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

Abstract

Burkholderia mallei is the etiological agent of glanders, a highly contagious and often fatal disease in equids. Due to the high genetic clonality of B. mallei, high-resolution typing assays are necessary to differentiate between individual strains. Here we report on the development and validation of a robust and reproducible core genome-based Multi Locus Sequence Typing Assay (cgMLST) for B. mallei, which is based on 3328 gene targets and enables high-resolution typing at the strain level. The assay was validated using a set of 120 B. mallei genomes from public databases and 23 newly sequenced outbreak strains from in-house strain collections. In this cgMLST analysis, strains from different geographic regions were clearly distinguished by at least 70 allele differences, allowing spatial clustering while closely related and epidemiologically related strains were separated by only zero to three alleles. Neither the different sequencing technologies nor the assembly strategies had an influence on the cgMLST results. The developed cgMLST is highly robust, reproducible and can be used for outbreak investigations, source tracking and molecular characterization of new B. mallei isolates.

Sequence-based detection and typing procedures for Burkholderia mallei: Assessment and prospects

“…The latter, which comprises the sequence analysis of seven highly conserved genes (MLST-7) ( 4 , 5 ), revealed only one sequence type (ST40) for nearly all B. mallei strains, e.g., out of 120 investigated B. mallei strains, 118 strains were classified as ST40 while merely two strains, NCTC 10247 and NCTC 10260 isolated in Turkey, were identified as ST100 ( 31 ). Thus, this method is unable to depict the global diversity of B. mallei strains, merely allowing an identification of the organism at the species level.…”

“…Regarding Burkholderia sp., the first cgMLST schemes have been developed for B. pseudomallei and B. stabilis ( 70 , 71 ). Recently, two cgMLST schemes were published for B. mallei ( 31 , 59 ). Both used the type strain genome B. mallei ATCC 23344 as seed genome, but different sets of query genomes, resulting in the identification of different numbers of target genes: 3,328 and 2,838 core genome genes (66.2 and 56.5 % of the seed genome genes), respectively.…”

“…For example, three different lineages were detected in India, as well as different clusters in Bahrain and Dubai. On the other hand, the B. mallei population in Pakistan proved to be highly homogenic, which reflects the movement of equids between provinces ( 31 , 59 ). Appelt et al ( 31 ) additionally defined a three-allele threshold as the number of maximum allele differences between strains of the same outbreak.…”

“…A drawback of cgMLST analysis is the necessity of assembled genomes as basis for the analysis. Although Appelt et al ( 31 ) state that assembly strategy would not change the cgMLST analysis result, it can be assumed that sequencing quality has a major impact on the assembly quality and thus also on a number of cgMLST targets. This was merely tested for two strains and has to be further assessed with a large number of sequencing data.…”

“…Likewise, incorrect database entries regarding strain identity and metadata have to be corrected. Appelt et al(31) identified five entries where strain designation did not match the genotyping analysis result, which included two presumed type strains ATCC 23344 and Budapest (= NCTC 10229) (Table…”