Cellular Lipid and Fatty Acid Compositions of Burkholderia pseudomallei Strains Isolated from Human and Environment in Viet Nam

Viet nam is known as an endemic area of melioidosis but its etiologic agent originated in Viet nam was not extensively studied. For the first time, we analyzed the cellular lipid and fatty acid compositions of 15 Vietnamese isolates of Burkholderia pseudomallei, 10 from humans and 5 from the environment. Cellular lipid compositions were analyzed by two-dimensional thin-layer chromatography on silica gel G plates. Cellular fatty acid methyl esters were analyzed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). The major lipids in all the isolates were phosphatidylglycerol (PG), two forms of phosphatidylethanolamine (PE-1 and PE-2), and two forms of ornithine-containing lipid (OL-1 and OL-2). PE-1 contained non-hydroxy fatty acids at both sn-1 and ?2 positions, while PE-2 possessed 2-hydroxy fatty acids and non-hydroxy fatty acids in a ratio of 1: 1. Since snake venom phospholipase A₂ digestion of PE-2 liberated 2-hydroxy fatty acids, it was confirmed that these acids are at the sn-2 position of glycerol moiety. In both OL-1 and OL-2, amide-linked fatty acid was 3-hydroxy palmitic acid (3-OH-C16: 0), while ester-linked fatty acids were non-hydroxy acids in OL-1 and 2-hydroxy acids in OL-2. The total cellular fatty acid compositions of the test strains were characterized by the presence of 2-hydroxy palmitic (2-OH-C16: 0), 2-hydroxy hexadecenoic (2-OH-C16: 1), 2-hydroxy octadecenoic (2-OH-C18: 1), 2-hydroxy methylene octadecanoic (2-OH-C19CPA), 3-hydroxy myristic (3-OH-C14: 0) and 3-hydroxy palmitic (3-OH-C16: 0) acids. There were significant differences in the concentration of hexadecenoic (C16: 1), methylene hexadecanoic (C17CPA), octadecenoic (C18: 1) and methylene octadecanoic (C19CPA) acids among the Vietnamese isolates of B. pseudomallei. However, no significant difference was observed in cellular lipid and fatty acid components between strains of human and environmental origins.     

Burkholderia pseudomallei interferes with inducible nitric oxide synthase (iNOS) production: a possible mechanism of evading macrophage killing

Burkholderia pseudomallei is a causative agent of melioidosis, a life threatening disease which affects humans and animals in tropical and subtropical areas. This bacterium is known to survive and multiply inside cells such as macrophages. The mechanism of host defense against this bacterium is still unknown. In this study, we demonstrated that B. pseudomallei exhibited unique macrophage activation activity compared with Escherichia coli and Salmonella typhi. The mouse macrophage cell line (RAW 264.7) infected with B. pseudomallei at MOI of 0.1:1, 1:1 and 10:1 did not express a detectable level of inducible nitric oxide synthase (iNOS). Moreover, the B. pseudomallei infected cells released TNF-alpha only when they were infected with high MOI (10:1). Unlike the cells infected with B. pseudomallei, the cells infected with E. coli, and S. typhi expressed iNOS even at MOI of 0.1:1. These infected cells also released a significantly higher level of TNF-alpha at the low MOI ratio. The cells that were preactivated with IFN-gamma prior to being infected with B. pseudomallei exhibited an enhanced production of iNOS and TNF-alpha release. The increased macrophage activation activity in the presence of IFN-gamma also correlated with the restriction of the intracellular bacteria survival. Moreover, IFN-gamma also prevented cell fusion and multinucleated cell formation induced by B. pseudomallei, a phenomenon recently described by our group. Altogether, these results indicate that internalization of B. pseudomallei failed to trigger substantial macrophage activation, a phenomenon which could prolong their survival inside the phagocytic cells and facilitate a direct cell to cell spreading of B. pseudomallei to neighboring cells.     

Detection and differentiation of Burkholderia pseudomallei, Burkholderia mallei and Burkholderia thailandensis by multiplex PCR

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.     

Enzymatic activity of cell-free extracts from Burkholderia oxyphila OX-01 bio-converts (+)-catechin and (−)-epicatechin to (+)-taxifolin

This study characterized the enzymatic ability of a cell-free extract from an acidophilic (+)-catechin degrader Burkholderia oxyphila (OX-01). The crude OX-01 extracts were able to transform (+)-catechin and (?)-epicatechin into (+)-taxifolin via a leucocyanidin intermediate in a two-step oxidation. Enzymatic oxidation at the C-4 position was carried out anaerobically using H₂O as an oxygen donor. The C-4 oxidation occurred only in the presence of the 2R-catechin stereoisomer, with the C-3 stereoisomer not affecting the reaction. These results suggest that the OX-01 may have evolved to target both (+)-catechin and (?)-epicatechin, which are major structural units in plants.     

Antigenic Differences between Clinical and Environmental Isolates of Burkholderia pseudomallei

Burkholderia pseudomallei is a free-living organism that causes the potentially lethal tropical infection melioidosis. The disease is endemic in many parts of eastern Asia and northern Australia. The presence of two distinct biotypes in soil can be reliably distinguished by their ability to assimilate l -arabinose. Whereas some soil isolates could utilize this substrate (Ara⁺), the remaining soil isolates and all clinical isolates tested so far could not (Ara^?). Only the Ara^? isolates were virulent in animal models. We have raised a murine monoclonal antibody (MAb) that can readily distinguish Ara^? from Ara⁺ biotypes. The MAb reacted with a high molecular weight component present only on the Ara^? biotype. With this MAb, clinical and soil Ara^?isolates gave identical positive reactions in agglutination, immunofluorescence, ELISA and immunoblot assays. Using these same assay systems, the soil Ara⁺ biotype did not react with the MAb. Similar but distinct immunoblot patterns were also noted when these two Ara biotypes were probed with sera from patients with melioidosis or with polyclonal immune rabbit sera. These data showed that the Ara^? biotype from both clinical and environmental isolates is antigenically different from its Ara⁺ environmental counterpart. The SDS-PAGE protein and lectin-binding profiles of both groups of Ara^? isolates were also found to be different from those of the Ara⁺ biotype.     

Characterization of Plant-Growth-Promoting Effects and Concurrent Promotion of Heavy Metal Accumulation in the Tissues of the Plants Grown in The Polluted Soil by Burkholderia Strain LD-11

Plant-growth-promoting (PGP) bacteria especially with the resistance to multiple heavy metals are helpful to phytoremediation. Further development of PGP bacteria is very necessary because of the extreme diversity of plants, soils, and heavy metal pollution. A Burkholderia sp. strain, numbered LD-11, was isolated, which showed resistances to multiple heavy metals and antibiotics. It can produce indole-3-acetic acid, 1-aminocyclopropane-1-carboxylic acid deaminase and siderophores. Inoculation with the LD-11 improved germination of seeds of the investigated vegetable plants in the presence of Cu, promoted elongation of roots and hypocotyledonary axes, enhanced the dry weights of the plants grown in the soils polluted with Cu and/or Pb, and increased activity of the soil urease and the rhizobacteria diversity. Inoculation with the LD-11 significantly enhanced Cu and/or Pb accumulation especially in the roots of the plants grown in the polluted soils. Notably, LD-11 could produce siderophores in the presence of Cu. Conclusively, the PGP effects and concurrent heavy metal accumulation in the plant tissues results from combined effects of the above-mentioned multiple factors. Cu is an important element that represses production of the siderophore by the bacteria. Phytoremediation by synergistic use of the investigated plants and the bacterial strain LD-11 is a phytoextraction process.     

Stable marker on flagellin gene sequences related to arabinose non-assimilating pathogenic Burkholderia pseudomallei

Using PCR-based isolation and sequence analysis of the flagellin gene from two distinct biotypes of Burkholderia pseudomallei, a 15-bp deletion was found within the variable domain of the gene in isolates capable of assimilating arabinose (Ara+). This finding led to the development of a PCR-based method in order to differentiate and identify pathogenic B. pseudomallei for epidemiological study. A pair of specific primers was designed covering the 15-bp deletion region at the variable domain. PCR-amplification products of 176 and 191 bp in size were detected from 41 Ara+ isolates and 39 Ara - isolates of B. pseudomallei, respectively. Moreover, flagellin gene fragments of other bacterial species tested in this study were not amplified using these primers. The results suggest that the flagellin gene sequences of both B. pseudomallei biotypes in this region are stable and distinct. This method can be applied and useful for the epidemiological study of B. pseudomallei.     

A possible pitfall in the identification of Burkholderia mallei using molecular identification systems based on the sequence of the flagellin fliC gene

Amotile Burkholderia mallei and motile Burkholderia pseudomallei display a high similarity with regard to phenotype and clinical syndromes, glanders and melioidosis. The aim of this study was to establish a fast and reliable molecular method for identification and differentiation. Despite amotility, the gene of the filament forming flagellin (fliC) could be completely sequenced in two B. mallei strains. Only one mutation was identified discriminating between B. mallei and B. pseudomallei. A polymerase chain reaction-restriction fragment length polymorphism assay was designed making use of the absence of an AvaII recognition site in B. mallei. All seven B. mallei, 12 out of 15 B. pseudomallei and 36 closely related apathogenic Burkholderia thailandensis strains were identified correctly. However, in three B. pseudomallei strains a point mutation at gene position 798 (G to C) disrupted the AvaII site. Therefore, molecular systems based on the fliC sequence can be used for a reliable proof of strains of the three species but not for the differentiation of B. mallei and B. pseudomallei isolates.