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The aim of the study was to identify and compare potential determinants of aminoglycoside resistance in gentamicin susceptible Burkholderia pseudomallei strains.Materials and methods. A bioinformatics analysis of whole genome shotgun sequences of three B. pseudomallei strains having different levels of sensitivity to gentamicin was carried out.Results and discussion. B. pseudomallei is intrinsically resistant to aminoglycosides. Such strains, as a rule, are not taken into account in the classical scheme of isolation and identification. At the same time, there were no significant differences in the clinical manifestations of melioidosis during infection with gentamicin-resistant and sensitive strains. In B. pseudomallei strains of different sequence types (ST70, ST948, and ST1566), point missense mutations were found in the genes of three efflux pumps of the RND family: AmrAB-OprA, BpeAB-OprB, BpeEF-OprC, and one with unknown functions, as well as in the gene aminoglycoside-6’-N-acetyltransferase AAC(6’)-III. All three strains had amino acid substitutions in the AmrA periplasmic linker: ARG160SER, Arg116Gln and Gly237Arg, Thr317Lys, respectively. In moderately sensitive strains (ST948 and ST1566), an identical Val222Met substitution was found in the repressor of the AmrAB-OprA operon, AmrR. It is likely that the intermediate level of sensitivity to gentamicin in the studied strains is mediated by the constitutive expression of the AmrAB-OprA operon, which partially compensates for the structural defects. It is also possible that a dinucleotide deletion in the AAC (6’)-III aminoglycoside-6’-N-acetyltransferase gene, as well as detected mutations in the homologues of the periplasmic linker (BPSL2234) of an uncharacterized efflux operon of the RND family, are involved in the loss of resistance to gentamicin.

The aim of the study was to analyze differential gene expression in Burkholderia pseudomallei strains with different survival rates under cold stress conditions.Materials and methods. Three strains of B. pseudomallei (sequence types ST 46, ST 70, and ST 85) were used as model strains. The RNA was isolated using the membrane columns method and stabilized through dehydration. The cDNA was sequenced on the Illumina MiSeq platform. Gene functions were classified using the KEGG PATHWAY database.Results and discussion. Based on the analysis of transcriptomes of B. pseudomallei strains after prolonged exposure to cold stress, the molecular mechanisms of B. pseudomallei adaptation to low temperatures have been described for the first time ever. It was shown that adaptation of B. pseudomallei to cold stress is associated with regulatory processes leading to a significant decrease in the total transcriptional activity. Two strategies of adaptation to low temperatures have been found: 1) modulation of regulatory processes leading to suppression of gene expression of the main metabolic pathways to the minimum level that ensures cell viability and activation of the minimum required set of stress response genes, and 2) less noticeable suppression of general metabolism in combination with activation of expression of an extended range of genes for cold and heat shock, general, osmotic, and universal stresses. Both mechanisms provide the causative agent of melioidosis with survival under conditions of prolonged cold stress at low positive temperatures. The first strategy showed greater efficiency at negative temperatures. The transition of B. pseudomallei to a viable but uncultivated state occurs in the long term (at least 2 years). While with the second strategy, this happens within 2 months. Assessment of the potential and molecular mechanisms of adaptation of this bacterium to cold stress is necessary to understand the degree of risk in case of a possible introduction of B. pseudomallei into regions with a temperate climate and to develop effective measures to ensure the biosafety of the environment.

Background: The correct identifying of pathogenic Burkholderia spp. using available commercial biochemical systems is a certain problem due to metabolic plasticity and variable enzymatic profile of isolates. Objectives: The current study aimed at using specific PCR and conventional multi-locus sequence typing (MLST) scheme to confirm the uncertain identification results for Burkholderia cepacia clinical isolate. Methods: Multilocus species-specific PCR and MLST profiling of high-throughput sequencing data have been used to clarify the varied results of biochemical identification of the strain. Results: The strain isolated from a patient with septicemia was initially identified as B. pseudomallei by Vitek 2 GN system but has an uncharacteristic antimicrobial resistance pattern and colony morphology. A species-specific multilocus PCR and whole-genome sequence profiling, according to the MLST scheme, allowed to identify an isolate as B. cepacia. Conclusions: The obtained results demonstrate the preference of molecular tests for correct identifying of pathogenic Burkholderia, considering that misidentification of those may lead to improper treatment or increase of biosafety risk.