Background Approximately 106 million cases of gonorrhoea occur worldwide each year. Gonorrhea significantly affects reproductive health and increases transmission of HIV. Antibiotic treatment is a critical control measure; however, this strategy is threatened by the rapid evolution of resistance in Neisseria gonorrhoeae(Gc). Gc susceptibility to ceftriaxone, the last remaining option for antibiotic monotherapy, has decreased globally over the last decade. Recently Gc has been elevated to “superbug” status due to the emergence of ceftriaxone-resistant (CRO^R) strains. Dual antibiotic therapy is now recommended in the USA and Europe. Ceftriaxone resistance in Gc is conferred primarily by mosaic penA alleles that encode an altered penicillin-binding protein 2 with up to 70 amino acid substitutions. Whether acquisition of these mosaic alleles is accompanied by a fitness cost is unknown.

Methods and Results Here we examined the impact of mosaic penA alleles from two well-characterised CRO^R clinical isolates, H041 (MIC = 2–4 µg/ml) and F89 (MIC = 1–2 µg/ml), on Gcfitness in vitro and in vivo. The wild-type penA allele of laboratory strain FA19 (CRO^S) was replaced by penA41or penA89 to create mutants FA19 penA41 and FA19 penA89, respectively. Acquisition of the mosaic alleles increased ceftriaxone resistance ≥ 500-fold. Both mutants grew significantly slower than FA19 in liquid culture. When cultured competitively with the parent strain, FA19 penA41 and FA19 penA89 demonstrated a fitness defect, as measured by competitive index. Mutants were attenuated relative to the parent strain during competitive murine infection. However, only CRO^R bacteria were recovered at later time points from 3 of 7 mice co-inoculated with FA19 penA41andFA19, suggesting selection of compensatory mutations in vivo.

Conclusions Acquisition ofmosaic alleles significantly reduced fitness of Gc, but compensatory mutations can be selected in vivo that alleviate fitness defects while maintaining resistance. Our studies may be useful in predicting the national and international spread of CRO^R Gc.