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Burkholderia pseudomallei, a Gram-negative bacterial pathogen that causes melioidosis, is of public health importance in endemic areas including Malaysia. An investigation of the molecular epidemiology links of B. pseudomallei would contribute to better understanding of the clonal relationships, transmission dynamics and evolutionary change. Multilocus sequence typing (MLST) of 45 clinical B. pseudomallei isolates collected from sporadic melioidosis cases in Malaysia was performed. In addition, a total of 449 B. pseudomallei Malaysian strains submitted to the MLST database from 1964 until 2019 were included in the temporal analysis to determine the endemic sequence types (STs), emergence and re-emergence of ST(s). In addition, strain-specific distribution was evaluated using BURST tool. Genotyping of 45 clinical strains was resolved into 12 STs, and the majority were affiliated with ST46 (n = 11) and ST1342 (n = 7). Concomitantly, ST46 was the most prevalent ST in Malaysia, which was first reported in 1964. All the Malaysian B. pseudomallei strains were resolved into 76 different STs with 36 of them uniquely present only in Malaysia. ST1342 was most closely related to ST1034, in which both STs were unique to Malaysia and first isolated from soil samples in Pahang, a state in Malaysia. The present study revealed a high diversity of B. pseudomallei in Malaysia. Localized evolution giving rise to the emergence of new STs was observed, suggesting that host and environmental factors play a crucial role in the evolutionary changes in B. pseudomallei., (© 2021 Wiley-VCH GmbH.)

Burkholderia pseudomallei (B. pseudomallei) is an intracellular pathogen that causes melioidosis, a life-threatening infection in humans. The bacterium is able to form small colony variants (SCVs) as part of the adaptive features in response to environmental stress. In this study, we characterize the genomic characteristics, antimicrobial resistance (AMR), and metabolic phenotypes of B. pseudomallei SCV and wild type (WT) strains. Whole-genome sequence analysis was performed to characterize the genomic features of two SCVs (CS and OS) and their respective parental WT strains (CB and OB). Phylogenetic relationship between the four draft genomes in this study and 19 publicly available genomes from various countries was determined. The four draft genomes showed a close phylogenetic relationship with other genomes from Southeast Asia. Broth microdilution and phenotype microarray were conducted to determine the AMR profiles and metabolic features (carbon utilization, osmolytes sensitivity, and pH conditions) of all strains. The SCV strains exhibited identical AMR phenotype with their parental WT strains. A limited number of AMR-conferring genes were identified in the B. pseudomallei genomes. The SCVs and their respective parental WT strains generally shared similar carbon-utilization profiles, except for D,L-carnitine (CS), g-hydroxybutyric acid (OS), and succinamic acid (OS) which were utilized by the SCVs only. No difference was observed in the osmolytes sensitivity of all strains. In comparison, WT strains were more resistant to alkaline condition, while SCVs showed variable growth responses at higher acidity. Overall, the genomes of the colony morphology variants of B. pseudomallei were largely identical, and the phenotypic variations observed among the different morphotypes were strain-specific., Competing Interests: The authors have declared that no competing interests exist.

Background. Central intermediary metabolism (CIM) in bacteria is defined as a set of metabolic biochemical reactions within a cell, which is essential for the cell to survive in response to environmental perturbations. The genes associated with CIM are commonly found in both pathogenic and non-pathogenic strains. As these genes are involved in vital metabolic processes of bacteria, we explored the efficiency of the genes in genotypic characterization of Burkholderia pseudomallei isolates, compared with the established pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) schemes. Methods. Nine previously sequenced B. pseudomallei isolates from Malaysia were characterized by PFGE, MLST and CIM genes. The isolates were later compared to the other 39 B. pseudomallei strains, retrieved from GenBank using both MLST and sequence analysis of CIM genes. UniFrac and hierachical clustering analyses were performed using the results generated by both MLST and sequence analysis of CIM genes. Results. Genetic relatedness of nine Malaysian B. pseudomallei isolates and the other 39 strains was investigated. The nine Malaysian isolates were subtyped into six PFGE profiles, four MLST profiles and five sequence types based on CIM genes alignment. All methods demonstrated the clonality of OB and CB as well as CMS and THE. However, PFGE showed less than 70% similarity between a pair of morphology variants, OS and OB. In contrast, OS was identical to the soil isolate, MARAN. To have a better understanding of the genetic diversity of B. pseudomallei worldwide, we further aligned the sequences of genes used in MLST and genes associated with CIM for the nine Malaysian isolates and 39 B. pseudomallei strains from NCBI database. Overall, based on the CIM genes, the strains were subtyped into 33 profiles where majority of the strains from Asian countries were clustered together. On the other hand, MLST resolved the isolates into 31 profiles which formed three clusters. Hierarchical clustering using UniFrac distance suggested that the isolates from Australia were genetically distinct from the Asian isolates. Nevertheless, statistical significant differences were detected between isolates from Malaysia, Thailand and Australia. Discussion. Overall, PFGE showed higher discriminative power in clustering the nine Malaysian B. pseudomallei isolates and indicated its suitability for localized epidemiological study. Compared to MLST, CIM genes showed higher resolution in distinguishing those non-related strains and better clustering of strains from different geographical regions. A closer genetic relatedness of Malaysian isolates with all Asian strains in comparison to Australian strains was observed. This finding was supported by UniFrac analysis which resulted in geographical segregation between Australia and the Asian countries.