MALDI-TOF MS: a new tool to rapidly assess antibiotic susceptibility Sören Schubert, MD Max von Pettenkofer-Institut Ludwig-Maximilians-University (LMU) Munich, Germany
Learning Objectives After this presentation, you should know the principles of MALDI-TOF MS and its application in identification of microorganisms be able to identify and to trace the development of MALDI-TOF MS for the -lactamase activity testing of bacteria record and compare facts about different approaches using MALDI-TOF MS for comprehensive antibiotic resistance testing be able to identify the pros and cons of using MALDI- TOF MS for resistance testing in diagnostic laboratory and will retrace possible future perspectives
MALDI-TOF MS MALDI Biotyper Bruker Daltonics Vitek MS biomérieux
MALDI-TOF: principle Matrix Assisted Laser Desorption/Ionization, Time Of Flight
MALDI TOF Identification of bacteria and fungi Intens. [a.u.] 5000 4000 3000 2000 4364.06 5096.01 5380.64 6254.64 6315.49 6410.90 ribosomal Protein m/z RL36 4364,33 RS32 5095,82 RL34 5380,39 RL33meth. 6255,39 RL32 6315,19 RL30 6410,60 RL35 7157,74 RL29 7273,45 RL31 7871,06 RS21 8368,76 7157.65 7273.87 1000 7870.62 8368.99 0 4000 4500 5000 5500 6000 6500 7000 7500 8000 m/z E. coli
MALDI-TOF MS bacteria identification Workflow
Identification
Antibiotic resistance testing (AST) Disc Diff. Walkaway Vitek 2 Phoenix Time to result 12 24 h 12 48 h 7 18 h 8 18 h
MALDI-TOF MS 1. Direct detection of resistance factors
MRSA detection by MALDI-TOF MS?
MRSA
MALDI TOF detection of MRSA
Wolters et al., 2011
MALDI-TOF for detection of antibiotic resistance -lactamases (ESBL) carbapenemases
Problem
> 700 -lactamase - subtypes SHV-1 SHV-2 SHV-3 SHV-178 OXA-1 OXA-2 OXA-3 OXA-161 TEM-1 TEM-2 TEM-3 TEM-213 CTX-M-1 CTX-M-2 CTX-M-3 CTX-M-75 CMY-1 CMY-2 CMY-3 CMY-23 IMP-1 IMP-2 IMP-3 IMP-23
Conclusion 1: Direct detection of resistance factors Detection of clonal groups No reliable resistance identification -lactamases, PBP2a, Van A Van B
MALDI-TOF MS 1. Direct detection of resistance factors (e.g. PBP2a) 2. β-lactamase activity test
-lactamase activity +H 2 O + 18 Da - 44 Da A B C
Ampicillin + ESBL-E. coli Ampicillin + -Lact.-neg. E. coli C A B Sparbier et al., 2012
Procedure 1 colony of fresh o/n culture 10 µl antibiotic solution (e.g. 0.5 µg/ml CTX) 1-2 h incubation, 37 C Spin down bacteria Supernatant on MALDI-target plate Mass range 100 1000 Da Calibration with suitable molecules
396.0 414.1 456.1 x10 4 5 4 3 2 1 0 Intens. [a.u.] 460.9 369.9 414.0 445.0 x10 4 0.8 0.6 0.4 0.2 0.0 x10 4 Intens. [a.u.] 396.1 414.1 456.1 1.5 1.0 0.5 0.0 Cefotaxime (CTX) A B C 360 380 400 420 440 460 480 m/z CTX + ESBL- neg. E. coli CTX + ESBL-pos. E. coli CTX / CLAV + ESBL-pos. E. coli Intens. [a.u.]
Σ peak-intensity hydrolysed log Σ peak-intensity non-hydrolysed
CAZ 0.5 mg/ml CAZ in 10 mm NH 4 -hydrogen carbonate 2 h incubation 15 µl sup plus 5 µl 0.5 ng/µl reserpine 1.5 µl spotted 1.5 1.0 -lactamaseactivity 0.5 0.0-0.5 CAZ_09 2012-04-17 CAZ_16358 2012-04-17 CAZ_20 2012-04-17 CAZ_DH5a 2012-04-17 CAZ_K1 2012-04-17 logrq No cleavage spontaneous hydrolysis
Identification -lactamase activity
E. coli directly from positve blood cultures ampicillin 1 0.5 0-0.5 Ampicillin MIC >8 Ampicillin MIC 8 Jung et al., 2014 4 MIC>256 5 MIC 4 9 MIC 8 10 MIC >256 11 MIC >256 16 MIC >256 18 MIC >256 20 MIC 3 21 MIC 4 22 MIC 3 25 MIC 4 30 MIC 6 31 MIC 4 37 MIC 6 39 MIC 2 40 MIC >256 41 MIC 3 42 MIC >256 45 MIC >256 46 MIC 4 47 MIC>256 51 MIC>256 52 MIC 4 54 MIC >256 55 MIC >256 56 MIC >256 57 MIC >256 60 MIC >256 61 MIC >256 62 MIC 4 66 MIC >256 67 MIC >256 69 MIC 4 72 MIC >256 73 MIC >256 74 MIC >256 76 MIC >256 81 MIC >256 82 MIC 6 84 MIC >256 85 MIC 3 86 MIC >256 87 MIC 8 90 MIC 2 91 MIC >256 92 MIC >256/4 93 MIC 1,5 94 MIC >256 E. coli ATCC 25922 logrq
Conclusion 2: MALDI-TOF -lactamase activity test Rapid test 1.5 3 h Automated analysis Directly from positive blood cultures Restricted to certain antibiotic resistances -lactamases, e.g. ESBL, carbapenemases
MALDI-TOF MS 1. Direct detection of resistance factors (e.g. PBP2a) -lactamase activity test 3. Antibiotic susceptibility test - phenotypic assays MBT-RESIST
Phenotypic Susceptibility Testing (MBT-RESIST) 13 C 6 15 N 2 -L-Lysin 8Da 8Da For all growing bacteria applicable Susceptible: No integration Resistant: Integration
Susceptibility testing using stable isotopes normal Lys Control 1 heavy Lys + antibiotic Susceptible Resistant heavy Lys Control 2 October 22, 2015 30
S. aureus mass spectra gel view MSSA OXA - susceptible strain MRSA OXA - resistant strain normal normal heavy + Oxa heavy + Oxa heavy heavy October 22, 2015 31
MRSA P. aeruginosa
MBT MS-RESIST / P. aeruginosa (Tobramycin- susceptible) Intens. [a.u.] 6000 5000 5213.353 5739.928 1025_N 0:B8 MS, BaselineSubtracted, Smoothed 4000 3000 2000 5116.874 normal 1000 0 6000 5212.952 1020_H_60 0:A10 MS, BaselineSubtracted, Smoothed 5000 4000 3000 2000 1000 5116.441 5739.242 heavy + TOB Intens. [a.u.] 0 6000 1020_H 0:A7 MS, BaselineSubtracted, Smoothed 4000 5021.891 5213.753 5117.392 5244.916 5740.349 5795.447 heavy 2000 0 5000 5100 5200 5300 5400 5500 5600 5700 5800 m/z Jung et al., EJCMID 2013 October 22, 2015 33
MS-RESIST/ P. aeruginosa (Tobramycin-resistant) Intens. [a.u.] 5000 4000 6050.312 4025_N 0:G10 MS, BaselineSubtracted, Smoothed 6679.371 3000 2000 5740.567 6351.691 normal 1000 0 6000 6113.220 6630.988 4020_H_60 0:G1 MS, BaselineSubtracted, Smoothed 5000 4000 3000 2000 1000 6049.902 5739.994 6113.019 5796.772 6351.301 6390.383 6678.694 6717.281 heavy + TOB 0 1.25 Intens. [a.u.]x10 4 6050.252 4020_H 0:F11 MS, BaselineSubtracted, Smoothed 6679.317 1.00 0.75 5740.398 6113.323 6351.650 6718.107 6390.951 heavy 0.50 5796.203 0.25 0.00 5600 5800 6000 6200 6400 6600 m/z Jung et al., EJCMID 2013 October 22, 2015 34
ciprofloxacin Pseudomonas aeruginosa meropenem tobramycin Jung et al., 2014
MALDI-TOF MS 1. Direct detection of resistance factors (e.g. PBP2a) -lactamase activity test 3. Antibiotic susceptibility test - phenotypic assays MBT-RESIST MBT-ASTRA
3. Antibiotic susceptibility testing by phenotypic assays Phenotypic assay without isotopes?
MALDI-TOF MS as a quantitative growth monitor MBT-ASTRA BHI McF 0.5 Incubation 37 C Species dependent time Antibiotic Cell lysis Lysis reagent with internal standard Target preparation Acquisition of MS profile spectra
spectra view Klebsiella pneumoniae Standard [M+ H] 2+ Standard [M+ H] 2+ Standard [M+ H] + Standard [M+ H] + BHI + Meropenem 8 µg/ml susceptible BHI only Meropenem resistant Lange et al., J Clin Microbiol. 2014
Conclusion (1) Direct detection of resistance factors Detection of clonal groups May help to identify distinct clonally distributed resistance factors False positive and negative results possible! (yet) no direct detection of -lactamases, PBP2a, Van A/B,
Conclusion (2) -lactamase activity test Rapid test 1.5 3 h Automated analysis Directly from positive blood cultures Restricted to certain resistances -lactamases All -lactamases detectable?
Conclusion (3) Phenotypic resistance test (MBT-RESIST / MBT-ASTRA) Rapid tests 2 3 h Automated analysis available Stable Isotopes: A rather complex workflow kits and cards are needed All bug-drug combinations analyzable?
MALDI-TOF MS for antibiotic resistance testing Pro Reduction of time to result: 12 h 2.5 h Phenotypic assay irrespective of underlying molecular mechanism High-throughput feasible Low costs of consumables Cons Initial culture necessary plus 12 24 h High costs of MALDIhardware (Yet) no determination of MIC values (Yet) no test kits available hands on time is high
Max von Pettenkofer-Institut Jette Jung Theresa Eberl Christina Popp Julia Walker Christina Hamacher Lukas Schmidt Birgit Gross Andreas Wieser ForBIMed FöFoLe