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

Abstract

The Bacillus cereus group, also known as B. 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 of B. 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 2,231 B. cereus s.l. genomes using a combination of in silico (i) average-nucleotide identity (ANI)-based genomospecies assignment, (ii) ANI-based subspecies assignment, (iii) seven-gene multi-locus sequence typing (MLST), (iv) single-locus panC group assignment, (v) rpoB allelic 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. For panC group assignment, we show that an adjusted, eight-group framework is largely, albeit not perfectly, congruent with the proposed eight-genomospecies taxonomy, as panC alone may not distinguish (i) B. luti from Group II B. mosaicus and (ii) B. paramycoides from Group VI B. mycoides . We additionally provide a list of loci that capture the topology of the whole-genome B. cereus s.l. phylogeny that may be used in future sequence typing efforts. For researchers with access to WGS, MLST, and/or panC data, we showcase how our recently released software, BTyper3 ( https://github.com/lmc297/BTyper3 ), can be used to assign B. cereus s.l. isolates to taxonomic units within this proposed framework with little-to-no user intervention or domain-specific knowledge of B. cereus s.l. taxonomy. We additionally outline a novel method for assigning B. 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.

Comparative phenotypic, genotypic and genomic analyses of Bacillus thuringiensis associated with foodborne outbreaks in France

“…Bt belongs to the large group of opportunistic bacteria Bacillus cereus sensu lato (Bc), whose classification is complex and regularly revisited [ 7 ]. This group contains at least eight species, spread across seven phylogenetic clades (I to VII), defined by a part of the panC gene sequence, with Bt distributed among four of them (II to VI) [ 8 , 9 ].…”

Characterization of Bacillus cereus Group Isolates From Human Bacteremia by Whole-Genome Sequencing

“…Recently, it has been shown that these methods are largely congruent in the B. cereus s.l. genomospecies attribution ( Carroll et al, 2020 ). In order to evaluate the presence of the main virulence factors, generally polymerase chain reaction (PCR) amplifications are performed for the identification of seven enterotoxigenic among the B. cereus virulence genes: hemolysin BL ( hbl A, hbl C, hbl D), enterotoxin non-hemolytic ( nhe A, nhe B, nhe C); cytotoxin K ( cyt K); enterotoxin FM ( ent FM), enterotoxin S ( ent S), and emetic toxin ( ces ) ( Fricker et al, 2007 ; Owusu-Kwarteng et al, 2017 ).…”

Keeping up with the Bacillus cereus group: taxonomy through the genomics era and beyond

“…Taxonomic and/or nomenclatural information obtained within the following contemporary frameworks is displayed in the box to the right of the phylogeny: (i) genomospecies assignments obtained using the standardized B. cereus s.l. genomospecies/subspecies/biovar framework proposed in 2020 (i.e., "Proposed 2020 Genomospecies") and BTyper3 v. 3.1.0 (Carroll, Cheng, and Kovac 2020;Carroll, Wiedmann, and Kovac 2020;Jain et al 2018); (ii) genomospecies assigned using the Genome Taxonomy Database (GTDB) Release 05-RS95 (17 July 2020) and GTDB-Tk v. 1.3.0 (i.e., "GTDB R95") (Chaumeil et al 2019;Eddy 2011;Hyatt et al 2010;Jain et al 2018;Matsen, Kodner, and Armbrust 2010;Ondov et al 2016;Parks et al 2020;Parks et al 2018;Price, Dehal, and Arkin 2010); (iii) phylogenetic marker gene-based operational taxonomic unit (mOTU)/specI cluster assignments (i.e., "specI v. 3/mOTU v. 2.5"); mOTUs were assigned using the mOTU taxonomy database v. 2.5 (Milanese et al 2019) and classify-genomes (accessed 3 June 2020; https://github.com/AlessioMilanese/classify-genomes. ), while specI clusters (Mende et al 2020;Mende et al 2013) were assigned using the specI v. 3 database and progenomes_classifier (accessed June 25, 2020; https:// github.com/AlessioMilanese/progenomes_classifier. )…”