Background The aetiological bacterial agent of gonorrhoea, Neisseria gonorrhoeae, has become resistant to each of the first-line antibiotics used to treat it, including ciprofloxacin. One diagnostic approach to identify ciprofloxacin-susceptible isolates is to determine codon 91 in the gene encoding the A subunit of DNA gyrase, gyrA, where coding for the wild-type serine (gyrA91S) is associated with ciprofloxacin susceptibility and phenylalanine (gyrA91F) with resistance. The aim of this study was to investigate the possibility of diagnostic escape from gyrA susceptibility testing. Methods We used bacterial genetics to introduce pairwise substitutions in GyrA positions 91 (S or F) and 95 (D, G, or N), which is a second site in GyrA associated with ciprofloxacin resistance, into five clinical isolates of N gonorrhoeae. All five isolates encoded GyrA S91F, an additional substitution in GyrA at position 95, substitutions in ParC that are known to cause an increased minimum inhibitory concentration (MIC) to ciprofloxacin, and GyrB 429D, which is associated with susceptibility to zoliflodacin (a spiropyrimidinetrione-class antibiotic in phase 3 trials for treatment of gonorrhoea). We evolved these isolates to assess for the existence of pathways to ciprofloxacin resistance (MIC >= 1 pg/mL) and measured MICs for ciprofloxacin and zoliflodacin. In parallel, we searched metagenomic data for 11 355 N gonorrhoeae clinical isolates with reported ciprofloxacin MICs that were publicly available from the European Nucleotide Archive for strains that would be identified as susceptible by gyrA codon 91-based assays. Findings Three clinical isolates of N gonorrhoeae with substitutions in GyrA position 95 associated with resistance (G or N) maintained intermediate ciprofloxacin MICs (0 center dot 125-0 center dot 5 pg/mL), which has been associated with treatment failure, despite reversion of GyrA position 91 from phenylalanine to serine. From an in-silico analysis of the 11 355 genomes from N gonorrhoeae clinical isolates, we identified 30 isolates with gyrA codon 91 encoding a serine and a ciprofloxacin resistance-associated mutation at codon 95. The reported MICs for these isolates varied from 0 center dot 023 pg/mL to 0 center dot 25 pg/mL, including four with intermediate ciprofloxacin MICs (associated with substantially increased risk of treatment failure). Finally, through experimental evolution, one clinical isolate of N gonorrhoeae bearing GyrA 91S acquired ciprofloxacin resistance through mutations in the gene encoding for the B subunit of DNA gyrase (gyrB) that also conferred reduced susceptibility to zoliflodacin (ie, MIC >= 2 pg/mL). Interpretation Diagnostic escape from gyrA codon 91 diagnostics could occur through either reversion of the gyrA allele or expansion of circulating lineages. N gonorrhoeae genomic surveillance efforts might benefit from including gyrB, given its potential for contributing to ciprofloxacin and zoliflodacin resistance, and diagnostic strategies that reduce the likelihood of escape, such as the incorporation of multiple target sites, should be investigated. Diagnostics that guide antibiotic therapy can have unintended consequences, including novel resistance determinants and antibiotic cross-resistance. Funding US National Institutes of Health National Institute of Allergy and Infectious Diseases, National Institute of General Medical Sciences, and the Smith Family Foundation. Copyright (c) 2023 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license.
