Open Access

Clear distinction between Burkholderia mallei and Burkholderia pseudomallei using fluorescent motB primers

Acta Veterinaria Scandinavica201557:13

https://doi.org/10.1186/s13028-015-0104-4

Received: 22 October 2014

Accepted: 24 February 2015

Published: 7 March 2015

Abstract

Background

A frame-shift mutation in the flagellum motor gene motB coding for the chemotaxis MotB protein of Burkholderia mallei has been utilized to design a conventional duplex PCR assay with fluorescent labelled primers.

Findings

Species specificity was tested with a panel of 13 Burkholderia type strains. A total of 41 B. mallei field strains, 36 B. pseudomallei field strains, and 1 B. thailandensis field strain from different geographic regions were tested and correctly identified. Testing of 55 non-Burkholderia bacterial species revealed 100% specificity of the assay. The minimum detection limit was 1 pg DNA or 160 GE for B. mallei and 130 GE for B. pseudomallei, respectively.

Conclusions

This assay enables the clear distinction between B. mallei and B. pseudomallei/B. thailandensis.

Keywords

Duplex PCR Fluorescent primers Burkholderia

Findings

Despite Burkholderia mallei, B. pseudomallei and B. thailandensis being genetically closely related Gram negative bacteria, they display significant differences in pathogenicity and habitat. B. mallei, a facultative intracellular, non-motile, equine pathogen, is the causative agent of glanders, a highly contagious and frequently fatal zoonotic disease of the upper respiratory tract and lungs [1]. The disease has a 95% case fatality rate in untreated humans with septicaemia and a 50% case fatality rate in antibiotic treated individuals [1].

B. pseudomallei, a facultative intracellular, motile bacterium found in contaminated water and soil, is the etiological agent of melioidosis, an infectious disease in man and animal in the tropics [2]. The clinical picture in animals and humans resembles that of glanders in horses. Human infection usually develops after inhalation, ingestion, or cutaneous uptake of the pathogen [2,3]. Melioidosis has a case fatality rate of 39.5%, and untreated septicaemia is fatal in up to 80% of cases [4]. Both B. mallei and B. pseudomallei are considered potential bioweapons and are listed as category B biothreat agents by the U.S. Centers for Disease Control and Prevention [5]. B. thailandensis is generally considered a weakly pathogenic, motile soil bacterium, rarely causing disease in man or animal [6]. Glanders and melioidosis may cause diagnostic problems in endemic regions because of their clinical, morphologic and genetic similarity, and even more so in non-endemic countries, due to the lack of awareness of these diseases. In order to initiate appropriate patient treatment, rapid species identification is necessary, especially in view of the intrinsic resistance of both agents to many commonly used antibiotics and their differing susceptibilities [7,8].

Based on the results from a previous study [9], a frame-shift mutation in the flagellum motor gene motB coding for the chemotaxis MotB protein [GenBank:BMA2861] of B. mallei (ATCC 23344) was utilized to design a simple conventional duplex PCR assay with fluorescent labelled primers enabling the distinction between B. mallei and B. pseudomallei/B. thailandensis. Bacterial strains were obtained from the strain collection of the National and OIE Reference Laboratory for Glanders at the Friedrich-Loeffler-Institute in Jena, Germany (Tables 1 and 2). All Burkholderia strains were cultured at 37°C on calf blood agar containing 3% (v/v) glycerol. All other bacteria were grown on standard media and appropriate atmospheric conditions.
Table 1

Panel of Burkholderia mallei and B. pseudomallei field strains used for validation

Origin

B. mallei

B. pseudomallei

Africa

-

2

Arabian Peninsula

3

-

Asia

-

1

East Asia

1

4

South Asia

17

5

Southeast Asia

-

13

Europe

3

4

Indonesia

1

-

South America

5

2

Transcontinental Europe/Asia

2

-

Unknown

9

5

Total

41

36

Table 2

Panel of non-Burkholderia strains used for specificity testing

Species

Strain

Species

Strain

Actinobacillus pleuropneumoniae

ATCC 27088

Legionella pneumophila sub. pneumophila

DSM 7513

Bacillus atrophaeus

ATCC 9372

Mannheimia haemolytica

ATCC 33396

Bacillus brevis

ATCC 8246

Ochrobactrum anthropi

CCUG 1047

Bacillus cereus

ATCC 10876

Oligella urethralis

DSM 7531

Bacillus megaterium

DSM 90

Pasteurella multo ssp.multo

ATCC 43137

Bacillus mycoides

ATCC 6462

Pasteurella multocida

DSM 5281

Bacillus subtilis

ATCC 6633

Proteus mirabilis

DSM 4479

Bacillus thuringiensis

ATCC 10792

Pseudomonas aeruginosa

ATCC 9027

Bartonella henselae

DSM 28221

Pseudomonas alcaligenes

ATCC 14909

Bartonella quintana

DSM 21441

Pseudomonas fluorescens

ATCC 13525

Bordetella bronchiseptica

ATCC 19395

Pseudomonas polymyxa

ATCC 842

Brucella abortus

ATCC 23448

Pseudomonas putida

ATCC 12633

Brucella melitensis

ATCC 23456

Rhodococcus equi

DSM 20307

Brucella suis

ATCC 23444

Salmonella enteritidis

147 (95)

Campylobacter coli

DSM 4689

Salmonella typhumirium

9098 (221)

Campylobacter jejuni subsp. jejuni

DSM 4688

Staphylococcus aureus subsp. aureus

DSM 6732

Chlamydia abortus

07 DC0059

Stenotrophomonas maltophilia

ATCC 13637

Chlamydia pecorum

06 DC0055

Streptococcus agalactiae

DSM 6784

Chlamydia psittaci

C1/97

Streptococcus equi subsp. equi

ATCC 9528

Clostridium baratii

ATCC 25782

Streptococcus equi subsp. zooepidemicus

ATCC 700400

Clostridium botulinum A

NCTC 7272

Streptococcus equinus

DSM 20558

Clostridium botulinum B

NCTC 7273

Streptococcus parauberis

DSM 6631

Escherichia coli

DSM 30083

Taylorella equigenitalis

DSM 10668

Francisella tularensis sub. holarctica

LVS

Yersinia enterocolitica subsp. enterocolitica

ATCC 9610

Francisella tularensis sub. tularensis

FSC 237 (SchuS4)

Yersinia enterocolitica subsp. enterocolitica

DSM 9499

Haemophilus influenzae

ATCC 9006

Yersinia enterocolitica subsp. palearctica

DSM 13030

Klebsiella pneumoniae subsp. pneumoniae

DSM 30104

Yersinia pseudotuberculosis

IP32953

Lactobacillus ruminis

DSM 20403

  

Genomic DNA was prepared from culture material using the High Pure PCR Template Preparation Kit according to the manufacturer’s instructions (Roche, Mannheim, Germany). All DNA samples were quantified using a NanoDrop 1000 spectrophotometer (Fisher Scientific, Schwerte, Germany). The duplex polymerase chain reaction (PCR) was designed using the forward primer MBF04 (5′- CGTCAAGCGGGTGAACCA -3′), the 6-FAM labelled reverse primer MBR04-FAM (5′-6-FAM-GTCGTCCTCGCTCTTTCGC -3′), and the ATTO565 labelled reverse primer MBR10-ATTO565 (5′-ATTO565-GTCCTCGCTCTTCTTCGCG-3′). Primers were designed with the Genious software package (Ver. 6.1), to generate a specific 6-FAM labelled 326 bp DNA fragment for B. mallei and an ATTO565 labelled 325 bp DNA fragment for B. pseudomallei/B. thailandensis, respectively. Labelled primers were obtained from Microsynth (Balgach, Switzerland), the unlabelled primer from Jena Bioscience (Jena, Germany). PCR was conducted in a 20 μL reaction containing 0.3 μM of the primers (MBF04, MBR04-FAM, and MBR10-ATTO565), 1 × 5-Prime HotMasterMix (VWR, Darmstadt, Germany), 2.5% DMSO and 10 ng template (total DNA). The PCR was performed in a Mastercycler pro S™ (Eppendorf, Germany) under the following conditions: initial denaturation at 95°C for 1 min; 40 cycles at 95°C for 10 s, 63°C for 15 s, 70°C for 30 s, and the final extension at 70°C for 5 min. 13.3 μL PCR reaction mixed with 2.7 μL 6 × Loading Dye (Fermentas, Schwerte, Germany) were analysed by electrophoresis on a 1.25% agarose gel (wt/vol) at 9 V/cm for 40 min. Images were captured after an exposure period of 30 s for each LED/filter set using the G-Box EF2 Gel Documentation System (Syngene Europe, Cambridge, UK): Blue-LED/Filt525 and Green-LED/Filt605 for the visualisation of 6-FAM and ATTO565 labelled PCR products, respectively. For optional ethidium bromide imaging (302 nm UV illuminator/FiltUV), the gel was stained after capturing the 6-FAM/ATTO565 images. Fragment sizes (326/327 bp) and correct labelling (6-FAM/ATTO565) of the amplicons were confirmed by means of capillary electrophoresis using a Genetic Analyzer 3130 with a G5 filter set (Applied Biosystems/Hitachi, Darmstadt, Germany). Species specificity was tested with a panel of 13 Burkholderia type strains. Additionally, a total of 41 B. mallei field strains from equines, 36 B. pseudomallei field strains from human and environmental origin, and one B. thailandensis field strain, all from different geographic regions were tested and correctly identified (Table 1). Testing of 55 non-Burkholderia bacterial species revealed 100% specificity of the assay (Table 2). The minimum detection limit was 1 pg DNA or 160 genome equivalents (GE) for B. mallei and 130 GE for B. pseudomallei, respectively. In order to compare the sensitivity of our assay with other assays used by the National and OIE Reference Laboratory for Glanders, several clinical B. mallei samples were tested by a conventional fliP PCR [10] and a real time PCR assay targeting fliC [11]. Despite the lower sensitivity we determined for our assay, it revealed comparable sensitivity to the conventional fliP PCR and a higher sensitivity than the real time fliC assay in the tested clinical samples (Additional file 1).

Fluorescent primers are widely used in real time PCR technology and several highly sophisticated and elegant PCR assays have been developed for the identification and differentiation of B. mallei and B. pseudomallei and other Burkholderia species in the past few years [12]. This study describes the design of a simple conventional duplex PCR with fluorescent labelled primers for amplifying species-specific amplicons of B. mallei and B. pseudomallei/B. thailandensis, respectively. These closely related species can cause considerable problems during the identification process in the laboratory as colony characteristics and routine biochemical tests are not sufficiently discriminative for species identification. The benefit of this assay is not only the unambiguous identification of B. mallei and the closely related species B. pseudomallei and B. thailandensis by fluorescence image capturing but also the possibility of detecting the B. mallei/pseudomallei/thailandensis complex on a standard ethidium bromide stained agarose gel.

Abbreviations

ATCC: 

American type culture collection

CCUG: 

Culture collection university of Göteborg

DSM: 

Deutsche Sammlung von Mikroorganismen

FAM: 

Fluorescein

FSC: 

Francisella strain collection, Sweden

GE: 

Genome equivalent

LED: 

Light-emitting diode

NCTC: 

National collection of type cultures

Declarations

Acknowledgements

Katja Fischer, Nadin Lemser and Peggy Marten are thanked for their excellent technical assistance. We appreciate the help of PD Dr. H. Scholz, Munich, Germany, of Prof. A. Pereira Lage, Minas Gerais, Brazil, of Dr. M. Saqib, Faisalabad, Pakistan, of PD Dr. R. Grunow, Berlin, Germany, Dr. U. Wernery, Dubai, UAE, and Dr. F. Al-Salloom, Kingdom of Bahrain, for providing sample material, strains and DNA preparations. This work was partially funded by the Federal Ministry for Education and research (BMBF #01KI1001A) and the EU (EAHC Grant Agreement No 2010 2102).

Authors’ Affiliations

(1)
Friedrich-Loeffler-Institut, Bundesforschungsinstitut für Tiergesundheit
(2)
National and OIE Reference Laboratory for Glanders, Friedrich-Loeffler-Institut, Bundesforschungsinstitut für Tiergesundheit

References

  1. Dvorak GD, Spickler AR. Glanders. J Am Vet Med Assoc. 2008;233:570–7.View ArticlePubMedGoogle Scholar
  2. Cheng AC, Dance DA, Currie BJ. Bioterrorism Glanders and Melioidosis. Euro Surveill. 2005;10:3.Google Scholar
  3. Wiersinga WJ, Currie BJ, Peacock SJ. Melioidosis. N Engl J Med. 2012;367:1035–44.View ArticlePubMedGoogle Scholar
  4. Zysk G, Splettstösser WD, Neubauer H. A review on melioidosis with special respect on molecular and immunological diagnostic techniques. Clin Lab. 2000;46:119–30.PubMedGoogle Scholar
  5. Rotz LD, Khan AS, Lillibridge SR, Ostroff SM, Hughes JM. Public health assessment of potential biological terrorism agents. Emerg Infect Dis. 2002;8:225–30.View ArticlePubMed CentralPubMedGoogle Scholar
  6. Brett PJ, DeShazer D, Woods DE. Burkholderia thailandensis sp. nov., a Burkholderia pseudomallei-like species. Int J Syst Bacteriol. 1998;48:317–20.View ArticlePubMedGoogle Scholar
  7. Thibault FM, Hernandez E, Vidal D, Girardet M, Cavallo JD. Antibiotic susceptibility of 65 isolates of Burkholderia pseudomallei and Burkholderia mallei to 35 antimicrobial agents. J Antimicrob Chemother. 2004;54:8.View ArticleGoogle Scholar
  8. Gilad J. Burkholderia mallei and Burkholderia pseudomallei: the causative micro-organisms of glanders and melioidosis. Recent Pat Antiinfect Drug Discov. 2007;2:233–41.View ArticlePubMedGoogle Scholar
  9. Schmoock G, Ehricht R, Melzer F, Rassbach A, Scholz HC, Neubauer H, et al. DNA microarray-based detection and identification of Burkholderia mallei, Burkholderia pseudomallei and Burkholderia spp. Mol Cell Probes. 2009;23:178–87.View ArticlePubMedGoogle Scholar
  10. Scholz HC, Joseph M, Tomaso H, Al Dahouk S, Witte A, Kinne J, et al. Detection of the reemerging agent Burkholderia mallei in a recent outbreak of glanders in the United Arab Emirates by a newly developed fliP-based polymerase chain reaction assay. Diagn Microbiol Infect Dis. 2006;54:241–7.View ArticlePubMedGoogle Scholar
  11. Tomaso H, Scholz HC, Al Dahouk S, Pitt TL, Treu TM, Neubauer H. Development of 5′ nuclease real-time PCR assays for the rapid identification of the Burkholderia mallei//Burkholderia pseudomallei complex. Diagn Mol Pathol. 2004;13:247–53.View ArticlePubMedGoogle Scholar
  12. Lowe W, March JK, Bunnell AJ, O’Neill KL, Robison RA. PCR-based methodologies used to detect and differentiate the Burkholderia pseudomallei complex: B. pseudomallei, B. mallei, and B. thailandensis. Curr Issues Mol Biol. 2013;16:23–54.PubMedGoogle Scholar

Copyright

© Schmoock et al.; licensee BioMed Central. 2015

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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