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Antibiosis Activities againstBurkholderia pseudomalleiby OtherBurkholderiaSpeciess

Table OF CONTENTS

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Table OF CONTENTS

I

List OF ABBREVIATIONS

Two

List OF TABLES

Three

Summary

Four

  1. Introduction

1

  1. LITERATURE REVIEW

2.1Burkholderia pseudomallei

2.1.1 Features

2.1.2 Epidemiology

2.1.3 Pathogenicity

2.2 Antibiosis Activities ofBurkholderia pseudomallei

2.2.1 Microbial Hostility

2.2.2 Bacteriocin

2

2

3

4

4

3.0 MATERIALS AND METHOD

3.1 List of Materials

3.2 Preparation of Agar Media

3.2.1 Ashdown’s Agar

3.2.2 Mueller Hinton ( MH ) Agar

3.3 Antibiosis Screening Method

3.3 Acquirement and Further Antagonistic Detection of Antibiosis Compound in a Cell Free State ( CFS )

3.4 Word picture of the Antibiosis Compound

5

6

6

7

7

8

4.0 EXPECTED OUTCOME

9

Work Agenda

10

Mentions

11

List OF ABBREVIATIONS

BLIS

Bacteriocin-like inhibitory substance

CDC

Centers for Disease Control and Prevention

Californium

Cell free supernatant

CFU

Colony-forming unit

GenR

Genetically opposition

Gens

Genetically sensitive

HCl

Hydrochloric acid

Pound

Luria Bertani

MH

Mueller Hinton

NaCl

Sodium chloride

NaOH

Sodium hydrated oxide

National trust

Northern Territory

rRNA

Ribosomal ribonucleic acid

spp.

Speciess ( plural )

UHQ

Ultra High Quality

ZOI

Zone of suppression

µl

Microlitre

List OF TABLES

Table

Title

Page

3.1.1

List of “target” strain and “challenger” strain isolates that will be used in the experimental undertaking.

5

Antibiosis Activities againstBurkholderia pseudomalleiby OtherBurkholderiaSpeciess

Nur Ezzah binti Sainei

Resource Biotechnology

Department of Molecular Biology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

Summary

Burkholderia pseudomalleiis known as a important agent to the distribution of melioidosis within the tropical portion of the universe and a various species of bacteriums which inhabit a assortment of ecological niches including dirt and H2O beginning. By analyzing and understanding the nature of antibiosis activities againstB. pseudomalleiby other counterBurkholderiaspecies, we will able to place the predominantBurkholderiaspp. and the grade of their capablenesss to suppress the growing ofB. pseudomallei. To analyse the microbic antibiosis in this undertaking, a selected antibiosis testing method will be conducted followed by the aggregation and word picture of the antibiosis compounds. The expected result of this undertaking will include the observation of antibiosis activities in the signifier of suppression zone ( ZOI ) and the successful extraction and word picture of the merchandise of microbic hostility.

Keywords: Agar diffusion method, antibiosis, bacteriocin,Burkholderia pseudomallei, melioidosis.

1.0 Introduction

Burkholderia pseudomalleiis a species of bug that inhabits a assortment of natural environments including H2O and dirt. The environmental prevalence of the micro-organism is significantly related to the outgrowth of an endemic tropical unwellness known as melioidosis ( Marshall et al. , 2010 ) . The distribution ofB. pseudomalleiin diverse ecological niches is extended yet inconsistent due to the dispersion of otherBurkholderiaspecies within the same niche. The co-existence of differentBurkholderiaspp. in a specific home ground allows the happening of antibiosis activities of certainBurkholderiaspp. againstB. pseudomallei. By transporting out this undertaking, we will able to place the predominantBurkholderiaspp. that are able to hinder the growing ofB. pseudomalleiand to analyze the grade of their inhibitory capablenesss by detecting the presence and size of ZOI that surrounded the counter settlements within a lawn ofB. pseudomallei. Furthermore, we will able to understand the cardinal features of the agent which is responsible to the growing suppression ofB. pseudomalleiby qualifying the collected counter compound from the antibiosis activities.

The proposed aims for this undertaking are:

  1. To detect the antibiosis activities against the growing ofB. pseudomalleiby otherBurkholderiaspecies through the conductivity of suggested antibiosis testing method ;
  2. To obtain the antibiosis compounds from the microbic counter activities ;
  3. To qualify the antibiosis compounds in term of their sensitiveness to pH and heat stableness.

2.0 LITERATURE REVIEW

2.1Burkholderia pseudomallei

2.1.1 Features

Burkholderia pseudomalleiis classified under the genusBurkholderiawith other former members of rRNA homology group II pseudomonads ( Coenye & A ; Vandamme, 2003 ; Inglis & A ; Sagripanti, 2006 ) . The morphological visual aspect of the bacteria is described as a thin, vacuolated signifier which possesses rounded terminals and often expounded to look in the form of a “safety pin” through bipolar staining ( Cheng & A ; Currie, 2005 ) . It is categorized as a Gram-negative, saprophytic B which is capable of motility and surviving as a facultative anaerobe ( Brett & A ; Woods, 2000 ) . When cultured on selective agars, the settlements ofB. pseudomalleishow variable morphologies from the initial signifier of smooth settlements to the visual aspect of dry and wrinkly settlements on farther incubation up to several yearss ( Cheng & A ; Currie, 2005 ; Inglis & A ; Sagripanti, 2006 ; Chantratita et al. , 2007 ) . The various feature ofB. pseudomalleiallows the micro-organism to accommodate to diverse ecological niches from moist dirt and dead H2O beginnings in tropical and semitropical states to the interior of animate beings and worlds ( Inglis & A ; Sagripanti, 2006 ; Kaestli et al. , 2009 ; Goodyear et al. , 2013 ) .

2.1.2 Epidemiology

B. pseudomalleiis the chief aetiologic agent of melioidosis, an emerging unwellness which is endemic to northern Australia and southeast Asia correlated with other tropical and semitropical states within the latitude of 20EsN and 20EsS ( Godoy et al. , 2003 ; Cheng & A ; Currie, 2005 ; Kaestli et al. , 2009 ) . Due to the world-wide infection with no available accredited vaccinums or proper medical therapies,B. pseudomalleiis labeled as class B being by CDC for its possible potency as a biowarfare agent ( Thibault, 2004 ; Massey, 2014 ) . The highest figure of instances of melioidosis-infected patients was documented in Ubon Ratchathani, northeasterly Thailand whereby 20 % of the community possess blood poisoning which led to a important figure of mortality ( Cheng & A ; Currie, 2005 ; Kaestli et al. , 2009 ; Galyov et al. , 2010 ) . The species of bacteriums is able to infect human population via hurt tegument, direct injection, consumption and inspiration ( Limmathurotsakul et al. , 2010 ) .

2.1.3 Pathogenicity

B. pseudomalleiinfection manifests in the signifier of abscesses in internal variety meats and in terrible instances, the visual aspect of infected daze and ague pneumonia ( Galyov et al. , 2010 ) . In chronic instances, the unwellness can take to demise. Harmonizing to Nandi and Tan ( 2013 ) , the genomic Deoxyribonucleic acid ofB. pseudomalleiwas discovered to be surprisingly big, dwelling of two round chromosomal Deoxyribonucleic acid that reach up to 7.2 Mb in size which are comprised of more than 5,600 protein-coding cistrons and a huge aggregation of virulency cistrons that code for adhesins, toxins, bunchs of capsular polyose, and diverse secernment systems of type III and IV.

2.2 Antibiosis Activities againstBurkholderia pseudomallei

2.2.1 Microbial Hostility

Microbial hostility or antibiosis is defined as an active biological activity between at least two closely related species of bacteriums whereby the counter bacterium is capable of prosecuting in competition for foods against another species of bacteriums, hindering the reproduction of the bacteriums by bring forthing toxins or antibiotics ( A?ivkoviA‡ et al. , 2010 ) . In this instance, the counter bacteriums are the counterBurkholderiaspp. which will vie against the growing ofB. pseudomallei. To screen for the presence of antibiosis activities, there are several available methods established which include the direct topographic point on lawn and somersault run methods ( Marshall et al. , 2010 ) , agar diffusion trial every bit good as Transwell civilization system ( Lin et al. , 2011 ) . The showing method that will be used in the undertaking will be a modified version of agar diffusion trial by Marshall et Al. ( 2010 ) due to the simpleness, efficaciousness and dependability of the method.

2.2.2 Bacteriocin

Bacteriocin is identified as an antibacterial polypeptide which is produced to eliminate or impeding the growing of a species of bacteriums by counter micro-organism ( Wilson, 2010 ) . Harmonizing to Marshall et Al. ( 2010 ) , the antibiosis activities againstB. pseudomalleiby otherBurkholderiaspp. particularlyB. ubonensisindicated that bacteriocins or BLIS ( bacteriocin-like inhibitory substance ) were produced by the counterBurkholderiaspp.

3.0 MATERIALS AND METHODS

3.1 List of Materials

  1. Isolates ofBurkholderiaspecies andBurkholderiaclosely related sp.

Table 3.1.1: List of “target” strain and “challenger” strain isolates that will be used in the experimental undertaking.

Target strain (Burkholderia pseudomallei)

Challenger strain

Sarawak strain ( GENR)

Burkholderia ubonensis( Sarawak )

Sabah strain ( GENR)

B. ubonensis( NT, Australia )

Sarawak strain ( GENs)

Burkholderia thailandensis

NT ( Australia ) strain ( GENR)

Burkholderia pyroccinia

Burkholderia cepacia

Ralstoniaspecies

Burkholderia multivorans

  1. Ashdown’s selective agar ( contains tryptone, glycerin, 1 % aqueous impersonal ruddy, 0.1 % crystal violet, agar pulverization, UHQ H2O, Garamycin )
  2. Mueller Hinton ( MH ) agar ( contains beef infusion, casein hydrolysate, amylum, agar )
  3. Luria Bertani ( LB ) stock ( contains tryptone, barm infusion, Na chloride ( NaCl ) )
  4. 6 M Hydrochloric acid ( HCl )
  5. 1 M Sodium hydrated oxide ( NaOH )
  6. Spectrophotometer ( Shimadzu UV-VIS Spectrophotometer/UVmini 1240, Japan )
  7. Hot home base with scaremonger
  8. Centrifuge ( ALC Centrifuge 4206, Italy )
  1. Preparation of Agar Media

3.2.1 Ashdown’s Agar

In the method of fixing Ashdown’s agar, all the ingredients will be assorted together and autoclaved. The sterilised mixture will so be cooled to 55 EsC followed by the add-on of 13 milligram of Garamycin ( 8 mg/L ) . After that, the mixture will be poured onto the Petri dishes and will be allowed to chill before being stored at 4 EsC.

3.2.2 Mueller Hinton ( MH ) Agar

Harmonizing to Acumedia ( 2011 ) , all the ingredients will be assorted and suspended in 1 L of purified H2O. The mixture will be heated with changeless shaking and boiled for a minute. After that, it will be autoclaved for 15 proceedingss at 121 EsC and cooled to the room temperature. The concluding pH value will be ensured to achiever the scope within 7.3 ± 0.1 at 25 EsC before the mixture will be poured into Petri dishes.

  1. Antibiosis Screening Method

The selected “target” strain ( B. pseudomallei ) will be streaked across a home base and allowed to turn at 37 EsC overnight. The cell suspension of the “target” strain will be prepared on the following twenty-four hours by reaping 1-2 settlements off the agar home base and re-suspending them in 0.85 % saline to 0.5 McFarland Standard turbidness or OD of ? 0.13 at 600 nanometer or about 108to 109cells/ml. A unfertile cotton swab will be used to plunge into the cell suspension and streaked in three waies across the surface of the MH agar. The MH home base will be left within the biosafety cabinet to let the extra liquid to the full absorbed. Then, 10 ul of cell suspension of the “challenger” strains will be inoculated onto the lawn of index cells. The home bases will be incubated at 37 EsC overnight and the presence of ZOI will be observed and recorded daily.

3.4 Acquirement and Further Antagonistic Detection of Antibiosis Compound in a Cell Free State ( CFS )

The process will affect the aggregation of aliquots from the civilization of selected counter “challenger” strain which will be grown at 30 EsC in prepared LB stock ( Marshall et al. , 2010 ) . The samples will be centrifuged for half an hr at 10,000g before the filter sterilisation will be carried out on the samples to fling the cells ( Marshall et al. , 2010 ) . Method of good diffusion check will be conducted to mensurate the antibiosis activity in the CFS by ab initio inoculating a suspension ofB. pseudomalleiover the overall surface of an MH home base ( Marshall et al. , 2010 ) . After that, the civilization will be dried followed by the film editing of 6 millimeter Wellss in diameter into the agar which will function to incorporate about 100 µl of CFS ( Marshall et al. , 2010 ) . The home bases will be incubated at the temperature of 30 EsC for about 48 hours and the indicant of the ZOI visual aspect will be measured whereby the diameter of the ZOI that is more than 6 millimeter will be indicated as the lift province of antibiosis activity while the ZOI which is recorded as 6 millimeter and less in diameter will match to the absence of antibiosis activity ( Marshall et al. , 2010 ) .

3.5 Word picture of the Antibiosis Compound

The acquired antibiosis substance will be farther analyzed by qualifying the biochemical compound in term of the sensitiveness in pH value and heat stableness. In finding the stableness of the compound to heat, the aliquots of the CFS will be heated for three different continuance periods which will be for 10, 30, and 60 proceedingss at three assorted temperatures such as 50, 70, and 100 EsC, resulting in nine different interventions of heat ( Marshall et al. , 2010 ) . The sample in control will be treated under the temperature of 37 EsC. In finding the stableness of the compound in pH fluctuation, two aliquots will ab initio be adjusted to pH 4.0 by blending in 6 M HCl before one of the two aliquots will be readjusted to pH 7.0 with the add-on of 1 M NaOH ( Marshall et al. , 2010 ) . Another two aliquots will be adjusted to pH 9.0 utilizing the same 1 M NaOH followed by the readjustment of one of the aliquots to pH 7.0 with 6 M HCl ( Marshall et al. , 2010 ) . One of the two samples in control will have no intervention while the other will be treated with an tantamount sum of H2O as the volume of acid and base.

4.0 EXPECTED OUTCOME

At the terminal of this undertaking, we will anticipate that the proposed method of the antibiosis showing will be conducted successfully. We will besides anticipate to detect and enter the antibiosis activities againstBurkholderia pseudomalleiby otherBurkholderiaspecies. Furthermore, we will expect the skill of the antibiosis compound to be accomplished every bit good as the word picture of the compound in term of its pH sensitiveness and heat stableness.

Work Agenda

Undertaking Activities

2014

2015

Jul

Aug

Sep

Oct

Nov

Dec

Jan

Feb

Mar

Apr

May

Data aggregation

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Proposal composing and presentation

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Benchwork and sample processing

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Progress study

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Datas analysis

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Data proof: statistical analysis

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Report authorship and presentation

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