1. A NEW RAPID RESAZURIN-BASED MICRODILUTION ASSAY FOR ANTIMICROBIAL SUSCEPTIBILITY TESTING OF NEISSERIA GONORRHOEAE Sunniva Förster 1,3,4, Valentino Desilvestro 2, Lucy Hathaway 3, Nicola Low 1, Christian Althaus 1, Magnus Unemo 4 1 Institute of Social and Preventive Medicine (ISPM), University of Bern, Switzerland 2 World Trade Institute (WTI), University of Bern, Switzerland 3 Institute for Infectious Diseases, University of Bern, Switzerland 4 WHO Collaborating Centre for Gonorrhoea and other STIs, Örebro University, Sweden IUSTI, Marrakesh, 11 May 2016

2. 2 Background Etest No standardized liquid broth MIC assay for N. gonorrhoeae! Source: Adapted from Brook et. al, 2012 Agar dilution Easy to learn Not available for all drugs Depends on 1:2 dilutions Slow Visual readout High flexibility Laborious Visual readout Extensive training Slow

3. 3 Objectives Develop Liquid Broth MIC Assay  Rapid  Reproducible  Reduced bias Step 1: Use 8 WHO reference strains and 7 antimicrobials to develop reproducible method Step 2: Apply method to 84 blinded strains

4. 4 Graver Wade broth for MIC testing  Chemically defined liquid broth (Wade and Graver, 2007).  Transparent, colourless.  Supports the growth of all tested auxotypes and phylogenetically different clinical isolates from very low inocula.  Cost efficient and easy to produce (~15 USD/L).  Good medium for MIC testing? Source: Förster et. al 2015 Time [h] Bacteria [CFU/ml] WHO G

5. 5 Experimental workflow Prepare dilution series for each antimicrobial Prepare bacterial suspension Add bacteria to the antimicrobials and incubate (6 h) Add resazurin and incubate (1 h) Measure fluorescence (560/590 nm) and calculate MIC Dilution series pink blue

6. 6 Dose response relationship for antimicrobials EC50 EC90 EC10 Is there a relationship between the EC and Etest MIC? Etest MICs tend higher than Effective concentrations (EC) measured in liquid broth!

7. 7 Correlation of EC values with Etest MIC EC [%] Pearson’s R

8. 8 Regression analysis for each antimicrobial

9. 9 Predicted MIC Deviation from Etest [doubling dilutions] Predicted MIC [mg/L] Etest MIC [mg/L] Predicted MIC [mg/L] Etest MIC [mg/L]

10. 10 Comparison of category agreement with Etest I = Intermediate resistance, R = resistance, S = susceptible (EUCAST 2016 breakpoints) Etest Predicted MIC Correct I -> R/S (no error) S -> R (minor error) R -> S (major error) AzithromycinCiprofloxacin Cefixime Ceftriaxone Tetracycline Penicillin G Spectinomycin

11. 11 Summary & Conclusions  Graver Wade medium can be used for MIC testing in liquid broth  EC values from fluorescent readouts can be used to predict MICs  Fluorescent method allows to define MIC values on a continuous scale  Method can classify strains according to EUCAST categories with high accuracy in less than eight hours  Method can be readily used for high-throughput screening of antimicrobials against N. gonorrhoeae (e.g. Checkerboard testing) The predicted MIC has to be validated with more strains for diagnostic purposes to recognize resistance.

12. 12 Acknowledgements  Magnus Unemo  Valentino Desilvestro  Lucy Hathaway  Nicola Low  Christian Althaus