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:

iProfessor : Suliman M.EISanous

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)

i

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

ii

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

iii

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

iv

No. Title Page

Title……………………………………………. i. Preface………………………………………………………. ii. Dedication……………………………………………………. iii. Acknowledgment……………………………………………………… iv. Abstract…………………………………………………. v. Objectives………………………………………………………….. vi..vii ..…………………………………………………… االطروحةملخص 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

LIST OF CONTENTS

v

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

Staphylococcus spp …….. 30

2.7.1 Preparation of metabolite……………………………………………. 30 2.8 Methods for detection the inhibitory effect of Bacillus spp on

Staphylococcus spp……………………………………………….. 31

vi

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

metabolite filtrate…………………………………………………. 32

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

against Staphylococcus…………………………………….. 34

3.2 Determination of inhibitory activity unit……………………........... 34 3.4 Determination the bacteriostatic and bactericidal activity of

metabolite filtrate………………………………………………… 35

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

vii

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

viii

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

ix

اهللا الرحمن الرحيمبسم

األطروحةملخص

م ون ت ان وثالث ن عزل إثن ن م رام، م صبغة ج ه ل يلية الموجب صيات الباس صادر الع م

ة زارع الباآتريولوجي ان الم صادر آ ذة الم م ه ة، أه صيات . مختلف ذه الع ن ه ة م ثالث

ورات العن آان المعزوله الباسيلية واع المك ة لها المقدرة علي إحباط نمو بعض أن قودي

العنقودية الذهبية والمكورات البشرية آالمكورات

صيات ان الع ي آ يلية الت ا الباس ي له ة ه ورات العنقودي و المك اط نم ي إحب درة عل الق

ليشنيفورمس ثم تم عزل هذه المواد والباسيلس سبتلس والباسيليالباسيلس سيرس

صيات ا الع ي تفرزه يليةالت يلية المز الباس صيات الباس ن الع ورة م ةآ ض لدراس بع

ة ة والفيزيائي ها الكيميائي ة . خواص ي ذائب واد ه ذه الم ين أن ه يوتب ي ف اء وف الم

زيم للبكترياالميثانول آما أنها حساسة للحراره وتبين أيضًا أن لها تأثير قاتل آما أن ان

. علي نفكيك وتحليل هذه المواديعمل Kبروتينيز

x

Objectives

The objectives of this work were:-

1- Isolation and identification of different species of the genus Bacillus.

2- Studies the bactericidal effect of Bacillus metabolites on Staphylococcus

spp.

3- Study of physical and chemical properties of these metabolites.

xi

List of Table

Table (no) page

3.2 Determination of inhibitory activity unit in metabolite

filtrate

34

3.3 Theromstabilty……………………………………………. 35

3.4 Stabability of metabolite against enzymes……………….. 36

xii

List Of Figure

Figure

(no)

page

Figure (1) Detection of the inhibitory effect of Bacillus subtilis

metabolite on Staphylococcus epidermidis. The Disc

method

38

Figure (2) Detection of the inhibitory effect of Bacillus subtilis

metabolite on Staphylococcus epidermidis .The Strip

method

39

Figure (3) Detection of the inhibitory effect of Bacillus cerues.

metabolite on Staphylococcus aureus .The Disc method

40

1

1

Introduction

The use of microorganisms for biological purposes has become an

effective alternative to control plant and animal pathogens.

Bacillus are well known as bacterioin producer. However there is a

direct relationship between the growth condition (limitation in nutrition) and

capacity of production of these substances. The biological control of plant and

animal pathogens is of paramount importance nowadays, since the

conventional use of chemical treatment has been seriously questioned because

of environmental and human hazards.

There is direct relation between production of these active metabolites

from Bacillus spp and sporulation of the Bacillus cell. The multiplicity of

biologically active substances excreted by spore former into their culture

medium is well known, but remain unexplained . In bacilli, which will be

considered first, these are mainly antibiotics and exoenzymes , in clostridia

they include toxins . This active extracellular products include beside

antibiotics protease which is produced by Bacillus subtilis ,Bacillus

licheniformis and Bacillus cereus. It has strong proteolytic activity Also there

is amylase Asingle exoenzyme α amylase is responsible for the amylotic

activity. Two types of RNA degrading enzymes are found in culture filtrate

,and wall lytic enzyme .So we have many extracellular products ,that may

responsible for the inhibition of Staphylococcus spp :antibiotics and protease

and RNA degrading enzymes and wall lytic enzyme and amylase

2

This research refers to isolation of metabolites with bactericidal effect from

bacillus spp against staphylococcus and studying their chemical and physical

properties.

The objectives of this work were:-

1- Isolation and identification of different species of the genus Bacillus.

2- STUDY The bactericidal effect of Bacillus metabolites on Staphylococcus

spp.

3- Study of physical and chemical properties of these metabolites.

3

CHAPTER ONE

LITERATURE REVIEW

1.1The genus Bacillus

The organisms which belong to the group of aerobic spore bearing

bacilli are cylindrical , usually flagellated grow in chain and produce large

colonies on solid media(Merchant and Packer,1967) .Practically all species

liquefy gelatin .Only one species Bacillus anthracis is pathogenic(Merchant

and Packer,1967). They are found in soil and decaying vegetation where they

are most active in bringing about decomposition of organic substance

particularly those change grouped together under term ammonification. They

are, therefore, create an important in maintenance of soil fertility(Merchant

and Packer,1967) .Because of resistance of spore to high temperature and

desiccation these organisms are among those which frequently contaminate

culture media in laboratories. (Merchant and Packer,1967) .

The various species are differentiated from each other by size , shape

and position of spores and tendency to chain formation ,mobility by

differences in culture and physiologic characteristic ,and pathogenisity .Beside

Bacillus anthracis a few of other species which have been known to produce

pathogenic condition, Bacillus subtilis may cause panaophthalmitis (Merchant

and Packer,1967).

4

Species of the genus Bacillus are mainly Gram-positive rods motile

(some non motile forms occur) and non acid fast. They produce heat resistant

spores under aerobic conditions (Gordon et al., 1973).

Bacillus species are widely disturbed in the environment mainly because of

their highly resistant endospores. In soil endospores of Bacillus anthracis

prototype of the genus, can survive for more than 50 years, able to tolerate

extremely adverse conditions such as desiccation and high temperatures

(Quinn et al., 2002).

1.1.2Classification and Identification of the genus Bacillus:

The genus Bacillus, the type genus of the family Bacillaceae, included more

than 60 species (Sneath, 1986). Classification of the genus Bacillus was made

on the basis of aerobic growth of spore forming rods (Smith et al., 1952).

Generally the Bacillus species are identified by methods based on spore

morphology and biochemical reactions. Molecular methods such as DNA

homology (Skey et al., 1978 and Krych et al., 1980), pyrolysis gas

chromatography and pyrolysis mass spectrometry (O'Donnell et al., 1980)

were also used in the identification of Bacillus species.

There is considerable evidence that Bacillus thuringiensis (Bt) and Bacillus

sphericus (Bsph) should be considered a single species (Bauman et al., 1984).

Many reports reviewed that classical biochemical and morphological methods

5

of classifying bacteria have consistently faled to distinguished Bacillus

thuringiensis from Bacillus sphericus (Kampfer, 1991).

1.1.3 Habitat

Bacillus species are ubiquitous, inhibiting soil, water, and airborne dust

(Schnepf et al, 1998). Thermophilic and psychrophilic members of the genus

can grow at temperatures ranging between (5 - 58)Cْ and can flourish at

extremes of acidity and alkalinity ranging from pH 2 - 10 (Gordon et al.,

1973). Therefore Bacillus species can be recovered from a wide variety of

ecological niches. Some species may be part of the normal intestinal

microflora of humans and other animals (Elmer et al., 1997).

Most Bacillus species encountered in the laboratory are saprophytic

contaminants or members of the normal flora (Smith et al., 1952).

1.1.4Morphology and cultural characteristics

The Bacillus cell ranges from 0.5 - 1.2µm length in diameter (Merchant et al.,

1967). Catalase is produced by most species and sporulation is not inhibit by

most incubation temperatures, positive characteristics that aid in

distinguishing genus Bacillus from bacteria that can grow aerobically on

nutrient medium at 37Cْ, although the optimal temperature for growth of these

bacteria, termed mesophiles, is at 37Cْ. They can grow at temperatures ranging

between20-45Cْ. Those with an optimal incubation temperature of 15Cْ, are

6

termed psychrophiles and those with an optimal incubation temperature close

to 60Cْ, are termed thermophiles (Quinn et al., 2002).

1.1.5 Pathogenicty:

Bacterial pathogens of insects has been used to control crop and forests pest

for almost five decades. However, it has been used as an effective agent for its

importance to public health, primarily in mosquitoes and blackfiles control

(Tuason et al., 1979). Numerous Bacillus spp. have been identified as insect

pathogens except Bacillus. Anthracis the causative agent of human and animal

anthrax (Terrnova and Blake, 1978).

Bacillus cereus was found associated with food poisoning and other

opportunistic in human (Bergdoll, 1981). Other Bacillus species which may be

viewed as potential opportunistic pathogens, include Bacillus sphericus

Bacillus megaterium, Bacillus pumilus, Bacillus circulans, Bacillus

licheniforms Bacillus, mycoides Bacillus macerans, Bacillus coagulans and

Bacillus. thuringiensis (Sliman et al., 1987) .

1.1.6Bacteriocin production

Production of antimicrobial compounds seem to be a general

phenomenon for most bacteria ( Sugita, 1998). An admirable array of

microbial defense systems are produced, including broad-spectrum classical

antibiotics, metabolic by-products such as organic acids, and lytic agents such

as lysozyme. In addition, several types of protein exotoxins, and bacteriocins,

7

which are biologically active peptide moieties with bactericidal mode of

action, were described (Sugita, 1998). This biological arsenal is remarkable in

its diversity and natural abundance, since some substances are restricted to

some bacterial groups while other are widespread produced (Riley and Wertz.,

2002).

The search for new antimicrobial agents is a field of utmost importance.

The prevalence of antimicrobial resistance among key microbial pathogens is

increasing at an alarming rate worldwide (Singer et al., 2003). Current

solutions involve development of a more rationale approach to antibiotic use

and discover of new antimicrobials, but the problem of antibiotic resistance is

increasing globally and may render the current antimicrobial agents

insufficient to control at least for some bacterial infections (Bhavnani and

Ballow, 2000). Many bacterial species produce peptide antibiotics, called

bacteriocins, that often have an antimicrobial effect on closely related

organisms (Naclerio et al 1993). These compounds have been extensively

studied because of their potential applications in the food industry as natural

biopreservatives and in pharmaceuticals as antimicrobials (Milles et,al 1992).

Bacteriocins produced by lactic-acid bacteria have been the focus of many

investigations because of their particular importance in the dairy industry

(Jack et,al. 1995).

1.1.7 Bacteriocins classification

Based on their chemical structures, stability, and mode of action,

bacteriocins have been classified as:(i) antibiotics (ii) small heat-stable

peptides (iii) large heat-labile proteins and (iv) complex proteins that require

carbohydrate or lipid moieties for activity (Klaenhammer ,1993).

8

1.1.8. Mechanisms of action

The mechanisms of action of peptide antibiotics are diverse, but the

bacterial membrane is the target for most bacteriocins (Klaenhammer ,1993).

Sahl and Bierbaum, (1998) reported that antibiotics such as nisin, subtilin and

epidermin have novel secondary modes of action in addition to their well-

established lytic activity (Sahl and Bierbaum, 1998). This dual antibiotic

activity of a single molecule no doubt plays an important role in the

antimicrobial mechanism of some bacteriocins (McCafferty et,al .,1999).

Several bacteriocins or bacteriocin-like molecules have been described for

Bacillus spp., including coagulin (Hyronimus et., al 1998), cerein 7 (Oscariz

et.al., 1999). Subtilosin (Zheng and Slavik, 1999). And thuricin 7 (Cherif

et.al 2001), and some of them have a broad spectrum of antibacterial activity.

Bacillus cereus, isolated from soils of native woodlands of southern Brazil,

has antagonistic action against several pathogenic and food-spoilage

microorganisms, such as bacteria involved in bovine mastitis (Bizani and

Brandelli, 2002). Treatment with proteases and trichloroacetic acid

inhibits..Bacillus cereus antimicrobial activity (Bizani and Brandelli ,2002),

suggesting that it produces a bacteriocin-like substance.

Rodriguez et.al (2000) reported that Almost 70% of the isolates

exhibited antimicrobial activity against one or more indicator bacteria.

Among lactic acid bacteria isolated from raw milk 82 of 298 strains (27.5%)

displayed bacteriocin activity (Jaki et.al.,1999), while 8.7% of Staphylococcus

aureus strains isolated from cattle produced antimicrobial substances (Oliveria

et.al1998). (De Vuyst ,et.al.,2003) found that 122 of 426 Enterococcus strains

(28.6%) of various origins produced enterocin. Antimicrobial evaluation of

cyanobacteria demonstrated that 16.3% were active against Gram-positive and

9

5.8% against Gram-negative bacteria, while 10.5% possessed antifungal

activity (Jaki , et. al 1999). The high proportion of antimicrobial producing

strains may be associated with an ecological role, playing a defensive action to

maintain their niche, or enabling the invasion of a strain into an established

microbial community (Riley and Wertz , 2002)

The inhibitory effect was mostly against Gram-positive bacteria. Most

strains inhibited Bacillus cereus(Riley and Wertz. , 2002). The antimicrobial

spectra, essentially restricted to Gram-positive bacteria, may suggest that the

produced antimicrobial substances are related to a specific feature of Gram-

positive bacteria. It is well known that activity of bacteriocins produced by

Gram-positive bacteria is restricted to other Gram-positive bacteria (Riley and

Wertz , 2002).

Riley and Wertz (2002)reported that The pH values of the crude

antimicrobial substances indicate that the inhibitory effect was not due to

production of organic acids. Most of these substances were partially or

completely inactivated by proteases and TCA, suggesting that a protein

moiety is involved in the activity. This may indicate that bacteriocin-like

substances are implied in antimicrobial activity. These substances showed

high thermal resistance and low molecular weight, which are characteristics of

small hydrophobic peptides that constitute class II bacteriocins (Riley and

Wertz,2002).

A variety of antimicrobial compounds are produced by members of the

genus Bacillus, many of these were identified as peptides, lipopeptides and

phenolic derivatives (Nakano and Zuber, 1990). A wide range of antimicrobial

substances produced by Bacillus spp. isolated from arthropods were recently

described, including aromatic acids, acetylamino acids (amino acid analogs),

10

and peptides (Gebhardt et al,2002). Bacteriocin-like substances have been

related from Bacillus spp. isolated from soil (Bizani and Bradelli ,2002) and

vegetal tissues (Bechard, et al,1998). A siderophore with wide range of

antibacterial spectrum is produced by Bacillus spp. from fish intestine (Sugita,

1998) and aminopolyol antibiotic by soil isolates of Bacillus cereus (Stabb et

al.,1994).

Bacteriocins are inhibitory peptides or proteins, produced by different

groups of bacteria, which have bactericidal effects on micro-organisms closely

related to the producer (Jack et al., 1995). They have received increasing

interest, especially those produced by lactic acid bacteria (LAB), because of

their potential use as food additives and their efficiency for the biological

control of spoilage and pathogenic organisms (Delves-Broughton 1990).

Klaenhammer (1993) reported that the using of nisin in processed

cheese spreads has greatly stimulated research on the use of bacteriocins as

antimicrobial agents in food systems in the USA. (Klaenhammer 1993).

For the last decade, much research has focused on the bacteriocins

produced by lactic acid bacteria (LAB) (Klaenhammer, 1993 and Jack et al.

1995).

Elsanousi and other In 1978 observed that the isolation of Closterdium

chauvoei from Calves suspected to have died of blackleg disease was difficult.

At the same time the frequent isolation of bacillus spp from specimens

received from the field was suspected to have possible role played by bacillus

ssp in inhibition of Clostridium chauvoei. The same authors tested the invetro

inhibition of Clostridium chauvoei. Strain CH3by bacillus ceruse(Elsanoousi

et al,1978).

11

1.1.9 Bacteriocinogenic species

The genus Bacillus encompasses a number of bacteriocinogenic species

such as Bacillus subtilis which produces subtilin (Jansen and Hirschmann,

1944) and subtilosin (Zheng and Slavik ,1999). Bacillus coagulans which

produces coagulin (Hyronimus et al., 1998). Bacillus megaterium which

produces megacin (Von Tersch and Carlton ,1983) . Unlike some bacteriocins

produced by LAB, which can show a broad spectrum of activity against

Gram-positive bacteria including non-bacteriocinogenic LAB strains, Bacillus

spp. bacteriocins generally show a narrower inhibitory activity (Beegle and

Yamamoto ,1992).

Bacillus thuringiensis is widely used in agriculture for the control of

many insect pathogens (Beegle and Yamamoto ,1992). It is characterized by

the production of crystal proteins (α-endotoxins) with a specific activity

against certain insect species (Beegle and Yamamoto ,1992). Moreover, a

number of extracellular compounds are produced by Bacillus thuringiensis,

including phospholipases, chitinases, proteases (Lovgren et al. 1990) .

β-exotoxins secreted vegetative insecticidal proteins and antibiotic

compounds with antifungal activity (Stabb et al. 1994). The safe worldwide

use of Bacillus thuringiensis for over three decades on many agricultural

crops for protection from several insect parasites argues strongly that the

micro-organism can be harnessed for application in agriculture and industry

for food and feed production (Drobniewski , 1993).

Closely related to Bacillus thuringiensis are Bacillus cereus and

Bacillus weihenstephanensis which are frequently involved in the

contamination of food products such as boiled rice, raw milk and dairy

products (Drobniewski, 1993). The consumption of food contaminated with

12

Bacillus cereus can cause diseases associated with enterotoxins and emetic

toxins (Drobniewski, 1993). Because of the close phenotypic and genotypic

relation to Bacillus cereus and, Bacillus thuringiensis was considered to be a

potential source of bacteriocins active against the two pathogenic species.

Despite the wide biotechnological importance of Bacillus thuringiensis

and its versatility as a biological control agent the selection of

bacteriocinogenic strains has attracted little attention. Only four bacteriocins

have been partially characterized from Bacillus thuringiensis/Bacillus cereus

thuricin (950 kDa) and tochicin (10·5 kDa) from Bacillus thuringiensis strains

HD2 and HD868, respectively (Favret and Yousten 1989, Paik et al. 1997);

and cerein (9 kDa) and cerein 7 (3·9 kDa) from Bacillus cereus strains GN105

and BC7, respectively (Naclerio et al. 1993) (Oscariz et al., 1999).

1.2. Staphylococcus

1.2.1. General characteristics:

Staphylococcus are Gram-positive spherical bacteria that occur in microscopic

clusters resembling grapes (Kenneth, 2002).

The staphylococcus are divided into two groups on the basis of their ability to

clot plasma (the coagulase reaction), the coagulase positive and coagulase

negative staphylococcui (Kenneth, 2002). Nearly all strains of Staph aureus

produce the enzyme coagulase and all strains of Staph epidermidis lack this

enzyme (Kenneth, 2002).

1.2.2 History, taxonomy and declassification of Staphylococcus:

Staphylococci were observed in pus and cultivated by koch and Pasteur

independently in 1878 and 1880, Ogston is credited with applying the name

(Staphylococcus) in 1881 (Riemann, 1969).

13

In 1884 Rosenbach described the two pigmented colony types of

Staphylococci and proposed the appropriate nomenclature: Staphylococcus

aureus (yellow) and Staphylococcus albus (white).

The latter species in now named Staphylococcus epidermidis (Kenneth, 2002).

The genus staphylococcus is currently composed of 33 species, 17 of which

may be encountered human clinical specimens (Koneman et al, 1997).

As discussed before Staphylococci divided into two groups on the basisi of

their ability to clot blood plasma by the action of Staphylococcui coagulase,

the coagulase-positive species: Staph aureus, Staph intermedius and Staph

delphini and the coagulase variable species Staph hyicus (Kloos, 1990).

Coagulase negative species: Staph epidermidis, Staph lugdunensis and Staph

saprophyticus beside 30 more species (Kloos, 1990).

Staphylococci are generally found on the skin and mucous membranes of

humans and other animals (Koneman et al., 1997). Staphylococi, which found

in man and nonhuman primates are Staph aureus, Staph epidermidis, Staph

saprophyticus, Staph haemolyticus, Staph warneri, Staph hominis, Staph

simulans, Staph lugdunensis, Staph capitis, Staph scheiferi, Staph pasteuri,

Staph auricularis, Staph cohnii, Staph xylosus, Staph saccharolyticus, Staph

caprae, Staph pulvereri and staphylococci found in other animals are Staph

gallinarum, Staph lentus, Staph equorum, Staph vitulus, and the other

Staphylococcal species (mostly environmental) are Staph carnosus, Staph

caseolyticus, Staph kloosii and Staph arlettae ( koneman et al, 1997 ). Baron

et al., (1994) reported that, Staph lugdunensis, Staph schleiferi, and Staph

intermedius may also produce coagulase or clumping factor.

14

1.2.3 Morphology of staphylococci:

Staphylococci are perfectly spherical cells about 1 micrometer in diameter

they grow in clusters because staphylococci divide in two planes (Kenneth,

2002). Although organism in clinical material may appear as a single cell,

paris, or short chains (Murray et al., 1998). Young cocci stain strongly as

Gram positive on aging many cells become Gram-negative (Brooks et al,

1995).Capsules may be formed in very young cultures, but they disappear in a

few hours (Riemann, 1969).

1.2.4 Growth and cultural characteristics:

Staphylococci grow readily on most bacteriological media under aerobic or

micro aerophiliuc conditions (Brooks et al., 1995). They grow most rapidly at

37Cْ but from pigment best at room temperature (20 – 25Cْ) (Brooks et al.,

1995).

The colonies, usually 1 – 3 mm in diameter, are smooth, circular, low convex,

glistening, raised, and butyrous (Duguid, 1989).

They are distinguished from those of most other bacteria by their golden or

white pigmentation and greater opacity for their size (Duguid, 1989).

Staph aureus usually from grey to deep golden yellow colonies (Brooks et

al., 1995). Staph epidermidis colonies usually are grey to white on primary

isolation many colonies develop pigments only upon prolonged incubation as

he reported. No pigments produced anarobically or in broth (Brooks et al.,

1995). Various degrees of haemolysis are produced by Staph aureus and

occasionally by other species (Brooks et al., 1995).

Hemolytic patterns of species can be used in tracing back the source of

infection (Devriese and Deryche, 1979). Staphylococci are aerobic and

15

faculatatively anaerobic, catalse-positive, non-motile, non-spore-forming, and

fermentative (Carter et al., 1986).

All Staphylococcus species are oxidase-negative except for strains of Staph

caseolyticus, Staph sciuri, Staph lentus, and Staph vitulus (Koneman et al.,

1997).

Under the influence of drug; like penicillin; staphylococci are lysed, also

staphylococci slowly ferment many carbohydrates, producing lactic acid but

not gas and proteolytic activity varies greatly from one strain to another

(Brooks et al., 1995).

Pathogenic staphylococci produce many extraccelluar substances, some of

these are toxic (Brooks et al., 1995). Many of the toxins are under both

chromosomal and extra chromosomal control (brook et al., 1995).

Toxins are divided in to exotoxins, Leukocidin, Exfoliatuve toxin, Toxic shok

syndrome toxin, and enterotoxins. The enterotoxins are heat stable (they resist

boiling for 30 minutes) and resist the action of gut enzymes (Brokks et al.,

1995).

Important causes of food poisoning, enterotoxins are produced when Staph

aureus grow in carbohydrate and protein foods (Brooks et al., 1995).

Staphylococci are initially recognized by the appearance of their characteristic

colonies in a culture of Blood agar, Nutrient agar or MacConkey's agar

(Duguid, 1989). Blod agar plates give rise to typical colonies in 18 hours at

37Cْ (Brooks et al., 1995). They may form zones of heamolysis on blood agar

(Duguid, 1989) .

16

Staphylococci can grow in a selective medium, which has a component added

to it, inhibit and/or prevent the growth of certain types or species of bacteria or

to promote the growth of the desired species (Kenneth, 1997).

Baird parker's medium is selective media which is mainly used for coagulase-

positive pathogenic staphylococci(Baird Parker,1962).On this medium

coagulase-positive staphylococci growth is generally delayed (Saeed, 1995)

and they reduce tellurite with production of black colonies (Jawetz et al.,

1980). Coagulase-negative species show delayed growth forming brownish

black colonies (Saeed, 1995).

Staphylococcus No. 110 medium is a selective and diagnostic medium which

selects on the basis of salt tolerance. On this medium all strains of Staph

aureus can grow (Chapman, 1946). Most of Staphylococci tested on this

medium showed well growth (Saeed, 1995).

Mohammed (1997) found that Staphylococci varied in growth between weak

moderate and failed to grow. Mannitol salt agar (M.S.A) is recommended for

the detection and enumeration of staphylococcus species from food. Staph

aureus yields colonies surrounded by a yellow hallo caused by the

acidification of mannitol. Other Staphylococci may also ferment mannitol and

thus resemble Staph aureus on M.S.A. (Baron et al., 1994).

Staphylococci are relatively resistant to drying and heat. They withstand 50Cْ

for 30 minutes (Brooks et al., 1995). Also they reported that a culture of

staphylococci contains some bacteria that differ from the bulk of the

population in expression of colony characteristics (colony size, pigment,

haemolysis), in enzyme elaboration, in drug resistance, and in pathogencity.

Under standard conditions all Staphylococci are V.P. positive except Staph

intermedius, Staph hyicus and Staph Simulans.

17

1.2.5. Staphylococcus infection:

Staphylococci generally are found on the skin and mucous membranes of

humans and other animals (Koneman et al., 1997).

Many species of Staphylococci are opportunitistic pathogens that cause

infection when host defenses breakdown (Baron et al., 1994). Staphylococcus

is an important pathogen in human that can cause a wide spectrum of diseases,

including life threatening system maladies, cutaneous infections, opportunistic

infections, and urinary tract disease (Muttay et al., 1998).

Staphylococci among the most frequently isolated organisms in the clinical

laboratory (Koneman et al, 1997).

Staphylococcus aureus should always be considered a potential pathogen

(Kenneth, 2002). Staph epidermidis are non pathogenic and may even play a

protective role in their hosts as a normal flora, but may be pathogen in the

hospital environment (Kenneth, 2002).

No vaccine is available to that stimulates active immunity against

Staphylococcal infection in humans (Kenneth, 2002).

The coagulase negative Staphylococci are normal human flora and some

time cause infection, often associated with implanted appliances and devices,

specially in very young, old, and immuno-compromised persons (Brooks et al,

1995). Approximately 75% of these infections caused by coagulase-negative

staphylococci are due to Staph.

18

1.2.6 Staphylococcus aureus

Staphylococcus aureus is one of the major causes of hospital-acquired

infection (Kenneth,1998). One study ranked it fourth in a listing of the

“Pathogens Most Frequently Isolated from hospitalized patients when all

anatomic sites are considered (Kenneth, 1998).

Approximately 40% of the general population and 50 – 90% of health

care practitioners harbor an Staphylococcus aureus colony in their anterior

nasal passage. Infection becomes a problem when bacteria migrate from their

normal habitat, especially in individuals already suffering from a

compromised immunologic response. (Kenneth,1998)

Staphylococcus aureus most commonly causes a localized skin

infection, although it can also infect the eye, nose, throat, urethra, vagina, and

gastrointestinal tract (Kenneth,1998).

In addition, Staphylococcus aureus can cause more serious disease

when it enters the bloodstream, such as pneumonia, osteomyelitis, arthritis

endocarditis, myocarditis, brain abscesses and meningitis (Kenneth,1998).

The toxins most relevant to disease causing symptoms in humans are

the superantigens and α-toxins(Kenneth,1998).

The α-toxins oligomerize to form pores in the host cellular membrane,

allowing cellular contents to leak into the extracellular matrix.

(Kenneth,1998) The superantigens, consisting of enterotoxins and the toxic

shock syndrome toxin, are responsible for Staphylococcus aureus-related

food poisoning and toxic shock syndrome, respectively. Individuals with

damaged or severely compromised immune systems, such as recent surgical

recoveries or burn victims, IV drug users, insulin-dependent diabetics and

19

hemodialysis patients are more susceptible to Staphylococcus infection than

healthy individuals(Kenneth,1998).

Individuals identified with Staphylococcus infections are most

commonly found in hospital intensive care units, burn units, and dermatology

and surgical units, reflecting the increased susceptibility of these individuals

to Staphylococcus infections due to compromised immune function.

Treatment of Staphylococcus aureus infections showing no signs of broad-

antibiotic resistance is achieved with a cocktail of antibiotics, including some

of the following: gentamycin, rifamicin, fusidic acid, erythromycin,

vancomyin, and cefotaxime. However, 90% of Staphylococcus aureus strains

are penicillin resistant. In these scenarios, methicillin and vancomycin are

the only available treatment options(Kenneth,1998).

1.2.7 Staphylococcos epidermidis

Staphylococcus epidermidis is a coagulase-negative bacteria (Coppoc,

1996). Like Staphylococcus aureus, Staphylococcus epidermidis is a common

cause of nosocomial infections, particularly blood infections which can lead to

complications of the central nervous system. (Coppoc, 1996).

Staphylococcus epidermidis resides primarily in the skin, leaving those

who have frequent contact with needles and other invasive health care

appliances particularly susceptible to illness Staphylococcus epidermidis is

very likely to contaminate patient-care equipment and environmental surfaces,

possibly explaining the high incidence of Staphylococcus epidermidis in

hospital settings.

While there is extensive information concerning Staphylococcus

aureus virulence factors, there is relatively little known about Staphylococcus

20

epidermidis mode of action Infection is treated primarily with vancomycin or

rifampin (Coppoc,1996).

1.2.8 S Staphylococcus saprophyticus

Staphylococcus saprophyticus is a common cause of urinary tract

infections in men and women. Risk factors and treatment guidelines are the

same as those described for Staphylococcus aureus (Coppoc,1996)

1.2.9 A closer Look at Vancomycin Resistance

Staphylococcus aureus strains acquired in the hospital are often

resistant to many types of antibiotics (Bogle, 1997) . A significant portion of

Staphylococcus aureus strains are now methicillin-resistant, in addition to

being resistant to other penicillin-derived drugs (Bogle,1997) . For these

methicillin-resistant strains, vancomycin is the only available, effective drug

for treatment (Bogle, 1997) . However, in 1996, there were reports of strains

of Staphylococcus aureus with decreased susceptibility to vancomycin,

labelled glycopeptide intermediate-resistant Staphylococcus aureus, or

vancomycin-intermediate Staphylococcus aureus (GISA and VISA,

respectively). (Bogle, 1997). In the early 1990s, vancomycin-resistant

enterococci (VRE) emerged. Laboratory tests have shown that resistance,

coded in the VanA, VanB and VanC genes, can be transferred from

Enterococci to Staphylococcus and other Gram-positive bacteria. For such

transfer to occur in a non-laboratory controlled environment, there would be

no FDA-approved, effective means of combating multi-drug resistant

Staphylococcus infections (Bogle,1997) .

Staphylococcus aureus can acquire resistance through

extrachromosomal plasmids , through additional genetic information delivered

by transposons, and through mutations in chromosomal genes. Resistance is

21

achieved through a variety of mechanisms, including enzymatic inactivation

of the drug (as with penicillinase , which cleaves the beta-lactam ring of

penicillins), alteration of the drug target to prevent binding, and enhanced

removal of the drug from the host tissue (Bogle, 1997) .

A mutation in the MecA gene of Staphylococcus will provide resistance

to all beta-lactams (Bogle, 1997) . This gene codes for Penicillin Binding

Protein a mutation in the gene prevents penicillin from effectively binding

foreign particles. Exposing a bacterial culture to oxacillin tests resistance to

beta-lactams. If the bacteria is resistant to oxacillin, it will be resistant to all

beta-lactams, and quite possibly also resistant to erythromycin, gentamicin,

tetracycline, and clindamycin (Bogle, 1997).

Vancomycin resistance has been, to date, documented in enterococci,

but not in staphylococcus (Bogle, 1997). Enterococci are Gram positive

pathogens, listed as the second most common cause of hospital-acquired

bacterial infections. They are part of the normal bacterial-makeup of the

human gastrointestinal tract, and like Staphylococci, cause infection when

they enter the bloodstream. Enterococci can cause urinary tract infections,

wound infections, septicaemia and endocarditis. They are naturally resistant

to cephalosporins, aminoglycosides and clindamycin, but were, until recently,

susceptible to vancomycin. The vanA and vanB genes are responsible for

encoding vancomycin resistance in the majority of Enterococci infections.

Enterococci are transmitted via person-to-person contact (i.e. on the hands of a

health care provider), or through indirect contact (i.e. through contaminated

environmental surfaces). The vanA and vanB genes are responsible for

resistance transmittance vanC is a chromosomal gene that cannot be

transmitted between Gram-positive organisms (Bogle, 1997).

22

1.3 Antimicrobial Chemotherapy

Antimicrobial drugs exploit differences in structure or biochemical function

between host and parasite (Zee and Hirsh, 1999 ) .

Modern chemotherapy is traced to the work of Paul Ehrich, who devoted his

life to discovering agents that possessed selective toxicity (Zee and

Hirsh1999)

The first clinically successful broad-spectrum antibacterial drugs were the

sulfonamides, developed in 1935 as a result of Ehrlich's work with synthetic

dyes. It was, however, the discovery of further antibiotics, led to the

subsequent discovery of further antibiotics, chemical substances produced by

microorganisms that at low concentrations inhibit or kill other microorganisms

(Zee and Hirsh, 1999 ).

The chemical modification of many of the drugs discovered early in the

antibiotic revolution has led to the development of new and powerful

antimicrobial drugs with properties distinct from their parents.

Antibiotics and their derivatives have more importance as antimicrobial

agents than do the fewer synthetic antibacterial drugs (Zee and Hirsh1999 ).

1.3.1 Spectrum of action of antimicrobial Drugs

Antimicrobial drugs can be classified as narrow or broad spectrum based on

the range of bacteria on which they act (Zee and Hirsh, 1999).

23

1.3.1.1Classification of microorganism

Penicillin is narrow spectrum because they inhibit only Gram positive

bacteria. Choramphramphenicol are broad spectrum because they inhibit both

Gram poitive and gram Negative bacteria. Polyenes only inhibit fungi (Zee

and Hirsh ,1999

1.3.1.2Antibacterial activity

Some antibiotics are narrow spectrum in that they inhibit only Gram –positive

bacitracin (Vancomycin) or Gram negative bacteria (polymyxin) whereas

broad spectrum drugs such as tetracyclines inhibit both Gram positive and

Gram negative bacteria. Other drugs such as penicillin G are most active

against Gram –positive bacteria but will inhibit some Gram –negatives (Zee

and Hirsh, 1999).

1.3.1.3 Bacteriostaic and bactericidal effect of antimicrobial

. This distinction is an approximation that depends on drug concentrations and

the organism involved. For example, penicillin is bactericidal at high

concentrations and bacteriostatic at lower ones. (Zee and Hirsh, 1999).

The distinction between bactericidal and bacteriostatic is critical in certain

circumstances, such as the treatment of meningitis or septicemia in

neutropenic patients (Zee and Hirsh, 1999).

1.3.2Mechanism of action of antimicrobial drugs:

The marked structural and biochemical differences between eukaryotic and

prokaryotic cells give greater opportunity for selective toxicity of antibacterial

drugs compared to antifungal drugs because fungi, like mammalian cells, are

eukaryotic. Developing selectively toxic antiviral drugs is particularly difficult

24

because viral replication depends largely on the metabolic pathways of the

host cell (Zee and Hirsh, 1999).

The mechanism of action of antibacterial drugs fall into four categories: 1)

inhibition of cell wall synthesis, 2) damage to cell membrane function, 3)

inhibition of nucleic acid synthesis or function, and 4) inhibition of protein

synthesis (Zee and Hirsh, 1999).

25

CHAPTER TOW

MATERIALS AND METHODS

2.1 Organisms

Thirty two isolate of Gram-positive bacilli were screened for their

antibiotic-like production .They were isolated from contaminated cultures.

The isolates were cultured in blood agar for purification and stored in cooked

meat media at 4Cْ. Identification was based on morphology staining and

biochemical characters following Barrow and Feltham (1993) and (Sneaths et

al.1986).

2.2 Equipments

The equipment used for isolation, storage, identification of different

sample of bacillus and Staphylococcus are:

Slides, wire loop (standard loop with 4mm internal diameter, oil of

immersion for lenses (Hopkin William, England) graduate pipettes of

different size (Pyrex, England ) Pasteur pipettes , wooden and metal racks ,

media Bottles of different sizes bijou , McCartney , universal and large sizes

disposable Petri dishes ( Sterling ltd. England ) glass Petri dishes ( Pyrex

England) syringes , 20 ml , 1ml , Haste, Italy ) filter paper 1.25 cm (England

graduated glass cylinder of different size , conical flat bottles (Pyrex ,

England) , round flask (Pyrex , England ) Durham’s tubes membrane filter

0.22Mic

Microscope (Olympus, Tokyo, Japan) pH-meter (corning model 10)

Thermostatically controlled incubator 37˚C,Admiral refrigerator, Autoclave

26

(Baird and Tatlock ltd. London, England),Centrifuge, Millipore Filter holder

(Millipore filter corp. Bedford Mass. U.S.A)

2.3 Media

Media used for cultivation and identification of Bacillus Spp. and

Staphylococcus spp

2.3.1Nutrient broth(Oxoid)

The medium was prepared by dissolving 13g of the medium in one liter

of distilled water .The pH was adjusted to 7.4 and distributed into screw-

capped boottles 5ml each and sterilized at 121°C for 15 minutes

2.3.2 Nutrient agar(Oxoid)

The medium was prepared by dissolving 28grams of powder in one liter

of distilled water by boiling .The medium was sterilized by autoclaving

(121°C for 15 minutes, cooled to 55°C and then distributed into sterile Petri

dishes 20 ml in each

2.3.3Blood agar(Oxoid)

Forty grams of blood agar base (Oxoid )were suspended in liter of distilled

water , steamed to dissolved ,cooled and the PH adjusted to 7.4 , it was then

sterilized by autoclaving at 121°C for 15 minutes after cooling to about 45°C

, defibrinated sheep blood was added aseptically in 7-10٪ concentration

before pouring into plates in 15 ml amounts

2.3.4Indole Medium(Oxoid)

The indol medium was prepared according to Barrow and Feltham

(1993)from the following (G/L, W/V) Bacto peptone (Oxoid), 20g, sodium

chloride (B.D.H), 5g and D.W 1000 ml. The tested microorganisms were

27

inculcated in peptone water then incubated at 37°C for 48 hours. Three drops

of kovacs reagent were added to culture and shaken well. Production of pink

co lour on upper layer of the reagent was considered indol positive

2.3.5 Voges -Proskauer test (V P)

V P medium was prepared according to Barrow and Feltham (1993) as

follows :( G/L, W/V), peptone 10g, glucose 5g, distilled water1000 ml.

This test was performed to detect the production of acetylmethyl carbinol .

Glucose phosphate broth was inoculated with tested organisms and incubated

at 37 ْC for 48 hours, then O.6 ml of alpha-naphthol solution followed by 0.2

ml of40% potassium hydroxide solution were added to one ml of culture,

mixed well and examined after 15 minutes and one hour .Bright red colour

indicated positive result

2.3.6Casein agar (milk agar) medium(Oxoid)

Prepared according to Barrow and Feltham (1993) as follows :whole

fresh milk was stored overnight in refrigerator .The creamy layered was

removed ,and the milk was steamed for one hour and cooled in refrigerator

.Sufficient litmus solution was added to give a bluish –purple colour and the

medium was sterilized at 115°C for 10 minutes .It was then cooled to about 50 ْ

C and added to double strength nutrient agar which was melted and cooled to

50-55°C, mixed and distributed in Petri-dishes .Casein agar plate was

inculated with tesed cultured and incubated at 37°C for 24-48 hour for

clearing the medium around the bacterial growth

2.3.7Starch medium

Starch medium was prepared according to Barrow and feltham (1993)

as follows: Fifty grams of starch were triurated with water to smooth cream

28

.And then added to molten nutrient agar .The mixture was sterilized at 115° C

for 15 minutes and distributed into sterilized Petri dishes 20ml in each .Starch

agar plate was inoculated with tested culture and incubated at 37°C for

24hour .Plate flooded with lugol”s iodine solution .Hydrolysis was indicated

by clear co lour less zones .Starch which had not been hydrolyzed turned blue

2.3.8Gelatin agar medium (Oxoid)

Gelatin agar medium was prepared according to Barrow and Feltham

(1993) as follows:

Gelatin 4g, D.W.50ml, nutrient agar 1000 ml the gelatin was soaked in the

water and when thoroughly softened was added to melted nutrient agar .mixed

and sterilized at 115°C for 10 min.

2.3.9 Citrate medium (Oxoid)

Twenty three grams of powder were dissolved in to 1000 ml distilled

water by boiling. The pH was adjusred to 7.0, and then the medium was

sterilized by autoclaving at 121°C for 15 minutes and distributed in to sterile

Scrow –caped bottle and allowed to solidify in slope position.The citrate

medium was inculated with tested organisms, incubated at 37°C and examined

daily for up to seven days .The devoloment of blue colour in the medium was

consider positive result

2.3.10 Ammonium salt sugar(Oxoid)

Ammonium salt sugar was prepared according to Barrow and Feltham

(1993) as follows:

This medium consisted of ammonium phosphate chloride magnesium

sulphate, Yeast extract agar and bromcresol purple .It was prepared by adding

29

the solids to 1000ml distilled water, dissolved completely by boiling and

sterilized at 115°C for 20 minutes .The medium was allowed to cool to about

55°Cand the appropriate sugar was added as sterile solution to give a final

concentration 1% the medium mixed and distributed aseptically in to sterile

tubes, then allowed to set in slope position

2.3.11Motility medium (Oxoid)

Thirteen grams of dehydrated nutrient broth were added to 4grams of

agar and dissolved in one liter of distilled water by boiling and the pH was

adjusted to 7.4, distributed in 5ml amounts in tests tubes containing craig-

tubes and sterilized by autoclaving at 121°Cfor 15 minutes. The carigie tube

was used to detect the motility of the organisms. The growth of the

microorganism outside the tube indicated that it is motile organisms

2.3.12Hugh and Leifsons (O\F) medium (Oxoid)

The medium was prepared by dissolving 10.3grams of solid in liter of

distilled water by heating ,and the pH adjusted to 7.1 .Filtered bromothymol

blue (.2%aqueous solution) was added and then sterilized at 115°C for 20

minutes .Sterile solution of glucose was added aseptically in to sterile tubes.

Two tubes of Hugh and Leifson’s medium were inoculated with tested

organism. One of them was covered with a layer of sterile paraffin, all tubes

were incubated at 37°C and examined daily for seven days .Fermentative

organisms produced a yellow color on both tubes while oxidative organisms

produced a yellow colour only on open tube

2.3.13 Nitrate Broth (Oxoid)

One gram of nitrate was dissolved in one liter of nutrient broth ,then

distributed in to tubes and sterilized by autoclaving at 115°C for 15 minute

30

.The tested microorganism was grown in nitrate broth and incubated at 37°C

for 5 days .one ml of nitrate reagent A was added followed by one ml of

reagent .A deep red color produced was considered as positive reaction .Tubes

that did not show red color ,zinc powder was added and followed to stand

.Formation of red color indicate that nitrate was present and that tested

organism did not reduce it

2.4. Buffers

2.4.1 Normal Saline

Normal saline was prepared as described by Barrow and Feltham

(1993) from the following (G/L , W/V) sodium chloride (B.D.H) 8.5g,

distilled water one liter .the salt was dissolved in distilled water ,the mixer was

then distributed in bottles of different sizes ,autoclaved at 121°C for 20 min.

and store at room temperature for later use

2.4.2 Distilled Water

Tap water was distilled using distillator (Elga England)

2.5 Test reagents

2.5.1 Hydrogen peroxide

The reagent was prepared as described by Barrow and Feltham (1993)

from hydrogen peroxide (H) (Analar) 3% aqueous solution (10 volumes).

2.5.2 Nitrate test reagents

The reagents were prepared as described by Barrow and Feltham (1993)

solution A sulphanilic acid 33%in 5N- acetic acid, dissolved by gentle heat

solution B: Dimethyl -naphthylamin 0.6%in 5N-acetic acid .The complete

reagent was used to detect nitrate reduction .

31

2.5.3 Neutral red Reagent

The reagent was prepared according to Barrow and Feltham (1993) as

follow :from neutral red (B.D.H) 1g and D.W. 100ml .the reagent was

prepared by dissolving 1g of neutral red in 100 ml D.W .to form o.1% solution

. The indicator was autoclaved at 121° C for 15 minutes and kept at 4°C.

2.5.4 Kovac’s reagent

The reagent was prepared as described by Barrow and Feltham (1993)

this reagent of paradimethyl- aminobenzaldehyde, amylalcohol and

concentrated hydrochloric acid .after preparation the reagent was steored in

the refrigerator at 4°C.

2.5.5 Methyl red

The indicator was prepared according to Barrow and Feltham (1993)

from the following :Methyl red (B.D.H),1g ethanol (analar) ,300ml and

D.W.200ml the ingredients were mixed and kept at 4°c

2.5.6 Bromocresol purple

The reagent was prepared as described by Barrow and Feltham (1993)

Bromocresol purple solution was prepared as 0.9% solution. It was used in

ammonium salt sugar

2.5.7 Polyethylene glycol

Ready –made Flakes were obtained commercially (B.D.H)

2.5.8 Xylene

Ready –made solution was obtained commercially (B.D.H)

32

2.5.9Sodium chloride

In solution (NaOH) (B.D.H) was prepared by dissolving 40g of powder

in one litre D.W to form NAOH solution

2.5.10 Hydrochloric acid

Hydrochloric acid solution was prepared by dissolving 36.5 ml

concentration Hydrochloric acid in one litre D.W

2.5.11 Oxidase test reagent

This was prepared as 1%tetramethyl-p-phenylene diamine

dihydrochloride aqueous solution it was kept at 4°c in stoppered glass bottle

and was protected from light.

2.5.12 Andrads indicator

The indicator was prepared as described by Barrow and Feltham

(1993).This was prepared by dissolving 5g of acid fuchsin in one liter of

distilled water ,then 150ml of alkali solution (NaOH).It was used in peptone

sugar medium

2.6 stains

2.6.1 Gram Stain

Standerd stains as described by Barrow and Feltham (1993) were used

2.7 Selection of Bacillus spp which produce active metabolite

Fresh cultures of Staphylococcus grown in nutrient broth were sown

on the surface of nutrient agar. The plates were left to dry. A drop of Bacillus

SPP cultures was applied on the surface of the sown agar, left to dry and

incubated at 37°C over nigh t.This criteria will lead to select Bacillus Spp

which produce metabolite that can inhibit Staphylococcus.

33

2.7. Preparation of metabolite

Nutrient broth was cultured with fresh Bacillus spp and incubated for

8hours at37 ْ C then the culture was centrifuged and the supernatant was

removed aseptically and placed in dialysis tube in poly ethylene glycol in

order to concentrate the metabolite .The supernatant was then filtered with

0.22Mm filter and kept in sterile tubes and stored at 4°C.

2.8. Methods for detection of the inhibitory effect of Bacillus SPP.

metabolite on Staphylococcus

2.8.1The Disc method

Staphylococcus spp. were cultured in nutrient agar plate using wire loop

(Streaking ) . Filter paper discs were prepared in the lab by soaking them in

the filtrates of 3 bacilli metabolite .then they were placed on nutrient agar

surface in similar manner to antibiotic sensitivity discs. Figure (1)

(Personal method .Professor Suliman M. Elsanousi)

2.8.2 Scraping method

Fresh cultures of Bacillus spp were grown as blob inoculation method in

nutrient agar .They were then incubated at 37°C for an over night. The growth

was scraped off using bent sterile glass rod . A few drops of chloroform were

spreaded all over the plate .The plate were allowed to dry. Staphylococcus spp

were cultured in a perpendicular manner across the place on which Bacillus

spp were grown .The plates were incubated at 37°C for an overnight to study

the inhibitory effect of the metabolites secreted by Bacillus spp on

Staphylococcus. (Personal method .Professor Suliman M. Elsanousi)

34

2.8.3 Strip method

Fresh cultures of Staphylococcus in nutrient broth incubated for 24 hours were

spreaded on surface of nutrient agar, Then a strip filter paper soaked in the

filtrates of bacilli metabolite was placed on nutrient agar surface and the plates

were incubated at 37°C for overnight. Figure (2) (personal method .Proffessor

Suliman M. Elsanousi)

2.9 Determination of inhibitory activity unit (IAU) of metabolite in

filtrates

The filtrates were sequentially diluted with normal saline 5 times. Eeach

of these dilution was tested against Staphylococcus spp, in order to establish

the minimum inhibitory concentration of the metabolite against

Staphylococcus .One (IAU)was defined as higher dilution of filtrates that

inhibits in vitro the growth of target pathogen .Table (1)

2.10 Determination the bacteriostatic and bactericidal activity of

antibiotic metabolite in filtrates

From the inhibitory zones of Staphylococcus, Swabs were taken from surface

of the agar where no Staphylococcus growth was observed , then the swabs

were cultured on agar surface and incubated for 48hours in 37°C . Growth or

no growth, made differences between bacteriostatic and bactericidal activity

of filtrates. (Personal method .Professor Suliman M. Elsanousi)

2.11 Thermostability

1.5 ml of filtrates was dispensed in Eppendorf sterile tubes .The tubes

were treated as follows 1) maintained at room temperature 25ºC 2) water bath

heated at 100°C during 15 min and 3) autoclaved at 121°C during 20 min

35

.from each set of tubes, 0.5 ml were tested against staphylococcus for

inhibitory activity as shown in Table (2)

2.12 Solubility in methanol of metabolite in filtrates

One volume of antibiotic metabolite filtrate mixed with one volume of

methanol

2.13 Stability of metabolite against enzymes

One ml of metabolites filtrate were placed in two tubes, one tube was

treated with proteinase K, and the other one with trypsin and incubated at

37°C for two hours, to let enzyme work, then the enzymes were inactivated by

heating at 60°C, before they tested against Staphylococcus as shown in

Table(3)

36

CHAPTER THREE

RESULT

3.1 Thirty two isolates of Gram positive bacilli were screened for their

production of metabolites against Staphylococci.

Three of them were positive for production of metabolites active against

Staphylococci.(Their metabolite completely inhibited the Staphylococcus

growth in vitro )They were Bacillus cereus ,Bacillus licheniformis and

Bacillus subtilis

The Staphylococcus spp. which were sensitive to these metabolites produced

by Bacillus were Staphylococcus aureus, Staphylococcus epidermidis and

Staphylococcus saprophyticus

3.2 Determination of inhibitory activity unit (IAU) of metabolite in

filtrates

Different metabolite dilutions were tested against Staphylococcus spp in

order to determine the inhibitory activity unit which is higher dilution of

metabolite filtrate that inhibits in vitro the growth of Staphylococcus as shown

in Table (1)

The inhibitory activity unit which is higher dilution of metabolite

filtrate that inhibits in vitro the growth of Staphylococcus was 1x10-1

37

Table (1)

Determination of inhibitory activity unit (IAU) of

metabolite in filtrates

Size of inhibition zone (cm) Dilution factor

0.5 1x10-1

0 1x10-2

0 1x10-3

0 1x10-4

0 1x10-5

3.4 Determination the bacteriostatic and bactericidal activity of

metabolite in filtrates

No growth was observed in nutrient agar surface that mean:

The metabolite has bactericidal effect against some Staphylococcus spp.

3.5Thermostability

The metabolite filtrates were treated with three different temperatures

(Room temperature 25°C, 37°C-100°C water bath and autoclaving 121°C )

giving the following result .The result showing that the metabolites were

unstable in high temperatures (Heat sensitive) as shown in table (2)

38

Table (2)

Thermostability of metabolite after treated by different temperatures

Diameter of inhibition zone (cm) Treatments

1.5 Room temperature (25 ْ C ±2 ْ C)

20 minutes

0.5 Water bath (100 ْ C ±2 ْ C) 20 minutes

0 Autoclaving (121 ْC±1 ْ C) 20 minutes

3.6 Solubility of active metabolite filtrates in methanol

The metabolites were soluble in methanol

3.7Stability of the active metabolite against enzymes

To establish stability of the active metabolite against enzymes ,the

metabolites were treated with protienase K and Trypsin enzyme .The

metabolites were denatured with protieanse K and no effect was detected with

Trypsin Enzyme. (Table 3)

39

Table( 3 )

Stability of the active metabolite against enzymes

The detection of the inhibitory effect of Bacillus subtilis metabolites on

Staphylococcus epidermidis is shown in fig( 1)

Inhibition zone size (cm) Enzyme

0 Protienase K

1.5 Trypsin

40

Figure (1) Demonstration of the inhibitory effect of Bacillus subtilis. metabolite on

Staphylococcus epidermidis (The Disc method)

41

Figure (2) Methods for detection of the inhibitory effect of Bacillus subtilis.

Metabolite on Staphylococcus epidermidis

Strip method

42

43

Figure no (3) detection of the inhibitory effect of Bacillus cerues. Metabolite on Staphylococcus aureus The Disc method

44

CHAPTER FOUR

DISCUSSION

This study has dealt in its object with three aspects .The first one was

the isolation and identification of different species of the genus Bacillus that

might produce a metabolite which has inhibitory effect on Staphylococcus

spp .The second one was the study of the inhibitory effect of these Bacilli and

their products on staphylococcus spp. The third one was to study the physical

and chemical properties of these metabolites.

Synthesis of peptide antibiotic usually starts at the end of exponential

growth reaching maximum concentration after cell growth has ceased

(Bodanszky and Perlman ,1969) The factor triggering the onset of antibiotic

synthesis is more likely the exhaustion of limiting nutrient required for cell

growth. This limitation usually stimulates differentiation which for the case of

Bacilli, means endospore formation. Sporulation is associated with synthesis

of new cell wall and degradation of that in mother cell .This cell wall like

antibiotics contains D-aminoacids. It is suggested that an altered metabolism

for cell wall synthesis could furnish the precursors for antibiotic

synthesis(Besson et al, 1987)

Bioassay based on clearing zone on agar plate was used to determine

the bactercidial activity of metabolite produced by Bacillus spp against

Staphylococcus. Inhibition zone revealed the production of strong antagonist

by Bacillus spp. These results are interesting, because the antibiotic produced

inhibit the growth of both prokaryotes and eukaryotes ,as in case of other

antibiotic synthesis by Bacillus subtilis (Maget –Dana and Pypoux, 1994).

45

According to our result the antibiotic produced by Bacillus spp has

bactericidal and probably fungicidal activity .The target site for iturin A and

bacillomycin L antibiotic produced by Bacillus subtilis in yeast is in plasma

membrane. Both antibiotics promote sphreroplast and rapidly increase

permeability to K+ which has been associated with its fungicidal activity

(Besson et al. 1984). The ability of iturin antibiotic to increase permeability of

target microorganism is due to the formation of ion channels on cell

membrane. In this context a tertiary structure iturin –phospholipid –sterol was

suggested to be the biologically efficient structure (Maget_Dana and

peypoux, 1994).

Balassa (1964) reported that there is direct correlation between

sporulation and production of some exoenzyme , antibiotic and exotoxin .

It was already known in 1949 that bacilli produce many antibiotics

(Abraham and Florey., 1949 ) . At least some of these antibiotics are species

specific and one of species may produce several ones . Bacillus subtilis is

agood example in addition to antibacterial antibiotics (Abraham and Florey.,

1949 ).

No attempt was made here to review the problem of the mode action of

these antibiotics on sensitive bacteria , beyond saying that many cell function

are affected by one or another ,cell wall synthesis ,oxidative phosphorylation

,membrane permeability (Bernolohr and Novelli,1959)

Bernolohr and Novelli (1959 ) reported that in the case of bacteriocin

produce by Bacillus licheniformis antibiotic molecule is subunit of spore coat

protein is incorporated as such in the late stage of spore formation.

We recommend the search for more bacteriocin – producers bacilli and

their effect on various type of bacteria

46

CHAPTER FIVE

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