Development of Rapid Multilocus Amplicon Sequencing Assay for the Detection of Antimicrobial Resistance Markers in Burkholderia pseudomallei

Burkholderia pseudomallei, the causative agent of melioidosis, is a select agent due to its threat not only to public health but also as a potential bioterrorism agent. In recent years, amplicon sequencing has proven a useful... [ view full abstract ]

Burkholderia pseudomallei, the causative agent of melioidosis, is a select agent due to its threat not only to public health but also as a potential bioterrorism agent. In recent years, amplicon sequencing has proven a useful tool in rapidly detecting and characterizing B. pseudomallei due to its high throughput format and ability to detect very low target loads in clinical and environmental sample types. Previously, we developed a 68 amplicon sequencing assay that allowed for detection of and differentiation within B. pseudomallei, as well as differentiation from B. mallei and other near neighbor species. These 68 loci also allowed for characterization of virulence genes in B. pseudomallei. We have now developed a 10-plex Multi-locus Amplicon Sequencing (AmpSeq) assay that targets antimicrobial resistance (AMR) mechanisms. These genetic mechanisms include enzymatic inactivation, efflux pump substrate affinities and gene expression modulation. These genetic changes confer resistance to multiple different antibiotics, including Ceftazidime, Co-trimoxazole, Trimethoprim, and Amoxicillin-clavulanic acid. This amplicon sequencing system employs a universal amplicon indexing system along with multiplex amplification reactions allowing for sequencing of hundreds of samples across numerous targets within a single Illumina MiSeq sequencing run. The AMR targets were validated via screening across a strain collection with both known and unknown resistance profiles. These targets were subsequently run across a larger set of 540 Burkholderia spp. Isolates for further analysis. Rapid detection of antibiotic resistance in B. pseudomallei could lead to improved diagnostics in the clinic, and therefore a more effective treatment.