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We tested the activity of ETX0914 against 187 Neisseria gonorrhoeae isolates from men with urethritis in Nanjing, China, in 2013. The MIC50, MIC90, and MIC range for ETX0914 were 0.03 μg/ml, 0.06 μg/ml, and ≤0.002 to 0.125 μg/ml, respectively. All isolates were resistant to ciprofloxacin, and 36.9% (69/187) were resistant to azithromycin. Of the isolates, 46.5% were penicillinase-producing N. gonorrhoeae (PPNG), 36% were tetracycline-resistant N. gonorrhoeae (TRNG), and 13% (24 isolates) had an MIC of 0.125 μg/ml for ceftriaxone. ETX0914 may be an effective treatment option for gonorrhea.
In the absence of an effective gonococcal vaccine, the prevention and control of gonorrhea depend on early diagnosis and effective antimicrobial therapy for patients at risk for acquiring infection and their sex partners. Currently, the first-line agents for the treatment of gonorrhea in most countries are extended-spectrum cephalosporins (ESCs), such as cefixime and ceftriaxone. However, gonococcal strains with reduced susceptibility or frank resistance to ESCs have emerged, and treatment failures with the oral agent cefixime (1,–5) and injectable ceftriaxone have been documented in several countries (6,–10).
ETX0914 (also known as AZD0914) is a novel spiropyrimidinetrione bacterial DNA gyrase/topoisomerase inhibitor, which functions by inhibiting DNA biosynthesis and stabilizing cleaved covalent complexes of gyrase and double-stranded broken DNA. This results in the blockage of religation of the double-strand cleaved DNA to form fused circular DNA (11). ETX0914 has displayed potent in vitro antibacterial activity against N. gonorrhoeae, including multidrug-resistant strains, certain Gram-positive organisms, such as Staphylococcus aureus and Streptococcus pneumoniae, and fastidious Gram-negative organisms, such as Haemophilus spp. and Moraxella catarrhalis, and also atypical and anaerobic organisms (12,–15). Cross-resistance between ETX0914 and fluoroquinolones has not been observed (16). The rates of resistance in N. gonorrhoeae to ciprofloxacin and other antimicrobials have been high in China (17,–21). Our previous study showed that 99.8% of the N. gonorrhoeae strains isolated from symptomatic men attending a sexually transmitted diseases (STD) clinic in Nanjing, China, between April 2011 and December 2012 were resistant to ciprofloxacin (22). Here, we investigated the in vitro activity of ETX0914 against 187 clinical gonococcal strains isolated from men with gonococcal urethritis attending this clinic a year later in 2013, and we compared the activity of ETX0914 to that of other currently or previously used antimicrobials administered for the treatment of gonorrhea.
The Gram stains of 177/187 urethral exudates showed polymorphonuclear leukocytes (PMNs) and Gram-negative intracellular diplococci, criteria that are highly specific (>99%) for gonococcal infection (23–24). Urethral swab specimens from men with urethritis were also inoculated onto Thayer-Martin medium (DL Biotech, China) and cultured in candle jars at 36°C. Gonococcal isolates were identified by colonial morphology, Gram stain, and oxidase testing. The MICs (in micrograms per milliliter) of N. gonorrhoeae to ETX0914, penicillin, tetracycline, ciprofloxacin, spectinomycin, azithromycin, cefixime, and ceftriaxone were determined by agar gel dilution. N. gonorrhoeae ATCC 49226, WHO references A, G, and J, and a ceftriaxone-resistant strain (25) were used as quality controls. MIC breakpoints for assigning susceptible, intermediate, and resistance statuses to N. gonorrhoeae isolates for penicillin, tetracycline, ciprofloxacin, spectinomycin, cefixime, and ceftriaxone were determined according to CLSI standards (26). Interpretative criteria from the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (www.eucast.org) (27) were used for azithromycin. The production of β-lactamase was identified by filter paper acidometric testing that used bromocresol purple as a pH indicator (28). IBM SPSS Statistics version 22.0 was used for statistical analysis. The Mann-Whitney test was used for pairwise comparisons between groups.
All isolates of N. gonorrhoeae were inhibited by concentrations of ≤0.125 μg/ml of ETX0914; the MIC50, MIC90, and MIC range of ETX0914 were 0.03 μg/ml, 0.06 μg/ml, and ≤0.002 to 0.125 μg/ml (Table 1), respectively. The results of susceptibility testing of other antibiotics are shown in Table 2. All isolates were resistant to ciprofloxacin (MIC, ≥1 μg/ml); the MIC50 and MIC90 were both ≥8 μg/ml. The MICs of gonococcal strains for ETX0914 were lower than those for ciprofloxacin (P < 0.0001), with a median 256-fold difference. The MIC distributions of ETX0914 and ciprofloxacin for all isolates are shown in Fig. 1.
Of the isolates, 36.9% (69/187) were resistant to azithromycin (MIC, ≥1 μg/ml), and nine displayed high-level azithromycin resistance (MIC, ≥256 μg/ml). Also, 74.3% (139/187) and 87.2% (163/187) of isolates were resistant to penicillin and tetracycline, respectively. Of the isolates, 46.5% (87/187) were penicillinase-producing N. gonorrhoeae (PPNG), and 36.4% (68/187) were tetracycline-resistant N. gonorrhoeae (TRNG). The MIC50 and MIC90 for ETX0914 were 0.03 μg/ml and 0.06 μg/ml, respectively, for β-lactamase-producing isolates, which are the same as or lower than those for β-lactamase-negative strains. Of the isolates, 28.9% (54/187) were resistant to ciprofloxacin, penicillin G, tetracycline, and azithromycin.
All isolates were susceptible to spectinomycin (MIC, ≤32 μg/ml), cefixime (MIC, ≤0.25 μg/ml), and ceftriaxone (MIC, ≤0.125 μg/ml). However, 5 isolates (2.7%) had elevated MICs for cefixime (≥0.125 μg/ml), and 24 isolates (12.8%) had an MIC of 0.125 μg/ml for ceftriaxone (the breakpoint). The MIC90s for ETX0914 and cefixime were the same (0.06 μg/ml), but the MIC90 for ceftriaxone was greater (0.125 μg/ml). The MIC for ETX0914 for the Spanish ceftriaxone-resistant strain was 0.06 μg/ml.
We tested the antimicrobial activity of a new spiropyrimidinetrione molecule, ETX0914 (also known as AZD0914), against clinical gonococcal isolates. A high prevalence of resistance was observed for antimicrobials that had been used in the past but that have been put aside. Moreover, 28.8% of the isolates showed multidrug resistance, i.e., resistance to ciprofloxacin, penicillin, tetracycline, and azithromycin; however, all isolates were inhibited by ≤0.125 μg/ml ETX0914. Isolates of N. gonorrhoeae with borderline high MICs for ceftriaxone (0.125 μg/ml) had corresponding MICs for ETX0914 that were 8- to 21-fold lower (0.016 to 0.06 μg/ml). The in vitro results of this study demonstrated that the antimicrobial activity of ETX0914 was superior to that of ciprofloxacin and several other antimicrobials currently or previously recommended for the treatment of gonorrhea. ETX0914, like ciprofloxacin, is a type II topoisomerase poison, but the interactions with DNA gyrase subunits differ between the two antibiotics (11).
Our results concur with those of other studies (13, 14) that have shown ranges of MICs, MIC50, and MIC90 of ETX0914 for international reference strains of N. gonorrhoeae and clinical isolates from European countries to be 0.002 to 0.25 μg/ml, 0.064 to 0.125 μg/ml, and 0.125 to 0.25 μg/ml, respectively.
In conclusion, ETX0914 has excellent antimicrobial activity against clinical isolates of N. gonorrhoeae, including high-level ciprofloxacin-resistant and otherwise multiresistant strains. ETX0914 may be an effective treatment option for gonorrhea.
We thank J. Camara for providing the ceftriaxone-resistant N. gonorrhoeae isolate.
X.-H.S. directed the project, supervised the bacterial isolate collection, and prepared the manuscript. W.-J.L., Y.-R.L., C.W., and S.L. carried out the antimicrobial susceptibility testing and identification of the tetM- and β-lactamase gene-producing plasmid. B.-X.W. directed the project and consulted on manuscript preparation. P.A.R., R.A.A., and J.P.M. consulted on the project and manuscript preparation. All authors read and approved the final manuscript.
We declare no conflicts of interest.
This work was supported in part by NIH/NIAID grant no. U19AI084048.