Development and Validation of a Burkholderia pseudomallei Core Genome Multilocus Sequence Typing Scheme To Facilitate Molecular Surveillance

Abstract

Objectives: Burkholderia pseudomallei causes the severe disease melioidosis. Whole genome-sequencing (WGS) based typing methods currently offer the highest resolution for molecular investigations of this genetically diverse pathogen. Still, its routine application in diagnostic laboratories is limited by the need for high computing power, bioinformatic skills and variable bioinformatic approaches, the latter affecting the results. We therefore aimed to establish and validate a WGS-based core genome multilocus sequence typing (cgMLST) scheme, applicable in routine diagnostic settings. Methods: A soft defined core genome was obtained by challenging the B. pseudomallei reference genome K96243 with 469 environmental and clinical genomes, resulting in 4,221 core and 1,359 accessory targets. The scheme was validated with 320 WGS datasets. We compared our novel typing scheme with single nucleotide polymorphism based-approaches investigating closely and distantly related strains. Finally, we applied our scheme for tracking the environmental source of a recent infection. Results: The validation of the scheme detected >95% good cgMLST target genes in 98.4% of the genomes. Comparison with existing typing methods revealed very good concordance. Our scheme proved to be applicable to investigate not only closely related strains, but also the global B. pseudomallei population structure. We successfully utilized our scheme to identify a sugar cane field as the presumable source of a recent melioidosis case. Conclusion: We developed a robust cgMLST typing scheme which integrates high resolution, maximized standardization and fast analysis for the non-bioinformatician. Our typing scheme has the potential to serve as a routinely applicable classification system in B. pseudomallei molecular epidemiology.

Sequencing-Based Genotyping of Pakistani Burkholderia mallei Strains: A Useful Way for Investigating Glanders Outbreaks

“…However, the Burkholderia-specific MLST scheme [21] fails to differentiate the highly clonal B. mallei strains while an MLVA scheme [22] provides higher resolution, but the investigated repeat regions are prone to homoplasy. Recently, a cgMLST scheme for B. pseudomallei, the assumed progenitor of B. mallei, was proposed [32]. As this scheme could also not sufficiently differentiate B. mallei strains (unpublished data), a B. mallei-specific cgMLST scheme was developed in the present study.…”

“…As in the present study, the true epidemiologic connection between isolates remained elusive, and further studies are required to determine this value for B. mallei. Such a threshold is also not known yet for B. pseudomallei cgMLST analyses, as even two alleles separate outbreak strains of a single transmission event from unrelated isolates and epidemiological connections were merely assumed for isolates differing by one allele [32]. A similarly strict differentiation could be expected for B. mallei.…”

Genetic diversity and spatial distribution of Burkholderia mallei by core genome-based multilocus sequence typing analysis

“…Whereas a cgMLST assay has been published for B . pseudomallei recently [ 26 ], for B . mallei no cgMLST assay was available.…”

Environmental Factors Associated With Soil Prevalence of the Melioidosis Pathogen Burkholderia pseudomallei: A Longitudinal Seasonal Study From South West India

“…The only two samples which became culture-positive were collected from a grassland in Udupi and a paddy field in Shimoga during the monsoon and showed remarkably high loads by direct qPCR (Figure 5). Whole genome sequencing of those isolates and subsequent cgMLST (Lichtenegger et al, 2021) revealed clustering in the same group with other Indian isolates and strains from Sri Lanka, but also showed remarkable genetic difference between each other (Figure 9).…”

“…Illumina short reads of the environmental B. pseudomallei isolates from this study were submitted to the Sequence Read Archive under the NCBI accession ERR9146399 (IND_S3) an ERR9138530 (IND_S14). Whole genome sequencing data of B. pseudomallei strains were analyzed in SeqSphere (Ridom GmbH, Germany) using our previously published B. pseudomallei core genome MLST (cgMLST) scheme (Lichtenegger et al, 2021). A UPGMA tree and a minimum spanning tree were constructed based on the allelic profiles of the isolates.…”