RNA markers enable phenotypic test of antibiotic susceptibility in Neisseria gonorrhoeae after 10 minutes of ciprofloxacin exposure

Abstract

Antimicrobial-resistant Neisseria gonorrhoeae is an urgent public-health threat, with continued worldwide incidents of infection and rising resistance to antimicrobials. Traditional culture-based methods for antibiotic susceptibility testing are unacceptably slow (1–2 days), resulting in the use of broad-spectrum antibiotics and the further development and spread of resistance. Critically needed is a rapid antibiotic susceptibility test (AST) that can guide treatment at the point-of-care. Rapid phenotypic approaches using quantification of DNA have been demonstrated for fast-growing organisms (e.g. E. coli) but are challenging for slower-growing pathogens such as N. gonorrhoeae. Here, we investigate the potential of RNA signatures to provide phenotypic responses to antibiotics in N. gonorrhoeae that are faster and greater in magnitude compared with DNA. Using RNA sequencing, we identified antibiotic-responsive transcripts. Significant shifts (>4-fold change) in transcript levels occurred within 5 min of antibiotic exposure. We designed assays for responsive transcripts with the highest abundances and fold changes, and validated gene expression using digital PCR. Using the top two markers (porB and rpmB) we correctly determined the antibiotic susceptibility and resistance of 49 clinical isolates after 10 min exposure to ciprofloxacin. RNA signatures are therefore promising as an approach on which to build rapid AST devices for N. gonorrhoeae at the point-of-care, which is critical for disease management, surveillance, and antibiotic stewardship efforts.

Impact of population structure in the design of RNA-based diagnostics for antibiotic resistance in Neisseria gonorrhoeae

“…This pattern also held true for all transcripts regardless of their fold change in abundance between isolates (Supplementary Figure 1). Interestingly, at baseline (T0), the two markers previously reported as being diagnostic of ciprofloxacin resistance (3), porB and rpmB , diverged transcriptionally with increasing evolutionary distance between isolates as well (Supplementary Figure 1).…”

“…Black circles indicate all significant transcripts for each model contrast, and red dots indicate the four candidate diagnostic markers ultimately chosen for RT-qPCR validation. Most of the observed response occurred (1) in susceptible strains between the control and the drug exposed conditions, (2) between resistant and susceptible isolates in the antibiotic exposure condition, and (3) having a significant interaction term indicating differential response between susceptible and resistance isolates across treatments.…”

“…Some of the earliest cellular responses to antibiotic exposure are mediated through transcriptional changes, which has recently been demonstrated in Escherichia coli , Pseudomonas aeruginosa , Klebsiella pneumoniae , Acinetobacter baumannii , and Neisseria gonorrhoeae (1–4). These RNA-based tests are particularly attractive as POC diagnostics as drug sensitive and resistant bacteria proceed down distinct physiological pathways indicative of cellular stress or normal cell growth in as little as 10 minutes (3) – much faster than the current ‘gold standard’ for resistance phenotyping via minimum inhibitory concentration (MIC) by agar dilution, e-test, or disk diffusion which can take days to months depending on bacterial growth rate.…”

“…After defining candidate diagnostic markers for azithromycin resistance, we then verify them via RT-qPCR in an expanded panel of isolates. We also assess the previously described diagnostic markers for ciprofloxacin resistance in N. gonorrhoeae , porB and rpmB (3), in a phylogenetically representative panel of isolates.…”

Rapid antimicrobial susceptibility test using spatiotemporal analysis of laser speckle dynamics of bacterial colonies

“…These techniques also require complex readout systems, and their compatibility with standard methods should be demonstrated for point-of-care use. Biochemical ASTs have also been suggested using Raman spectroscopic biomarkers 21 , DNA probes 22,23 , and RNA markers 24 . Although these methods offer rapid ASTs, the universal application of probe molecules to multiple strains and antibiotics remains to be addressed.…”