P21 A novel, fast, impedance-based antimicrobial susceptibility test for Neisseria gonorrhoeae
Authors: Daniels Caitlin 
Published: January 2023

Abstract

Objectives
To demonstrate that the impedance-based fast antimicrobial susceptibility test (iFAST) can define N. gonorrhoeae susceptibility for a range of frontline antibiotics, and determine time taken to see responses.

Background
Gonorrhoea is the second most common bacterial sexually transmitted infection in the UK. It can cause complicated infection and infertility if not treated effectively. Resistance to frontline treatments and MDR N. gonorrhoeae infections (‘super gonorrhoea’) are increasing, fuelled by inappropriate selection of antibiotics. The current gold-standard antimicrobial susceptibility test (AST) for N. gonorrhoeae is MIC agar dilution, which is time-consuming (∼7 days) and labour-intensive. Unemo et al. 2020 (https://doi.org/10.1093/jac/dkw288) published detailed resistance profiles of WHO-reference N. gonorrhoeae strains. This project aimed to test whether these strain sensitivities against front line antibiotics ceftriaxone, ciprofloxacin and azithromycin could be defined faster using iFAST, which calculates the opacity and electrical size of individual cells by measuring change in electrical currents as single cells flow between micro-electrodes. Antibiotic-treated susceptible bacteria change electrical radius (surrogate measure of cell size) and opacity (based on properties of bacterial membranes as resistors), compared with untreated or resistant cells (Spencer et al. 2020, 10.1038/s41467-020-18902-x).

Methods
N. gonorrhoeae WHO-reference strains F and X were incubated overnight on standard chocolate agar plates at 37°C, 3%–5% CO2. 10 viable plate colonies were selected and inoculated in 37°C brain heart infusion media, supplemented with 0.42% sodium bicarbonate and 1% Isovitalex. After 30 min incubation at 37°C with shaking, the cell concentration was measured on iFAST and added to five concentrations of ceftriaxone, ciprofloxacin, and azithromycin (at the published agar MIC and ±2 × 2-fold concentration) or at EUCAST susceptibility breakpoint concentrations (strains F, X, O, V, K, Y), at a final cell concentration of 5 × 105 cells/mL. Cells were exposed at 37°C 250 rpm for 2, 3 and 4 h. 60 µL of 1:10 dilutions of sample were read on iFAST. MATLAB analysis software created contours around 50% of untreated cells and overlayed this on exposed population scattergrams.

Results
Electrical MICs (eMICs) showed differences in cell count, electrical radius and opacity between cells exposed to concentrations ≥MIC, subinhibitory concentrations, or untreated cells for all three frontline drugs. These differences were detectable after 2 h of antibiotic exposure but were clearest after 4 h. When tested at breakpoint concentrations only, iFAST accurately detected susceptibility of tested N. gonorrhoeae reference strains after 4 h exposure. Generally, susceptible cells had reduced electrical cell size and increased opacity distribution (out of contour shift) for all compounds, though there was variation due to differing modes of action. Ciprofloxacin and azithromycin had more decreased electrical cell radius with increased opacity spread. Comparatively, ceftriaxone had smaller changes in electrical radius and a larger spread of opacity readings.

Conclusions
iFAST is potentially a rapid and accurate method for determining N. gonorrhoeae strain susceptibility for ceftriaxone, ciprofloxacin, and azithromycin. With additional validation, it may be useful for future N. gonorrhoeae AST development, as well as investigating the effect of novel antimicrobial compounds.