To evaluate the application of metagenomic sequencing in identification and tracking of drug-contaminating bacteria.

Both metagenomic sequencing and isolation culturing method were employed to identify and genotype bacteria in two batches of possibly contaminated gel medicines as well as the environmental samples from testing laboratory. Bacteria isolated by culturing method were identified through Gram’s stain，biochemical reaction，MALIDI-TOF-MS，16srDNA sequencing and Bacillus specific PCR. The detected Bacillus cereus were typed by multilocus sequence typing (MLST) .The metagenomic sequencing results were analyzed at the strain level using MetaPhlan software.

Both MLST and MetaPhlan analysis revealed a substantial contamination of Bacillus cereus in gel medicines as well as environmental samples，with the presence of the same Bacillus cereus group. Both metagenomic sequencing and culturing methods detected Paenibacillus，Brevibacillus and Neobacillus in gel medicines，but amplicon sequencing also detected these bacteria in environment which might be the source of contamination.

These findings indicate that metagenomic sequencing technology is reliable and has the advantage over isolation culturing in comprehensively detecting potentially hazardous bacterial species and accurately performing strain-level traceability analysis. It holds significant application value in pharmaceutical contamination identification and traceability.