Neisseria gonorrhoeae diagnostic escape from a gyrA-based test for ciprofloxacin susceptibility and the effect on zoliflodacin resistance: a bacterial genetics and experimental evolution study.
Neisseria gonorrhoeae diagnostic escape from a gyrA-based test for ciprofloxacin susceptibility and the effect on zoliflodacin resistance: a bacterial genetics and experimental evolution study.
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淋病的奈瑟氏菌诊断从基于GYRA的环丙沙星易感性以及对Zoliflodacin耐药的影响的逃脱：一种细菌遗传学和实验进化研究。
DOI：
10.1016/s2666-5247(22)00356-1
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发表时间：
2023-04
期刊：
影响因子：
38.2
通讯作者：
Grad, Yonatan H.
中科院分区：
文献类型：
--
作者：
Rubin, Daniel H. F.;Mortimer, Tatum;Grad, Yonatan H.
淋病的病原体淋病奈瑟氏菌已经对用于治疗它的每一种一线抗生素产生耐药性，包括环丙沙星。鉴定环丙沙星敏感分离株的一种诊断方法是确定编码DNA促旋酶A亚基gyrA的基因中的密码子91，其中编码野生型丝氨酸（gyrA 91 S）与环丙沙星敏感性相关，编码苯丙氨酸（gyrA 91 F）与耐药性相关。本研究的目的是调查gyrA药敏试验诊断逃逸的可能性。我们使用细菌遗传学在GyrA位点91（S或F）和95（D、G或N）（GyrA中与环丙沙星耐药性相关的第二个位点）中引入成对取代，进入5个淋病奈瑟菌临床分离株。所有五个分离株编码GyrA S91 F，GyrA第95位的额外取代，ParC中已知导致对环丙沙星的最小抑制浓度（MIC）增加的取代，以及GyrB 429 D，其与对唑氟沙星（治疗淋病的3期试验中的螺嘧啶三酮类抗生素）的敏感性相关。我们对这些分离株进行了进化，以评估是否存在环丙沙星耐药途径（MIC ≥1 μg/mL），并测量了环丙沙星和唑氟沙星的MIC。与此同时，我们检索了11 355 N淋病临床分离株的宏基因组数据，这些分离株具有报告的环丙沙星MIC，这些MIC可从欧洲核苷酸档案中公开获得，用于通过基于gyrA密码子91的测定鉴定为敏感的菌株。尽管GyrA 91位从苯丙氨酸逆转为丝氨酸，但3株GyrA 95位置换与耐药性（G或N）相关的淋病奈瑟菌临床分离株维持了中等水平的环丙沙星MIC（0.125 - 0.5 μg/mL），这与治疗失败相关。通过对11355个淋病奈瑟菌临床分离株基因组的计算机模拟分析，我们确定了30个分离株，其中gyrA密码子91编码丝氨酸，密码子95处存在环丙沙星耐药相关突变。报告的这些分离株的MIC范围为0.023 μg/mL至0.25 μg/mL，包括4株中等环丙沙星MIC（与治疗失败风险大幅增加相关）。最后，通过实验进化，1株携带GyrA 91 S的淋病奈瑟菌临床分离株通过编码DNA促旋酶B亚基（gyrB）的基因突变获得了环丙沙星耐药，这也降低了对唑氟沙星的敏感性（即MIC ≥2 μg/mL）。gyrA密码子91诊断的诊断逃逸可通过gyrA等位基因的逆转或循环谱系的扩增而发生。淋病奈瑟菌基因组监测工作可能受益于包括gyrB，鉴于其潜在的贡献环丙沙星和唑氟沙星耐药性，和诊断策略，减少逃逸的可能性，如纳入多个靶位点，应进行调查。指导抗生素治疗的诊断可能会产生意想不到的后果，包括新的耐药决定因素和抗生素交叉耐药性。美国国立卫生研究院、国家过敏和传染病研究所、国家普通医学科学研究所和史密斯家庭基金会。
The aetiological bacterial agent of gonorrhoea, Neisseria gonorrhoeae, has become resistant to each of the first-line antibiotics used to treat it, including ciprofloxacin. One diagnostic approach to identify ciprofloxacin-susceptible isolates is to determine codon 91 in the gene encoding the A subunit of DNA gyrase, gyrA, where coding for the wild-type serine (gyrA91S) is associated with ciprofloxacin susceptibility and phenylalanine (gyrA91F) with resistance. The aim of this study was to investigate the possibility of diagnostic escape from gyrA susceptibility testing. We used bacterial genetics to introduce pairwise substitutions in GyrA positions 91 (S or F) and 95 (D, G, or N), which is a second site in GyrA associated with ciprofloxacin resistance, into five clinical isolates of N gonorrhoeae. All five isolates encoded GyrA S91F, an additional substitution in GyrA at position 95, substitutions in ParC that are known to cause an increased minimum inhibitory concentration (MIC) to ciprofloxacin, and GyrB 429D, which is associated with susceptibility to zoliflodacin (a spiropyrimidinetrione-class antibiotic in phase 3 trials for treatment of gonorrhoea). We evolved these isolates to assess for the existence of pathways to ciprofloxacin resistance (MIC ≥1 μg/mL) and measured MICs for ciprofloxacin and zoliflodacin. In parallel, we searched metagenomic data for 11 355 N gonorrhoeae clinical isolates with reported ciprofloxacin MICs that were publicly available from the European Nucleotide Archive for strains that would be identified as susceptible by gyrA codon 91-based assays. Three clinical isolates of N gonorrhoeae with substitutions in GyrA position 95 associated with resistance (G or N) maintained intermediate ciprofloxacin MICs (0·125–0·5 μg/mL), which has been associated with treatment failure, despite reversion of GyrA position 91 from phenylalanine to serine. From an in-silico analysis of the 11 355 genomes from N gonorrhoeae clinical isolates, we identified 30 isolates with gyrA codon 91 encoding a serine and a ciprofloxacin resistance-associated mutation at codon 95. The reported MICs for these isolates varied from 0·023 μg/mL to 0·25 μg/mL, including four with intermediate ciprofloxacin MICs (associated with substantially increased risk of treatment failure). Finally, through experimental evolution, one clinical isolate of N gonorrhoeae bearing GyrA 91S acquired ciprofloxacin resistance through mutations in the gene encoding for the B subunit of DNA gyrase (gyrB) that also conferred reduced susceptibility to zoliflodacin (ie, MIC ≥2 μg/mL). Diagnostic escape from gyrA codon 91 diagnostics could occur through either reversion of the gyrA allele or expansion of circulating lineages. N gonorrhoeae genomic surveillance efforts might benefit from including gyrB, given its potential for contributing to ciprofloxacin and zoliflodacin resistance, and diagnostic strategies that reduce the likelihood of escape, such as the incorporation of multiple target sites, should be investigated. Diagnostics that guide antibiotic therapy can have unintended consequences, including novel resistance determinants and antibiotic cross-resistance. US National Institutes of Health National Institute of Allergy and Infectious Diseases, National Institute of General Medical Sciences, and the Smith Family Foundation.