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. 2012 Jun 13;2(2):148–156. doi: 10.1556/EuJMI.2.2012.2.8

Discrimination of Burkholderia mallei/pseudomallei from Burkholderia thailandensis by sequence comparison of a fragment of the ribosomal protein S21 (rpsU) gene

H Frickmann 1,2,1,2,*, N Chantratita 3,3, Y P Gauthier 4,4, H Neubauer 5,5, R M Hagen 6,1
PMCID: PMC3515745  EMSID: EMS50548  PMID: 23227305

Abstract

Discrimination of Burkholderia (B.) pseudomallei and B. mallei from environmental B. thailandensis is challenging. We describe a discrimination method based on sequence comparison of the ribosomal protein S21 (rpsU) gene.

The rpsU gene was sequenced in ten B. pseudomallei, six B. mallei, one B. thailandensis reference strains, six isolates of B. pseudomallei, and 37 of B. thailandensis. Further rpsU sequences of six B. pseudomallei, three B. mallei, and one B. thailandensis were identified via NCBI GenBank. Three to four variable base-positions were identified within a 120-base-pair fragment, allowing discrimination of the B. pseudomallei/mallei-cluster from B. thailandensis, whose sequences clustered identically. All B. mallei and three B. pseudomallei sequences were identical, while 17/22 B. pseudomallei strains differed in one nucleotide (78A>C). Sequences of the rpsU fragment of ‘out-stander’ reference strains of B. cepacia, B. gladioli, B. plantarii, and B. vietnamensis clustered differently.

Sequence comparison of the described rpsU gene fragment can be used as a supplementary diagnostic procedure for the discrimination of B. mallei/pseudomallei from B. thailandensis as well as from other species of the genus Burkholderia, keeping in mind that it does not allow for a differentiation between B. mallei and B. pseudomallei.

Keywords: Burkholderia mallei, Burkholderia pseudomallei, Burkholderia thailandensis, glanders, melioidosis, rpsU

Introduction

Melioidosis is a rare but hazardous infectious disease that is caused by Burkholderia (B.) pseudomallei, an opportunistic saprophyte that is endemic in tropical and subtropical regions ranging from latitudes 20° north to 20° south, particularly in South-East Asia [1–5]. The disease manifests as respiratory tract infection, acute septicemia, and abscess formation in multiple organs [3, 6]. B. mallei is an obligate parasite of solipeds causing the rare but often fatal zoonosis glanders [7, 8]. B. mallei and B. pseudomallei were initially considered to be two distinct species. However, DNA–DNA-hybridization studies suggested that they are merely pathovars of one single species [9, 10]. Sequence analysis of the gene of the filament-forming flagellin, fliC, corroborated this theory [11].

Bacteria of both species share many phenotypic characteristics. The syndromes associated with both diseases are variable but indistinguishable by clinical and histopathological examinations [12–14].

Both B. mallei and B. pseudomallei have been classified as potential category B biological threat agents by the Centers for Disease Control [12]. Thereby, the horse-adapted parasite B. mallei has an even higher impact on public health than B. pseudomallei [15].

At the end of the last century, the new species B. thailandensis was assigned from B. pseudomallei-like arabinose-fermenting soil isolates [16–18]. This new member of the ‘pseudomallei’-group displays low virulence in laboratory animals and is considered to be a facultative pathogen with clinical relevance only in seriously compromised patients [18–21]. The ‘pseudomallei’-group is an interesting model for studying the evolution from saprophyte to parasite or from an environmental to a highly pathogenic agent.

A reliable discrimination of the biosafety level 3 (BSL3) agents B. mallei and B. pseudomallei from the nearly non-pathogenic B. thailandensis is of great importance for decisions regarding both treatment and implementation of safety measures in clinic and laboratory [4]. Such laboratory safety measures include isolation on selective media such as Pseudomonas cepacia (PC) agar or Ashdown’s agar, investigation of motility, and biochemical testing under BSL3 conditions [4, 22], making these procedures even more time-consuming.

Discrimination of B. mallei/pseudomallei from B. thailandensis by phenotypic and molecular methods is challenging due to close homology. Distinguishing of the bacteria by microscopy or culture-morphology [4, 8, 12] is not possible. Identifications by biochemical identification systems such as API 20NE (bioMérieux, Nürtingen, Germany), RapID™ NF Plus (Thermo Fisher Scientific, Lenexa, KS), automated systems such as Phoenix (BD, Heidelberg, Germany), VITEK (bioMérieux, Nürtingen, Germany), or latex agglutination are not reliable [4, 23–26]. A MALDI-TOF mass spectrometry technology based discrimination is possible, if a specialized bio-terrorism database [27] is purchased.

Due to the lacking reliability of phenotypic methods for the discrimination within the Burkholderia genus, various molecular methods have been developed, including conventional, real-time, and multiplex polymerase chain reactions (PCRs), loop-mediated isothermal amplification (LAMP) procedures, sequence-based approaches, DNA micro-arrays, and fluorescence in situ hybridization [28–35].

However, these more or less complex procedures are rarely established in routine laboratories, while classical 16S rRNA gene sequencing is nowadays broadly performed. Nevertheless, the mere comparison of the 16S rRNA gene sequence may be non-reliable, because the 1488-bp sequences of the 16S rRNA genes of B. mallei and B. pseudomallei differ at only eight single nucleotide positions, while the similarity to the 16S rRNA gene sequence of B. thailandensis is more than 99% [29, 36]. Therefore, confirmation of 16S rRNA gene sequencing results by alternative easy-to-perform sequencing procedures is desirable.

DeShazer et al. [37] identified an open reading frame (ORF) by upstream sequencing of the fliC gene of the B. pseudomallei strain 1016b. The predicted protein product of the ‘rpsU gene’ with a length of 70 amino acids showed a high similarity to the ribosomal subunit protein S21 of E. coli and several other bacterial species. This small protein has only been found in eubacteria to-date and belongs to the macromolecular synthesis (MMS) operon which is responsible for the initiation of protein, DNA, and RNA synthesis. In a previous study, our group identified species-specific variations of the rpsU gene sequence in a small number of Burkholderia reference strains [28].

In this report, we describe the sequencing of the rpsU gene in reference strains as well as in clinical and environmental isolates of B. mallei, B. pseudomallei, and B. thailandensis. Based on the rpsU sequences, we propose a simple polymerase chain reaction (PCR) strategy with subsequent sequencing for the discrimination of the BSL3 agents B. mallei/pseudomallei from other species of the genus Burkholderia.

Materials and methods

Bacterial strains

In total, 6 B. mallei strains, 16 B. pseudomallei strains, and 38 B. thailandensis strains as well as single reference strains of the ‘out-standers’ B. cepacia, B. gladioli, B. vietnamensis, and B. plantarii were grown for consecutive sequencing of the rpsU gene. Antigen preparation of all strains comprised growth on nutrient agars (Columbia agar enriched with 5% sheep blood, BD, Heidelberg, Germany), swabbing from the plates and dilution in sterile 0.9% NaCl solution, adjusting to 108–109 bacteria per milliliter and heat-inactivation as previously described [28]. Bacterial cultures of B. mallei and B. pseudomallei were performed in BSL3 laboratories.

The sequenced B. mallei strains included the reference strains ATCC (American Type Culture Collection) 15310, ATCC 23344, NCTC (National Collection of Type Cultures) 10248, Mukteswar, Zagreb, and Bogor. Heat-inactivated preparations of the ATCC strains were provided by Dr. Niederwöhrmeier, WIS (German Armed Forces Research Institute for Protective Technologies and NBC Protection, ‘Wehrwissenschaftliches Institut für Schutztechnologien’), Munster, Germany. The strains Bogor, Mukteswar, and Zagreb were isolated from horses and are still used to prepare mallein for serological testing in horses. Heat inactivated antigen was purchased from ID-DLO, Lelystad, The Netherlands. These strains have to be considered as ‘museum’ strains because they are more than 30 years old.

Sequence analyzed B. pseudomallei strains included ATCC 15682, ATCC 23343, 6068VIR, NCTC 4845, HumA, 551A, 603A, Mal 6, E 38, 844 (‘museum strains’), UE 3 (isolated from soil in North-East Thailand), UE 4, UE 9, UE 10, UE 16, and UE 19 (isolated from clinical samples in North-East Thailand).

Sequenced B. thailandensis strains included the type strain ATCC 700388 (DSM (Germany strain collection, ‘Deutsche Stammsammlung für Mikroorganismen’) 13276) as well as isolates from soil in North-East Thailand (UE 2, UE 5, UE 8, UE 11, UE 17, UE 20, UE 23, UE 26, UE 29), in Central Thailand (E 27, E 32, E 111, E 202, E 216, E 217, E 221, E 229, E 236, E 287, E 290, E 296, E 305, E 306), in Vietnam (S1–S7), and in Laos (L 1, L 3, L 6, L 7, L 13, L 15, L 16).

Biochemical identification of all clinical and environmental isolates from South-East Asia was performed under BSL3 conditions by API 20E (bioMérieux, Nürtingen, Germany) with arabinose (Ara) fermentation as the key reaction for the discrimination of B. pseudomallei (Ara(−)) from B. thailandensis (Ara(+)) [16–18]. Differentiation beyond the species level, e.g. by molecular typing, was not performed.

Sample preparation from heat-inactivated bacterial suspensions for PCR

Crude bacterial DNA preparations were obtained according to the following protocol: 10-µl aliquots of the heat-inactivated bacterial suspensions were re-suspended in 200 μl of lysis buffer containing 0.5% Tween 20™ (ICI, American Limited, Merck, Hohenbrunn, Germany), 2 mg ml/l proteinase K (Roche, Mannheim, Germany), 3.5 mM MgCl2 (Sigma-Aldrich, Steinheim, Germany), 15 mM ammonium sulfate (Sigma-Aldrich, Steinheim, Germany), and 60 mM Tris–HCl (pH 8.5) (Sigma-Aldrich, Steinheim, Germany). The suspension was incubated at 56 °C for 2 h yielding a clear lysate that was boiled for additional 10 min.

The PCR mixture contained 0.5 U Taq-polymerase (Perkin Elmer, Waltham, Massachusetts, USA), 1 µM of primers fup-1 (5´-GTG-GAG-CTT-CTT-CGG-CAG-CAT-3´) and fup-2 (5´-ATG-ACG-ACG-ATT-CTT-TTG-AA-3´), 1 mM dNTP, 2.5 mM MgCl2, 7.5 mM ammonium sulfate, and 30 mM Tris–HCl (pH 8.5). The PCR procedure consisted of an initial denaturation step for 10 min at 94 °C that was followed by 35 cycles of denaturation at 94 °C for 60 s, annealing at 59 °C for 60 s and elongation at 72 °C for 60 s. An additional elongation step for 10 min at 72 °C was added. Amplicon identification was performed by agarose gel electrophoresis and ethidium bromide staining.

Specificity testing of the PCR with a panel of bacteria which might produce pyogenic or granulomatous lesions, closely related species, possible sample contaminants, or other bacteria was previously described in detail [28].

Sequencing of the rpsU gene and multiple sequence alignment

The rpsU gene sequences of the following strains were obtained from NCBI GenBank (accession number in brackets) (http://www.ncbi.nlm.nih.gov/genbank/): B. pseudomallei 1026b (U73848), MSHR 346 (CP001408), 1106a (CP000572), 1710b (CP000124), K96243 (BX571965), 668 (CP000570), B. mallei NCTC 10247 (CP000548), NCTC 10229 (CP000546), SAVP1 (CP000526), B. thailandensis E264 (CP000086) or from previous studies of the authors [11, 28]: B. pseudomallei ATCC 15682 (AF084812), ATCC 23343 (AF084813), and 6068VIR (AF447447), B. mallei ATCC 15310 (AF084814), and ATCC 23344 (AF08A815), B. thailandensis ATCC 700388 (AF447448), Burkholderia cepacia ATCC 25416 (AF447444), Burkholderia gladioli ATCC10248 (AF447445), Burkholderia vietnamiensis LMG 10929 (AF447450), Burkholderia plantarii LMG 9035 (AF447446).

All amplicons were sequenced on an ABI 377 Prism™ Dye Sequencing Apparatus using the ABI Prism Dye Terminator Cycle Sequencing Ready Reaction Kit™ according to the manufacturer’s instructions (Perkin Elmer, Weiterstadt, Germany). Multiple sequence alignment and neighbor-joining analysis [38] were performed using the open source software ClustalW2 version 2.1 and ClustalX2 version 2.1 (http://www.clustal.org) [39]. The phylogenetic tree based on the ClustalX2 analysis was printed using the open source NJplot software (http://pbil.univ-lyon1.fr/software/njplot.html).

Sequences of newly sequenced isolates were clustered with previously known sequences from the NCBI GenBank database. There was no redundant submission of previously deposited species-specific sequences to NCBI GenBank.

Results

Positive PCR results with a predicted amplicon size of 210 bp were obtained from all reference strains and isolates of the ‘pseudomallei’-group and the ‘out-standers’ B. cepacia, B. gladioli, B. plantarii, and B. vietnamensis. The complete rpsU genes were sequenced resulting in 210 readable nucleotides including a variable 120-base-pair fragment ranging from base 50 to base 169 (Table 1). All detected sequences were 100% identical with previously published sequences in NCBI GenBank.

Table 1.

Alignment of the sequences of the variable 120-base-pair fragment of the rpsU gene of B. mallei, B. pseudomallei, B. thailandensis, and other Burkholderia spp.

graphic file with name EuJMI-02-148-g02.jpg
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The DNA sequences of all B. mallei and three B. pseudomallei reference strains (ATCC 15682, 551A, and NCTC 4845) were identical. In contrast, 13 B. pseudomallei reference strains (603A, 668, 844, 1026b, 1106a, 1710b, 6068VIR, ATCC 23343, E 38, HumA, K96243, Mal 6, and MSHR346) and the clinical B. pseudomallei isolates UE 4, UE 9, UE 10, UE 16, and UE 19 from North-East Thailand differed in one base pair at position 78 of the open reading frame (78A>C). Two single shifts in the published sequences of B. pseudomallei 1026b at position 63 (63C>T) and of B. pseudomallei 668 at position 144 (144C>T) could not be observed in any other B. pseudomallei strain. There was an obvious sequence heterology in the variable 120-base-pair fragment of the rpsU sequence when compared to the corresponding sequence of B. thailandensis (Table 1) with three to four nucleotide substitutions, respectively. All 37 environmental B. thailandensis isolates from different regions of South-East Asia (North-East Thailand, Central Thailand, Vietnam and Laos) showed an identical rpsU gene sequence. The ‘out-standers’ from the genus Burkholderia were associated with different and species-specific sequences.

A phylogenetic tree based on the variable 120-base-pair fragment of the rpsU gene sequences shows the percent nucleotide divergence and the similarity of this section of the rpsU gene of members of the ‘pseudomallei’-group in comparison with other species of the Burkholderia genus (Fig. 1).

Fig. 1.

Fig. 1.

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Phylogenetic tree based on the variable 120-base-pair fragment of the rpsU gene sequences constructed by the neighbor-joining method, using the open source software ClustalX2 (version 2.1) and NJPlot. The scale indicates the percentage of base difference (percent divergence)

Despite the different DNA sequences, the predicted amino acid sequence obtained by transcription of the reverse complement of the whole rpsU gene is identical for all investigated Burkholderia species (MTT-ILL-KEN-EPF-EVA-IRR-FRR-AIE-KNG-LIA-ELR-ERQ-AYE-KPT-AVR-KRK-KAA-AVK-RLH-KRL-RSQ-MLP-KKL-H).

Discussion

The motile B. pseudomallei and the non-motile B. mallei cause severe infections in both humans and animals, melioidosis, and glanders, respectively [8, 15]. Though yet rare, B. pseudomallei has a clear tendency of becoming a cosmopolite leaving its natural tropical habitat just as B. mallei did hundreds or thousands of years before by using the horse as vehicle. Intriguingly, B. pseudomallei has appeared in Italy where it is regularly isolated from drinking water and in France in riding schools, thus becoming an increasing environmental risk [40, 41].

Because of the high pathogenicity of B. mallei and B. pseudomallei, their rapid and reliable discrimination from the environmental saprophytic low-pathogenic B. thailandensis [18–21, 42] is essential in order to allow initiation of appropriate therapy for the patient or for a rapid enforcement of procedures against the spread of bacteria by implementation of barrier nursing, post-exposure procedures, and handling of the bacteria in a laboratory under BSL3 conditions [4].

In this study, we demonstrate the variations of the ribosomal protein S21 (rpsU) gene of B. mallei and B. pseudomallei in comparison to B. thailandensis and chosen other species of the genus Burkholderia. The protein S21 belongs to the macromolecular synthesis (MMS) operon. This gene is usually found as the first gene in the rpsU–dnaG–rpoD operon, and the gene products are responsible for the initiation of protein, DNA, and RNA synthesis in eubacteria. This sequence of genes is conserved in many Gram-negative bacteria [36, 43].

While all investigated B. mallei and B. thailandensis strains from different geographic origins were associated with species-specific sequences, B. pseudomallei strains showed one single mutation at position 78. We could identify the same sequence as in B. mallei strains in three B. pseudomallei reference strains (B. pseudomallei ATCC 15682, 551A, and NCTC 4845). Thirteen B. pseudomallei reference strains and all six isolates from Thailand showed a 78A>C shift. The strain 844 and the isolates UE 16 and UE 19, which have the same fliC sequence as B. mallei strains as previously demonstrated [11], were identical regarding the rpsU sequence with the other investigated B. pseudomallei isolates with the 78A>C shift. Three to four mutations in the nucleotide sequence of the rpsU gene of B. thailandensis allow for a differentiation of this non-pathogenic saprophyte from the dangerous agents B. mallei and B. pseudomallei as well as from other pathogenic species as B. cepacia and B. gladioli. However, these base substitutions have no effect on the amino acid sequence of the protein S21.

The stability of the target gene in general and the primer regions in particular is a major factor influencing the reliability of diagnostic PCR assays. We showed that the rpsU gene is well conserved within the genus Burkholderia and can consequently be used for sequence comparison based diagnostic assays. The described PCR assay was able to amplify DNA from strains of various geographic origins and times of isolation demonstrating the genetic stability of the selected primer sequences. The sequence heterology in the inner variable 120-base-pair fragment of the rpsU gene of the investigated Burkholderia species allows the use of this PCR with subsequent sequencing as a reliable diagnostic molecular tool for the discrimination of the pathovars B. mallei and B. pseudomallei from environmental B. thailandensis and other, genetically even more distinct Burkholderia spp. outside of the ‘pseudomallei’-complex. Though a further discrimination of B. mallei and B. pseudomallei is not possible based on rpsU sequencing, a preliminary identification of a clinical isolate as B. mallei/pseudomallei allows for conclusions on necessary therapeutic and hygiene approaches.

The assumption of our work, that the difference of pathogenicity of B. mallei, B. pseudomallei, and B. thailandensis might be connected to variations in the ribosomal protein S21, proved to be incorrect. Only few point mutations were found which do not affect the amino acid sequence. However, this finding confirms the hypothesis that the three members of the ‘pseudomallei’-group, the highly adapted parasite B. mallei and the saprophytes B. pseudomallei and B. thailandensis, originated from a common ancestor. There is a need for further investigations to elucidate the evolution of virulence and pathogenicity of these closely related species to find new strategies for therapeutic intervention and vaccine development.

Conclusions

In summary, sequencing of the rpsU gene and subsequent comparison of the variable 120-base-pair fragment with published sequences allows for an easy-to-perform, rapid, and reliable discrimination of the highly pathogenic B. mallei and B. pseudomallei from the environmental B. thailandensis. Considering the importance of a correct identification of BSL3 Burkholderia spp., however, a diagnosis on species level has to be enforced by another method. If bio-terrorism is suspected, molecular typing procedures such as MLST, RFLP, or VNTR analysis have to supplement the mere identification of B. mallei or B. pseudomallei on species level [12].

Acknowledgments

We thank Mrs. C. Lodri for excellent technical assistance. We thank V. Wuthiekanun and N.J. White, Welcome Research Unit, Bangkok, for providing Burkholderia spp. strains.

Contributor Information

H. Frickmann, 1Department of Tropical Medicine at the Bernhard Nocht Institute, German Armed Forces Hospital Hamburg, Hamburg, Germany; 2Institute for Medical Microbiology, Virology and Hygiene, University of Rostock Hospital, Rostock, Germany.

N. Chantratita, 3Department of Microbiology and Immunology, and Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.

Y. P. Gauthier, 4Institut de Recherches Biomédicales des Armées Département de Microbiologie, La Tronche, France.

H. Neubauer, 5Friedrich Loeffler Institute, Federal Research Institute for Animal Health, Jena, Germany.

R. M. Hagen, 1Department of Tropical Medicine at the Bernhard Nocht Institute, German Armed Forces Hospital Hamburg, Hamburg, Germany.

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