<i>In Vivo</i> -Selected Compensatory Mutations Restore the Fitness Cost of Mosaic <i>penA</i> Alleles That Confer Ceftriaxone Resistance in <i>Neisseria gonorrhoeae</i> (2018)

Resistance to ceftriaxone in <i>Neisseria gonorrhoeae</i> is mainly conferred by mosaic <i>penA</i> alleles that encode penicillin-binding protein 2 (PBP2) variants with markedly lower rates of acylation by ceftriaxone. To assess the impact of these mosaic <i>penA</i> alleles on gonococcal fitness, we introduced the mosaic <i>penA</i> alleles from two ceftriaxone-resistant (Cro<sup>r</sup>) clinical isolates (H041 and F89) into a Cro<sup>s</sup> strain (FA19) by allelic exchange and showed that the resultant Cro<sup>r</sup> mutants were significantly outcompeted by the Cro<sup>s</sup> parent strain <i>in vitro</i> and in a murine infection model. Four Cro<sup>r</sup> compensatory mutants of FA19 <i>penA41</i> were isolated independently from mice that outcompeted the parent strain both <i>in vitro</i> and <i>in vivo</i> One of these compensatory mutants (LV41C) displayed a unique growth profile, with rapid log growth followed by a sharp plateau/gradual decline at stationary phase. Genome sequencing of LV41C revealed a mutation (G348D) in the <i>acnB</i> gene encoding the bifunctional aconitate hydratase 2/2 methylisocitrate dehydratase. Introduction of the <i>acnB</i><sub><i>G348D</i></sub> allele into FA19 <i>penA41</i> conferred both a growth profile that phenocopied that of LV41C and a fitness advantage, although not as strongly as that exhibited by the original compensatory mutant, suggesting the existence of additional compensatory mutations. The mutant aconitase appears to be a functional knockout with lower activity and expression than wild-type aconitase. Transcriptome sequencing (RNA-seq) analysis of FA19 <i>penA41 acnB</i><sub><i>G348D</i></sub> revealed a large set of upregulated genes involved in carbon and energy metabolism. We conclude that compensatory mutations can be selected in Cro<sup>r</sup> gonococcal strains that increase metabolism to ameliorate their fitness deficit.<b>IMPORTANCE</b> The emergence of ceftriaxone-resistant (Cro<sup>r</sup>) <i>Neisseria gonorrhoeae</i> has led to the looming threat of untreatable gonorrhea. Whether Cro resistance is likely to spread can be predicted from studies that compare the relative fitnesses of susceptible and resistant strains that differ only in the <i>penA</i> gene that confers Cro resistance. We showed that mosaic <i>penA</i> alleles found in Cro<sup>r</sup> clinical isolates are outcompeted by the Cro<sup>s</sup> parent strain <i>in vitro</i> and <i>in vivo</i> but that compensatory mutations that allow ceftriaxone resistance to be maintained by increasing bacterial fitness are selected during mouse infection. One compensatory mutant that was studied in more detail had a mutation in <i>acnB</i>, which encodes the aconitase that functions in the tricarboxylic acid (TCA) cycle. This study illustrates that compensatory mutations can be selected during infection, which we hypothesize may allow the spread of Cro resistance in nature. This study also provides novel insights into gonococcal metabolism and physiology.