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

Abstract

Burkholderia (B.) mallei is a host-adapted equine pathogen that causes glanders, a re-emerging zoonotic disease, which is endemic in Pakistan and other developing countries and seriously impacts the global equine movement. Due to globalization, the geographical restriction of diseases vanishes and the lack of awareness of and experience with eradicated diseases in industrialized countries also promotes the re-introduction of infections in these regions. Owing to the high equine population, the Pakistani province Punjab is a potential hotspot where several glanders outbreaks have been seen over last two decades. For determining the genomic diversity of B. mallei in this and other equine-populated prefectures, the genomes of 19 B. mallei strains isolated between 1999 and 2020 in different locations were sequenced and their genotypes were determined. Particularly, for genetically highly homogenous pathogens like B. mallei genotyping techniques require a high discriminatory power for enabling differentiation on the strain level. Thus, core-genome single nucleotide polymorphism (cgSNP) analysis was applied for distinguishing the highly similar strains. Furthermore, a whole-genome sequence-based core genome multi locus sequence typing (cgMLST) scheme, specific to B. mallei, was developed and additionally applied to the data. It was found that B. mallei genotypes in Pakistan persisted over time and space and genotype clusters preferred connection with a time point rather than the place of isolation, probably due to frequent equine movement, which promotes the spread of glanders. The cgMLST approach proved to work in accord with SNP typing and may help to investigate future glanders outbreaks.

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

“…The congruence of SNP and cgMLST analyses was demonstrated for a set of global B. mallei strains, which both methods clustered identically ( 59 ) ( Figure 5 ). However, by employing SNP analysis, a more detailed strain differentiation is possible, as one cgMLST allele difference can reflect several base changes in the target.…”

“…While this is commonly done for some pathogens, such as Brucella sp. ( 57 ), this approach has limitations for other bacteria such as Bacillus anthracis ( 58 ) and also B. mallei ( 59 ). Indeed, MLVA profiles obtained in silico from genomes of Pakistani B. mallei strains differed markedly from previously published profiles, disqualifying this in silico approach from sequencing data obtained by Illumina technology.…”

“…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.…”

“… Comparison of clustering generated by cgMLST and cgSNP analysis ( 59 ) (modified). Colors indicate country of origin of the strain as registered with the corresponding BioSample entry.…”