Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, United States of America.
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, United States of America.
PLoS Biol. 2020 Mar 19;18(3):e3000651. doi: 10.1371/journal.pbio.3000651. eCollection 2020 Mar.
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.
淋病奈瑟菌（Ng）快速抗生素药敏试验（AST）对于对抗广泛的抗生素耐药性至关重要。在基因 AST 中检测核酸可以很快，但对于β-内酰胺类药物（用于治疗 Ng 的最大抗生素类别）却没有成功。Ng 的快速表型 AST 受到病原体缓慢倍增时间和缺乏快速定量病原体对β-内酰胺类药物反应的方法的挑战。在这里，我们提出了两个问题：（1）是否可以使用核酸定量来非常快速地测量 Ng 的β-内酰胺药敏表型，使用的抗生素暴露时间比 Ng 的 1-2 小时倍增时间短得多？（2）这种短期抗生素暴露是否可以预测 Ng 在多天的平板生长测定中测量的抗生素耐药谱？为了回答这些问题，我们设计了一种创新的快速表型 AST 方法，该方法在暴露于β-内酰胺类药物后测量外源性核酸酶对 DNA 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-内酰胺类药物后可及性增加，并且选择得当的表面活性剂可使外膜穿孔并增强这种效应。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 分钟后的 nuc-aAST 结果与经过数天的基于培养的金标准 AST 结果之间具有良好的一致性。这些结果为开发 Ng 和其他全球健康威胁急需的表型 AST 提供了新途径。
