Mobilization using incompatibility group P1 plasmids in strains of Pseudomonas pseudomallei and Pseudomonas mallei as potential vectors for DNA cloning]

The cells of Pseudomonas pseudomallei and Pseudomonas mallei have been shown to serve as recipients for the plasmid RSF1010 and its recombinant derivatives pVA1 and pVA4. The conjugative plasmids RP1 and pTH10 of the incompatibility group P1 are able to mobilize the nontransmissive vector plasmids for conjugation transfer into Pseudomonas pseudomallei and Pseudomonas mallei strains. The SmR determinant of the plasmid RSF1010 is expressed in the latter strains. These data makes the mentioned vector plasmids the candidates for DNA cloning in these strains.     

Alanine racemase mutants of Burkholderia pseudomallei and Burkholderia mallei and use of alanine racemase as a non-antibiotic-based selectable marker

Burkholderia pseudomallei and Burkholderia mallei are category B select agents and must be studied under BSL3 containment in the United States. They are typically resistant to multiple antibiotics, and the antibiotics used to treat B. pseudomallei or B. mallei infections may not be used as selective agents with the corresponding Burkholderia species. Here, we investigated alanine racemase deficient mutants of B. pseudomallei and B. mallei for development of non-antibiotic-based genetic selection methods and for attenuation of virulence. The genome of B. pseudomallei K96243 has two annotated alanine racemase genes (bpsl2179 and bpss0711), and B. mallei ATCC 23344 has one (bma1575). Each of these genes encodes a functional enzyme that can complement the alanine racemase deficiency of Escherichia coli strain ALA1. Herein, we show that B. pseudomallei with in-frame deletions in both bpsl2179 and bpss0711, or B. mallei with an in-frame deletion in bma1575, requires exogenous D-alanine for growth. Introduction of bpsl2179 on a multicopy plasmid into alanine racemase deficient variants of either Burkholderia species eliminated the requirement for D-alanine. During log phase growth without D-alanine, the viable counts of alanine racemase deficient mutants of B. pseudomallei and B. mallei decreased within 2 hours by about 1000-fold and 10-fold, respectively, and no viable bacteria were present at 24 hours. We constructed several genetic tools with bpsl2179 as a selectable genetic marker, and we used them without any antibiotic selection to construct an in-frame ΔflgK mutant in the alanine racemase deficient variant of B. pseudomallei K96243. In murine peritoneal macrophages, wild type B. mallei ATCC 23344 was killed much more rapidly than wild type B. pseudomallei K96243. In addition, the alanine racemase deficient mutant of B. pseudomallei K96243 exhibited attenuation versus its isogenic parental strain with respect to growth and survival in murine peritoneal macrophages.     

Transformation of pathogenic pseudomonas by plasmid DNA]

The possibility has been shown of the genetical transformation of Pseudomonas mallei strains by the purified DNA of the plasmids RSF1010, pES154, pBS222 and pBR325. The frequency of transformation varied from 1.2 x 10(1) to 2.0 x 10(2) depending on the plasmid DNA and transformation technique used in the experiments. Pseudomonas pseudomallei cells could not be transformed by the methods described in the paper.     

Burkholderia thailandensis: biological properties, identification and taxonomy

Burkholderia pseudomallei-like microorganisms have been isolated from soil and water in regions with endemic melioidosis. These strains have biochemical and antigenic profiles identical to melioidosis agents, except that they differ by virulence and L-arabinose (vir-, ara+). There are minor differences between these species by rRNA sequence. DNA hybridization and, more so, positive transformation of DNA auxotrophic mutants of B. pseudomallei by cell lysates of B. thailandensis and B. mallei confirmed the homology of these species' genomes. These members of the Burkholderia genus (pseudomallei, mallei, and thailandensis) can be regarded as a supraspecies taxon: pseudomallei group. B. thailandensis strains are not virulent for guinea pigs and slightly virulent for golden hamsters. Immunization with live cultures of B. thailandensis protected more than 50% guinea pigs challenged with 200 LD50 B. pseudomallei 100. B. thailandensis is suggested as a potential melioidosis vaccine.     

Molecular and Physical Characterization of Burkholderia mallei O Antigens

Burkholderia mallei lipopolysaccharide (LPS) has been previously shown to cross-react with polyclonal antibodies raised against B. pseudomallei LPS; however, we observed that B. mallei LPS does not react with a monoclonal antibody (Pp-PS-W) specific for B. pseudomallei O polysaccharide (O-PS). In this study, we identified the O-PS biosynthetic gene cluster from B. mallei ATCC 23344 and subsequently characterized the molecular structure of the O-PS produced by this organism.     

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.     

Antigenic relatedness between Burkholderia pseudomallei and Burkholderia mallei

Burkholderia pseudomallei and Burkholderia mallei are causative agents of distinct diseases, namely, melioidosis and glanders, respectively. The two species are very closely related, based on DNA-DNA homology, base sequence of the 16S rRNA, and phenotypic characteristics. Based on the use of polyclonal antisera, B. pseudomallei and B. mallei are also found to be antigenically closely related to one another. We previously reported the production of monoclonal antibodies (MAbs) against B. pseudomallei antigens; one group was specific for the 200-kDa exopolysaccharide present on the surface of all B. pseudomallei isolates, and the other was specific for the lipopolysaccharide (LPS) structure present on more than 95% of the B. pseudomallei tested. In the present study, we showed that the MAbs against 200-kDa antigen of B. pseudomallei cross-reacted with a component present also in some B. mallei isolates (3/6), but the positive immunoblot reaction was noted below the 200-kDa position. On the other hand, none of the six B. mallei isolates reacted with the MAb specific for B. pseudomallei LPS. It was of interest to observe that only the 3 exopolysaccharide-positive B. mallei isolates reacted with a commercial MAb against B. mallei LPS. The data presented suggest that B. mallei can be classified antigenically into two types based on their reactivities with different MAbs, i.e., the presence or absence of exopolysaccharide and the types of lipopolysaccharide. The heterogeneity of the LPS from these two closely related organisms is most likely related to the differences in its O-polysaccharide side chain.     

BurkDiff: a real-time PCR allelic discrimination assay for Burkholderia pseudomallei and B. mallei

A real-time PCR assay, BurkDiff, was designed to target a unique conserved region in the B. pseudomallei and B. mallei genomes containing a SNP that differentiates the two species. Sensitivity and specificity were assessed by screening BurkDiff across 469 isolates of B. pseudomallei, 49 isolates of B. mallei, and 390 isolates of clinically relevant non-target species. Concordance of results with traditional speciation methods and no cross-reactivity to non-target species show BurkDiff is a robust, highly validated assay for the detection and differentiation of B. pseudomallei and B. mallei.     

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.     

Burkholderia thailandensis E125 harbors a temperate bacteriophage specific for Burkholderia mallei

Burkholderia thailandensis is a nonpathogenic gram-negative bacillus that is closely related to Burkholderia mallei and Burkholderia pseudomallei. We found that B. thailandensis E125 spontaneously produced a bacteriophage, termed phiE125, which formed turbid plaques in top agar containing B. mallei ATCC 23344. We examined the host range of phiE125 and found that it formed plaques on B. mallei but not on any other bacterial species tested, including B. thailandensis and B. pseudomallei. Examination of the bacteriophage by transmission electron microscopy revealed an isometric head and a long noncontractile tail. B. mallei NCTC 120 and B. mallei DB110795 were resistant to infection with phiE125 and did not produce lipopolysaccharide (LPS) O antigen due to IS407A insertions in wbiE and wbiG, respectively. wbiE was provided in trans on a broad-host-range plasmid to B. mallei NCTC 120, and it restored LPS O-antigen production and susceptibility to phiE125. The 53,373-bp phiE125 genome contained 70 genes, an IS3 family insertion sequence (ISBt3), and an attachment site (attP) encompassing the 3' end of a proline tRNA (UGG) gene. While the overall genetic organization of the phiE125 genome was similar to lambda-like bacteriophages and prophages, it also possessed a novel cluster of putative replication and lysogeny genes. The phiE125 genome encoded an adenine and a cytosine methyltransferase, and purified bacteriophage DNA contained both N6-methyladenine and N4-methylcytosine. The results presented here demonstrate that phiE125 is a new member of the lambda supergroup of Siphoviridae that may be useful as a diagnostic tool for B. mallei.     

Immunobiological properties of Burkholderia pseudomallei and Burkholderia mallei capsular substances

The biopolymer composition, immunotropic and immunogenic properties of the fractions of B. pseudomallei and B. mallei were under study. The first two capsular fractions of these agents were found to be similar in their biopolymer composition that was indicative of their close relations. At the same time the causative agents of glanders proved to have decreased content of high molecular glycoproteids and LPS fragments. In the causative agents of melioidosis, capsular fractions K3 and K4 were characterized by the domination of proteins with a molecular weight of 42-25 kD. Fraction K4 in B. pseudomallei and fraction K1 in B. mallei had pronounced immunosuppressing properties ensuring the protection of encapsulated microbial cells in the body. The biopolymers forming fractions K1, K2, K3 in B. pseudomallei and fraction K2 in B. mallei were characterized by immunomodulating properties.     

Diversity of 16S-23S rDNA internal transcribed spacer (ITS) reveals phylogenetic relationships in Burkholderia pseudomallei and its near-neighbors

Length polymorphisms within the 16S-23S ribosomal DNA internal transcribed spacer (ITS) have been described as stable genetic markers for studying bacterial phylogenetics. In this study, we used these genetic markers to investigate phylogenetic relationships in Burkholderia pseudomallei and its near-relative species. B. pseudomallei is known as one of the most genetically recombined bacterial species. In silico analysis of multiple B. pseudomallei genomes revealed approximately four homologous rRNA operons and ITS length polymorphisms therein. We characterized ITS distribution using PCR and analyzed via a high-throughput capillary electrophoresis in 1,191 B. pseudomallei strains. Three major ITS types were identified, two of which were commonly found in most B. pseudomallei strains from the endemic areas, whereas the third one was significantly correlated with worldwide sporadic strains. Interestingly, mixtures of the two common ITS types were observed within the same strains, and at a greater incidence in Thailand than Australia suggesting that genetic recombination causes the ITS variation within species, with greater recombination frequency in Thailand. In addition, the B. mallei ITS type was common to B. pseudomallei, providing further support that B. mallei is a clone of B. pseudomallei. Other B. pseudomallei near-neighbors possessed unique and monomorphic ITS types. Our data shed light on evolutionary patterns of B. pseudomallei and its near relative species.     

Molecular-genetic approaches to diagnosis and intraspecific typing of causative agents of glanders and melioidosis

Pathogenic Burkholderia--Burkholderia mallei and Burkholderia pseudomallei--are causative agents of glanders and melioidosis, severe infectious diseases of man and animals. They are regarded as potential agents of bioterrorism. The existing bacteriological and immunological methods of identification of B. mallei and B. pseudomallei are not efficient enough for the rapid diagnosis and typing of strains. Described in the paper are molecular methods of detection of the agents by PCR, hybridization and strain typing made on the basis of bacterial total cell protein profiles, RAPD, ribotyping as well as of plasmid and DNA microrestriction analyses.     

Human single-chain Fv antibodies against Burkholderia mallei and Burkholderia pseudomallei

Much effort has been devoted to the development of mouse monoclonal antibodies that react specifically with Burkholderia mallei and Burkholderia pseudomallei for diagnostic and/or therapeutic purposes. Our present study focused on the screening of a phage-displayed nonimmune human single-chain Fv (scFv) antibody library against heat-killed B. mallei and B. pseudomallei for the generation of human scFv antibodies specific to the two pathogenic species of bacteria. Using two different panning procedures, we obtained seven different scFv phage antibodies that interacted with the heat-killed whole bacterial cells of B. mallei and B. pseudomallei. Our results demonstrate that panning of a human scFv antibody library against heat-killed whole bacterial cells may provide a valuable strategy for developing human monoclonal antibodies against the highly pathogenic bacteria.     

Effect of cultivation temperature on the extrachromosomal hereditary factors of the causative agent of melioidosis

The cultivation temperature of Burkholderia pseudomallei has been shown to determine both the direction of morphological dissociation and the prophage induction rate. Inheriting plasmid replicons was found to depend on the temperature conditions during the growth of these bacteria. No influence of B. pseudomallei plasmids pPM1 and pCM2 on the lysogenic state of the bacterial cell and the formation of different B. pseudomallei variants was noted.     

Identification of the causative agents of glanders and melioidosis by polymerase chain reaction

Burkholderia mallei and B. pseudomallei are causative agents of glanders and melioidosis, respectively, i.e. severe and fatal infection diseases of man and animal. The computer-based analysis of the 23S rRNA gene sites was used for selecting the primers. Two pairs of primers were chosen for the identification of B. mallei and Bpseudomallei. DNAs from 48 B. pseudomallei and 15 strains of B. mallei, unlike from other geterological bacteria, were positively amplified. Therefore, the method of polymerase chain reaction can be used in laboratory diagnosis of glanders and melioidosis.     

Comparative genomics and an insect model rapidly identify novel virulence genes of Burkholderia mallei

Burkholderia pseudomallei and its host-adapted deletion clone Burkholderia mallei cause the potentially fatal human diseases melioidosis and glanders, respectively. The antibiotic resistance profile and ability to infect via aerosol of these organisms and the absence of protective vaccines have led to their classification as major biothreats and select agents. Although documented infections by these bacteria date back over 100 years, relatively little is known about their virulence and pathogenicity mechanisms. We used in silico genomic subtraction to generate their virulome, a set of 650 putative virulence-related genes shared by B. pseudomallei and B. mallei but not present in five closely related nonpathogenic Burkholderia species. Although most of these genes are clustered in putative operons, the number of targets for mutant construction and verification of reduced virulence in animal models is formidable. Therefore, Galleria mellonella (wax moth) larvae were evaluated as a surrogate host; we found that B. pseudomallei and B. mallei, but not other phylogenetically related bacteria, were highly pathogenic for this insect. More importantly, four previously characterized B. mallei mutants with reduced virulence in hamsters or mice had similarly reduced virulence in G. mellonella larvae. Site-specific inactivation of selected genes in the computationally derived virulome identified three new potential virulence genes, each of which was required for rapid and efficient killing of larvae. Thus, this approach may provide a means to quickly identify high-probability virulence genes in B. pseudomallei, B. mallei, and other pathogens.     

Comparative analysis of total cell protein electrophoregram of pathogenic Burkholderia

Whole-cell proteins of 22 strain of Burkhoderia pseudomallei, including 13 B. mallei, 5 B. cepacia strains and 14 strains of opportunistically pathogenic Pseudomonas defined by 1D SDC-PAAG electrophoresis. Electrophoregrams contained 35 to 45 protein fractions sized 19 to 130 kDa, which were highly reproductive. On the basis of computer-aided comparative analysis of protein patterns the interspecies and intraspecies grouping of studied microorganisms was made. The cluster analysis of the similarity matrix of protein spectra made it possible to allocate two groups of strains at the level of similarity of 78%. Group I was formed by Burkholderia species that previously belonged to the II RNA-DNA homology group of Pseudomonas: B. pseudomallei, B. mallei, B. cepacia. All Pseudomonas species were added to the 2nd Group: P. aeruginosa, P. stutzeri, P. testosterone, P. fluorescens, P. putida, P. mendocina. Four phenons were isolated among the strains of B. pseudomallei and 2 phenons--among the strains of B. mallei at the threshold similarity level (89%). The authors conclude that the comparative analysis of electrophoregrams of whole-cell proteins can be useful in the identification and typing of pathogenic Burkholderia.     

Genome-wide expression analysis of iron regulation in Burkholderia pseudomallei and Burkholderia mallei using DNA microarrays

Burkholderia pseudomallei and B. mallei are the causative agents of melioidosis and glanders, respectively. As iron regulation of gene expression is common in bacteria, in the present studies, we have used microarray analysis to examine the effects of growth in different iron concentrations on the regulation of gene expression in B. pseudomallei and B. mallei. Gene expression profiles for these two bacterial species were similar under high and low iron growth conditions irrespective of growth phase. Growth in low iron led to reduced expression of genes encoding most respiratory metabolic systems and proteins of putative function, such as NADH-dehydrogenases, cytochrome oxidases, and ATP-synthases. In contrast, genes encoding siderophore-mediated iron transport, heme-hemin receptors, and a variety of metabolic enzymes for alternative metabolism were induced under low iron conditions. The overall gene expression profiles suggest that B. pseudomallei and B. mallei are able to adapt to the iron-restricted conditions in the host environment by up-regulating an iron-acquisition system and by using alternative metabolic pathways for energy production. The observations relative to the induction of specific metabolic enzymes during bacterial growth under low iron conditions warrants further experimentation.     

Evaluating Burkholderia pseudomallei Bip proteins as vaccines and Bip antibodies as detection agents

Burkholderia pseudomallei is a biothreat agent and an important natural pathogen, causing melioidosis in humans and animals. A type III secretion system (TTSS-3) has been shown to be critical for virulence. Because TTSS components from other pathogens have been used successfully as diagnostic agents and as experimental vaccines, it was investigated whether this was the case for BipB, BipC and BipD, components of B. pseudomallei's TTSS-3. The sequences of BipB, BipC and BipD were found to be highly conserved among B. pseudomallei and B. mallei isolates. A collection of monoclonal antibodies (mAbs) specific for each Bip protein was obtained. Most recognized both native and denatured Bip protein. Burkholderia pseudomallei or B. mallei did not express detectable BipB or BipD under the growth conditions used. However, anti-BipD mAbs did recognize the TTSS needle structures of a Shigella strain engineered to express BipD. The authors did not find that BipB, BipC or BipD are protective antigens because vaccination of mice with any single protein did not result in protection against experimental melioidosis. Enzyme-linked immunosorbent assay (ELISA) studies showed that human melioidosis patients had antibodies to BipB and BipD. However, these ELISAs had low diagnostic accuracy in endemic regions, possibly due to previous patient exposure to B. pseudomallei.