Impact of Fluoroquinolone Resistance Mutations on Gonococcal Fitness and In Vivo Selection for Compensatory Mutations

(See the editorial commentary by Dillon and Parti, on pages 1775–7.) Neisseria gonorrhoeae is a Gram-negative diplococcus that plays a major role in urogenital tract and perinatal infections [1]. Gonorrhea is the second most frequently reported bacterial sexually transmitted infection (STI) in the United States, with an estimated 700 000 new cases each year. The rate of gonorrhea is highest among females aged <25 years old, especially those that engage in high-risk sexual behaviors [2]. The high prevalence of gonorrhea is particularly concerning because it is a major cause of pelvic inflammatory disease and can lead to serious outcomes such as ectopic pregnancy, infertility, and chronic pelvic pain. Gonorrhea is also a cofactor in human immunodeficiency virus transmission [3]. Antibiotic resistance emerges rapidly in N. gonorrhoeae, which challenges treatment options and threatens current control measures. Currently, only a single class of antibiotics, the third-generation cephalosporins, is recommended for routine treatment of gonorrhea in the Centers for Disease Control and Prevention (CDC) sexually transmitted disease guidelines [2]. The status of N. gonorrhoeae as a “superbug” continues to increase with the recent isolation of a ceftriaxone-resistant strain [4]. Fluoroquinolones were removed from the CDC list of recommended first-line antibiotics for treatment of gonorrhea in 2007 [5]. First used clinically in the mid-1980s [6], over 80% of gonococcal isolates in the western Pacific region were ciprofloxacin resistant (CipR) by 2002 [7, 8]. A steady increase in quinolone-resistant N. gonorrhoeae (QRNG) followed in the United States, with 891 of 6009 (14.8%) clinical isolates identified as QRNG in 2007 [9]. Resistance to this relatively inexpensive class of antibiotics, which target topoisomerase II (DNA gyrase) and topoisomerase IV [6], in N. gonorrhoeae is due to amino acid substitutions in the quinolone resistance–determining regions (QRDRs) of the A subunits of DNA gyrase (GyrA) and ParC, the A subunit of DNA topoisomerase IV [10–13]. Traditionally, antibiotic resistance is accompanied by a fitness loss, particularly when it occurs via mutation of genes that are important for basic cellular functions [14]. Some resistance mutations, however, provide a growth benefit in vitro or increase fitness when tested in pathogenesis models [15–19]. This phenomenon can be due to compensatory mutations that balance the detrimental effects of resistance mutations [20]. Increased microbial fitness during infection can also be a direct consequence of the resistance mutation, as shown for mutations that increase the efflux of antimicrobial substances [15, 18]. For example, in N. gonorrhoeae, multitransferable resistance (mtr) mutations, defined here as mutations in the mtrR repressor gene or its promoter region, increase resistance to macrolide antibiotics and high levels of penicillin G through overexpression of the MtrC-MtrD-MtrE active efflux pump. The mtr mutations also confer a fitness advantage during experimental infection of female mice [18], which is most likely due to increased resistance to host innate effectors that are also substrates of the MtrC-MtrD-MtrE active efflux pump [21–23]. Whether fluoroquinolone resistance mutations also promote increased in vivo fitness of N. gonorrhoeae is not known. This question warrants testing based on the wide prevalence of QRNG strains and reports that gyrA and gyrA, parC mutations in some pathogens confer a fitness advantage in animal models [16, 17]. To further explore the rapid spread of QRNG worldwide, here we introduced commonly isolated gyrA and parC mutations into a wild-type N. gonorrhoeae strain and tested the consequence on gonococcal fitness in vitro and in the mouse infection model. We also examined the effect of these mutations in an mtr mutant of the same strain background to determine if the fitness benefit conferred by overproduction of the MtrC-MtrD-MtrE active efflux pump is lost or further increased in QRNG.