A NEW RAPID RESAZURIN-BASED MICRODILUTION ASSAY FOR ANTIMICROBIAL SUSCEPTIBILITY TESTING OF NEISSERIA GONORRHOEAE Sunniva Förster 1,3,4, Valentino Desilvestro.

Slides:



Advertisements
Similar presentations
An MIC is an MIC is an MIC, isnt it? Gunnar Kahlmeter Clinical microbiology Växjö, Sweden.
Advertisements

The Critical Challenge of Antibiotic Resistance: Are Wastewater Treatment Plants a Concern? Kourtney Brown 1, Stefan Walston 2, Channah Rock 2, & Jean.
Evaluation of the FUNGIFAST ® AFG yeast antifungal susceptibility test : comparison with EUCAST and the E-Test method INTERNAL STUDY - (ELITECH GROUP -
DISK ASSAYS Concentration of EXEG 1706 (  g/ml) OD 605nm
Lecture 3 Antimicrobials and Susceptibility tests Dr. Abdelraouf A. Elmanama Islamic University-Gaza Medical Technology Department.
Screening for new antibiotics
Plasmids Chromosome Plasmid Plasmid + Transposon Plasmid + integron Plasmid+transposon +intergron Chromosome Chromosome + transposon Chromosome + transposon.
Mannitol Salt Agar-Cefoxitin Combination as a Screening Medium for MRSA SMYTH, RW and KAHLMETER, G Dept. of Clinical Microbiology, Central Hospital, S-351.
PHT 381 Lab # 8. MIC: MIC: It is the lowest concentration of the antimicrobial agent that inhibits the growth of the test organism but not necessarily.
Analysis of mutations within multiple genes associated with resistance in clinical isolate of Neisseria gonorrhoeae with reduced ceftriaxone susceptibility.
Trevor Winstanley Rebecca Clarke Department of Microbiology
Melioidosis: Current Diagnostic Approach and Role in Clinical practice Rob Baird Royal Darwin Hospital.
IN THE NAME OF ALLAH ALMIGHTY THE MOST COMPASSIONATE THE MERCIFUL.
PK/PD of Antibiotics in relation to resistance Otto Cars MD Department of Medical Sciences Infectious diseases Uppsala University Sweden.
Rapid Emergence of Gonococcal Fluoroquinolone Resistance in Men Who Have Sex with Men in King County, WA WLH Whittington MR Golden KK Winterscheid SA Wang.
In Vitro Testing of Antimicrobial Agents Maryam Monajemzadeh, Pathologist Children Medical Center Hospital Tehran University Of Medical Sciences.
Mic 224 Lab 6 Streak Plate Technique and Antibiotic Sensitivity.
KIRBY – BAUER MINIMUM INHIBITORY CONCENTRATION MINIMUM BACTERIOCIDAL CONCENTRATION.
International Standards and Antimicrobial Susceptibility Testing Antimicrobials SIG Workshop ASM Canberra, 12 July, 2015 Peter Taylor, SEALS Microbiology,
ANTIBIOTICS SUSCEPTIBILITY TESTING
ANALYTICAL PROCESS CONTROL
References Summary Background Quality Control Parameters for Omadacycline Minimum Inhibitory Concentration (MIC) Susceptibility Tests Using Fresh Media.
Methods for detecting resistance Goal: To determine whether organism expresses resistances to agents potentially used for therapy Designed to determine.
IN THE NAME OF ALLAH ALMIGHTY THE MOST COMPASSIONATE THE MERCIFUL.
Susceptibility (Sensitivity) Testing: Results Pharmaceutical Microbiology – Practical Course Semester One_ Sensitivity Results & MIC Broth Dilution/
PHT 226 Lab no 9. MIC: It is the lowest concentration of the antimicrobial agent that inhibits the growth of the test organism but not necessarily kills.
Antibiotics Basmah almaarik
Developed by JALAL SHEIKH, Ph.D.
Lab: 6 ANTIBIOTIC SENSITIVITY TESTING
Lab # 1. Antimicrobial Therapy  Natural antibiotic agents:  Produced by microorganisms:  Penicillium notatum – penicillin  Semi-synthetic antibiotic.
Antimicrobial susceptibility patterns of Polish invasive isolates of Neisseria meningitidis in the years Marcin Kadłubowski 1, Anna Skoczyńska.
Efficacy of gentamicin in combination with other different antimicrobials: optimism to treat and combat superbug Neisseria gonorrhoeae Vikram Singh 1,
Quality Control of Antimicrobial Susceptibility Tests
Development of a Novel Antibody-Based Assay for Simultaneous Identification of a Pathogen and Determination of its Antimicrobial Susceptibility Jonathan.
Introduction & Purpose Results Conclusions
Candida species distribution and antifungal susceptibility testing according to European Committee on Antimicrobial Susceptibility Testing and new vs.
Antimicrobial Susceptibility Testing (AST)
Are Vitek2 system and E-test relevant and reliable for determining susceptibility to temocillin? Visée C.1, Frippiat F1, Descy J.2, Meex C.2, Melin P.2,
R. Leclercq  Clinical Microbiology and Infection 
Antimicrobial susceptibility testing of colistin – evaluation of seven commercial MIC products against standard broth microdilution for Escherichia coli,
Correspondence: Bactericidal Activity of Fosfomycin against NDM-1 producing Enterobacteriaceae M. Albur, A. Noel, K. Bowker, A.
AST SC’s Perspective on the Development and Use of ECVs
D-735/178 50th ICAAC Sept , 2010 Boston
Linking Pneumocystis jiroveci sulfamethoxazole resistance to the alleles of the DHPS gene using functional complementation in Saccharomyces cerevisiae 
Antibiotic Susceptibility Testing
Antibiogram By:Dr. S. S. Khoramrooz In the name of God
P1257 Pharmacodynamics of Amikacin Inhale studied in an in vitro pharmacokinetic model of infection KE Bowker, AR Noel, SG Tomaselli, MLG Attwood, AP.
MIC Where does that number come from?
Role of inoculum and mutant frequency on fosfomycin MIC discrepancies by agar dilution and broth microdilution methods in Enterobacteriaceae  M. Ballestero-Téllez,
Antibiotics AMANY NIAZY
Neisseria gonorrhoeae: the first untreatable infection
Cryptococcus neoformans var
ANTIBIOTIC RESISTANCE
P. Moreillon, J.M. Entenza  Clinical Microbiology and Infection 
D.E. Low  Clinical Microbiology and Infection 
…monitoring trends in antimicrobial resistance in Canada
Candida species distribution and antifungal susceptibility testing according to European Committee on Antimicrobial Susceptibility Testing and new vs.
Zhangqi Shen, Yang Wang, Yingbo Shen, Jianzhong Shen, Congming Wu 
C. -J. Soussy, J. Nguyen, F. Goldstein, H. Dabernat, A. Andremont, R
S. Bengtsson, C. Bjelkenbrant, G. Kahlmeter 
Antibiotic susceptibility testing
Jacques Sirot, Patrice Courvalin, Claude-James Soussy 
Antibiotics AMANY NIAZY
Development of the EUCAST disk diffusion antimicrobial susceptibility testing method and its implementation in routine microbiology laboratories  E. Matuschek,
A. Carannante, G. Prignano, M. Cusini, A. Matteelli, I. Dal Conte, V
…monitoring trends in antimicrobial resistance in Canada
…monitoring trends in antimicrobial resistance in Canada
Recommended scheme for determination of heteroresistance and interpretation criteria. Recommended scheme for determination of heteroresistance and interpretation.
Activity of linezolid against Gram-positive cocci isolated in French hospitals as determined by three in-vitro susceptibility testing methods  C. Muller-Serieys,
History of discovered and recommended antimicrobials and evolution of resistance in Neisseria gonorrhoeae, including the emergence of genetic resistance.
Presentation transcript:

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

8 Regression analysis for each antimicrobial

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