Rapid antibiotic susceptibility testing (AST) for Neisseria gonorrhoeae (Ng) is critically needed to counter widespread antibiotic resistance. Detection of nucleic acids in genotypic AST can be rapid, but it has not been successful for β-lactams (the largest antibiotic class used to treat Ng). Rapid phenotypic AST for Ng is challenged by the pathogen’s slow doubling time and the lack of methods to quickly quantify the pathogen’s response to β-lactams. Here, we asked two questions: (1) Is it possible to use nucleic acid quantification to measure the β-lactam susceptibility phenotype of Ng very rapidly, using antibiotic-exposure times much shorter than the 1- to 2-h doubling time of Ng? (2) Would such short-term antibiotic exposures predict the antibiotic resistance profile of Ng measured by plate growth assays over multiple days? To answer these questions, we devised an innovative approach for performing a rapid phenotypic AST that measures DNA accessibility to exogenous nucleases after exposure to β-lactams (termed nuclease-accessibility AST [nuc-aAST]). We showed that DNA in antibiotic-susceptible cells has increased accessibility upon exposure to β-lactams and that a judiciously chosen surfactant permeabilized the outer membrane and enhanced this effect. We tested penicillin, cefixime, and ceftriaxone and found good agreement between the results of the nuc-aAST after 15–30 min of antibiotic exposure and the results of the gold-standard culture-based AST measured over days. These results provide a new pathway toward developing a critically needed phenotypic AST for Ng and additional global-health threats.

迫切需要对淋病奈瑟氏菌（Ng）进行快速抗生素敏感性测试（AST），以应对广泛的抗生素耐药性。基因型AST中的核酸检测可以快速进行，但对于β-内酰胺类（用于治疗Ng的最大抗生素类）而言，尚未成功。Ng的快速表型AST受病原体缓慢的倍增时间和缺乏快速定量病原体对β-内酰胺反应的方法的挑战。在这里，我们问了两个问题：（1）是否可以使用核酸定量法非常快速地测量Ng的β-内酰胺敏感性表型，而抗生素的暴露时间要比Ng的1至2小时加倍时间短得多？（2）这样的短期抗生素暴露可以预测Ng的抗生素耐药性吗通过平板生长测定法在多天内进行测量？为了回答这些问题，我们设计了一种创新的方法来执行快速表型AST，该方法测量暴露于β-内酰胺后DNA对外源核酸酶的可及性（称为核酸酶可及性AST [nuc-aAST]）。我们表明，抗生素敏感细胞中的DNA在暴露于β-内酰胺时具有更高的可及性，并且明智地选择的表面活性剂可渗透外膜并增强这种作用。我们测试了青霉素，头孢克肟和头孢曲松，发现抗生素暴露15-30分钟后nuc-aAST的结果与几天内测量的基于金标准培养物的AST的结果之间有很好的一致性。这些结果为开发针对Ng的急需表型AST提供了新途径 以及其他全球健康威胁。