Non-Toxin-Producing Bacillus cereus Strains Belonging to the B. anthracis Clade Isolated from the International Space Station

Abstract

In an ongoing Microbial Observatory investigation of the International Space Station (ISS), 11 Bacillus strains (2 from the Kibo Japanese experimental module, 4 from the U.S. segment, and 5 from the Russian module) were isolated and their whole genomes were sequenced. A comparative analysis of the 16S rRNA gene sequences of these isolates showed the highest similarity (>99%) to the Bacillus anthracis-B. cereus-B. thuringiensis group. The fatty acid composition, polar lipid profile, peptidoglycan type, and matrix-assisted laser desorption ionization-time of flight profiles were consistent with the B. cereus sensu lato group. The phenotypic traits such as motile rods, enterotoxin production, lack of capsule, and resistance to gamma phage/penicillin observed in ISS isolates were not characteristics of B. anthracis. Whole-genome sequence characterizations showed that ISS strains had the plcR non-B. anthracis ancestral "C" allele and lacked anthrax toxin-encoding plasmids pXO1 and pXO2, excluding their identification as B. anthracis. The genetic identities of all 11 ISS isolates characterized via gyrB analyses arbitrarily identified them as members of the B. cereus group, but traditional DNA-DNA hybridization (DDH) showed that the ISS isolates are similar to B. anthracis (88% to 90%) but distant from the B. cereus (42%) and B. thuringiensis (48%) type strains. The DDH results were supported by average nucleotide identity (>98.5%) and digital DDH (>86%) analyses. However, the collective phenotypic traits and genomic evidence were the reasons to exclude the ISS isolates from B. anthracis. Nevertheless, multilocus sequence typing and whole-genome single nucleotide polymorphism analyses placed these isolates in a clade that is distinct from previously described members of the B. cereus sensu lato group but closely related to B. anthracis. IMPORTANCE The International Space Station Microbial Observatory (Microbial Tracking-1) study is generating a microbial census of the space station's surfaces and atmosphere by using advanced molecular microbial community analysis techniques supported by traditional culture-based methods and modern bioinformatic computational modeling. This approach will lead to long-term, multigenerational studies of microbial population dynamics in a closed environment and address key questions, including whether microgravity influences the evolution and genetic modification of microorganisms. The spore-forming Bacillus cereus sensu lato group consists of pathogenic (B. anthracis), food poisoning (B. cereus), and biotechnologically useful (B. thuringiensis) microorganisms; their presence in a closed system such as the ISS might be a concern for the health of crew members. A detailed characterization of these potential pathogens would lead to the development of suitable countermeasures that are needed for long-term future missions and a better understanding of microorganisms associated with space missions.

Keywords: Bacillus; Bacillus anthracis; Bacillus cereus; ISS; genomics; spores.

FIG 1

Electron micrographs of ISSFR-003 spores. The inset is a transmission electron micrograph exhibiting the exosporium characteristics of the B. cereus group of species. The scanning electron micrograph clearly shows the presence of pili that were also reported in some strains of B. cereus.

FIG 2

Phylogenetic tree based on GyrB sequences (~1,900 bases) and generated by the neighbor-joining method showing the phylogenetic relationship between 11 ISS isolates and members of the B. cereus group. Bootstrap values from 500 replications are shown at branch points. Bar, 0.02 substitution per site.

FIG 3

MLST analysis of Bacillus strains of this study and related species of the B. cereus sensu lato group. The genomic contigs of ISS isolates obtained were searched for glpF, gmk, ilvD, pta, pur, pycA, and tpi gene sequences, which are standardized for use in MLST of the B. cereus sensu lato group of species (43). The sequences retrieved were compared with the STs deposited in the B. cereus MLST database (44), concatenated according to the MLST scheme. It was found that all 11 ST sequences derived from the ISS isolates are identical and do not correspond to any known allele combination. The reconstruction was based on the RAxML algorithm (45), and the bootstrap values were calculated by using 500 replicates. The bar indicates 1% sequence divergence.

FIG 4

Placement of the ISS isolates into a core genome SNP phylogeny. We used 461 B. cereus sensu lato genomes publicly available in the NCBI database to generate a 193,732-character SNP matrix with BWA-MEM and GATK in conjunction with NASP. The tree was inferred with IQ-TREE. The SNPs were called from a 628,820-character core genome alignment. The phylogeny retention index (0.94) and the consistency index (0.31) were calculated with Phangorn. The B. cereus sensu lato phylogeny is separated into three major clades. The expanded view on the right provides a closer look at the portion of the tree within clade 1 that includes the ISS isolates. The ISS isolates are closely related to B. anthracis but are in a distinct and separate clade.