Role Selective and Nonselective Media for Isolation of Burkholderia

Species from Patients with Suspected Melioidosis

R. Lia Kusumawati

, Mirzan Hasibuan

, Afrinayanti W. Siregar

and Tryna Tania

Department of Microbiology, Faculty of Medicine, Universitas Sumatera Utara, Jl. Universitas No.1 Kampus USU Medan

20155, Indonesia

University of Sumatera Utara Hospital, Jl. Dr. T Mansyur No. 66 Kampus USU Medan 20154, Indonesia

Resident in Departement of Microbiology, Faculty of Medicine, Universitas Indonesia, Jl. Pegangsaan Timur 16 Cikini

Jakarta 10320, Indonesia

Keywords: Burkholderia Species, Ashdown’s Selective Agar, Melioidosis

Abstract: Melioidosis is an infectious disease caused by the Burkholderia bacteria species, especially Burkholderia

pseudomallei and Burkholderia cepacia, disease is endemic in Southeast Asia and Northern Australia. As a

tropical country like Indonesia, this is a very serious global threat. This study aims to compare selective and

non-selective media in diagnosing Melioidosis based on the results of culture of clinical specimens of

patients with suspected Melioidosis. The results showed that as many as 112 (100%) of suspected clinical

samples of bacterial melioidosis were grown on nonselective media, Mac-Conkey agar and 110 (98.2%)

bacteria growing on Columbia agar agar medium. While on Ashdown’s Selective Agar (ASA) there is

bacterial growth of 10 samples (8.9%.). Where 7 of 10 samples are is Burkholderia species (Burkhoderia

cepacia 5 and Burkholderia pseudomallei 2). The results of this study indicate that Ashdown's media plays

an important role in selecting Burkholderia species as the cause of Melioidosis.

1 INTRODUCTION

The Burkholderia genus is made up of a variety of

species, Gram-negative bacilli, saprophytes in soil

and water reservoirs, endemic to tropical and

subtropical regions such as Southeast Asia and

Northern Australia. Some species of the

Burkholderia genus are widely used in

biotechnology, bioremediation, biocontrol and

agricultural industries (Estrada-De et.al, 2001).

Three known species from this genus as an etiologic

agent and cause fatal diseases in humans and

animals are Burkholderia pseudomallei,

Burkholderia mallei, and Burkholderia cepacia

[2]

The disease that caused by these three species are

known as melioidosis. Melioidosis is an infectious

disease that has a complex spectrum such as local

skin lesions, sub acute pneumonia, abscess on

infected organ, musculoskeletal infections, and

fulminant pneumonia (Cheng et.al, 2005).

Burkholderia pseudomallei has been known for

causing severe sepsis that leading to mortality of the

patient (Currie et.al, 2010).

This situation is a serious global threat,

especially for a tropical country, such as Indonesia.

The clinical diagnosis of melioidosis remains

difficult since the disease has no pathognomonic

signs and symptoms (Wiersinga et.al, 2006). Current

standard diagnostics are routine culture on non-

selective media such as blood agar and selective

gram-negative Mac-Conkey. However, both media

are not selective for Burkholderia species, so

Burkholderia bacterial colonies are difficult to

distinguish from other bacterial colonies. Therefore,

selective media is needed to overcome difficulties of

the diagnosis. Selective media such as Ashdown's

Selective Agar (ASA) as a standard to establish

laboratory diagnosis of melioidosis. This study

focused on the role of selective and nonselective

media for culture clinical samples collected from

patients suspected with Melioidosis.

582

Kusumawati, R., Hasibuan, M., Siregar, A. and Tania, T.

Role Selective and Nonselective Media for Isolation of Burkholderia Species from Patients with Suspected Melioidosis.

DOI: 10.5220/0010079305820585

In Proceedings of the International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches (ICOSTEERR 2018) - Research in Industry 4.0, pages

582-585

ISBN: 978-989-758-449-7

2 METHOD

A descriptive study, comparing the role of selective

and nonselective media in growing Burkholderia

species bacteria. Samples were collected by using

total sampling method between January and June

2018, based on inclusion and exclusion criteria.

Identification of bacterial colonies was

phenotypically tested by using Vitek 2 Compact.

2.1 Specimen Collection

samples were collected from all clinical specimens

that sent to Universitas Sumatera Utara Hospital

Microbiology Laboratory from patients suspected

with melioidosis based on the physician diagnosis on

the Clinical Microbiology laboratory request form.

Several types of specimens collected in the form

were throat swab, blood, urine, pus, sputum and

respiratory secretions.

2.2 Non-selective Media Columbia

Agar and Mac-Conkey Agar

Columbia agar media preparation are made by

dissolving 3.8 gram of Columbia into 100 mL of

sterile aquadest and sterilized by using autoclave at

121

C for 15 minutes. After sterilization, put

solution at room temperature until medium

temperature reaches 40-45

C, 5% blood sheep was

added and homogenized, then poured on sterile

petridish. For Mac-Conkey agar media, 5.15 gram

are dissolved into 100 mL of sterile aquadest and

autoclaved. After sterilization process, put the

solution at room temperature until medium

temperature reaches 50

C and finally poured on

sterile petridish.

2.3 Selective Media Ashdown's

Selective Agar (ASA)

Selective media preparation by weighing the

composition of media consisting of Tryptone soya

broth 10 gram, agar bacterial 15 gram, 40 ml

glycerol, 5 mL of 0.1% crystal violet, 5 mL of 1%

neutral red and 950 mL of distilled water, all of the

material were dissolved into an Erlenmeyer and

sterillized by using an autoclave at 121

C for 15

minutes. Put the solution at room temperature until

the temperature reaches 50

C, gentamycin was

added with concentration 4mg/liter and

homogenized, then the media is poured on sterile

petridish.

2.4 Bacterial Culture

Bacterial culture was done on by using selective

ASA medium, as well as on routine media,

Columbia Blood Agar and Mac-Conkey Agar.

Bacterial culture was incubated at 35°C for 24-48

hours. Microscopic and macroscopic identification

were done for every grown colonies.

2.5 Identification

The identification was started with macroscopic

observation of bacterial colonies by performing a

morphological selection of the suspected

Burkholderia species bacteria. Followed by

microscopic observation with Gram staining. The

suspected Burkholderia species underwent

identification stage by using the Vitek 2 Compact.

Phenotypically, this tool can identify Burkholderia

species bacteria using GN card and simultaneously

performing antibiotic susceptibility test by using

AST GN card.

2.6 Data Analysis

Data of the comparison between selective and non-

selective media in growing Burkholderia species

was analysed. All results were presented in the

tabulation and percentage.

3 RESULTS AND DISCUSSIONS

Based on the results of culture of clinical specimens

from patients suspected with Melioidosis on routine

or non-selective media (Columbia Agar and Mac-

Conkey Agar) for 24 hours found the growing

bacteria Escherichia coli, Klebsiella pneumonia,

Pseudomonas putida, Pseudomonas aeruginosa,

Pseudomonas stutzeri, Pseudomonas fluorescens,

Serratia marcescens and Acinetobacter baumannii.

While Burkholderia colony species were not seen

yet, the incubation time on nonselective media

therefore was extended to 48 hours. Based on

observed colonies of Burkholderia species after 48

hours, it was seen in streaks thus continued to

subculture and identification stage. However, in both

media colonies of Burkhokderia species bacteria

were not typical, making it a little difficult to do the

selection.

Columbia or blood agar was used as a

nonselective medium to evaluate the vitality of the

Role Selective and Nonselective Media for Isolation of Burkholderia Species from Patients with Suspected Melioidosis

583

strain. Almost all bacteria can grow on this medium

so there will be a competition for growth between

species of bacteria (Edler, et.al, 2017). Whereas

Mac-Conkey was more selective towards Gram-

negative bacteria, but the growth of Burkholderia

species requires accuracy and a longer incubation

time (> 48 hours) to ensure the presence or absence

of Burkholderia species colonies.

In contrast to Ashdown's agar selective

media, at 24 hours the bacteria had grown and

shown a distinctive colony morphology such as

round, convex, absorbed little of the red pigment,

and wavy surfaces. Based on the identification

results using the GN identification card Vitek 2

Compact, colonies that grow on Ashdown's selective

media were Burkholderia pseudomallei and

Burkholderia cepacia. While other colonies that

grew on this selective media were Pseudomonas

putida and Pseudomonas stutzeri. The results of the

growth comparison on selective and nonselective

media can be seen in table 1.

Table 1: Growth of clinical strains at 48 hours on selective

and nonselective media

Bacterial growth Nonselectiv

Selective

CA MCA ASA

Escherichia coli + + -

lebsiella pneumoniae + + -

Pseudomonas putida + + +

Pseudomonas

aeruginosa

+ + -

Pseudomonas stutzeri + + +

Pseudomonas

luorescens

+ + +

Burkholderia

pseudomallei

+ + +

urkholderia ce

acia + + +

Serratia marcescens + + -

Acinetobacter

baumannii

+ + -

The results of culture from 112 specimens of

patients suspected with Melioidosis showed bacterial

growth in Columbia Agar Blood medium which was

110 (9.8%) and in Mac-Conkey media. While on

Ashdown's selective media, bacterial growth was

seen in 10 samples, consisted of Burkholderia

pseudomallei 2 (1.8%), Burkholderia cepacia 5

(4.4%), Pseudomonas stutzeri 2 (1.8%) and

Pseudomonas putida 1 (0.9%). All bacterial growth

from all clinical samples of patients suspected with

Melioidosis were presented in Table 2.

Table 2: Other microorganisms growth seen on media

Bacterial

species

No. (%) growth of bacterial

isolates in each media

(n=112)

MCA

(n=112)

ASA

(n=112)

Escherichia

coli

8(7.1) 8(7.1) 0(0)

neumoniae 28

(

)

(

)

(

)

utida 1

(

0.9

)

(

0.9

)

(

0.9

)

P. aeruginosa 54(48) 54(48) 0(0)

P. stutzeri 2(1.8) 2(1.8) 2(1.8)

luorescens 3

(

2.6

)

(

2.6

)

(

)

seudomallei 1

(

0.9

)

(

0.9

)

(

1.8

)

.cepacia 1(0.9) 3(2.6) 5(4.4)

S. marcescens 2

(

1.8

)

(

1.8

)

(

)

. baumannii 10

(

8.9

)

(

8.9

)

(

)

Total : 110(98.2

)

112(100

)

10(8.9)

Based on the results of selective and

nonselective media comparison, it can be seen that

Ashdown's selective media has a higher selection

rate for the growth of Burkholderia. This findings

indicated the use of media has of great significance

role in establishing diagnosis of Melioidosis.

Although the media is very selective, Pseudomonas

growth was also seen in this study. It is possible that

Pseudomonas-type bacteria were found to be

resistant to Gentamycin so that it grew in selective

Ashdown’s Media agar. So the composition of the

media needs to be modified to the ASA medium.

Modification of ASA media media was

needed as Burkholderia pseudomallei selective agar

(BPSA), this medium was designed to improve

recovery from strains that easier to be inhibited by

Burkholderia pseudomallei, Burkholderia cepacia,

and Pseudomonas aeruginosa, used to determine the

selectivity and sensitivity of BPSA. The purpose of

BPSA was to inhibit the growth of Pseudomonas

aeruginosa making the identification of

Burkholderia species easier to characterize because

of the typical morphology of the colony Howard,

et.al, 2005).

The sensivity was equal between ASA and

BPSA based on their sensitivity ratio, although

BPSA selectivity was found to be lower than ASA

medium. Culture from 86 of 155 clinical specimens

showed growth in at least one selective medium

(range, 1 to 4 positive samples per patient) (Peacock

et.al, 2005). BPSA has advantages in showing

typical morphology such as crinkled and undulating

colonies, while in ASA media it was seen to be

smooth surface and convex colonies. Both media did

not show any significant difference overall (Howard,

et.al, 2005). ASA media supported the growth of

ICOSTEERR 2018 - International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches

584

Burkholderia species including Burkholderia mallei

and suitable for screening during situation when

Burkholderia pseudomallei and/or Burkholderia

mallei were suspected (Peacock et.al, 2005).

ASA media has an important role to support

the diagnosis of melioidosis at the University

Hospital of North Sumatra. This study findings

demonstrated that to use only the current routine or

nonselective media as the standard diagnostic tools

of culture was definitely not enough to establish the

diagnosis of melioidosis from clinical samples. The

use and application of ASA as standard culture

media in clinical microbiology laboratory routine

services to support and establish the challenging

diagnosis of melioidiosis from clinical samples

collected from patients suspected with melioidosis

was imperative.

4 CONCLUSIONS

This study showed that the use of ASA media was

necessary especially in clinical microbiology

laboratory settings and has high selectivity rate for

Burkholderia. In this study, 7 of 10 types of bacteria

that had successfully grown on ASA media were

found to be Burkholderia species (5 of Burkholderia

cepacia and 2 of Burkholderia pseudomallei).

ACKNOWLEDGEMENTS

Authors are gratefully acknowledged to the

Research Institute of Universitas Sumatera Utara for

providing the funds from TALENTA 2018 based on

the scheme of Research Development of University

of Sumatera Utara Hospital (PPRSU) with contract

number: 72/2.3.1/PPM/ KP-TALENTA USU/2018.

REFERENCES

Estrada-De Los Santos, P., Bustillos-Cristales, R. and

Caballero-Mellado, J., 2001. Burkholderia, a genus

rich in plant-associated nitrogen fixers with wide

environmental and geographic distribution. Applied

and Environmental Microbiology. American Society

Micorbiology.

Cheng, A.C. and Currie, B.J., 2005. Melioidosis:

epidemiology, pathophysiology, and management.

Clinical Microbiology Reviews. US National Library

of Medicine National Institutes of Health.

Currie, B.J., Ward, L. and Cheng, A.C., 2010. The

epidemiology and clinical spectrum of melioidosis:

540 cases from the 20 year Darwin prospective study.

PLoS Neglected Tropical Diseases. US National

Library of Medicine National Institutes of Health.

Wiersinga WJ, van der Poll T, White NJ, Day NP,

Peacock SJ. 2006. Melioidosis: insights into the

pathogenicity of B.pseudomallei. Nature Review

Microbiology. US National Library of

Medicine National Institutes of Health.

Howard, K and Inglis TJJ. 2003. Novel Selective Medium

for Isolation of Burkholderia pseudomallei. Journal of

Clinical Microbiology. American Society

Micorbiology.

Peacock, SJ., Chieng, G., Allen, C., Cheng., Dance, D.,

Amornchai, p.,1 Wongsuvan, G., Teerawattanasook,

N. Chierakul, W., Day, NP and Wuthiekanun, V.

2005. Comparison of Ashdown’s Medium,

Burkholderia cepacia Medium, and Burkholderia

pseudomallei Selective Agar for Clinical Isolation of

Burkholderia pseudomallei. Journal of Clinical

Microbiology. American Society Micorbiology.

Edler, C.,

Derschum, H., Kohler, M., Neubauer,

H., Frickmann, H and Hagen, RM. 2017. Comparison

of Mast Burkholderia Cepacia, Ashdown +

Gentamicin, and Burkholderia Pseudomallei Selective

Agar for the Selective Growth of Burkholderia Spp.

European Journal of Microbiology and Immunology.

US National Library of Medicine National Institutes of

Health.

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