Surfactant-enhanced DNA accessibility to nuclease accelerates phenotypic β-lactam antibiotic susceptibility testing of Neisseria gonorrhoeae

Abstract

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.

Machine Learning Establishes Single-Cell Calcium Dynamics as an Early Indicator of Antibiotic Response

“…This limitation could be overcome by leveraging Nernstian dyes or other live-cell, staining-based measurements of cellular physiology which changes in response to antibiotic stress. For example, stains which measure changes in DNA accessibility in response to beta-lactamases [ 64 ] could be explored. Integrating multiple signals of dynamic physiology, akin to a dynamic version of cell painting [ 65 ] developed for eukaryotic cells, will further enrich our understanding of drug mechanisms of action while simultaneously increasing the speed at which computers can identify resistant pathogens in the clinic.…”

Bridging the gap between development of point-of-care nucleic acid testing and patient care for sexually transmitted infections

“…Recently, Savela and colleagues developed a new strategy for accelerating pheno-molecular AST by measuring DNA accessibility to exogenous nucleases after exposure to β-lactams (the largest antibiotic class used to treat NG). 106 The authors showed that genomic DNA in antibiotic-susceptible cells has increased accessibility upon exposure to β-lactams such as penicillin, cefixime, and ceftriaxone and that a judiciously chosen surfactant permeabilized the outer membrane and enhanced this effect. Moreover, the authors employed digital PCR and digital loop mediated isothermal amplification (LAMP) for precise quantification of DNA.…”

Leapfrogging laboratories: the promise and pitfalls of high-tech solutions for antimicrobial resistance surveillance in low-income settings

“…Even the most innovative 'tortoise' advancements speed up or eliminate one or more incubation steps but typically still require some growth. [18][19][20] More sensitive or alternate growth-detection technologies can drastically reduce turn-around times and represent longer stride tortoise strategies, often with unfortunately hefty price tags. Automated culture, identification and sensitivity testing systems such as BACTEC, BacT/ Alert or VersaTREK (blood culture) and VITEK and VITEK-MS, Phoenix, Bruker MALDI Biotyper or Sensititre (identification and/or susceptibility testing), as well as DNA-leakage or early growth-detection tests [18][19][20] accomplish this and, in some cases, are easier to quality assure than traditional protocols.…”