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. Electronic supplementary material The online version of this article (doi:10.1186/s13028-015-0104-4) contains supplementary material, which is available to authorized users.

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. 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   [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.

Additional file
Additional file 1: Comparison of the motB PCR assay to the conventional fliP and real time fliC PCR assays in clinical samples (Burkholderia type strains ATCC 23343 T, ATCC 23344T).