Abstract

Burkholderia pseudomallei, a Gram-negative bacterium that causes melioidosis may be differentiated from closely related species of Burkholderia mallei that causes glanders and non-pathogenic species of Burkholderia thailandensis by multiplex PCR. The multiplex PCR consists of primers that flank a 10-bp repetitive element in B. pseudomallei and B. mallei amplifying PCR fragment of varying sizes between 400–700 bp, a unique sequence in B. thailandensis amplifying a PCR fragment of 308 bp and the metalloprotease gene amplifying a PCR fragment of 245 bp in B. pseudomallei and B. thailandensis. The multiplex PCR not only can differentiate the three Burkholderia species but can also be used for epidemiological typing of B. pseudomallei and B. mallei strains.

Introduction

Burkholderia pseudomallei, a Gram-negative bacterium, causes in humans and animals a glanders-like disease called melioidosis. The bacterium is a soil organism found mainly in Southeast Asia and Northern Australia. In Singapore, there were 59 cases of melioidosis reported in 2001 compared to 77 cases in 2000 [1]. Singapore is a predominantly urban environment where melioidosis occurs mainly among older adults with underlying illnesses such as diabetes mellitus. Even some healthy young servicemen of the Singapore Armed Forces (SAF) have been afflicted with melioidosis after acquiring the infection while training in the field [2].

B. pseudomallei can be isolated from soil, muddy water and rice paddy fields in the endemic regions. Melioidosis can be acquired through skin abrasions or wounds, or by inhalation and ingestion. Patients present with a variety of clinical manifestations ranging from acute septicemia with high mortality rate to chronic infections with localized lesions in multiple organs. The organism is highly resistant to a wide range of antibiotics.

Burkholderia mallei causes a disease called glanders primarily in horses, mules and donkeys. It can potentially cause serious disease in human as documented in laboratory cases [3]. Glanders were eradicated from domestic animals in most countries in the West but the disease can still be found occasionally in a few countries in Central and Southeast Asia, Middle East such as Turkey and parts of Africa [4,5]. DNA–DNA homology, cellular lipid and fatty acid composition revealed that B. pseudomallei and B. mallei are very closely related to one another [6]. The high homology between the two species in DNA sequences is shown in genes, such as flagellin and 16S ribosomal RNA, thus making it difficult to differentiate them by molecular identification systems [7,8]. Both B. pseudomallei and B. mallei have been classified by Centers for Disease Control and Prevention, USA as category B agents in the list of potential biological terrorism agents based on their potential for large scale dissemination with resultant illness [9].

Recently, two distinct biotypes of B. pseudomallei strains have been defined based on their ability to assimilate l-arabinose and their difference in pathogenicity [10–13]. Both biotypes have been found in soil of areas endemic for melioidosis in Thailand. The Ara+B. pseudomallei is much less virulent than AraB. pseudomallei, being found in areas with very few melioidosis cases [11,12]. Subsequently, a distinct new species, Burkholderia thailandensis, has been proposed for the Ara+B. pseudomallei strain [14]. Since two of the three Burkholderia species are capable of causing severe disease, it would be important to distinguish them from each other in clinical diagnosis and epidemiological surveillance.

The present study utilizes a repetitive DNA element, 5′-CGACGCAGGC-3′ found in B. pseudomallei and B. mallei isolates [15]. This can be used as a genetic marker to differentiate B. pseudomallei and B. mallei from B. thailandensis in epidemiological studies. Nucleotide sequence analysis revealed that the number of the DNA element, 5′-CGACGCAGGC-3′ contained in the individual repeats was highly variable among B. pseudomallei and B. mallei isolates from human, animal and soil. In contrast, this repetitive element was not found in the isolates of B. thailandensis although some flanking sequences of the repetitive element were conserved in all B. pseudomallei, B. mallei and B. thailandensis isolates. PCR primers based on the flanking sequences of the repetitive element were designed, and PCR amplified products were sequenced for all the isolates. Primers that were subsequently designed using the sequences obtained were used to differentiate B. pseudomallei and B. mallei from B. thailandensis isolates in a multiplex PCR. In addition, the multiplex PCR could also differentiate the B. mallei and B. pseudomallei isolates by the presence or absence of protease gene.

Materials and methods

Bacterial isolates and culture conditions

Sixty B. pseudomallei isolates from clinical, animals, environmental sources and ATCC 23343 and 15682 were used in the study (Table 1). Nine B. mallei and 16 B. thailandensis isolates were also used in the study. The B. thailandensis isolates were obtained from soil collected from different areas in Northeastern and Central Thailand [13]. Strains of other bacterial species used to determine the specificity of the multiplex PCR were B. cepacia ATCC 25416 and 17616, Pseudomonas aeruginosa ATCC 27853, B. gladioli NC012378, B. pickettii NC 011149, B. caryophylli ATCC 25418, Ralstonia pickettii ATCC 27511, R. solanacearum ATCC 33193, Staphlyococcus aureus ATCC 25923, Yersinia enterocolitica ATCC 9610, Erwinia amylovora ATCC 19382, Campylobacter jejuni ATCC 43911, Clostridium difficile ATCC 9689, Xanthomonas axonopodis ATCC 43911. Total chromosomal DNA was extracted and purified with a Qiagen Genomic DNA Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Alternatively, a colony boiling method was used in which a loopful of bacteria cultured on LB agar (DIFCO, Becton Dickinson and Company, Sparks, MD, USA) was suspended in 100 µl sterile distilled water, followed by heat inactivation at 99 °C for 15 min. One microliter of each bacterial strain was used for PCR amplification.

Table 1

Sources of B. pseudomallei, B. mallei and B. thailandensis isolates

Bacterial species Reference no. Source Country/Origin Total number 
B. pseudomallei 1, 2, 4, 5, 7–12, 14, 15, 18–24, 26, 28, 30, 31, 33–40, 59, Jumbari Human Singapore 60 
 9-D38465, 0-D10468, 9-A57203, 3-D85239, 358, 4-589580, 4-D82316 Human Malaysia 
 23343 Human ATCC 
 115 Chimpanzee Singapore 
 413 Cassowary 
 488 Gorilla 
 490 Bird 
 612 Crown pigeon 
 363, 679 Guinea pig Malaysia 
 216, 408 Monkey 
 3/96, 6/96, 21/96, 28/96, 476/96 Pig Malaysia 
 15682 Monkey ATCC 
 153 Mueller gibbon Singapore 
 77/96, 78/96, 79/96, 109/96, 15/10 Soil 
     
B. thailandensis 700388 Soil ATCC 16 
 TRF681, TRF682, TRF683, TRF686, TRF666, E229, E236, E246, E256, E201, E202, E257, E287, E305 Soil Thailand 
 S95019 Human  
     
B. mallei 10399 Horse ATCC 
 23344 Human 
 10229 – NCTC 
 10230 – 
 10247 – 
 10248 Human 
 10260 Human 
 120 – 
 3708 Mule 
Bacterial species Reference no. Source Country/Origin Total number 
B. pseudomallei 1, 2, 4, 5, 7–12, 14, 15, 18–24, 26, 28, 30, 31, 33–40, 59, Jumbari Human Singapore 60 
 9-D38465, 0-D10468, 9-A57203, 3-D85239, 358, 4-589580, 4-D82316 Human Malaysia 
 23343 Human ATCC 
 115 Chimpanzee Singapore 
 413 Cassowary 
 488 Gorilla 
 490 Bird 
 612 Crown pigeon 
 363, 679 Guinea pig Malaysia 
 216, 408 Monkey 
 3/96, 6/96, 21/96, 28/96, 476/96 Pig Malaysia 
 15682 Monkey ATCC 
 153 Mueller gibbon Singapore 
 77/96, 78/96, 79/96, 109/96, 15/10 Soil 
     
B. thailandensis 700388 Soil ATCC 16 
 TRF681, TRF682, TRF683, TRF686, TRF666, E229, E236, E246, E256, E201, E202, E257, E287, E305 Soil Thailand 
 S95019 Human  
     
B. mallei 10399 Horse ATCC 
 23344 Human 
 10229 – NCTC 
 10230 – 
 10247 – 
 10248 Human 
 10260 Human 
 120 – 
 3708 Mule 

ATCC, American Type Culture Collection, Rockville, MD, USA; TRF, Thailand Research Fund; NCTC, National Collection of Type Cultures, UK.

Design of primers for PCR amplification of B. pseudomallei, B. mallei and B. thailandensis

Primers SR1 and SR5, located 79-bp upstream and 134-bp downstream of a repetitive DNA element, were used to amplify the genomic DNA of B. pseudomallei and B. mallei[15]. As reported previously, the primers flanking the repetitive element amplified fragments of 400–700 bp in size depending on the number of 10-bp repetitive elements present in the isolates. These primers amplified only a single 402-bp fragment in all the B. thailandensis isolates as reported previously.

Primer SRT3, located at nucleotide positions 75–94 of the 361-bp fragment B. thailandensis (GenBank Accession No. AF325538), was designed to amplify a specific fragment of 308 bp with the primer SR5 in B. thailandensis that was absent in B. pseudomallei and B. mallei (Fig. 1).

Figure 1

Nucleotide sequences for 361-bp fragment used to design specific primer for B. thailandensis. The location of the primers SR1, SRT3 and SR5 are indicated in boxes.

Figure 1

Nucleotide sequences for 361-bp fragment used to design specific primer for B. thailandensis. The location of the primers SR1, SRT3 and SR5 are indicated in boxes.

The primers 14F5 and 14R5, located at nucleotide positions 1640–1663 and 1862–1884 of the serine metalloprotease gene MprA (GenBank Accession No. AF254803), were designed to amplify a 245-bp fragment in B. pseudomallei and B. thailandensis that was absent in B. mallei[16].

PCR amplification

PCR amplification was performed in a total volume of 25 µl containing 100 ng of bacterial genomic DNA, 200 µM of each dNTP, 0.2 µM of each primer (SR1: 5′ ACC GCG TAT GAA GGG ATG TC 3′, SR5: 5′ ACG CGC ACG CAC CTG CTG AAC 3′, 14F5: 5′ ACC TGC TGC CGG GCT ACG ACT TCA 3′, 14R5: 5′ CAC CTT GCC GAC CCA CGA GAT GC 3′ and SRT3: 5′ AAA GCT GCG CGC TCG GCA TC 3′), 1 unit of enzyme mix and 1×buffer from the GC-rich PCR Kit (Roche Diagnostics GmbH, Mannheim, Germany). The reaction mix was denatured initially at 95 °C for 3 min followed by 35 cycles of 95 °C for 30 s, 58 °C for 30 s and 72 °C for 1 min, and a final extension at 72 °C for 7 min. PCR products were analyzed on a 1.5% agarose gel.

Results

Multiplex PCR to differentiate B. pseudomallei, B. mallei and B. thailandensis isolates

The five primers, SR1, SR5, SRT3, 14F5 and 14R5 were multiplexed into a single PCR to detect and differentiate B. pseudomallei, B. mallei and B. thailandensis isolates. Fig. 2 shows a 245-bp fragment amplified from the metalloprotease gene, MprA, and a fragment of variable size ranging from 400–700 bp resulting from amplification of the 10-bp repetitive element in the B. pseudomallei isolates. The PCR amplification results for the 10-bp variable-number of tandem repeats for the B. pseudomallei isolates have previously been reported [15].

Figure 2

Multiplex PCR analysis of B. mallei, B. thailandensis and B. pseudomallei isolates. Lane 1–2 B. mallei ATCC 10399 and ATCC 23344; Lanes 3–9 B. thailandensis ATCC 700388, TRF681, TRF682, TRF683, TRF686, TRF666, S95019; Lanes 10–17 B. pseudomallei 2, 4, 10, 20, 22, 37, 40; 59; Lane 18 B. cepacia ATCC 25416; Lane 19 P. aeruginosa ATCC 27853; Lane 20 distilled water; Lane M 100bp Marker.

Figure 2

Multiplex PCR analysis of B. mallei, B. thailandensis and B. pseudomallei isolates. Lane 1–2 B. mallei ATCC 10399 and ATCC 23344; Lanes 3–9 B. thailandensis ATCC 700388, TRF681, TRF682, TRF683, TRF686, TRF666, S95019; Lanes 10–17 B. pseudomallei 2, 4, 10, 20, 22, 37, 40; 59; Lane 18 B. cepacia ATCC 25416; Lane 19 P. aeruginosa ATCC 27853; Lane 20 distilled water; Lane M 100bp Marker.

Results from PCR amplification of the nine B. mallei isolates showed that only one fragment of variable size was amplified from the 10-bp repetitive element. The amplified 10-bp repetitive elements were sequenced for all the B. mallei isolates. There were 5–34 copies of the 10-bp repeats in the B. mallei isolates (Table 2 and Fig. 3).

Table 2

Categories of variable-number tandem repeats in B. mallei isolates

Reference no. Repeat type 
ATCC 10399 24X 
ATCC 23344 27X 
NCTC 10229 34X 
NCTC 10230 34X 
NCTC 10247 19X 
NCTC 10248 22X 
NCTC 10260 25X 
NCTC 120 5X 
NCTC 3708 8X 
Reference no. Repeat type 
ATCC 10399 24X 
ATCC 23344 27X 
NCTC 10229 34X 
NCTC 10230 34X 
NCTC 10247 19X 
NCTC 10248 22X 
NCTC 10260 25X 
NCTC 120 5X 
NCTC 3708 8X 

Repeat types are designated according to the copy number of 5′-CGACGCAGGC-3′ in the tandem repeats; X represents the copy number.

Figure 3

Multiplex PCR analysis of B. mallei isolates prepared by colony boiling method. Lane 1–9 B. mallei ATCC 10399, ATCC 23344, NCTC120, NCTC10229, NCTC10230, NCTC10247, NCTC10248, NCTC10260 and NCTC3708; Lane 10 B. mallei NCTC10229 genomic DNA; Lane 11 distilled water; Lane M 100bp Marker.

Figure 3

Multiplex PCR analysis of B. mallei isolates prepared by colony boiling method. Lane 1–9 B. mallei ATCC 10399, ATCC 23344, NCTC120, NCTC10229, NCTC10230, NCTC10247, NCTC10248, NCTC10260 and NCTC3708; Lane 10 B. mallei NCTC10229 genomic DNA; Lane 11 distilled water; Lane M 100bp Marker.

In B. thailandensis isolates, a 245-bp fragment was amplified from a sequence similar to the B. pseudomallei metalloprotease gene. In addition, two other fragments of 402 and 308 bp were obtained (Fig. 2).

Sixty B. pseudomallei isolates from clinical, animal and environmental samples, nine B. mallei isolates and 16 B. thailandensis isolates were amplified using the multiplex PCR. The sources of the isolates are shown in Table 1. PCR amplification results with the expected fragments were obtained from these isolates. Results from the PCR amplification of some of the isolates are shown in Fig. 2.

Specificity and sensitivity

The multiplex PCR assay was specific for B. pseudomallei, B. mallei and B. thailandensis. The assay failed to amplify any PCR fragments in a wide range of other bacteria such as B. cepacia, P. aeruginosa, B. gladioli, B. pickettii, B. caryophylli, R. pickettii, R. solanacearum, S. aureus, Y. enterocolitica, E. amylovora, C. jejuni, C. difficile and X. axonopodis.

The sensitivity of the assay was determined by serial dilution of genomic DNA isolated from B. pseudomallei, B. mallei and B. thailandensis. The limit of detection of the assay was 10 ng for B. pseudomallei, 100 pg for B. mallei and 10 ng for B. thailandensis. Each of the primers pair used in the assay had slightly different optimal annealing temperatures for the PCR amplification and this could have resulted in differences in sensitivity of the primer pairs seen when they are multiplexed in a single assay. Though the assay is not as sensitive as other PCR assays that can detect a few genome-equivalents of the bacteria (about 10 fg) [17,18], the assay is still a useful and rapid method to discriminate the three species when cultures are obtained from clinical and environmental samples. The colonies grown in overnight culture can be used directly for PCR amplification after heat inactivation, thus avoiding the need to extract the genomic DNA.

Discussion

The need to differentiate between B. mallei and B. pseudomallei is increasingly vital since both bacteria have been considered as potential bioterrorism threat agents. In countries where B. pseudomallei is endemic, it would be useful to differentiate the two Burkholderia species in order to ascertain whether infection is acquired naturally or otherwise. It is also important to be able to differentiate the avirulent B. thailandensis isolates from the virulent B. pseudomallei and B. mallei isolates in control measures for the prevention of infection from exposure to the bacteria.

With completion of the whole genome sequence of B. pseudomallei and B. mallei by Sanger Institute (http://www.sanger.ac.uk/) and The Institute of Genome Research (http://www.tigr.org/), the differences found between the genomes could be tapped to design better diagnostics to differentiate between the two species. In the meantime, the multiplex PCR developed in this study would be useful to differentiate the three Burkholderia species as well as to strain type the B. mallei and B. pseudomallei isolates. The 10-bp repetitive element is highly polymorphic in B. pseudomallei and B. mallei isolates and its inclusion into the multiplex PCR will not only detect the B. pseudomallei and B. mallei isolates but also allow strain typing to be done in a single multiplex reaction.

Acknowledgments

This work was supported by a grant from the Singapore Armed Forces Medical Corps, Singapore. We thank Drs. Sirirurg Songsivilai, Tararaj Dharakul and Visanu Thamlikitkul from the Department of Immunology and the Department of Internal Medicine, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand and Vanaporn Wuthiekanun from Wellcome Unit, Faculty of Tropical Medicine, Bangkok, Thailand for the providing the B. thailandensis isolates, and Dr. S.D. Puthucheary from Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur for providing B. pseudomallei isolates from Malaysia.

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