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Objectives: Although meropenem is widely used to treat Burkholderia infections, the response of Burkholderia pathogens to this antibiotic is largely unexplored.Methods: Burkholderia thailandensis, a model for Burkholderia spp., particularly Burkholderia mallei and Burkholderia pseudomallei, was challenged with a lethal level of meropenem and survivors were isolated. The genomes of two of the isolates were analysed to identify mutated genes and these genes were then specifically examined in more isolates to profile mutation diversity. Mutants were characterized to investigate the biological basis underlying survival against meropenem.Results: One of two genes associated with tRNA metabolism [metG or trmD, encoding methionyl-tRNA synthetase or tRNA (guanine-N1)-methyltransferase, respectively] was found to be mutated in the two survivors. A single nucleotide substitution and a frameshift mutation were found in metG and trmD, respectively. Five different substitution mutations affecting methionine- or tRNA-binding sites were found in metG during further screening. The mutants exhibited slowed growth and increased tolerance not only to meropenem but also various other antibiotics. This tolerance required intact RelA, a key stringent response.Conclusions: Specific mutations affecting the tRNA pool, particularly those in metG, play a pivotal role in the B. thailandensis response to meropenem challenge. This mechanism of antibiotic tolerance is important because it can reduce the effectiveness of meropenem and thereby facilitate chronic infection by Burkholderia pathogens. In addition, specific mutations found in MetG will prove useful in the effort to develop new drugs to completely inhibit this essential enzyme, while preventing stringent-response-mediated antibiotic tolerance in pathogens. [ABSTRACT FROM AUTHOR]

Aims To develop a simple gas chromatography-mass spectrometry ( GC- MS) method for the detection and differentiation of Burkholderia pseudomallei and Burkholderia mallei from each other, Burkholderia thailandensis and several members of the Burkholderia cepacia complex. Methods and Results Biomarkers were generated by one-step thermochemolysis ( TCM) and analysed using a GC- MS system. Fragments of poly-3-hydroxybutyrate- co-hydroxyvalerate [poly(3 HBA- co-3 HVA)] produced by TCM were useful biomarkers. Several cellular fatty acid methyl esters were important in differentiating the various Burkholderia species. A statistical discrimination algorithm was constructed using a combination of biomarkers. The identities of four B. pseudomallei strains, four B. mallei strains and one strain of each near neighbour were confirmed in a statistically designed test using the algorithm. The detection limit for this method was found to be approximately 4000 cells. Conclusions The method is fast, accurate and easy to use. The algorithm is robust against different growth conditions (medium and temperature). Significance and Impact of the Study This assay may prove beneficial in a clinical diagnostic setting, where the rapid identification of B. pseudomallei is essential to effective treatment. This method could also be easily employed after a biological attack to confirm the presence of either B. pseudomallei or B. mallei. [ABSTRACT FROM AUTHOR]

5′-Methylthioadenosine/ S-adenosylhomocysteine nucleosidase ( MTAN) plays crucial roles in the production of autoinducers and methionine metabolism. Putative genes encoding MTAN and Ado Hcyase from Burkholderia thailandensis were cloned and characterized. The Km values of MTAN for 5′-methylthioadenosine ( MTA) and S-adenosylhomocysteine ( SAH) were 19 and 58 μM, respectively. The catalytic efficiency of MTAN for SAH was only 0.004% of the value for MTA, indicating an almost complete substrate preference of MTAN for MTA. The results of autoinducer-2 assay of B. thailandensis and recombinants indicated that LuxS enzyme activity was lacking in Burkholderia species. Instead, Ado Hcyase hydrolysed SAH directly to homocysteine and adenosine in the activated methyl cycle. Meanwhile, the Km value of Ado Hcyase for SAH was determined to be 40 μM. Sequence analysis revealed that MTAN had much higher diversity than Ado Hcyase, which likely contributes to its substrate preference for MTA. Furthermore, the phylogenetic tree of MTAN sequences revealed that LuxS+ bacteria could be discriminated from LuxS− bacteria. These results suggested that the substrate preference of MTAN for MTA and SAH degradation pathway evolved with the bacterial-activated methyl cycle. [ABSTRACT FROM AUTHOR]

Burkholderia pseudomallei is the etiologic agent of the tropical disease melioidosis and is considered to be a potential biological weapon. Two B. pseudomallei-like species, Burkholderia oklahomensis and Burkholderia thailandensis, have been described in the literature. Infections with both of these microorganisms have occurred in the United States, but little is known about the relative virulence of these isolates in animal models of infection. In this study, B. oklahomensis and B. thailandensis CDC2721121 were determined to be avirulent in hamsters and mice at all challenge doses examined. The virulence of B. thailandensis CDC3015869, on the other hand, was more similar to the virulence of isolates of B. thailandensis from Southeast Asia. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

Summary: Burkholderia pseudomallei is an agent of melioidosis and is closely related to avirulent B. thailandensis. Burkholderia thailandensis has a 15-bp deletion within the variable region of the flagellin gene fliC compared with B. pseudomallei. The difference in the fliC gene might be related to virulence. In the present study, the invasion, internalization and intracellular replication of both phagocytic (mouse magcrophage cell line RAW264.7) and non-phagocytic cells (human lung epithelial cell line A549) of B. pseudomallei fliC knockout mutant (MM35) complemented with its own fliC (Cp) or with B. thailandensis fliC (Ct) was compared with those of the wild-type strains of B. pseudomallei (1026b) and B. thailandensis (E257). In phagocytic cells, there was no significant difference in bacterial uptake between Cp and Ct, but MM35 was internalized significantly less compared with 1026b, Cp, Ct and E257. The results suggest that flagella are involved in macrophage invasion. In non-phagocytic cells, Cp and Ct showed similar invasive capacities while 1026b, Cp and Ct showed significantly higher invasiveness than MM35, suggesting that flagella facilitate the non-phagocytic cell invasion. However, the invasive capacity of MM35 was significantly higher than that of E257, suggesting that in addition to the flagella, B. pseudomallei may need other factor(s) to facilitate invasion in non-phagocytic cells. [Copyright &y& Elsevier]

Summary: The molecular mechanisms involved in the development of a high level of resistance to a wide range of antimicrobials in Burkholderia pseudomallei and closely related species have not been sufficiently investigated. In the present study, the properties of B. pseudomallei, B. mallei and B. thailandensis mutants with increased resistance to fluoroquinolones and cephalosporins were analysed. Resistance to pefloxacin, ofloxacin and ceftazidime in B. pseudomallei and B. thailandensis was accompanied by an increased resistance to aminoglycosides, β-lactams, macrolides and chloramphenicol, whereas mutants of B. mallei were characterized by a narrower spectrum of resistance. With the use of the differential display technique, we demonstrated that multiple resistant variants of B. pseudomallei, B. mallei and B. thailandensis had an increased expression of putative efflux transporters belonging to the resistance-nodulation division superfamily and the major facilitator superfamily. With the application of PCR-single-stranded conformational polymorphism (PCR-SSCP) and sequencing, point mutations in gyrA quinolone-resistance determining region were detected in the part of multiple resistant B. pseudomallei and B. mallei mutants. These results indicate that various molecular mechanisms are involved in the development of multiple drug resistance in pathogenic Burkholderia and may be useful for further studying the adaptability of this microorganism and optimization of treatment. [Copyright &y& Elsevier]