BY Maha Mustafa Ahmed Mohammed

69 Pages·2016·1.14 MB·English

Studies on Bacillus Species metabolites and
their bactericidal effect against
Staphylococcus species

BY
Maha Mustafa Ahmed Mohammed

B.SC. (F. of science and Technology )
ElNEElAIN UNIVERSTY
(2002)

Supervisor:
Professor : Suliman M .EISanousi

A thesis submitted to the University of Khartoum
in partial fulfillment of the requirements of master
Degree of in Microbiology

Department of Microbiology
Faculty of Veterinary Medicine
University of Khartoum

(2007)
PREFACE

This work described in this thesis was carried out in the Department of
Microbiology, Faculty of Veterinary Medicine University, of Khartoum
under supervisition of Prof .Suliman Mohamed Elsanousi

i
Dedication
This thesis is dedicated to my father,
who taught me that the best kind of
knowledge to have is that which is
learned for its own sake.
It is also dedicated to my mother,
who taught me that even the largest
task can be accomplished if it is done
one step at a time.
It is also dedicated to my brother ,
Your friendship is a treasure beyond
compare
It is also dedicated to my beloved
husband Ahmed
None of this would be possible without
your love and support

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Acknowledgment

I would like to express my sincere thanks and gratitude to
my supervisor professor Suliman Mohammed Elsanousi for
his Advice, interest, Leadership, and paitient guidance.
Thanks also extended to the staff of the Department of

microbiology including head department, Technicians for

their cooperation and assistant

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LIST OF CONTENTS
No. Title Page

Title……………………………………………. i.
Preface………………………………………………………. ii.
Dedication……………………………………………………. iii.
Acknowledgment……………………………………………………… iv.
Abstract…………………………………………………. v.
Objectives………………………………………………………….. vi.
ةحورطلاا صخلم …………………………………………………….. vii.
Table of contents ………………………………………………….. viii.
List of tables………………………………………………………… ix.
List of figures……………………………………………………… x.
Introduction………………………………………………………… xi.
Chapter one Literature review ……………………………………. 1
1.1 Genus Bacillus…………………………………………………….. 1
1.1.2 Classification and identification of the genus Bacillus ……………. 2
1.13 Habitat …………………………………………………………… 3
1.1.4 Morphological and cultural characteristic…………………………. 3
1.1.5 Pathogenicity ……………………………………………………… 4
1.1.6 Bacteriocin production……………………………………………. 4
1.1.7 Bacteriocin Classification ……………………………………….. 5
1.1.8 Mechanism of bacteriocin action…………………………………... 6
1.19 Bacteriocingenic species…………………………………………… 9
1.2 Staphylococcus………………………………………………...…… 10
1.2.1 General characteristic of Staphylococcus…………………………. 10
1.2.2 History and taxonomy and classification of Staphylococcus………. 10
1.2.3 Morphology of Staphylococcus ……………………………………. 12
1.2.4 Growth and cultural characteristic of Staphylococcus ………….. 12
1.2.5 Staphylococcus infection …………………………………………. 15
1.2.6 Staphylococcus aurues …………………………………………………. 16
1.2.7 Staphylococcus epidermidis ………………………………………………. 17
1.2.8 Staphylococcus saprophyticus …………………………………………. 18
1.2.9 Closer look at vancomycin resistance …………………………… 18
1.3 Antimicrobial chemotherapy ……………………………………… 20
1.3.1 Spectrum of action of antimicrobial drugs ……………………….. 20
1.3.1.1 Classification of microorganism…………………………………. 21
1.3.1.2 Antibacterial activity ……………………………………………… 21
1.3.1.3 Effect of antimicrobial Bactericidal or bacteriostatic 21
1.3.2 Mechanism of action of antimicrobial drugs………………………. 21
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2 Chapter two Material and method ………………………………… 23
2.1 Organisms …………………………………………………………. 23
2.2 Equipment …………………………………………………………. 24
2.3 Media………………………………………………………………. 24
2.3.1 Nutrient Broth …………………………………………………… 24
2.3.2 Nutrientagar 24
2.3.3 Blood agar 24
2.3.4 Indol medium ……………………………………………………. 24
2.3.5 Voges -Proskauer V.P test 25
2.3.6 Casein agar medium ……………………………………………….. 25
2.3.7 Starch Medium………………………………………………………. 25
2.3.8 Gelatin agar medium………………………………………............. 26
2.3.9 Citrate media………………………………………………........... 26
2.3.10 Ammonium salt agar……………………………………………… 26
2.3.11 Motility medium…………………………………………………… 27
2.3.12 Hugh and leifson (O.\F)medium………… ………..................... 27
2.3.13 Nitrate broth………………………………………………………... 28
2.4 Buffers……………………………………………………………… 28
2.4.1 Normal Saline………………………………………………............ 28
2.4.2 Distilled Water…………………………..…………………………. 28
2.5 Tests reagents……………………………………………………… 28
2.5.1 Hydrogen peroxide………………………………………….......... 28
2.5.2 Nitrate test reagent…………………………………………………. 28
2.5.3 Neutral red………………………………………………………… 28
2.5.4 Kovac's reagent……………………………………………………. 29
2.5.5 Methyl red………………………………………………………….. 29
2.5.6 Bromocresol purple…………………………..……………………. 29
2.5.7 polyethylene glycol………………………………………………… 29
2.5.8 Xylene……………………………………………………………… 28
2.5.9 Hydrochloric acid………………………………………………….. 29
2.5.10 Oxidase test reagent …………………………….. ………………… 30
2.5.11 Andrads indicator test reagent………………………………………. 30
2.6 Stains……………………………………………………………….. 30
2.6.1 Gram Stain…………………………………………………………. 30
2.7 Selection of Bacillus spp which produce metabolite against 30
Staphylococcus spp ……..
2.7.1 Preparation of metabolite……………………………………………. 30
2.8 Methods for detection the inhibitory effect of Bacillus spp on 31
Staphylococcus spp………………………………………………..
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2.8.1 Disc Method………………………………………………………... 31
2.8.2 Scraping Method…………………………………………………… 31
2.8.3 Strip method……………………..………………………………… 31
2.9 Determination of inhibitory activity unit of metabolite in filtrate . 32
2.10 Determination the bacteriosataic and bactericidal activity of 32
metabolite filtrate………………………………………………….
2.11 Thermostability of metabolite filtrate…………………………….. 32
2.12 Solubility of metabolite filtrate in methanol………… 32
2.13 Stability of metabolite filtrate against enzymes ………………….. 33
3 Chapter Three Result……………………………………………….. 34
3.1 Screening of Bacillus spp for their production of metabolite activity 34
against Staphylococcus……………………………………..
3.2 Determination of inhibitory activity unit……………………........... 34
3.4 Determination the bacteriostatic and bactericidal activity of 35
metabolite filtrate…………………………………………………
3.5 Thermostability result………………………………………………. 35
3.6 Solubility of metabolite filtrate in methanol……………………… 35
3.7 Stability of metabolite filtrate against enzymes…………………... 35
4 Chapter four Discussion…………………………………………… 41
5 References…………………………………………………………. 43

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Abstract

The present study was carried out to isolate and identify the different
type of Bacillus ssp from different sources.And to tested their metabolites as
bactericidal to Staphylococci
Thirty two Gram positive bacilli were isolated from different sources.
The main source was contaminants in bacteriological cultures.
The isolates were screened for their production of metabolites against
Staphylococcus (Staphylococcus aureus, Staphylococcus epidermidis and
Staphylococcus saprophytycus).
Three of these isolates were positive for production of metabolites that
completely inhibited Staphylococcus growth .The three positive isolates
were Bacillus cereus, Bacillus lichniformis and Bacillus subtilis
The metabolites were isolated and partially purified from these
species in order to study their chemical and physical properties. The result of
chemical and physical properties of the active metabolites filtrates revealed
the presence of an extracellular thermosensetive (the metabolite degrading
by high temperature ) ,but these metabolites could be kept at room
temperature for long time .
In order to establish the inhibitory activity unit different metabolite
filtrates dilutions were tested against Staphylococcus spp. The inhibitory
activity unit which is higher dilution of metabolite filtrate that inhibits in
vitro the Staphylococcus growth Was 1x10-1
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From plate assay to establish the bacteriostatic or bactericidal activity
of metabolite filtrate, the result revealed that the metabolites have
bactericidal effect against staphylococcus spp.
The metabolite filtrate stability test against enzymes revealed that the
metabolite filtrates were denatured by protease enzyme

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ىيحشنا ًٍحشنا الله ىغب
تحٔشطلأا صخهي
سداصي ٍي ،واشج تغبصن ّبجًٕنا تيهيعابنا ثايصعنا ٍي ٌٕثلاثٔ ٌاُثإ لضع ىت
ثايصعنا ِزْ ٍي تثلاث .تيجٕنٕيشتكابنا عساضًنا ٌاك سداصًنا ةزْ ىْأ ،تفهتخي
تيدٕقُعنا ثاسٕكًنا عإَأ ضعب ًَٕ طابحإ يهع ةسذقًنا آن ٌاك ّنٔضعًنا تيهيعابنا
تيششبنا ثاسٕكًنأ تيبْزنا تيدٕقُعنا ثاسٕكًناك
يْ تيدٕقُعنا ثاسٕكًنا ًَٕ طابحإ يهع ةسذقنا آن ٌاك يتنا تيهيعابنا ثايصعنا
دإًنا ِزْ لضع ىت ىث ظيسٕفيُشين ظهيعابنأ ظهتبع يهيعابنأ طشيع ظهيعابنا
ضعب تعاسذن ةسٕكضًنا تيهيعابنا ثايصعنا ٍي تيهيعابنا ثايصعنا اْصشفت يتنا
يفٔ ءاًنا يف تبئار يْ دإًنا ِزْ ٌأ ٍيبتٔ .تيئايضيفنأ تيئايًيكنا آصإخ
ىيضَا ٌأ اًك ايشتكبهن متاق شيثأت آن ٌأ ًاضيأ ٍيبتٔ ِساشحهن تعاغح آَأ اًك لَٕاثيًنا
.دإًنا ِزْ ميهحتٔ كيكفَ يهع مًعي K ضيُيتٔشب

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