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 1
MALDI-TOF: principle Matrix Assisted Laser Desorption/Ionization, Time Of Flight MALDI TOF Identification of bacteria and fungi Intens. [a.u.] 5 4364.6 4 538.64 6254.64 596.1 6315.49 3 2 641.9 7157.65 7273.87 1 787.62 8368.99 4 45 5 55 6 65 7 75 8 m/z E. coli MALDI-TOF MS bacteria identification Workflow 2
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 3
MRSA detection by MALDI-TOF MS? MRSA MALDI TOF detection of MRSA 4
Wolters et al., 211 MALDI-TOF for detection of antibiotic resistance -lactamases (ESBL) carbapenemases Problem 5
SHV-1 SHV-2 SHV-3 SHV-178 TEM-1 TEM-2 TEM-3 TEM-213 CTX-M-1 CTX-M-2 CTX-M-3 CTX-M-75 OXA-1 OXA-2 OXA-3 OXA-161 CMY-1 CMY-2 CMY-3 CMY-23 IMP-1 IMP-2 IMP-3 IMP-23 > 7 -lactamase - subtypes 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 6
-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., 212 Procedure 1 colony of fresh o/n culture 1 µl antibiotic solution (e.g..5 µg/ml CTX) 1-2 h incubation, 37 C Spin down bacteria Supernatant on MALDI-target plate Mass range 1 1 Da Calibration with suitable molecules 7
Cefotaxime (CTX) Intens. [a.u.] x1 4 5 4 3 2 1 396. 414.1 456.1 A CTX + ESBL- neg. E. coli Intens. [a.u.] x1 4.8.6.4.2 369.9 414. 445. 46.9 B CTX + ESBL-pos. E. coli Intens. [a.u.]. x1 4 C 1.5 1..5. 36 38 4 42 44 46 48 m/z 396.1 414.1 456.1 CTX / CLAV + ESBL-pos. E. coli Σ peak-intensity hydrolysed log Σ peak-intensity non-hydrolysed.5 mg/ml CAZ in 1 mm NH 4 -hydrogen carbonate 2 h incubation 15 µl sup plus 5 µl.5 ng/µl reserpine 1.5 µl spotted -lactamaseactivity No cleavage spontaneous hydrolysis 8
Identification -lactamase activity E. coli directly from positve blood cultures ampicillin 1 Ampicillin MIC >8.5 logrq Ampicillin MIC 8 -.5 4 MIC>256 5 MIC 4 9 MIC 8 1 MIC >256 11 MIC >256 16 MIC >256 18 MIC >256 2 MIC 3 21 MIC 4 22 MIC 3 25 MIC 4 3 MIC 6 31 MIC 4 37 MIC 6 39 MIC 2 4 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 6 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 9 MIC 2 91 MIC >256 92 MIC >256/4 93 MIC 1,5 94 MIC >256 E. coli ATCC 25922 Jung et al., 214 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 9
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, 215 3 1
5 51 52 53 54 55 56 57 58 m/z S. aureus mass spectra gel view MSSA OXA - susceptible strain MRSA OXA - resistant strain normal normal heavy + Oxa heavy + Oxa heavy heavy October 22, 215 31 MRSA P. aeruginosa MBT MS-RESIST / P. aeruginosa (Tobramycin- susceptible) Intens. [a.u.] 6 5 5213.353 5739.928 125_N :B8 MS, BaselineSubtracted, Smoothed 4 3 2 5116.874 normal 1 6 5212.952 12_H_6 :A1 MS, BaselineSubtracted, Smoothed 5 4 3 2 1 5116.441 5739.242 heavy + TOB Intens. [a.u.] 6 12_H :A7 MS, BaselineSubtracted, Smoothed 4 521.891 5213.753 5117.392 5244.916 574.349 5795.447 heavy 2 Jung et al., EJCMID 213 October 22, 215 33 11
MS-RESIST/ P. aeruginosa (Tobramycin-resistant) Intens. [a.u.] 5 4 65.312 425_N :G1 MS, BaselineSubtracted, Smoothed 6679.371 3 2 574.567 6351.691 normal 1 6 6113.22 663.988 42_H_6 :G1 MS, BaselineSubtracted, Smoothed 5 4 3 2 1 649.92 6351.31 5739.994 6113.19 639.383 5796.772 6678.694 6717.281 heavy + TOB 1.25 Intens. [a.u.]x1 4 65.252 42_H :F11 MS, BaselineSubtracted, Smoothed 6679.317 1..75.5 574.398 6113.323 5796.23 6351.65 6718.17 639.951 heavy.25. 56 58 6 62 64 66 m/z Jung et al., EJCMID 213 October 22, 215 34 ciprofloxacin Pseudomonas aeruginosa meropenem tobramycin Jung et al., 214 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 12
3. Antibiotic susceptibility testing by phenotypic assays Phenotypic assay without isotopes? MALDI-TOF MS as a quantitative growth monitor MBT-ASTRA BHI McF.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] + Standard [M+ H] 2+ Standard [M+ H] + BHI + Meropenem 8 µg/ml susceptible BHI only Meropenem resistant Lange et al., J Clin Microbiol. 214 13
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? 14
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 15