Antimicrobial activity of soil against bacterial diarrhoea treatise

ANTIMICROBIAL ACTIVITY OF SOIL INTHE EASTERN CAPE AGAINST INFECTIVE

DIARRHOEA(RESEARCH TREATISE)

BySITUMBEKO LIWELEYA(s213459531)

Submitted in fulfilment of therequirements for the degree ofBACHALOROUS TECHNOLOGIEA: BIOMEDICAL

TECHNOLOGYAt the

HEALTH SCIENCES FACULTY

AtNelson Mandela Metropolitan University

Port Elizabeth, 2013.

SUPERVISOR- PROFESSOR SMITH. N.

DECLARATION

I, the undersigned, hereby declare that the research work

contained in this study is my own original work, and all the

sources I have used or quoted have been indicated and

acknowledged by means of complete references.

Situmbeko Liweleya

ABSTRACTAnecdotes, both historical and recent, recount the curing of

skin infections, including diaper rash, by using red soils

from the Hashemite Kingdom of Jordan. Following inoculation of

red soils isolated from geographically separate areas of

Jordan, Micrococcus luteus and Staphylococcus aureus were rapidly

killed. Over the 3-week incubation period, the number of

specific types of antibiotic-producing bacteria increased, and

high antimicrobial activity (MIC, ∼10 μg/ml) was observed in

methanol extracts of the inoculated red soils. Antibiotic-

producing microorganisms whose numbers increased during

incubation included actinomycetes, Lysobacter spp.,

and Bacillus spp. The actinomycetes produced actinomycin C2 and

actinomycin C3. No myxobacteria or lytic bacteriophages with

activity against either M. luteus or S. aureus were detected in

either soil before or after inoculation and incubation. These

results suggest that the antibiotic activity of Jordan's red

soils is due to the proliferation of antibiotic-producing

bacteria.

(Falkinham, 2009)

A total of 51 Actinomycetes were isolated from different soil

samples of Palestine. Preliminary screening by cross-streak

method was carried out for all the 51 isolates. After

preliminary screening, 17 isolates which showed antimicrobial

(antibacterial, antifungal) activity were selected for further

study. Among these 17 isolates tested, 5 isolates which were

found to be promising were subjected to detailed taxonomic

iii

studies. A novel strain of S. albovinaceus (isolate 10/2) which

was found to be maximum antibiotic producer and which has

shown both broad spectrum antibacterial and antifungal

activities was isolated and is been selected for further

detailed optimization studies.

(Abdelghani, 2009)

A total of 29 Bacillus species isolated from the soil was

analysed using the agar diffusion method in terms of their

general inhibition effects to some test bacteria. It has been

found that isolates are effective against Gram-positive and

Gram-negative bacteria whereas their extensive inhibition

effect is particularly against Gram-positive bacteria. On the

other hand, B. cereus M15 strain has an inhibitory effect against

both Gram-positive and Gram-negative bacteria. Furthermore

some isolates are more effective against test bacteria when

compared to some antibiotics.

(Yilmaz, 2006)

Soil samples were mixed with human saliva, incubated in media

suitable for bacterial and fungal growth and filtered.

Eighteen bacterial and five fungal species were isolated and

identified. The bacterial and fungal filtrates as well as the

isolated species were evaluated for their antimicrobial

activities against some pathogenic microbes causing

dermatological diseases (Staphylococcus aureus, methicillin

resistant S. aureus (MRSA) and Aspergillus niger). The bacterial

filtrate showed significant antagonistic effect against S.

iv

aureus and methicillin resistant S. aureus (MRSA), whereas showed

non inhibitory action on the pathogenic fungus.

(Sheikh, 2010)

During screening for antibiotic producing microorganisms from

environmental soil samples, the supernatant of a bacterial

isolate was found to have antibacterial and antifungal

activity on the standard indicator species. The standard

cylinder-plate method was used to determine the inhibitory

effect of the crude supernatant of each isolate on 6 bacterial

and 3 fungal standard strains by measuring the diameter of

inhibition zone. The highest inhibition zone on Aspergillus niger

belonged to culture broth of isolate FAS1 by 25 mm, and this

isolate was the most efficient microorganism to inhibit

standard bacterial and fungal species. Based on morphological

and biochemical properties as well as 16S rDNA gene analysis,

the selected isolate (isolate FAS(1)) belonged to Bacillus

genus.

(Moshafi, 2011)

The main aim of this study included determining whether or not

soil bound microbes’ exhibit antimicrobial properties against

known bacteria of medical importance. The objectives of this

research included investigating the development of a new

antimicrobial agent from soil. The second objective of this

research was to reduce the expanding problem of microbial

resistance in treatment of bacterial infections such as

infective diarrhoea.

v

Three soil samples were collected from 3 different soil sites

(Summerstrand, NMMU North Campus main building round-about and

NMMU South Campus water pond) and cultured for microbial

growth on suitable culture media. Pure colonies isolated from

the mixed growth were categorised by gram stain, where all of

them were gram positive bacilli. The pure colonies were tested

for antimicrobial activity against Escherichia coli, Staphylococcus

aureus, Shigella flexneri, Bacillus cereus and Salmonella spp. All the

three pure colonies antimicrobial activity was resisted by the

bacteria tested against. Positive controls of antibiotics were

tested against the known microorganisms and demonstrated

antimicrobial activity by zones of inhibition on growth media.

Key words: Antimicrobial; Bacillus cereus ; Escherichia coli ;

Staphylococcus aureus ; Shigella flexneri; Salmonella spp.; pure colony.

vi

ACKNOWLEDGMENTS

I, the author, would like to express my sincerest gratitude

and appreciation to the following people who have all

contributed in their own special ways towards the completion

of this study:

Professor N. Smith her invaluable guidance, encouragement,

support, assistance and patience with me during the duration

of this project.

Mrs. L Beyleveldt for the ordering of supplies.

The entire Department of Biochemistry and Microbiology for

their invaluable assistance and encouragement.

My Beautiful Wife Taonga, My Mother and family for their

unconditional love, perseverance and endurance during the time

when I was away from them.

My Supervisor, Mr. K. Mwiinga, Collegues, Mazabuka District

Hospital Human Resource Office and the entire Hospital

Administration for granting me the permission to undertake

this study.

vii

My fellow students of the Department of Biochemistry and

Microbiology for their invaluable assistance and

encouragement.

My heavenly Father, for being my Rock and Shelter and without

whom nothing is possible and for leaving me with the gift of a

peace of mind and heart throughout the study period.

TABLE OF CONTENT

SABSTRACT.........................................................iiiACKNOWLEDGMENTS...................................................vi

TABLE OF FIGURES................................................viiiLIST OF TABLES....................................................ix

LIST OF ABBREVIATIONS..............................................xCHAPTER ONE........................................................1

1.1 INTRODUCTION.................................................11.2 AIM..........................................................3

1.3 OBJECTIVES...................................................3CHAPTER 2..........................................................4

2.1 LITERATURE REVIEW............................................42.2 MATERIALS AND METHODS........................................6

2.2.1 SOIL SAMPLES.............................................6

viii

2.2.2 ISOLATION OF PURE CULTURE OF MICROBES....................72.2.3 SCREENING OF ANTIMICROBIAL ACTIVITIES OF PURE ISOLATES. . .9

2.2.4 TEST ORGANISMS...........................................92.2.5 RESULTS AND DISCUSSION...................................9

CHAPTER 3.........................................................143.1 CONCLUSION..................................................14

3.2 RECOMMENDATIONS.............................................16REFERENCES........................................................17

TABLE OF FIGURES

FIGURE 2.1 1..................................................7

FIGURE 2.1 2..................................................8

FIGURE 2.1 3..................................................8

FIGURE 2.1 4.................................................11

FIGURE 2.1 5.................................................12

ix

LIST OF TABLES

TABLE 2.1 1...................................................6

TABLE 2.1 2..................................................10

TABLE 2.1 3..................................................11

TABLE 2.1 4..................................................12

TABLE 2.1 5..................................................13

x

LIST OF ABBREVIATIONS

WHO……………………………………………………………..World Health Organisation

MRSA…………………………………………………………….Methicillin-Resistant Staphylococcus

aureus

xi

MDR-TB…………………………………………………………Multi Drug Resistant Tuberculosis

CFU………………………………………………………………..Colony Forming Units

MH-A……………………………………………………………..Mueller-Hinton Agar

T-S………………………………………………………………….Tenby, Summerstrand

NMMU-S………………………………………………………..Nelson Mandela Metropolitan

University South Campus

NMMU-N………………………………………………………..Nelson Mandela Metropolitan

University North Campus

SPP………………………………………………………………….Species

R………………………………………………………………………Resistant

P………………………………………………………………………Partial

µg…………………………………………………………………….Micro gram

CIP…………………………………………………………………...Ciprofloxacin

C……………………………………………………………………….Chloramphenical

E……………………………………………………………………….Erythromycin

CFX……………………………………………………………………Cephalexin

GM……………………………………………………………………Gentamicin

AP……………………………………………………………………..Ampicillin

A……………………………………………………………………….Amoxicillin

xii

CHAPTER ONE

1.1 INTRODUCTION

The increasing incidence of antibiotic-resistant bacteria,

especially the methicillin-resistant Staphylococcus aureus in

communities and hospitals, has placed great emphasis on the

need for new antimicrobial agents to treat infectious

diseases. In an attempt to uncover such resources researchers

are exploring some historically recognized natural remedies

which are still being used in some communities as an

alternative to expensive pharmaceutical drugs. (Historical

Anecdote Of Jordan's Red Soils May Offer New Antibiotic, 2009)

Antimicrobial chemotherapy has conferred huge bene ts on humanfi

health. A variety of microorganisms were elucidated to cause

infectious diseases in the latter half of the 19th century.

Thereafter, antimicrobial chemotherapy made remarkable

advances during the 20th century, resulting in the overly

optimistic view that infectious diseases would be conquered in

the near future. However, in response to the development of

antimicrobial agents, microorganisms that have acquired

resistance to drugs through a variety of mechanisms have

emerged and continue to plague human beings. In Japan, as in

other countries, infectious diseases caused by drug resistant

bacteria are one of the most important problems in daily

clinical practice. In the current situation, where multidrug-

resistant bacteria have spread widely, options for treatment

with antimicrobial agents are limited, and the number of brand

new drugs placed on the market is decreasing. Since drug-1

resistant bacteria have been selected by the use of

antimicrobial drugs, the proper use of currently available

antimicrobial drugs, as well as efforts to minimize the

transmission and spread of resistant bacteria through

appropriate infection control would be the rst step in fi

resolving the issue of resistant organisms. (Saga, 2009)

In 1928, Fleming discovered penicillin. He found that the

growth of Staphylococcus aureus was inhibited in a zone

surrounding a contaminated blue mold (a fungus from the

Penicillium genus) in culture dishes, leading to the nding fi

that a microorganism would produce substances that could

inhibit the growth of other microorganisms. The antibiotic was

named penicillin, and it came into clinical use in the 1940s.

In 1944, streptomycin, an aminoglycoside antibiotic, was

obtained from the soil bacterium Streptomyces griseus. Thereafter,

chloramphenicol, tetracycline, macrolide, and glycopeptide

(e.g., vancomycin) were discovered from soil bacteria. The

synthesized antimicrobial agent nalidixic acid, a quinolone

antimicrobial drug, was obtained in 1962. (Saga, 2009)

Historical anecdotes of the red soils from the Hashemite

Kingdom of Jordan tell of people using the soils to treat skin

infections and diaper rash. A multinational group of

researchers suggest the healing power may be due to

antibiotic-producing bacteria they have found living in the

soil. This discovery may ultimately lead to new antibiotic

treatments against harmful pathogens such as Staphylococcus

aureus. (Historical Anecdote Of Jordan's Red Soils May Offer

New Antibiotic, 2009)2

1.2 AIM

The main purpose of this study is to investigate the

biological activity of soil. Challenges in

biological screening remain a key focus in drug discovery from

soil.

1.3 OBJECTIVES

To determine antimicrobial activity of soil against bacteria

of medical importance,

3

To combat the problem of antimicrobial resistance,

To reduce the cost in development of drugs by using soil as a

source,

4

CHAPTER 2

2.1 LITERATURE REVIEW

Soils typically contain 109 to 1010 microorganisms per gram

(dry weight), which may represent more than a million

bacterial species. However, characterization of the small

fraction of microbes that has been cultivated provides only a

glimpse of their potential physiological capacity and

influence on soil ecosystems. The absence of pure cultures or

genome sequences makes it difficult to ascertain the roles of

specific microbes in soil environments: this is particularly

true for bacteria in the phylum Acidobacteria, which are broadly

distributed in soils but poorly represented in culture.

(Eichorst, 2007)

Diarrhoea caused by Shigella species is estimated by World health

organisation (WHO) to cause 50% of dysentery cases. S. dysenteriae

serotype 1 is particularly virulent, causing endemic and

epidemic dysentery with high death rate. Salmonella organisms

are endemic in many tropical and developing countries, while

other salmonellas cause food poisoning and bacteraemia. It is

highly infectious and resistance to common available

antimicrobials is an increasing problem.

(Cheesbrough, 2000)

Infections caused by resistant microorganisms often fail to

respond to conventional treatment, resulting in prolonged

illness, greater risk of death and higher costs.

5

Tuberculosis strains resistant to isoniazid and rifampicin

(multidrug-resistance - MDR-TB) require treatment courses that

are much longer and less effective. World Health Organisation

estimates that there are about 630 000 MDR-TB cases in the

world.

A high percentage of hospital-acquired infections are caused

by highly resistant bacteria such as methicillin-resistant

Staphylococcus aureus (MRSA) or multidrug-resistant Gram-negative

bacteria.

New resistance mechanisms have emerged, making the latest

generation of antibiotics virtually ineffective.

The evolution of resistant strains is a natural phenomenon

that happens when microorganisms are exposed to antimicrobial

drugs, and resistant traits can be exchanged between certain

types of bacteria. The misuse of antimicrobial medicines

accelerates this natural phenomenon. Poor infection control

practices encourage the spread of AMR. Infections caused by

resistant microorganisms often fail to respond to the standard

treatment, resulting in prolonged illness and greater risk of

death. The death rate for patients with serious infections

treated in hospitals is about twice that in patients with

infections caused by non-resistant bacteria.

(Antimicrobial resistance, 2013)

Synthesis of medicinally important compounds is very difficult

and thus the cost of medicine is also high because of the non-

availability of source materials especially aromatic

compounds.

6

(Gopalakrishnan, 2011)

2.2 MATERIALS AND METHODS

2.2.1 SOIL SAMPLES

Soil samples were collected from the different places Port

Elizabeth, Eastern Cape. Samples were collected from various

depth of the earth surface, ranging from layers just beneath

the upper surface to 6 inches depth. They were collected in

the sterile small plastic tubes and properly labelled with the

date of collection. Nine soil samples were collected within a

period of 3 weeks with regard to different weather conditions

7

(After rain, on a sunny day and in on a cold day). The

collected soil samples were dried in a hot air oven at 60–65°C

for 3 hours and stored in 4°C until examined.

Sample

Number

Date of

collection

Collection

site

Depth Weather

1 3rd August Tenby,

Summerstrand

5 inches Sunny

2 5th August NMMU South

Campus pond

6 inches Cold

3 14th August NMMU North

Campus

Round- about

6 Inches After rains

Table 2.1 1

Showing collection sites, dates, weather at collection and

depth of soil from where the isolates were collected using

Nutrient agar media

8

2.2.2 ISOLATION OF PURE CULTURE OF MICROBES

Three microorganisms were isolated and obtained as pure

culture by using standard microbiological method. From each

soil sample, 1 gm of dried soil was suspended in 9 mL sterile

water, and successive serial dilutions were made by

transferring 1mL of aliquots to 2nd test tube containing 9 mL

of sterile water, and in this way dilutions up to 10−4 were

prepared. Each time the contents were vortexed to form uniform

suspension. An aliquot of 0.1 mL of each dilution was taken

and spread evenly over the surface of Nutrient-agar medium on

16 cm petri dishes. Plates were incubated at 37°C and

monitored for 24 hours. The colonies were carefully counted by

visual observation and c.f.u per gram of soil was determined.

Plates those gave 100–150 colonies were chosen for further

isolation in pure culture. Suitable colonies those that showed

an anonymous appearance under light microscope were re-

cultivated several times for purity on blood agar.

Figure 2.1 19

Shows the serial dilution technique used to dilute the 1 gram

soil specimens for each of the three samples

Figure 2.1 2

Showing colonies observation and c.f.u per gram of soil on

Nutrient agar

Figure 2.1 3

10

Showing isolation in pure culture on blood agar

2.2.3 SCREENING OF ANTIMICROBIAL ACTIVITIES OF PURE ISOLATES

Preliminary screening for antibiotic activity of the isolates

was done by using streak-plating technique Mueller Hinton-agar

medium. Each pure isolates were streaked individually on

different agar plates in a single line. The plates were then

incubated at 32°C for 5 days to allow the isolates to secrete

antibiotics into the medium. After the incubation period, the

properly diluted test organisms were cross-streaked along the

line of fully grown isolates. Each streaking was started near

the edge of the plates and streaked toward

the Streptomyces growth line. The plates were then incubated

for 12 hours at 37°C, for the zone of inhibition to be

observed.

2.2.4 TEST ORGANISMS

Five test organisms were used to test the antibiotic activity

of the isolates. Four of them were gram-positive and four were

gram-negative bacteria. Gram-positive species were Staphylococcus

aureus and Bacillus cereus. Gram-negative strains were Escherichia

coli, Shigella flexneri, and Salmonella spp. They were maintained in

nutrient agar medium and mackonkey agar medium respectively at

4°C.

11

2.2.5 RESULTS AND DISCUSSION

This study was performed with an aim of isolating soil

microbial strains with antimicrobial activities using the

selective isolation media. Three different gram positive cocci

strains were isolated from 3 soil samples collected from

different locations in the Eastern Cape in the year of 2013.

All of these strains were collected by using Nutrient-agar

media. Nutrient agar is a microbiological growth

medium commonly used for the routine cultivation of non-

fastidious bacteria. It is useful because it remains solid

even at relatively high temperatures. Also, bacteria grown in

nutrient agar grows on the surface, and is clearly visible as

small colonies.

All purified isolates grew on blood-agar media showing

morphology, small (<2mm), yellowish in colour, doom shaped,

mucoid and non-haemolytic. In addition, all colonies possessed

an earthy odour. All of the strains were acid fast negative

and gram positive.

All the isolated bacteria strains were screened for their

antibacterial activity on Mueller- Hinton-agar medium using

streak-plating technique. Known antibiotics were also used as

positive controls to the study, which exhibited antibacterial

activity and was observed on all the plates with known

microorganisms.

12

Source Colony

Appearance

Colour

and Odour

Colony Size

(Diameter)

1(T-S) Doom

Shaped,

Mucoid And

Non-

Haemolytic

Cream

White-

Yellow

And

Earthy

Odour

<2mm

2(NMMU-S) Doom

Shaped,

Mucoid And

Non-

Haemolytic

Cream

White-

Yellow

And

Earthy

Odour

<2mm

3(NMMU-N) Doom

Shaped,

Mucoid And

Non-

Haemolytic

Cream

White-

Yellow

And

Earthy

Odour

<2mm

Table 2.1 2

Describes the colonies of purified isolates grown on blood-

agar media with morphology; small (<2mm), yellowish in colour,

doom shaped, mucoid and non-haemolytic

13

Source Gram Stain Zielh Neelsen

Stain1(T-S) Gram Positive

Cocci

Negative

2(NMMU-S) Gram Positive

Cocci

Negative

3(NMMU-N) Gram Positive

Cocci

Negative

Table 2.1 3

Shows the microscopic results of the pure colonies from soil

14

Figure 2.1 4

Shows Streak-plating technique to screen the antibacterial

activity of isolated microbes on Mueller- Hinton agar media,

with a resistance of known microorganism to pure colony

antimicrobial activity

Known

Microorganisms

Soil 1 (T-S) Soil 2 (NMMU-

S)

Soil 3 (NMMU-

N)Staphylococcus

aureus

R R R

Bacillus cereus R R REscherichia coli R R RShigella flexneri R R RSalmonella spp R R R

15

Table 2.1 4

Showing antibacterial activity of the isolates against a wide

range of test bacteria, showing resistance (R)

Figure 2.1 5

Showing antibacterial activities exhibited by known

antibiotics against known microorganisms used as positive

controls to the study, confirming zones of inhibition

Staphylococc

us aureus

Bacillus

cereus

Escherichi

a coli

Shigella

flexneri

Salmonella

spp

Ciprofloxac 3+ 2+ 3+ 3+ 3+

16

in (CIP)-

Dose- 5µgChloramphen

ical

(C)- 30µg

2+ 1+ 2+ 3+ 1+

Erythromyci

n (E)- 5µg

R - 2+ - 2+

Cephalexin

(CFX)- 30µg

1+ R P R P

Gentamicin

(GM)- 10µg

R 1+ P 1+ P

Ampicillin

(AP)

Dose- 10µg

- - P P P

Amoxycillin

(A)

Dose- 10µg

P R R R R

Table 2.1 5

Table 2.1.5- Shows the results microbial inhibition on the by

known antibiotics against five known microorganisms of medical

importance

17

CHAPTER 3

3.1 CONCLUSION

Historically the most commonly isolated actinomycete genera

have been Streptomyces and Micromonospora. As a result, the

majority of metabolites identi ed in screening programmes fi

searching for new antibiotics were derived from a relatively

limited pool of organisms. The genus Streptomyces is in fact

known as one of the major sources of bioactive natural

products. In the last decades, the intensive screening for new

secondary metabolites has also focused on minor groups of

actinomycetes, including species that are dif cult to isolate fi

and culture, and those that grow under extreme conditions.

In an effort to improve a screening programme in search of new

secondary metabolites with antimicrobial activity, alternative

selective conditions of pH and salinity for the isolation of

minor groups of actinomycetes not usually recovered in neutral

and low osmolality conditions was tested. The effect of this

expanded range of isolation conditions on the patterns of

detection of antibiotic activity was then evaluated.

(Basilio, 2003)

Actinomycetes comprise an extensive and diverse group of Gram-

positive, aerobic, mycelial bacteria that play an important

ecological role in soil cycles. Many are well known for their

economic importance as producers of biologically active

substances, such as antibiotics, vitamins and enzymes. In

addition, they are one of the major communities of the

microbial population present in soil, and their occurrence is

18

greatly in uenced by the environmental conditions of humidity,fl

temperature, pH and vegetation.

Soil actinomycetes for the most part show their optimum growth

in neutral and slightly alkaline conditions, and isolation

procedures have been traditionally based on this neutrophilic

character. Previous works showed the existence of a large

diversity of acidophilic actinomycetes that differed

morphologically and physiologically from neutrophilic species.

(Basilio, 2003)

In a study, from the soil samples of Karanjal regions of

Sundarbans of Bangladesh, about 55 actinomycetes of different

genera were isolated and screened for antibacterial activity.

In their screening work, they found that 20 isolates were

active against the test organisms. In another study,

356 Streptomyces isolates were obtained from soils in the

Aegean and East Black Sea regions of Turkey, and 36% of the

isolates were found to be active against tested

microorganisms. In a recent study performed in 2010 by Dehand,

the antibacterial activity of streptomyces isolates from soil

samples of West of Iran was investigated. Out of 150

actinomycetes, only 20 isolates (13.30%) showed activity

against the test bacteria.

(Sheikh, 2010)

In this study, in-vitro antimicrobial susceptibility tests

were performed using a panel which included both clinical

pathogens and laboratory control strains. All bacteria used

19

for the tests were resistant to at least one known

antimicrobial agent. The active soil isolates exhibited no

inhibitory pattern against the test organisms Staphylococcus

aureus, Bacillus cereus, Shigella flexneri, Escherichia coli and Salmonella spp.

Comparing the above mentioned results of other soil

antimicrobial studies, with this study, we can conclude that

the soil samples of three sites of choice are not rich source

of actinomycetes or any other bacteria which produce

metabolites inhibitory to bacterial pathogens. We found that

none of the isolated colonies were active against the test

bacteria. The known antibiotic positive controls were very

active and showed very large zone of inhibition.

3.2 RECOMMENDATIONS

Future investigations with different soil samples of different

properties still have to be carried out since these results

indicate that the selected soil samples stimulate growth of

all the pathogens. For future reference, there is need of

using further microbial identification techniques on the soil

isolates, other than gram stain and Ziehl Neelsen stain. The

precise identification of these soil pure isolates may be

20

studied on other bacteria of medical importance. Also, more

research on their biological properties may be carried out.

It is also clear that the wrong use of antimicrobial agents

resulted in the selection of resistant bacteria. Since the

advent of new mighty drugs is highly dif cult, the proper use fi

of currently available antimicrobial agents as well as efforts

to minimize the spread of resistant bacteria through

appropriate infection control would be quite important, and

may represent a rst step in solving the issue of resistant fi

microorganisms.

(Saga, 2009)

21

REFERENCES

(2009, May 21). Retrieved September 2013, from Science daily: http://www.sciencedaily.com/releases/2009/05/090518222202.htm

(2013, May). Retrieved Sepetember 2013, from World Health

Organisation:

http://www.who.int/mediacentre/factsheets/fs194/en/

Abdelghani, K. P. (2009, April). Antibacterial activity of

bacterial isolates of soil bacteria collected from Palestine.

Current Trends in Biotechnology and Pharmacy, 3(2). Retrieved September

2013, from http://www.pharmainfo.net/articles/antibacterial-

activity-bacterial-isolates-soil-bacteria-collected-palestine

Basilio, G. V. (2003, August). Patterns of antimicrobial

activities from soil actinomycetes isolated under different

conditions of pH and salinity. Journal of Applied Microbiology, 95(4),

814-823. Retrieved October 2013, from

http://onlinelibrary.wiley.com/doi/10.1046/j.1365-

2672.2003.02049.x/pdf

Cheesbrough. (2000). District laboratory practice in tropical countries (Vol.

2). Cambridge, United Kingdom: Cambridge University Press.

Retrieved September 2013

Eichorst, B. S. (2007, April). Isolation and Characterization

of Soil Bacteria That Define Terriglobus gen. nov., in the

Phylum Acidobacteria. Applied and Enviromental Microbiology, 73(8),

2708-2713. Retrieved September 2013, from

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1855589/#fn1

22

Falkinham, W. T. (2009, March). Proliferation of Antibiotic-

Producing Bacteria and Concomitant Antibiotic Production as

the Basis for the Antibiotic Activity of Jordan's Red Soils.

American Society For Microbiology, 75(9), 2735-2741. Retrieved

September 2013, from http://aem.asm.org/content/75/9/2735

Gopalakrishnan, N. M. (2011). Antibacterial activity of azo

compounds synthesized from the natural. Journal of Chemical and

Pharmaceutical Research, 3(4), 490-497. Retrieved September 2013,

from http://jocpr.com/vol3-iss4-2011/JCPR-2011-3-4-490-497.pdf

Moshafi, F. A.-N. (2011, July). Antimicrobial activity of

Bacillus sp. strain FAS1 isolated from soil. Pakistan Journal of

Pharmaceutical science, 24(3), 269-275. Retrieved September 2013,

from http://www.ncbi.nlm.nih.gov/pubmed/21715259

Saga, Y. (2009, April). History of Antimicrobial Agents and

Resistant. Japan Medical Association, 52(2), 103-108. Retrieved

September 2013, from

http://www.med.or.jp/english/journal/pdf/2009_02/103_108.pdf

Sheikh, H. M. (2010, October 4). Antimicrobial activity of

certain bacteria and fungi isolated from soil mixed with human

saliva against pathogenic microbes causing dermatological

diseases. Saudi Journal of Biological Sciences, 17(4), 331-339. Retrieved

September 2013, from

http://www.sciencedirect.com/science/article/pii/S1319562X1000

0732

23

Yilmaz, M. (2006, February 13). Antimicrobial activities of

some Bacillus spp. strains isolated from the soil. Microbiological

Research, 161(2), 127-131. Retrieved September 2013, from

http://www.sciencedirect.com/science/article/pii/S094450130500

0704

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