INTRODUCTION. Neisseria gonorrhoeae has developed resistance to all available drugs, with sporadic reports of concerning multi-drug resistant clones circulating internationally. The World Health Organisation has identified N. gonorrhoeae as a priority organism for enhanced, quality-assured antimicrobial resistance (AMR) surveillance, given that detection of resistance and decreased susceptibility in circulating strains is critical to maintain effective antimicrobial stewardship. However, public health surveillance needs to balance cost with the level of data extracted. Amplicon panels offer the ability to perform wide AMR genomic surveillance using fewer resources than whole genome sequencing. Here we developed and assessed a molecular AMR screening workflow for N. gonorrhoeae characterisation, including antimicrobial resistance markers.
METHODS. In collaboration with New Zealand and Queensland government health departments, a multi-plex PCR was developed using primers targeting 15 genes from NG-MAST, Neisseria MLST, and NG-STAR typing schemes, and plasmid-mediated penicillin resistance. An automated pipeline was established to process NextSeq Illumina sequences and implement downstream bioinformatic analysis, specifically looking for N. gonorrhoeae antimicrobial resistance markers. Validation in silico and wet lab verification against local and global N. gonorrhoeae sequences and isolates showed that the method had 100% typeability across each target, able to generate concordant MLST, NG STAR and NG MAST genotypes across a genetically diverse sample set of 123 isolates. An automated sequencing pipeline was established to process NextSeq Illumina sequence data, and implement downstream bioinformatic analysis, specifically looking for N. gonorrhoeae resistance markers.
RESULTS. All 17 primers in 123 samples successfully yielded correct ST for NG-MAST, NG-STAR and MLST. Submission into globally maintained typing schemes allowed for prediction of resistance phenotype, and identification of novel resistance types. This method offers a scalable and cost-effective approach to performing molecular surveillance for N. gonorrhoeae, which is otherwise cost prohibitive to many public health laboratories.
CONCLUSION. This work demonstrates viability and utility of a broad but affordable AMR surveillance pipeline. This will be implemented into routine use as part of Queensland Health’s molecular surveillance of N. gonorrhoeae isolates. This pipeline will inform on emerging N. gonorrhoeae resistance to major antibiotics in Queensland, and underpin enhanced antimicrobial stewardship.