of

proteinaceous exudate and neutrophils. Desquamation of the epithelial

cells .Corneas treated with lysostaphin had similar appearance to

untreated corneas Figure (3-20).

Figure 3-22: Section of eye (retina layer) treated wih lysostaphin at

concentration (5.625 µg/ml) showing still there was degeneration(D) &damage of

retina layers after treatment , outer plexiform (OP) ,cones (C) , ganglion cells

(G),(200X), (H&E).

D

C

OP

G

Corneas treated with propolis-lysostaphin and propolis-ciprofloxacin

combination Figure(3-23)(3-24) had moderate degree of pathological

changes included moderate corneal ulceration, conjunctivel edema, and

minor accumulation of fibrin in the anterior chamber were the primary

changes noted in those treated groups, than untreated corneas Figure (3-

20) but were still inflamed compared with an uninfected cornea Fig.(3-

19). Neutrophils were present in the stroma and anterior chamber, but to a

lesser extent than in untreated corneas.

Figure 3-23: Section of eye (cornea and ciliary process layer layer showing mild

inflammatory cells(IN) infiltrate of ciliary process(CP) and oedema(O) after

treated with propolis (2µg/ml) and lysostaphin(5.625 µg/ml) ,(400X), (H&E).

CP

IN O

S

Figure 3-24:Section of eye (cornea) layer showing mild inflammatory cells(IN)

infiltrate ,odema (O),stroma (S), after treated with propolis (2µg/ml )and

ciprofloxacin(3%) ,(400X), (H&E).

The corneal epithelium essentially appeared intact, though the

superficial layer of cells looked as if they were beginning to desquamate.

Corneal endothelial cells seemed enlarged and rounded compared with

the flattened appearance of normal endothelial cells. Furthermore, the

presence of focal thickness suggested endothelial cell dysfunction.

The fulminant destruction of the cornea that infected with MRSA

S3 ocular infection is a result of bacterial toxic products (α-Toxin, a 34-

kDa oligomeric hemolysin, is present in readily detectable amounts in

approximately 75% of S. aureus strains) (Dajcs et al., 2002a ; Girgis et

al., 2005) and an exuberant inflammatory response (Neutrophils and

other leukocytes are involved in the cornea’s inflammatory response to

microbial proliferation and invasion. These host reactions account for

much of the edematous, infiltrative, and necrotizing changes in bacterial

keratitis (O’Callaghan et al., 2007).

IN

O

Thus, antibiotics can eliminate viable bacteria from the cornea but do

little to prevent the subsequent damage from released bacterial and

neutrophil-derived proteases and toxins. The increased metabolic activity

of polymorphonuclear leukocytes and the subsequent release of free

radicals into the corneal stroma can cause extensive damage even after

the bacteria have been killed with antibiotics. The ability to essentially

recover from S. aureus keratitis was occurred with equivalent numbers of

PMNs present in the infected corneas. The effects of bacterial toxin on

the eyes of rabbits differ with individual variation of experimental

animals suggest that a difference in the direct action of the toxin

determines the outcome of the infection. This concept is supported by the

difference in toxin susceptibility in the erythrocytes of young and aged

rabbits(O’Callaghan et al., 2007).

The present study agreed with O’Callaghan et al., (2007) described

differences between young and aged rabbits in their susceptibility to S.

aureus keratitis, to corneal administration of α-toxin, and to erythrocyte

lysis by α-

toxin in vitro. These results are consistent with the concept that the toxin

causes direct pathologic cellular effects, the extent of which could

determine both the amount of direct tissue damage and the amount of

immunopathology induced by S. aureus keratitis.

For all these reasons, propolis considered drug of choice in the

future, Marcio et al., (2012) mentioned the anti-inflammatory activity

was associated with propolis or compounds such as polyphenols

(flavonoids, phenolic acids and their esters), terpenoids, steroids and

amino acids. The main mechanisms underlying the anti-inflammatory

activity of propolis included the inhibition of cyclooxygenase and

consequent inhibition of prostaglandin biosynthesis, free radical

scavenging, inhibition of nitric oxide synthesis, reduction in the

concentration of inflammatory cytokines and immunosuppressive activity

(Tam-no et al., 2006; Rebiai et al., 2011). Propolis was found to exert an

anti-inflammatory activity in vivo and in vitro models of acute and

chronic inflammation, indicating its promising potential as anti-

inflammatory agent of natural origin and as a source of chemical

compounds for the development of new drugs (Moura et al., 2011).

Conclusions &

Recommendations

Conclusions

1- The isolation rate of S. aureus isolates was 38 % among different

clinical specimens, with high prevalence range of MRSA strains

(55.26%) among the clinical isolates of S. aureus ,The Methicillin

resistance Staphylococci isolates showed resistance towards

methicillin (multi-drug resistance pattern). 2- Propolis (2 µg/ml) have highly growth inhibition effects against

Multidrug resistant (MRSA) isolates of S. aureus, While

lysostaphin, vancomycin and Ciprofloxacin were less effective

than propolis.

3- Combination between propolis with ( ciprofloxacin,lysostaphin)

give synergistic effect .While Combination propolis- vancomycin

and combination lysostaphin- vancomycin give antagonistic effect.

4- MRSA isolates have the ability to produce a slime layer but with

differences in their quantities, propolis at MIC(2 µg/ml) were

significantly inhibited the biofilm formation ability of MRSA

isolates when using alone more than Lysostaphin, was less

effective. 5- Results showed synergistic activity between propolis and

(lysostaphin ,ciprofloxacin) at effected concentration as antibiofilm

formation ability by all selected isolates. 6- Histopathological change showed that propolis was more effective

in treatment of MRSA keratitis than lysostaphin ,while

combination propolis - ciprofloxacin more effective than

combination lysostaphin –propolis.

Recommendations:

1- Using PCR for detection of mecA gene in local isolates of

Staphylococcus spp. and investigate the correlation of this gene

with other genes that govern multi-drug resistance to other

antibiotics.

2- Using PCR technique in diagnosis of microbial keratitis in Iraq.

3- Investigating the antimicrobial activity of propolis against other

diseases caused by S. aureus.

4- Studying the effect of propolis combined with other antibiotics or

other natural antimicrobial agents against MRSA keratitis.

5- Trying to extract the more effective substances of propolis and

investigate their role and mechanism of action as antimicrobial

agents.

6- Investigating other genetical methods such as phylochip and FISH

technology for identification and specification of microorganism

causing microbial keratitis.

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for a specific enzymatic approach to infectious disease. Prog. Drug.

Rer . 16 :309-33.

Appendix

Appendix 1

Results of biochemical tests for the identification of Staphylococcus spp. Isolates

by vitak 2 Compact System

Biochemical tests Codes S. aureus Isolates

D-AMYGDALIN AMY -

Ala-Phe-Pro ARYLAMIDASE APPA -

Leucine ARYLAMIDASE LeuA -

Alanine ARYLAMIDASE AlaA -

D-Ribose dRIP -

NOVOBIOCIN RESISTANCE NOVO -

D-RAFFINOSE dRAF -

OPTOCHIN RESISTANCE OPTO) +

PHOSPHATIDYLINOSITOL PHOSPHOLIPASE C PIPLC -

CYCLODEXTRIN CDEX -

L-Proline ARYLAMIDASE ProA -

Tyrosine ARYLAMIDASE TyrA -

L-LACTATE alkalinisation ILATK +

GROWTH IN 6.5% Nacl NC6.5 +

O/129 RESISTANCE (comp.vibrio.) O129R +

D-XYLOSE dXYL -

L-Asparatate ARYLAMIDASE AsapA -

BETA –GLUCURONIDASE BGURr -

D-SORBITOL dSOR -

LACTOSE LAC +

D-MANNITOL dMAN +

SALICIN SAL -

ARGININE DIHYDROLASE 1 ADH 1 +

BETA GALACTOPYRANOSIDASE BGAR -

ALPHA CALACTOSIDASE AGAL -

UREASE URE +

N-ACETYL-D-GLUCOSAMINE NAG +

D-MANNOSE dMNE +

SACCHAROSE/SUCROSE SAC +

BETA –GALACTOSIDASE BGAL +

ALPHA-MANNOSIDASE AMAN -

L-Pyrrolidonyl-ARYLAMIDASE PyrA +

POLYMIXIN B RESISTANCE POLYB -

D-MALTOSE dMAL -

METHYL –B-D-GLUCOPYRANOSIDASE MBdG +

D-TREHALOSE dTRE +

ALPHA-GLUCOSIDASE AGLU -

PHOSPHATASE PHOS +

BETA-GLUCURONIDASE BGUR -

D-GALACTOSE dGAL +

BACITRACIN RESISTANCE BACL +

PULLULAN PUL -

ARGININE DIHYDROLASE 2 ADH2a +

Abbreviations: +: positive; -: negati

Appendix 2

Sample analysis by FTIR

Appendix 3

FTIR of stander

Appendix 4

Sample analysis by HPLC

Appendix 5

HPLC of stander

Appendix 6

The OD value of biofilm formation in TCP obtained by using two

tested media (BHI & BHIglu )

Isolates

OD 490(nm)

LSD value Growth media BHIB BHIBglu

S 10 0.278 ± 0.06 0.366 ± 0.02 0.042 * S15 0.284 ± 0.02 0.386 ± 0.04 0.051 * S3 0.311 ± 0.01 0.447 ± 0.06 0.044 *

S43 0.262 ± 0.04 0.399 ± 0.01 0.062 * S57 0.276 ± 0.03 0.412 ± 0.00 0.077 * S22 0.171 ± 0.03 0.213 ± 0.03 0.043 * S64 0.146 ± 0.03 0.198 ± 0.05 0.066 NS S26 0.161 ± 0.02 0.197 ± 0.02 0.055 NS S30 0.187 ± 0.04 0.202 ± 0.04 0.052 NS S35 0.151 ± 0.01 0.182 ± 0.04 0.059 NS S40 0.179 ± 0.01 0.188 ± 0.02 0.047 NS S56 0.184 ± 0.02 0.226 ± 0.03 0.050 NS S72 0.106 ± 0.02 0.189 ± 0.05 0.042 * S86 0.081 ± 0.03 0.191 ± 0.01 0.051 * S18 0.093 ± 0.03 0.175 ± 0.02 0.048 * S11 0.086 ± 0.05 0.169 ± 0.03 0.055 * S37 0.119 ± 0.01 0.204 ± 0.05 0.046 * S91 0.110 ± 0.03 0.117 ± 0.01 0.039 NS S66 0.099 ± 0.02 0.113 ± 0.01 0.044 NS S79 0.086 ± 0.01 0.088 ± 0.00 0.027 NS S50 0.0916 ± 0.02 0.113 ± 0.01 0.043 *

* P<0.05, ns: non-significant

الخالصة

المرضى الراقدين في مستشفى الكندي نعينة سريرية م۱۰۰شملت هذه الدراسة جمع

التعلمي و المختبرات التعليمية لمدينة الطب في بغداد للفترة من حزيران ولغاية كانون االول

) ، ۲۷)، والحروق (۱٦( )،و الجروح ۱۹.هذه العينات توزعت بين مسحات(أالنف (۲۰۱۲

٪) ۳۸(عزلة ۳۸ تحديد وتم . )٦) ،واالدرار(۱۰) ، والقرنية ( ۱٥) ،و القشع (۷والتقيحات (

موجبة منها كانت ٪) ۸۲.٦( عزلة ۳۸الدراسة ان اظهرتS.aureus .تعود الى جنس

في ))Coagulase-Positive Staphylococci) )COPSلفحص االنزيم المخثر للبالزما

(Coagulase-Negativeنتيجة سالبة ٪)۱۷.۳( ۸اظهرت حين

Staphylococci)CONS(( .

تم التحري عن مقاومة العزالت للمضادات الحيوية بواسطة طريقة انتشار القرص على

من العزالت كانت مقاومة للمثسلين في ٪) ٥٥.۲٦(االكار الصلب وقد اظهرت الدراسة ان

من العزالت كانت حساسة تجاه هذا المضاد وان اعلى معدالت مقاومة ٪)٤٤.۷۳(حين ان

لبكتريا المكورات العنقودية الذهبية هي البكتريا المعزولة من مسحات (الجروح والتقيحات)

.٪)۱۰۰(وبنسبة مقاومة

ولقد أظهرت النتائج التي تم الحصول عليها من اختبار الحساسية للمضادات الحيوية ان

مثسلين الحيوية األخرى باإلضافة إلى الكانت مقاومة للعديد من المضادات MRSAعزالت

متعددة المقاومة للمضادات الحيوية).(

استخدمت في هذه الدراسة عدة انواع من المضادات البكتريية الحياتية الطبيعية

كساسين ، والكيمائية وهي (المستخلص الخام لمادة العكبر، الاليسوستافين ، السبروفلو

(MRSAوالفانكومايسين )،اختبرت فعاليتها في تثبيط نمو بكتريا المكورات العنقودية من نوع

كشفت النتائج أن بكتريا المكورات العنقودية .)بصورة منفردة واخرى مجتمعة بطريقة االنتشار

لص من المستخ )مايكروغرام/مل ۲ (أعلى حساسية للتركيز ت) كانMRSA S-3الذهبية (

األخرى المستخدمة في هذه المضادات البكتريية الحياتية الكيمائيةمن خام لمادة العكبرال

الدراسة.

ذات `)MRSAاظهرت الدراسة ان بكتريا المكورات العنقودية المقاومة للمثسلين (

مقاومة عالية للفعالية الضد الميكروبية لكل من (الاليسوستافين ، الفانكومايسين ،و

ميكروغرام/مل ٥.٦۲٥( وكساسين)عند استخدامها تحت تراكيز منهمالسبروفل

،حيث لم تظهر اي فروق معنوية عند التحليل االحصائي وكانت )%۳و ميكروغرام/مل ،۱٦،

التوالي.مقارنة بمقاومة هذا العزلة للفعالية الضد على) ملي متر۱۰و۱۱ ،۱۰(اقطار التثيط

).P>0.05مستوى احتمال (مايكروبية لمستخلص العكبر الخام تحت

،)MRSA- S3( المختارة العزلة على المزيج هذا من التأثيرالتأزري اظهرت النتائج

نمو سبروفلوكساسين) حيث تم تثبيط مع عند خلط( العكبر وقد تم الحصول على نفس النتائج

اظهرت حين في التوالي، على) ملي متر۱۳ ،۱۱( إلى التثبيط منطقة ارتفاع مع البكتيريا

)MRSA- S3 (مع فانكومايسين من مجموعة ضد عالية مقاومة )بحيث) عكبر واليسوستافين

.الذهبية المكورات العنقودية لبكتريا كان تاثيرها متضاد

أن النتائج الطبقة اللزجة وأظهرت إنتاج على MRSAتم التحري عن قابلية العزالت

طريقةاالنابيب باختالف كمية المواد بواسطة اختبارها كانت لها القابلية على انتاجها عند جميعها

من٪ ٥۷ أن اكار أحمر الكونغو لطريقة وفقا النتائج أظهرت ذلك، ومع. الملتصقة بين العزالت

MRSA من٪ ٤۳ كانت لها القابلية القوية على تكوين الطبقة اللزجة و MRSA اعطت نتيجة

الطبق الزرعي على انتاج البايوفلم بواسطة MRSAالتحري من قابلية تم وبالمثل .سالبة

لها القابلية العالية والقوية على MRSA العزالت أن إلى النتائج وأشارت) TCP(النسيجي

ذلك إلى باإلضافة. )۰.۳۱۱-۰.۲٦۲بين( تراوحت بايوفلم تشكيل من OD وقيمة تكوين البايوفلم

.من الكلوكوزالى الوسط٪۱ عند إضافة مبايوفل لتكوين OD اظهرت زيادة كبيرة في قيمة

عن فعالية المركبات المستخدمة سابقا (العكبر ،الاليسوستافين التحري تم

،السبروفلوكساسين ،والفانكومايسين)ضد قابلية انتاج بكتريا المكورات العنقودية من نوع

MRSA))مادة العكبر كان مثبط مايكروغرام/مل)من ۲) للبايوفلم . اظهرت النتائج ان اقل تركيز

)حيث بلغت ODلقابلية البكتريا على انتاج طبقة البايوفلم مماادى الى انخفاض قراءة (

.نانو ميتر )۰.۳۸۷٥(السيطرة بمجموعة مقارنة) نانو ميتر۰.۰۲۷(

تاثير قليل لاليسوستافين تحت تركيز اإلحصائي التحليل أظهر

على تكوين البايوفلم حيث انخفضت )MRSA -S3( على قابلية ) مايكروغرام/مل٥.٦۲٥(

ناحية من). نانومتر ۰.۳۸۹( السيطرة بمجموعة مقارنة) نانومتر ۰.۳۱۲( إلى(OD) قراءة

في ) سيبروفلوكساسين اليسوستافين ،( التاثيرالتآزري للعكبر مع النتائج أظهرت أخرى،

.المختارة التركيزالعالجي ضد تكوين البايوفلم من قبل العزالت

الدراسة النسيجية درست فيها فعالية المركبات السابقة (العكبر ،الاليسوستافين

،اليبروفلوكساسين ،والفانكومايسين ) بتراكيز مختلفة اعتمادا على نتائج الدراسة المختبرية في

المختارة عالج التهاب القرنية الناتج من بكتريا المكورات العنقودية المقاومة للمثسلين للعزلة

)MRSA-S3 في الحيوانات المختبرية (االرانب ) .اظهرت نتائج الدراسة تاثيرا عاليا لمادة (

مايكروغرام/مل ) حيث ادت الى اختفاء عالمات التهاب القرنية بصورة تامة ۲العكبر بتركيز (

لعالج ذات مقارنة بمجموعة حيوانات السيطرة الطبيعية ،اذ كانت عيون الحيوانات المصابة بعد ا

مظهر طبيعي يخلو من اي ارتشاح الخاليا االلتهابية .

بينت الدراسة النسجية عدم تاثير الاليسوستافين في عالج الحيونات المصابة حيث

اظهرت المقاطع ارتشاح الخاليا االلتهابية مع تلف شديد ببطانة القرنية ،اذا لم يكن هنالك فرق

المصابة غير معالجة ، من ناحية اخرى اظهرت نتائج معنوي عند مقارنتها مع الحيوانات

الفحص ان مجموعة الحيوانات المعالجة بخليط (عكبر +الاليسوستافين ) و(عكبر

+سبروفلوكساسين ) تحسن بسيط بدرجة االصابة اذ لوحظ قلة نسبة تقرح العين اضافة الى تراكم

نتائج معنوية مقارنة بمجموعة الحيوانات طفيف في الليفين في حجرة العين االمامية،وكانت هذه ال

المصابة غير معالجة فيما كانت ذات تاثير بسيط عند مقارنتها بالحيوانات المعالجة بمادة العكبر

ومجموعة السيطرة غير مصابة .

جمهورية العراق وزارة التعليم العالي و البحث العلمي

جامعة بغداد كلية العلوم

دراسة تاثير المضادات الحيوية والعكبر على امراضية

المكورات العنقودية الذهبية المقاومة بكـتريا للمثسلين

ةرسالكلية العلوم / جامعة بغداد وهي جزء من متطلبات نيل مجلس مقدمة إلى

علم األحياء المجهرية -درجة الماجستير في علوم الحياة

من قبل الشيخايالف باسم نوري

علوم الحياة / االحياء المجهرية / كلية العلوم / جامعة بغداد سبكالوريو)۲۰۱۱(

بأشراف

.م.د. هناء سليم يوسفا

۲۰۱۳تشرين الثاني / ۱٤۳٥محرم /

study the effect of antibiotics and propolis on ...scbaghdad.edu.iq/library/biology/ms.c/2013/study...

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