Shotgun metagenome sequencing and informatics can accurately form a metagenome-assembled genome (MAG) of the bacterial tier 1 select agent Burkholderia pseudomallei for rapid public health response events

Shotgun metagenomics, when sufficient read depth exists for each taxon, enables capturing metagenome-assembled genomes (MAGs) directly from a microbial community. In 2021, an aromatherapy spray contaminated with Burkholderia pseudomallei caused an outbreak of melioidosis in the United States. Metagenome-assembled genome binning depends in part on different nucleotide compositions, and the contaminated aromatherapy spray contained other bacteria, including related species (e.g., Burkholderia cepacia, Burkholderia cenocepacia, Burkholderia multivorans, Burkholderia pseudomultivorans, Cupriavidus pauculus, and Pseudomonas aeruginosa with average nucleotide identity (ANI) to B. pseudomallei being 84.2%, 84.4%, 84.7%, 84.8%, 75.7%, and 72.3%, respectively, and AAI being 79.5%, 79.8%, 80.5%, 80.4%, 62.5%, and 52.9%, respectively). We performed metagenomic sequencing on the contaminated aromatherapy spray to determine if a public metagenomic pipeline (https://github.com/nf-core/mag) can form a MAG of B. pseudomallei. Upon completion of the pipeline, inter- and intracontig comparisons revealed few potential contaminants of related taxa. Conservative removal of those contigs was especially valuable, ultimately obtaining an ANI of 99.9% between the B. pseudomallei MAG and the genome of an isolate from the aromatherapy spray. This underscores the importance of quality checking recovered MAGs (e.g., for congeneric chimerism) for high-resolution objectives such as outbreak pathogenomics. Importantly, our analysis revealed that the identical conclusion was made possible with the B. pseudomallei MAG (as with its corresponding isolate genome), which was that the aromatherapy B. pseudomallei originated from South Asia (specifically India). Because rapid read-based (k-mer) taxonomic classification methods often report false positives, this operational framework could be valuable for rapid biothreat radar detection systems in public health surveillance.IMPORTANCEIn 2021, an imported aromatherapy spray caused a U.S. outbreak of melioidosis after contamination with Burkholderia pseudomallei. Using shotgun metagenomics, we reconstructed a near-complete genome of the pathogen directly from the product, despite the presence of other related microbes. The assembled genome showed 99.9% similarity to a cultured isolate. This work demonstrates that metagenomics can recover high-quality pathogen genomes from complex samples, supporting outbreak investigations and enhancing public health surveillance.