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

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, with 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. 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 1,351 accessory targets. The scheme was validated with 320 WGS data sets. 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. 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 investigating not only closely related strains but also the global B. pseudomallei population structure. We successfully utilized our scheme to identify a sugarcane field as the presumable source of a recent melioidosis case. In summary, we developed a robust cgMLST scheme that integrates high resolution, maximized standardization, and fast analysis for the nonbioinformatician. Our typing scheme has the potential to serve as a routinely applicable classification system in B. pseudomallei molecular epidemiology.

Stringent factor and proteolysis control of sigma factor RpoS expression in Vibrio cholerae.

Vibrio cholerae can colonize the gastrointestinal track of humans and cause the disease cholera. During colonization, the alternative sigma factor, RpoS, controls a process known as "mucosal escape response," defining a specific spatial and temporal response and effecting chemotaxis and motility. In this report, the expression and proteolytic control of RpoS in V. cholerae was characterized. To date, aspects of proteolysis control, the involved components, and proteolysis regulation have not been addressed for RpoS in V. cholerae. Similar to Escherichia coli, we find that the RpoS protein is subjected to regulated intracellular proteolysis, which is mediated by homologues of the proteolysis-targeting factor RssB and the protease complex ClpXP. As demonstrated, RpoS expression transiently peaks after cells are shifted from rich to minimal growth medium. This peak level is dependent on (p)ppGpp-activated rpoS transcription and controlled RpoS proteolysis. The RpoS peak level also correlates with induction of a chemotaxis gene, encoding a methyl-accepting chemotaxis protein, earlier identified to belong to the mucosal escape response pathway. These results suggest that the RpoS expression peak is linked to (p)ppGpp alarmone increase, leading to enhanced motility and chemotaxis, and possibly contributing to the mucosal escape response.