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MALDI-TOF MS for the Identification of Anaerobic Bacteria

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MALDI-TOF MS for the Identification of Anaerobic Bacteria University Medical Center Groningen Department Medical Microbiology and Infection Prevention Linda Veloo Expert Center Anaerobic Infections The Netherlands www.mmb-umcg.eu © by author ESCMID Online Lecture Library Matrix Assisted Laser Desorption/Ionization time-of-flight Mass Spectrometry (MALDI-TOF MS) Time of flight tube Target at 15-25 kV Detector Ion source © by author ESCMID- “Time of flight” Online of individual Lecture proteins is converted Library into mass information. - Spectrum is produced - Database is built Veloo et al. Anaerobe 2011; 17:211-212 Workflow Direct spotting of bacteria on target using a toothpick Add HCCA matrix Data acquisition © Databy analyses author ESCMID Online Lecture Library Log score: <1.7 no reliable identification 1.7 – 2.0 reliable genus identification ≥ 2.0 reliable species identification Obtained spectrum is unique for bacterial species Intensity © by author ESCMID Online Lecture Library Anaerobic culture Phenotypic pure culture primary incubation 2 days 2-7 days aerotolerance identification MALDI-TOF MS 1 day 2-14 days primary incubation 2-7 days © by author MALDI-TOF MS testing minutes ESCMID Online Lecture Library How many anaerobic bacteria can be identified using MALDI-TOF MS? UMCG 2011/2012 Total no. of strains 1000 Species ID 650 65% Genus ID 149 15% No ID © 201by author20% ESCMID Online Lecture Library Performance differs per genus Genus* % species ID % genus ID Clostridium sp. (n=149) 97 3 B. fragilis sp. (n=179) 97 3 Parabacteroides sp. (n=14) 93 7 GPAC (n=133) 85 15 Prevotella sp.(n=83) 78 12 Propionibacterium sp. (n=129) 64 36 Actinomyces sp. (n=28) 57 43 Fusobacterium sp. (n=16)© by author50 50 Campylobacter sp. (n=17) 41 59 Bilophila sp. (n=14) 0 100 ESCMIDDivers (n=26) Online Lecture77 Library13 * Only isolates which could be identified with MALDI- TOF MS (approx. 80% of all isolates) Example from literature Species identification of clinical Prevotella isolates by Matrix-Assisted Laser Desorption Ionization-Time of Flight mass spectrometry Wybo et al. J. Clin. Microbiol. 2012; 50:1415-1418 Strains: 102 sequenced clinical isolates Identification MALDI-TOF MS: 1. using the commercial reference database 2. using the commercial reference database + the addition of main spectral profiles (MSP)© ofby 23 referenceauthor strains and 7 sequenced clinical isolates ESCMID Online Lecture Library How to make a Main Spectral Profile (MSP) x104 Intens. [a.u.] Intens. 1.5 Intens. [a.u.] Intens. 4000 1.0 3000 0.5 © by author 2000 0.0 2000 4000 6000 8000 10000 12000 14000 16000 m/z ESCMID Online1000 Lecture Library Average of at least 20 spectra 0 5920 5930 5940 5950 5960 5970 5980 m/z Example from literature Commercial reference database: Commercial database + additions: - 63 % correct species identification - 83 % correct species identification - 11 % correct genus identification - 6 % correct genus identification - 26 % no identification - 11 % no identification 14 % due to the addition of© main by spectra author of species not yet present in database 6 % due to the addition of main spectra of species already present in ESCMIDdatabase Online Lecture Library Summary (I) 1. ± 70 % of the anaerobes can be identified using MALDI-TOF MS 2. Performance differs per species/genus 3. MALDI-TOF MS database needs optimizing for the identification of anaerobic bacteria - addition of spectra to existing database increases performance - reference strains - clinical© isolates by author ESCMID Online Lecture Library ENRIA European Network for the Rapid Identification of Anaerobes Lead: Prof. A.W. Friedrich (The Netherlands) Prof. E. Nagy (Hungary) Coördination: © by author Dr. A.C.M. Veloo (The Netherlands) Prof. A.J. van Winkelhoff (The Netherlands) ESCMID Online Lecture Library In collaboration with: Dr. W. Pusch (Bruker, Germany) Dr. M. Kostrzewa (Bruker Germany) EU INTERREG ENRIA project Expertise laboratories: - UMCG, Groningen, The Netherlands - University of Szeged, Szeged, Hungary - Odense University Hospital, Odense, Denmark - University Hospital Brussels, Brussels, Belgium - Health Protection Agency, London, England - Praticien Hospitalier, Montpellier, France - Public Health Wales, Cardiff, England © by author ESCMID Online Lecture Library ENRIA project Goal: To optimize the MALDI-TOF MS database for the most common encountered anaerobic bacteria At least 5 MSP’s present of each species Strains are sent to the UMCG by the expertise laboratories © by author UMCG: - Culture - Storage - Identification by sequencing ESCMID Online- Ethanol suspension Lecture Library Validation of the optimized database How does this look like in practice: © by author ESCMID Online Lecture Library Conditions Which preanalytical factors influence the quality of the spectrum of anaerobic bacteria??? - Reproducibility of spotting - Incubation time - Pretreatment method -© Direct by spotting author - On target extraction - Full extraction ESCMID -Online Exposure to oxygenLecture Library Conditions Reproducibility spotting Amount of bacteria is important: Too little no peaks with sufficient intensity Too much saturation of detector and only prominent peaks are measured © by author Optimal amount: 130 µg/µl ESCMID Online Lecture Library Two examiners: an experienced one and a less experienced one Reproducibility spotting results © by author ESCMID Online Lecture Library Conditions Incubation time 24 hours 48 hours 72 hours 96 hours Pre-treatment method: © by author - direct spotting - on target extraction with 70% formic acid ESCMID Online- full extraction Lecture Library Incubation time results Incubation time (hours) Species 24 48 72 96 Gram-negative bacteria Bacteroides thetaiotaomicron 2.23 2.21 2.17 2.19 Prevotella intermedia 1.95a 2.03 2.27 2.04 Fusobacterium necrophorum 2.27 2.18 2.31 2.29 Fusobacterium nucleatumb 2.33 2.19 2.22 2.17 Campylobacter ureolyticus 1.90 2.07 2.01 1.92a Veillonella parvula 2.29 2.45 2.38 2.37 % reliable species ID direct spotting 67 100 100 83 % reliable species ID total 67 100 100 83 Gram-positive bacteria Parvimonas micra 2.33 2.29 2.30 2.27 Finegoldia magna 2.51c 2.43c 2.23c 2.21 Peptoniphilus harei 2.02c 2.23 2.19c 2.20 Peptoniphilus ivorii 1.78c 1.86d 2.02d 1.88d Atopobium minutum 1.96a 2.26 2.29 2.25 Clostridium butyricum 2.29 2.13 2.17 2.08 Clostridium hathewayii© by author2.20d 2.36 2.26 2.36 Actinomyces israellii <1.7e <1.7e <1.7e 2.03 Actinomyces graevenitzii 2.09c 2.36 2.00d 2.11 Actinomyces meyeri 2.27 2.15 2.12 2.20 Bifidobacterium dentium 2.30 2.12 2.37 2.28 ESCMID Bifidobacterium Online longum Lecture 2.18c 2.08c 2.07 Library2.01 Propionibacterium acnes 2.14c 2.24 2.10 2.16 Eggerthella lenta 2.21c 2.20 2.20d 2.10d % reliable species ID direct spotting 29 71 57 86 % reliable species ID total 79 86 93 93 Conditions Exposure to oxygen 0 hour 6 hours 24 hours 48 hours © by author Pre-treatment method: - direct spotting ESCMID Online- on target Lecture extraction with Library 70% formic acid - full extraction Exposure to oxygen results Exposure to oxygen (hours) Species 0 1 6 24 48 Gram-negative bacteria Bacteroides thetaiotaomicon 2.14 2.24 2.10 2.17 2.35 Bacteroides stercoris 2.19 2.14 2.18 2.25 2.27 Parabacteroides johnsonii 2.23 2.32 2.29 2.31 2.28 Fusobacterium necrophorum 2.08 2.30 2.19 <1.7a <1.7a Fusobacterium nucleatumb 2.08 2.27 2.32 2.19 2.08 Prevotella intermedia 1.92 1.93 1.86 1.95c <1.7a Prevotella oris 2.08 2.17 2.01 2.35 2.29 Alistipes onderdonkii 2.21 2.24 2.29 2.29 2.21 Veillonella parvula 2.25 2.13 2.2 2.20 2.16 % reliable species ID direct spotting 89 89 89 78 78 % reliable species ID total 89 89 89 78 78 Gram-positive bacteria Finegoldia magna 2.24 2.05c 2.34 2.52 2.50 Peptoniphilus harei 2.15 2.00 2.13 2.23b 2.19 Peptoniphilus ivorii © by<1.7 d author1.81e <1.7d 1.76e 1.80e Clostridium hathewayii 2.36 2.23 2.29 2.07 2.19 Clostridium ramosum 2.03 2.19 2.27 2.06 2.17 Actinomyces graevenitzii 2.05c 2.03e 2.11 2.06e <1.7d Actinomyces meyeri 2.03 2.27 2.35 2.19 2.25 Bifidobacterium longum 2.00c 2.12c 1.99c 2.15 2.17 ESCMID Propionibacterium Online acnes 2.18 Lecture 2.14c 2.12 2.13 2.13 Library Eggerthella lenta 2.11 2.30 2.27 2.16 2.02e Collinsella aerofaciens 2.26c 2.16 2.23c 2.32c 2.28c % reliable species ID direct spotting 64 55 73 64 64 % reliable species ID total 91 82 82 91 82 Summary (II) The fact whether an unknown anaerobic bacterium can be identified using MALDI-TOF MS mainly depends on: - The person who is spotting - The type of colony - Amount of bacteria spotted © by author - If sufficient MSP’s are present in the MALDI-TOF MS database ESCMID Online Lecture Library Recommendations in order to obtain a good quality spectrum - perform the MALDI-TOF MS measurement after 48 hours of incubation in an anaerobic environment - spot the bacteria on the same day - perform an on target extraction with 70% formic acid for gram-positive anaerobic© bybacteria author - have the spotting performed by an experienced examiner ESCMID Online Lecture Library Accomplished so far: Collected: ± 650 strains © by author ESCMID Online Lecture Library Preliminary results ENRIA Misidentifications: Sequence ID MALDI-TOF MS ID Anaerococcus vaginalis Anaerococcus hydrogenalis Bacteroides dorei Bacteroides vulgatus Bacteroides cellulosilyticus Bacteroides intestinalis Parabacteroides merdae Parabacteroides johnsonii © by author Clostridium rectum/ Fusobacterium mortiferum Fusobacterium mortiferum ESCMID Online Lecture Library Moryella indoligenes Fusobacterium naviforme No MSPs present in the database Dendrogram Anaerococcus vaginalis/hydrogenalis © by author ESCMID Online
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