Genomic characterization of a pathogenic Bacillus licheniformis strain LSDY01: deciphering its genetic diversity and...

Bacillus licheniformis is an opportunistic pathogen in clinical settings. However, the emergence of clinical strains carrying horizontally acquired virulence determinants, including chromosomal genomic islands harboring yopX, a putative type IV secretion system (T4SS), and plasmids bearing toxin-antitoxin systems and additional virulence factors, poses a significant challenge to diagnosis and treatment. Moreover, the genetic basis of the pathogenicity of clinical isolates has not been comprehensively studied.

A pathogenic B. licheniformis strain (LSDY01) isolated from a skin infection was subjected to whole-genome sequencing and comparative genomic analyses. Phylogenetic reconstruction, pan-genome analysis, and detailed characterization of plasmid and chromosomal virulence determinants were performed. Antimicrobial susceptibility testing was performed according to standardized guidelines. Biofilm formation assays were also conducted. The cytotoxic effect of LSDY01 on HEK293 cells was evaluated using a CCK-8 assay.

Strain LSDY01 belonged to B. licheniformis ST20, differing by only one allele from the prevalent ST3. Its closest relatives were the Daqu-derived strains CP143961.1 and CP143962.1. A unique horizontally acquired genomic island (~157 27 kb, GC 33.03%) and a putative type IV secretion system (T4SS) gene cluster were identified on the chromosome of this strain. A novel plasmid (pLSDY01), which is highly similar to environmental plasmids, harbors yopX, a toxin-antitoxin system, pilT, and a pistol ribozyme. LSDY01 was susceptible to imipenem and vancomycin but resistant to penicillin, erythromycin, and chloramphenicol. The CCK-8 assay revealed a non-significant trend toward reduced HEK293 cell viability after co-culture with LSDY01 (p = 0.0545 at 2 h of CCK-8 incubation).

Our findings suggest that horizontal gene transfer, including plasmid acquisition and potential phage integration, may have enabled B. licheniformis to evolve into a pathogen, highlighting the need to reassess the safety of traditionally non-pathogenic microbes.