No Assembly Required: Using BTyper3 to Assess the Congruency of a Proposed Taxonomic Framework for theBacillus cereusGroup With Historical Typing Methods

TheBacillus cereusgroup, also known asB. cereus sensu lato(s.l.), is a species complex comprising numerous closely related lineages, which vary in their ability to cause illness in humans and animals. The classification ofB. cereus s.l.isolates into species-level taxonomic units is essential for facilitating communication between and among microbiologists, clinicians, public health officials, and industry professionals, but is not always straightforward. A recently proposed genomospecies-subspecies-biovar taxonomic framework aims to provide a standardized nomenclature for this species complex but relies heavily on whole-genome sequencing (WGS). It thus is unclear whether popular, low-cost typing methods (e.g., single- and multi-locus sequence typing) remain congruent with the proposed taxonomy. Here, we characterize 2,231B. cereus s.l.genomes using a combination ofin silico(i) average-nucleotide identity (ANI)-based genomospecies assignment, (ii) ANI-based subspecies assignment, (iii) seven-gene multi-locus sequence typing (MLST), (iv) single-locuspanCgroup assignment, (v)rpoBallelic typing, and (vi) virulence factor detection. We show that sequence types (STs) assigned using MLST can be used for genomospecies assignment, and we provide a comprehensive list of ST/genomospecies associations. ForpanCgroup assignment, we show that an adjusted, eight-group framework is largely, albeit not perfectly, congruent with the proposed eight-genomospecies taxonomy, aspanCalone may not distinguish (i)B. lutifrom Group IIB. mosaicusand (ii)B. paramycoidesfrom Group VIB. mycoides. We additionally provide a list of loci that capture the topology of the whole-genomeB. cereus s.l.phylogeny that may be used in future sequence typing efforts. For researchers with access to WGS, MLST, and/orpanCdata, we showcase how our recently released software, BTyper3 (), can be used to assignB. cereus s.l.isolates to taxonomic units within this proposed framework with little-to-no user intervention or domain-specific knowledge ofB. cereus s.l.taxonomy. We additionally outline a novel method for assigningB. cereus s.l.genomes to pseudo-gene flow units within proposed genomospecies. The results presented here highlight the backward-compatibility and accessibility of the recently proposed genomospecies-subspecies-biovar taxonomic framework and illustrate that WGS is not a necessity for microbiologists who want to use the proposed nomenclature effectively.