36 37 The unmet medical need for novel intervention strategies to treat Neisseria gonorrhoeae 38 infections is significant, and is increasing as rapidly emerging resistance in this pathogen is 39 threatening to eliminate currently available treatment options. AZD0914 is a novel bacterial 40 gyrase inhibitor that possesses potent in vitro activity against high-level ciprofloxacin and 41 extended-spectrum cephalosporin resistant isolates and is currently in clinical development 42 for the treatment of N. gonorrhoeae infections. The propensity to develop resistance against 43 AZD0914 was examined in N. gonorrhoeae and found to be extremely low, a finding 44 supported by similar studies in Staphylococcus aureus. Genetic characterization of both first 45 step and second step mutants that exhibited decreased susceptibility to AZD0914 identified 46 substitutions in the conserved GyrB TOPRIM domain, confirming DNA gyrase as the primary 47 target of AZD0914 and providing differentiation from fluoroquinolones. Analysis of available 48 bacterial gyrase and topoisomerase IV structures including those bound to fluoroquinolone 49 and non-fluoroquinolone inhibitors has allowed the rationalization of the lack of cross50 resistance that AZD0914 shares with fluoroquinolones. Microbiological susceptibility data 51 also indicate that the topoisomerase inhibition mechanisms are subtly different between 52 N. gonorrhoeae and other bacterial species. Taken together, these data support the 53 progression of AZD0914 as a novel treatment option for the oral treatment of 54 N. gonorrhoeae infections. 55 56 57 on Jne 4, 2017 by gest httpaac.asm .rg/ D ow nladed fom INTRODUCTION 58 Neisseria gonorrhoeae is the obligate human pathogen responsible for the sexually 59 transmitted disease gonorrhea, an extremely prevalent infection, with global estimates by the 60 World Health Organization in 2012 exceeding 100 million infections per year (1). Although 61 N. gonorrhoeae infections were once relatively easily treated with sulfonamides or penicillin, 62 the emergence and spread of multi-drug resistant isolates has increased the difficulty of 63 effective treatment and novel therapeutic alternatives are needed (2). 64 65 Several fluoroquinolone drugs, notably ciprofloxacin, were used widely for the treatment of 66 gonorrhea starting in the early 1990s. These classes of antibacterial agents are potent DNA 67 synthesis inhibitors that target the bacterial Type II topoisomerases, DNA gyrase and 68 Topoisomerase IV, which are essential for bacterial DNA replication, recombination, and 69 separation of daughter chromosomes. However, N. gonorrhoeae isolates that were resistant 70 to ciprofloxacin arose quickly and treatment guidelines needed to be amended as effective 71 treatment of infections was being compromised. Indeed, the global burden of ciprofloxacin72 resistant N. gonorrhoeae isolates is now significant and is between 35% and >95% in many 73 countries (3-10) although the level in the USA is slightly lower at 15% (11). Ciprofloxacin 74 resistance in N. gonorrhoeae, like in other species, is typically manifested by point mutations 75 in the quinolone-resistance-determining region (QRDR) of the A subunits of the Type II 76 topoisomerases (GyrA and ParC) (5, 12-14). However, although in other species like 77 Staphylococcus aureus and Escherichia coli where low level fluoroquinolone resistance can 78 be manifested through point mutations in the topoisomerase B subunits, these mutations are 79 rarely identified in N. gonorrhoeae (15, 16). The high resistance to fluoroquinolones has 80 resulted in the removal of this class of drug from treatment guidelines to manage 81 N. gonorrhoeae infections. Intramuscular ceftriaxone, an expanded-spectrum cephalosporin 82 that targets the penicillin-binding proteins essential for bacterial cell wall synthesis, now 83 represents the last treatment option for the management of N. gonorrhoeae infections. 84 However, organisms with decreased susceptibility to these cephalosporins are now 85 emerging with several reports of high-level resistance to these drugs (5, 17-19), essentially 86 removing the last option for empirical therapy. Furthermore, even though drugs like 87 ciprofloxacin are no longer widely used to treat gonorrhea, resistance to this drug persists in 88 these multi-drug-resistant pathogens and has resulted in patients having fewer effective 89 treatment options (3-10). Taken together, this has created an urgent need for the discovery 90 and development of novel, first-in-class oral agents that can successfully treat infections 91 caused by N. gonorrhoeae carrying pre-existing resistance caused by long term 92 fluoroquinolone and cephalosporin usage. 93 94 on Jne 4, 2017 by gest httpaac.asm .rg/ D ow nladed fom AZD0914 is a novel spiropyrimidinetrione that is active against N. gonorrhoeae via inhibition 95 of DNA synthesis (20) and is equally effective against isolates that are both resistant and 96 susceptible to existing agents, including international refernce strains and the 2008 WHO 97 reference panel (16). Given the propensity for N. gonorrhoeae to develop resistance, in this 98 study we examined the ability of AZD0914 to suppress the emergence of resistance in vitro 99 in N. gonorrhoeae isolates. Finally, we also evaluated the propensity of resistance 100 emergence in isolates that carried resistance to fluoroquinolones and cephalosporins to 101 understand if there was a variation in these populations that could impact the utility of this 102 drug in the future. 103 104 MATERIALS AND METHODS 105 Bacterial Strains. The recent N. gonorrhoeae clinical isolates (ARC4673, ARC4676, and 106 ARC4677) were obtained from a clinic in Nanjing, China. The high-level ceftriaxone107 resistant isolate, first described by Camara et al. (17) was kindly provided by Dr. Ardanuy 108 from the L'Hospitalet de Llobregat in Barcelona, Spain and is referred to as ARC4682. The 109 three Chinese clinical isolates all contained the same ’A’ nucleotide deletion in the mtrR 110 promoter region as has been described for the ceftriaxone-resistant isolate (17). The 111 reference isolate ATCC 49226 was obtained from the American Type Culture Collection, and 112 was found not to contain a deletion in the mtrR promoter region. 113 Antimicrobial susceptibility testing. The minimum inhibitory concentration (MIC) against 114 each isolate was determined using agar dilution methodology according to Clinical 115 Laboratory Standards Institute (CLSI) documents M07-A9 and M100-S23 (21, 22). An 116 appropriate reference compound was included in each test, and N. gonorrhoeae ATCC 117 49226 was used as the quality control reference isolate (The CLSI approved QC range for 118 AZD0914 has been determined to be 0.06 – 0.5 μg/mL). AZD0914 was dissolved at high 119 concentrations in dimethyl suloxide (DMSO) and diluted. Ciprofloxacin was obtained from 120 MP Biomedicals (Santa Ana, CA). 121 Frequency of resistance studies. A suspension of cells (~OD600 3.2) was made from a 122 freshly grown (24hr) chocolate agar plate. Dilutions were plated on compound-free plates to 123 determine the CFU/mL of the suspension. First step spontaneous resistance frequencies 124 were determined after plating 100μL aliquots onto 1x GC agar (BD, Franklin Lakes, NJ) plus 125 Isovitalex (BD, Franklin Lakes, NJ) containing AZD0914 at 2-fold increasing concentrations 126 of the agar dilution MIC. Plates were incubated at 36C  1 C in 5%CO2 for 48hr. The 127 number of colonies that grew were used to calculate resistance frequencies. If no colonies 128 were observed the frequency of resistance was expressed as less than the frequency of 129 obtaining one resistant variant. Subsequent selection experiments were performed on GC 130 agar supplemented with 1% hemoglobin (BD, Franklin Lakes, NJ) as a higher burden on 131 on Jne 4, 2017 by gest httpaac.asm .rg/ D ow nladed fom bacteria could be evaluated and were repeated on multiple test occasions to ensure that 132 mutants that were obtained represented independent events and were not siblings. 133 Whole genome sequencing and analysis. Genomic DNA purified on the Maxwell 16 134 platform (Promega, Madison WI) and quantitated using the Qubit fluormeter (Invitrogen Life 135 Technologies, Grand Island, NY) was used as input material for library construction. DNA 136 libraries were prepared using the Nextera library construction protocol (Illumina, San Diego, 137 CA, USA) following the manufacturer’s instructions and sequenced on a MiSeq Sequencer 138 (Illumina, San Diego, CA, USA). For each isolate, approximately 2.5 million 150 bp paired 139 end sequence reads were de novo assembled and analyzed using the CLCBio suite of 140 software tools (Cambridge, MA, USA). The Multi Locus Sequence Type (MLST) profiles of 141 the isolates were determined by comparison of the seven allele sequences against the 142 curated public database available at http://pubmlst.org/neisseria and they were also typed 143 using the NG-MAST database available at http://www.ng-mast.net 144 Molecular analysis and transformation. The gyrB gene was amplified from strain 4676145 D1 using the For (5’-CCACAAGGTGCGGACTGTTTTGGC-3’) and 146 Rev (5’-AAAGGAGACGCGGCAAGCTGCCCG-3’) oligonucleotides (Eurofins MWG Biotech, 147 Huntsville, AL) in a standard polymerase chain reaction (PCR) using the High Fidelity PCR 148 mix (Roche, Nutley, NJ). Cycling conditions were a 30 second denaturation at 94°C, a 30 149 second annealing step at 55°C, followed by a 3 minute extension at 72°C. The polymerase 150 chain reaction (PCR) product was purified using a QIAquick PCR Purification kit (Qiagen, 151 Valencia, CA). Transformation in N. gonorrhoeae was performed by spotting 20 μL of DNA 152 in water (representing between 1 μg and 10 μg DNA) onto a GC agar plate (BD) and, after 153 allowing the DNA solution to dry onto the plate, streaking the recipient N. gonorrhoeae 154 strain, ATCC 49