Background Pharmaceutical cleanrooms remain vulnerable to microbial contamination introduced through environmental and personnel-associated pathways, yet the genomic characteristics and transmission dynamics of these contaminants are not well defined. Methods In this study, whole-genome sequencing (WGS) was applied to characterize microbial populations across a sterile vaccine production line. We adopted an integrated strategy that combined routine environmental monitoring with targeted genomic investigation, mapping the overall distribution of microorganisms and prioritizing isolates showing abnormal or recurrent detection for sequencing. By integrating SNP-based phylogeny, sampling location metadata, and ARG/virulence profiling. we identified five dominant opportunistic species, each displaying distinct genomic signatures indicative of different introduction pathways. Results By combining species-specific genomic features with their sampling distributions, we reconstructed four plausible contamination chains: clonal inward dissemination of Burkholderia contaminans from Grade B to Grade A areas, repeated water-associated introductions of Ralstonia pickettii , intermittent personnel-mediated seeding of Staphylococcus epidermidis , and sporadic external incursions of Microbacterium laevaniformans . ARGs and VFs were heterogeneously distributed, with mobile resistance determinants and colonization- or immune-evasion factors concentrated in B. contaminans and R. pickettii , suggesting elevated persistence potential under disinfectant pressure. Conclusion These findings demonstrate that WGS enables precise source attribution and transmission-route reconstruction beyond the capability of conventional typing methods, thereby supporting real-time contamination control and evidence-based microbial-risk management in pharmaceutical manufacturing.
Li et al. (Wed,) studied this question.