Shotgun metagenome sequencing and informatics can accurately form a metagenome-assembled genome (MAG) of the bacterial tier 1 select agent <i>Burkholderia pseudomallei</i> 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 <i>Burkholderia pseudomallei</i> 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., <i>Burkholderia cepacia</i>, <i>Burkholderia cenocepacia</i>, <i>Burkholderia multivorans</i>, <i>Burkholderia pseudomultivorans</i>, <i>Cupriavidus pauculus</i>, and <i>Pseudomonas aeruginosa</i> with average nucleotide identity (ANI) to <i>B. pseudomallei</i> 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 <i>B. pseudomallei</i>. 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 <i>B. pseudomallei</i> 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 <i>B. pseudomallei</i> MAG (as with its corresponding isolate genome), which was that the aromatherapy <i>B. pseudomallei</i> 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 <i>Burkholderia pseudomallei</i>. 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.