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

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.

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 and 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.