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

Nathan G. Schoepp,Olusegun O. Soge,Jeffrey D. Klausner,Emily S. Savela,Rustem F. Ismagilov,Justin C. Rolando,Matthew M. Cooper,Tobias Bollenbach

doi:10.1371/journal.pbio.3000651

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.

Highlights

Gonorrhea, caused by N. gonorrhoeae (Ng), is the second most common notifiable sexually transmitted infection (STI) in the United States [1] and the third most common STI globally
Like other nucleic acid (NA)-based antibiotic susceptibility test (AST) methods, the nuc-aAST (Fig 1) relies on measuring changes in the quantity of pathogen-specific NAs in response to a treatment with an antibiotic; the nucaAST differs from existing NA-based ASTs in three aspects
DNA is accessible to DNase if it is released from the cells upon cell lysis or if the action of the antibiotic porates the cells and allows DNase to access the intracellular DNA

Summary

Introduction

Gonorrhea, caused by N. gonorrhoeae (Ng), is the second most common notifiable sexually transmitted infection (STI) in the United States [1] and the third most common STI globally. Antibiotic resistance in Ng emerged quickly and continues to spread unchecked because there is no rapid antibiotic susceptibility test (AST) to guide treatment. Lacking a rapid AST, clinicians are limited to making empiric prescriptions as recommended by the CDC [6] or World Health Organization (WHO) [7]. When resistance to a particular antibiotic exceeds 5%, treatment guidelines are updated and the recommended treatment protocol is escalated to the line of antibiotic [8,9]. The global prevalence and spread of resistant Ng infections has led the CDC to place Ng in its highest (“urgent”) category of antimicrobial-resistant pathogen threats [13] and WHO to label Ng as a high-priority pathogen [14]. Despite the threat of untreatable Ng [15] and an international call for rapid diagnostics [16,17,18], no phenotypic AST currently exists that can be performed rapidly enough for the point of care (POC)

Objectives

Results

Conclusion