Antibiosis Activities against Burkholderia pseudomallei by Other Burkholderia Species

Published:

Antibiosis Activities against Burkholderia pseudomallei by Other Burkholderia Species


TABLE OF CONTENTS

TABLE OF CONTENTS

I

LIST OF ABBREVIATIONS

II

LIST OF TABLES

III

SUMMARY

IV

  1. INTRODUCTION

1

  1. LITERATURE REVIEW

2.1 Burkholderia pseudomallei

2.1.1 Characteristics

2.1.2 Epidemiology

2.1.3 Pathogenicity

2.2 Antibiosis Activities of Burkholderia pseudomallei

2.2.1 Microbial Antagonism

2.2.2 Bacteriocin

2

2

3

4

4

Lady using a tablet
Lady using a tablet

Professional

Essay Writers

Lady Using Tablet

Get your grade
or your money back

using our Essay Writing Service!

Essay Writing Service

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 Characterization of the Antibiosis Compound

5

6

6

7

7

8

4.0 EXPECTED OUTCOME

9

WORK SCHEDULE

10

REFERENCES

11

LIST OF ABBREVIATIONS

BLIS

Bacteriocin-like inhibitory substance

CDC

Centers for Disease Control and Prevention

CFS

Cell free supernatant

CFU

Colony-forming unit

GENr

Genetically resistance

GENs

Genetically sensitive

HCl

Hydrochloric acid

LB

Luria Bertani

MH

Mueller Hinton

NaCl

Sodium chloride

NaOH

Sodium hydroxide

NT

Northern Territory

rRNA

Ribosomal ribonucleic acid

spp.

Species (plural)

UHQ

Ultra High Quality

ZOI

Zone of inhibition

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 project.

5

Antibiosis Activities against Burkholderia pseudomallei by Other Burkholderia Species

Nur Ezzah binti Sainei

Resource Biotechnology

Department of Molecular Biology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

SUMMARY

Burkholderia pseudomallei is known as a significant agent to the distribution of melioidosis within the tropical part of the world and a versatile species of bacteria which inhabit a variety of ecological niches including soil and water source. By studying and understanding the nature of antibiosis activities against B. pseudomallei by other antagonistic Burkholderia species, we will able to identify the predominant Burkholderia spp. and the degree of their capabilities to inhibit the growth of B. pseudomallei. To analyze the microbial antibiosis in this project, a selected antibiosis screening method will be conducted followed by the collection and characterization of the antibiosis compounds. The expected outcome of this project will include the observation of antibiosis activities in the form of inhibition zone (ZOI) and the successful extraction and characterization of the product of microbial antagonism.

Lady using a tablet
Lady using a tablet

Comprehensive

Writing Services

Lady Using Tablet

Plagiarism-free
Always on Time

Marked to Standard

Order Now

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

1.0 INTRODUCTION

Burkholderia pseudomallei is a species of microbe that inhabits a variety of natural environments including water and soil. The environmental prevalence of the microorganism is significantly related to the emergence of an endemic tropical illness known as melioidosis (Marshall et al., 2010). The distribution of B. pseudomallei in diverse ecological niches is extensive yet inconsistent due to the dispersal of other Burkholderia species within the same niche. The co-existence of different Burkholderia spp. in a specific habitat allows the occurrence of antibiosis activities of certain Burkholderia spp. against B. pseudomallei. By carrying out this project, we will able to identify the predominant Burkholderia spp. that are able to impede the growth of B. pseudomallei and to study the degree of their inhibitory capabilities by observing the presence and size of ZOI that surrounded the antagonistic colonies within a lawn of B. pseudomallei. Furthermore, we will able to understand the fundamental characteristics of the agent which is responsible to the growth inhibition of B. pseudomallei by characterizing the collected antagonistic compound from the antibiosis activities.

The proposed objectives for this project are:

  1. To observe the antibiosis activities against the growth of B. pseudomallei by other Burkholderia species through the conduction of suggested antibiosis screening method;
  2. To obtain the antibiosis compounds from the microbial antagonistic activities;
  3. To characterize the antibiosis compounds in term of their sensitivity to pH and heat stability.

2.0 LITERATURE REVIEW

2.1 Burkholderia pseudomallei

2.1.1 Characteristics

Burkholderia pseudomallei is classified under the genus Burkholderia with other former members of rRNA homology group II pseudomonads (Coenye & Vandamme, 2003; Inglis & Sagripanti, 2006). The morphological appearance of the bacterium is described as a lean, vacuolated form which possesses rounded ends and frequently expounded to appear in the shape of a “safety pin” through bipolar staining (Cheng & Currie, 2005). It is categorized as a gram-negative, saprophytic bacillus which is capable of motility and surviving as a facultative anaerobe (Brett & Woods, 2000). When cultured on selective agars, the colonies of B. pseudomallei display variable morphologies from the initial form of smooth colonies to the appearance of dry and wrinkled colonies on further incubation up to several days (Cheng & Currie, 2005; Inglis & Sagripanti, 2006; Chantratita et al., 2007). The versatile characteristic of B. pseudomallei allows the microorganism to adapt to diverse ecological niches from damp soil and stagnant water sources in tropical and subtropical countries to the inside of animals and humans (Inglis & Sagripanti, 2006; Kaestli et al., 2009; Goodyear et al., 2013).

2.1.2 Epidemiology

B. pseudomallei is the main etiological agent of melioidosis, an emerging illness which is endemic to northern Australia and southeast Asia correlated with other tropical and subtropical countries within the latitude of 20ËšN and 20ËšS (Godoy et al., 2003; Cheng & Currie, 2005; Kaestli et al., 2009). Due to the worldwide infection with no available licensed vaccines or proper medical therapies, B. pseudomallei is labeled as category B organism by CDC for its possible potential as a biowarfare agent (Thibault, 2004; Massey, 2014). The highest number of cases of melioidosis-infected patients was documented in Ubon Ratchathani, northeastern Thailand whereby 20% of the community possess septicemia which led to a significant number of mortality (Cheng & Currie, 2005; Kaestli et al., 2009; Galyov et al., 2010). The species of bacteria is able to infect human population via wounded skin, direct injection, ingestion and inhalation (Limmathurotsakul et al., 2010).

2.1.3 Pathogenicity

B. pseudomallei infection manifests in the form of abscesses in internal organs and in severe cases, the appearance of septic shock and acute pneumonia (Galyov et al., 2010). In chronic cases, the illness can lead to demise. According to Nandi and Tan (2013), the genomic DNA of B. pseudomallei was discovered to be surprisingly large, consisting of two circular chromosomal DNA that reach up to 7.2 Mb in size which are comprised of more than 5,600 protein-coding genes and a vast collection of virulence genes that code for adhesins, toxins, clusters of capsular polysaccharide, and diverse secretion systems of type III and IV.

Lady using a tablet
Lady using a tablet

This Essay is

a Student's Work

Lady Using Tablet

This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.

Examples of our work

2.2 Antibiosis Activities against Burkholderia pseudomallei

2.2.1 Microbial Antagonism

Microbial antagonism or antibiosis is defined as an active biological activity between at least two closely related species of bacteria whereby the antagonistic bacteria is capable of engaging in competition for nutrients against another species of bacteria, impeding the reproduction of the bacteria by producing toxins or antibiotics (Živković et al., 2010). In this case, the antagonistic bacteria are the antagonistic Burkholderia spp. which will compete against the growth of B. pseudomallei. To screen for the presence of antibiosis activities, there are several available methods established which include the direct spot on lawn and flip streak methods (Marshall et al., 2010), agar diffusion test as well as Transwell culture system (Lin et al., 2011). The screening method that will be used in the project will be a modified version of agar diffusion test by Marshall et al. (2010) due to the simplicity, efficacy and reliability of the method.

2.2.2 Bacteriocin

Bacteriocin is identified as an antibacterial polypeptide which is produced to annihilate or hindering the growth of a species of bacteria by antagonistic microorganism (Wilson, 2010). According to Marshall et al. (2010), the antibiosis activities against B. pseudomallei by other Burkholderia spp. especially B. ubonensis indicated that bacteriocins or BLIS (bacteriocin-like inhibitory substance) were produced by the antagonistic Burkholderia spp.

3.0 MATERIALS AND METHODS

3.1 List of Materials

  1. Isolates of Burkholderia species and Burkholderia closely related sp.

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

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

Ralstonia species

Burkholderia multivorans

  1. Ashdown’s selective agar (contains tryptone, glycerol, 1% aqueous neutral red, 0.1% crystal violet, agar powder, UHQ water, gentamicin)
  2. Mueller Hinton (MH) agar (contains beef extract, casein hydrolysate, starch, agar)
  3. Luria Bertani (LB) broth (contains tryptone, yeast extract, sodium chloride (NaCl))
  4. 6 M Hydrochloric acid (HCl)
  5. 1 M Sodium hydroxide (NaOH)
  6. Spectrophotometer (Shimadzu UV-VIS Spectrophotometer/UVmini 1240, Japan)
  7. Hot plate with stirrer
  8. Centrifuge (ALC Centrifuge 4206, Italy)
  1. Preparation of Agar Media

3.2.1 Ashdown’s Agar

In the method of preparing Ashdown’s agar, all the ingredients will be mixed together and autoclaved. The sterilized mixture will then be cooled to 55 ËšC followed by the addition of 13 mg of gentamicin (8 mg/L). After that, the mixture will be poured onto the Petri dishes and will be allowed to cool before being stored at 4 ËšC.

3.2.2 Mueller Hinton (MH) Agar

According to Acumedia (2011), all the ingredients will be mixed and suspended in 1 L of purified water. The mixture will be heated with constant shaking and boiled for a minute. After that, it will be autoclaved for 15 minutes at 121 ËšC and cooled to the room temperature. The final pH value will be ensured to achiever the range within 7.3 ± 0.1 at 25 ËšC before the mixture will be poured into Petri dishes.

  1. Antibiosis Screening Method

The selected “target” strain (B. pseudomallei) will be streaked across a plate and allowed to grow at 37 ËšC overnight. The cell suspension of the “target” strain will be prepared on the next day by harvesting 1-2 colonies off the agar plate and re-suspending them in 0.85% saline to 0.5 McFarland Standard turbidity or OD of ≈ 0.13 at 600 nm or approximately 108 to 109 cells/ml. A sterile cotton swab will be used to immerse into the cell suspension and streaked in three directions across the surface of the MH agar. The MH plate will be left within the biosafety cabinet to allow the excess liquid fully absorbed. Then, 10 ul of cell suspension of the “challenger” strains will be inoculated onto the lawn of indicator cells. The plates will be incubated at 37 ËšC 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 procedure will involve the collection of aliquots from the culture of selected antagonistic “challenger” strain which will be grown at 30 ËšC in prepared LB broth (Marshall et al., 2010). The samples will be centrifuged for half an hour at 10,000g before the filter sterilization will be carried out on the samples to discard the cells (Marshall et al., 2010). Method of well diffusion assay will be conducted to measure the antibiosis activity in the CFS by initially inoculating a suspension of B. pseudomallei over the overall surface of an MH plate (Marshall et al., 2010). After that, the culture will be dried followed by the cutting of 6 mm wells in diameter into the agar which will serve to contain about 100 µl of CFS (Marshall et al., 2010). The plates will be incubated at the temperature of 30 ËšC for about 48 hours and the indication of the ZOI appearance will be measured whereby the diameter of the ZOI that is more than 6 mm will be indicated as the elevation state of antibiosis activity while the ZOI which is recorded as 6 mm and less in diameter will correspond to the absence of antibiosis activity (Marshall et al., 2010).

3.5 Characterization of the Antibiosis Compound

The acquired antibiosis substance will be further analyzed by characterizing the biochemical compound in term of the sensitivity in pH value and heat stability. In determining the stability of the compound to heat, the aliquots of the CFS will be heated for three different duration periods which will be for 10, 30, and 60 minutes at three various temperatures such as 50, 70, and 100 ËšC, ensuing in nine different treatments of heat (Marshall et al., 2010). The sample in control will be treated under the temperature of 37 ËšC. In determining the stability of the compound in pH fluctuation, two aliquots will initially be adjusted to pH 4.0 by mixing in 6 M HCl before one of the two aliquots will be readjusted to pH 7.0 with the addition of 1 M NaOH (Marshall et al., 2010). Another two aliquots will be adjusted to pH 9.0 using 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 receive no treatment while the other will be treated with an equivalent amount of water as the volume of acid and base.

4.0 EXPECTED OUTCOME

At the end of this project, we will expect that the proposed method of the antibiosis screening will be conducted successfully. We will also expect to observe and record the antibiosis activities against Burkholderia pseudomallei by other Burkholderia species. Furthermore, we will anticipate the acquirement of the antibiosis compound to be accomplished as well as the characterization of the compound in term of its pH sensitivity and heat stability.

WORK SCHEDULE

Project Activities

2014

2015

Jul

Aug

Sep

Oct

Nov

Dec

Jan

Feb

Mar

Apr

May

Data collection

â-º

â-º

â-º

â-

Proposal writing and presentation

â-º

â-º

â-

Benchwork and sample processing

â-º

â-º

â-º

â-º

â-

Progress report

â-º

â-

Data analysis

â-º

â-º

â-º

â-

Data validation: statistical analysis

â-º

â-º

â-

Report writing and presentation

â-º

â-º

â-º

â-º

â-

REFERENCES

Acumedia. (2011). Mueller Hinton Agar (7101). Retrieved from www.neogen.com/Acumedia/pdf/ProdInfo/7101_PI.pdf.

Brett, P. J., & Woods, D. E. (2000). Pathogenesis of and immunity to melioidosis.Acta Tropica,74 (2), 201-210.

Chantratita, N., Wuthiekanun, V., Boonbumrung, K., Tiyawisutsri, R., Vesaratchavest, M., Limmathurotsakul, D., ... & Peacock, S. J. (2007). Biological relevance of colony morphology and phenotypic switching by Burkholderia pseudomallei.Journal of Bacteriology,189 (3), 807-817.

Cheng, A. C., & Currie, B. J. (2005). Melioidosis: epidemiology, pathophysiology, and management.Clinical Microbiology Reviews,18 (2), 383-416.

Coenye, T., & Vandamme, P. (2003). Diversity and significance of Burkholderia species occupying diverse ecological niches.Environmental Microbiology,5 (9), 719-729.

Galyov, E. E., Brett, P. J., & DeShazer, D. (2010). Molecular insights into Burkholderia pseudomallei and Burkholderia mallei pathogenesis.Annual Review of Microbiology,64, 495-517.

Godoy, D., Randle, G., Simpson, A. J., Aanensen, D. M., Pitt, T. L., Kinoshita, R., & Spratt, B. G. (2003). Multilocus sequence typing and evolutionary relationships among the causative agents of melioidosis and glanders, Burkholderia pseudomallei and Burkholderia mallei.Journal of Clinical Microbiology,41 (5), 2068-2079.

Goodyear, A., Strange, L., Rholl, D. A., Silisouk, J., Dance, D. A., Schweizer, H. P., & Dow, S. (2013). An improved selective culture medium enhances the isolation of Burkholderia pseudomallei from contaminated specimens.The American Journal of Tropical Medicine and Hygiene,89 (5), 973-982.

Inglis, T. J., & Sagripanti, J. L. (2006). Environmental factors that affect the survival and persistence of Burkholderia pseudomallei.Applied and Environmental Microbiology,72 (11), 6865-6875.

Kaestli, M., Mayo, M., Harrington, G., Ward, L., Watt, F., Hill, J. V., ... & Currie, B. J. (2009). Landscape changes influence the occurrence of the melioidosis bacterium Burkholderia pseudomallei in soil in northern Australia.PLoS Neglected Tropical Diseases,3 (1), e364.

Limmathurotsakul, D., Wuthiekanun, V., Chantratita, N., Wongsuvan, G., Amornchai, P., Day, N. P., & Peacock, S. J. (2010). Burkholderia pseudomallei is spatially distributed in soil in northeast Thailand.PLoS Neglected Tropical Diseases,4 (6), e694.

Lin, H. H., Chen, Y. S., Li, Y. C., Tseng, I., Hsieh, T. H., Buu, L. M., & Chen, Y. L. (2011). Burkholderia multivorans acts as an antagonist against the growth of Burkholderia pseudomallei in soil.Microbiology and Immunology,55 (9), 616-624.

Marshall, K., Shakya, S., Greenhill, A. R., Padilla, G., Baker, A., & Warner, J. M. (2010). Antibiosis of Burkholderia ubonensis against Burkholderia pseudomallei, the causative agent for melioidosis.Southeast Asian Journal of Tropical Medicine and Public Health,41 (4), 904-912.

Massey, S., Yeager, L. A., Blumentritt, C. A., Vijayakumar, S., Sbrana, E., Peterson, J. W., ... & Torres, A. G. (2014). Comparative Burkholderia pseudomallei natural history virulence studies using an aerosol murine model of infection.Scientific reports,4, 1-11.

Nandi, T., & Tan, P. (2013). Less Is More: Burkholderia pseudomallei and Chronic Melioidosis.mBio,4 (5), e00709-13.

Thibault, F. M., Hernandez, E., Vidal, D. R., Girardet, M., & Cavallo, J. D. (2004). Antibiotic susceptibility of 65 isolates of Burkholderia pseudomallei and Burkholderia mallei to 35 antimicrobial agents.Journal of Antimicrobial Chemotherapy,54 (6), 1134-1138.

Wilson, C. L. (Ed.). (2010).Intelligent and active packaging for fruits and vegetables. Boca Raton, USA: CRC Press.

Živković, S., Stojanović, S., Ivanović, Ž., Gavrilović, V., Popović, T., & Balaž, J. (2010). Screening of antagonistic activity of microorganisms against Colletotrichum acutatum and Colletotrichum gloeosporioides.Archives of Biological Sciences,62 (3), 611-623.

1