The Centers for Disease Control and the World Health Organization have issued a list of priority pathogens for which there are dwindling therapeutic options, including antibiotic-resistant Neisseria gonorrheae, for which novel oral agents are urgently needed. Zoliflodacin, the first in a new class of antibacterial agents called the spiropyrimidinetriones, is being developed for the treatment of gonorrhea. It has a unique mode of inhibition against bacterial type II topoisomerases with binding sites in bacterial gyrase that are distinct from those of the fluoroquinolones. Zoliflodacin is bactericidal, with a low frequency of resistance and potent antibacterial activity against N. gonorrheae, including multi-drug-resistant strains (MICs ranging from <= 0.002 to 0.25 mu g/mL). Although being developed for the treatment of gonorrhea, zoliflodacin also has activity against Gram-positive, fastidious Gram-negative, and atypical pathogens. A hollow-fiber infection model using S. aureus showed that that pharmacokinetic/pharmacodynamic index of fAUC/MIC best correlated with efficacy in in vivo neutropenic thigh models in mice. This data and unbound exposure magnitudes derived from the thigh models were subsequently utilized in a surrogate pathogen approach to establish dose ranges for clinical development with N. gonorrheae. In preclinical studies, a wide safety margin supported progression to phase 1 studies in healthy volunteers, which showed linear pharmacokinetics, good oral bioavailability, and no significant safety findings. In a phase 2 study, zoliflodacin was effective in treating gonococcal urogenital and rectal infections. In partnership with the Global Antibiotic Research Development Program (GARDP), zoliflodacin is currently being studied in a global phase 3 clinical trial. Zoliflodacin represents a promising new oral therapy for drug-resistant infections caused by N. gonorrheae.

Next-generation sequencing (NGS) enables clinical microbiology assays such as molecular typing of bacterial isolates which is now routinely applied for infection control and epidemiology. Additionally, feasibility for NGS-based identification of antimicrobial resistance (AMR) markers as well as genetic prediction of antibiotic susceptibility testing results has been demonstrated. Various bioinformatics approaches enabling NGS-based clinical microbiology assays exist, but standardized, computationally efficient and scalable sample-to-results workflows including validated quality control parameters are still lacking. Bioinformatics analysis workflows based on k-mers have been shown to allow for fast and efficient analysis of large genomics data sets as obtained from microbial sequencing applications. We here demonstrate applicability of k-mer based clinical microbiology assays for whole-genome sequencing (WGS) including variant calling, taxonomic identification, bacterial typing as well as AMR marker detection. The wet-lab and dry-lab workflows were developed and validated in line with Clinical Laboratory Improvement Act (CLIA) guidelines for laboratory-developed tests (LDTs) on multi-drug resistant ESKAPE pathogens. The developed k-mer based workflow demonstrated >= 99.39% repeatability, >= 99.09% reproducibility and >= 99.76% accuracy for variant calling and applied assays as determined by intra-day and inter-day triplicate measurements. The limit of detection (LOD) across assays was found to be at 20x sequencing depth and 15x for AMR marker detection. Thorough benchmarking of the k-mer based workflow revealed analytical performance criteria are comparable to state-of-the-art alignment based workflows across clinical microbiology assays. Diagnostic sensitivity and specificity for multilocus sequence typing (MLST) and phylogenetic analysis were 100% for both approaches. For AMR marker detection, sensitivity and specificity were 95.29 and 99.78% for the k-mer based workflow as compared to 95.17 and 99.77% for the alignment-based approach. Summarizing, results illustrate that k-mer based analysis workflows enable a broad range of clinical microbiology assays, potentially not only for WGS-based typing and AMR gene detection but also genetic prediction of antibiotic susceptibility testing results.