MALDI-TOF MS: a new tool to rapidly assess antibiotic susceptibility

Transcription

1 MALDI-TOF MS: a new tool to rapidly assess antibiotic susceptibility Sören Schubert, MD Max von Pettenkofer-Institut Ludwig-Maximilians-University (LMU) Munich, Germany

2 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

3 MALDI-TOF MS MALDI Biotyper Bruker Daltonics Vitek MS biomérieux

4 MALDI-TOF: principle Matrix Assisted Laser Desorption/Ionization, Time Of Flight

5 MALDI TOF Identification of bacteria and fungi Intens. [a.u.] ribosomal Protein m/z RL ,33 RS ,82 RL ,39 RL33meth. 6255,39 RL ,19 RL ,60 RL ,74 RL ,45 RL ,06 RS , m/z E. coli

6 MALDI-TOF MS bacteria identification Workflow

7 Identification

8 Antibiotic resistance testing (AST) Disc Diff. Walkaway Vitek 2 Phoenix Time to result h h 7 18 h 8 18 h

9 MALDI-TOF MS 1. Direct detection of resistance factors

10 MRSA detection by MALDI-TOF MS?

11 MRSA

12 MALDI TOF detection of MRSA

13 Wolters et al., 2011

14 MALDI-TOF for detection of antibiotic resistance -lactamases (ESBL) carbapenemases

15 Problem

16 > 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

17 Conclusion 1: Direct detection of resistance factors Detection of clonal groups No reliable resistance identification -lactamases, PBP2a, Van A Van B

18 MALDI-TOF MS 1. Direct detection of resistance factors (e.g. PBP2a) 2. β-lactamase activity test

19 -lactamase activity +H 2 O + 18 Da - 44 Da A B C

20 Ampicillin + ESBL-E. coli Ampicillin + -Lact.-neg. E. coli C A B Sparbier et al., 2012

21 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 Da Calibration with suitable molecules

22 x Intens. [a.u.] x x10 4 Intens. [a.u.] Cefotaxime (CTX) A B C m/z CTX + ESBL- neg. E. coli CTX + ESBL-pos. E. coli CTX / CLAV + ESBL-pos. E. coli Intens. [a.u.]

23 Σ peak-intensity hydrolysed log Σ peak-intensity non-hydrolysed

24 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 lactamaseactivity CAZ_ CAZ_ CAZ_ CAZ_DH5a CAZ_K logrq No cleavage spontaneous hydrolysis

25 Identification -lactamase activity

26 E. coli directly from positve blood cultures ampicillin Ampicillin MIC >8 Ampicillin MIC 8 Jung et al., MIC>256 5 MIC 4 9 MIC 8 10 MIC > MIC > MIC > MIC > 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 > MIC 3 42 MIC > MIC > MIC 4 47 MIC> MIC> MIC 4 54 MIC > MIC > MIC > MIC > MIC > MIC > MIC 4 66 MIC > MIC > MIC 4 72 MIC > MIC > MIC > MIC > MIC > MIC 6 84 MIC > MIC 3 86 MIC > MIC 8 90 MIC 2 91 MIC > MIC >256/4 93 MIC 1,5 94 MIC >256 E. coli ATCC logrq

27 Conclusion 2: MALDI-TOF -lactamase activity test Rapid test h Automated analysis Directly from positive blood cultures Restricted to certain antibiotic resistances -lactamases, e.g. ESBL, carbapenemases

28 MALDI-TOF MS 1. Direct detection of resistance factors (e.g. PBP2a) -lactamase activity test 3. Antibiotic susceptibility test - phenotypic assays MBT-RESIST

29 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

30 Susceptibility testing using stable isotopes normal Lys Control 1 heavy Lys + antibiotic Susceptible Resistant heavy Lys Control 2 October 22,

31 S. aureus mass spectra gel view MSSA OXA - susceptible strain MRSA OXA - resistant strain normal normal heavy + Oxa heavy + Oxa heavy heavy October 22,

32 MRSA P. aeruginosa

33 MBT MS-RESIST / P. aeruginosa (Tobramycin- susceptible) Intens. [a.u.] _N 0:B8 MS, BaselineSubtracted, Smoothed normal _H_60 0:A10 MS, BaselineSubtracted, Smoothed heavy + TOB Intens. [a.u.] _H 0:A7 MS, BaselineSubtracted, Smoothed heavy m/z Jung et al., EJCMID 2013 October 22,

34 MS-RESIST/ P. aeruginosa (Tobramycin-resistant) Intens. [a.u.] _N 0:G10 MS, BaselineSubtracted, Smoothed normal _H_60 0:G1 MS, BaselineSubtracted, Smoothed heavy + TOB Intens. [a.u.]x _H 0:F11 MS, BaselineSubtracted, Smoothed heavy m/z Jung et al., EJCMID 2013 October 22,

35 ciprofloxacin Pseudomonas aeruginosa meropenem tobramycin Jung et al., 2014

36 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

37 3. Antibiotic susceptibility testing by phenotypic assays Phenotypic assay without isotopes?

38 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

39 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

40 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,

41 Conclusion (2) -lactamase activity test Rapid test h Automated analysis Directly from positive blood cultures Restricted to certain resistances -lactamases All -lactamases detectable?

42 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?

43 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 h High costs of MALDIhardware (Yet) no determination of MIC values (Yet) no test kits available hands on time is high

44 Max von Pettenkofer-Institut Jette Jung Theresa Eberl Christina Popp Julia Walker Christina Hamacher Lukas Schmidt Birgit Gross Andreas Wieser ForBIMed FöFoLe