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The Technical and Biological Reproducibility of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

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The Technical and Biological Reproducibility of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry RESEARCH ARTICLE The Technical and Biological Reproducibility of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) Based Typing: Employment of Bioinformatics in a Multicenter Study a11111 Michael Oberle1, Nadia Wohlwend2, Daniel Jonas3, Florian P. Maurer4¤, Geraldine Jost5, Sarah Tschudin-Sutter6, Katleen Vranckx7, Adrian Egli8,9* 1 Clinical Microbiology, Cantonal Hospital of Aarau, Aarau, Switzerland, 2 Clinical Microbiology, labormedizinisches Zentrum Dr. Risch AG, Berne, Switzerland, 3 Institute for Environmental Health Medicine and Hospital Infection Control, University of Freiburg, Freiburg, Germany, 4 Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland, 5 Clinical Microbiology, Dianalabs, Geneva, OPEN ACCESS Switzerland, 6 Infectious Diseases and Hospital Epidemiology, University Hospital Basel, Basel, Citation: Oberle M, Wohlwend N, Jonas D, Maurer Switzerland, 7 Applied Maths NV, Sint-Martens-Latem, Belgium, 8 Clinical Microbiology, University Hospital Basel, Basel, Switzerland, 9 Applied Microbiology Research, University of Basel, Basel Switzerland FP, Jost G, Tschudin-Sutter S, et al. (2016) The Technical and Biological Reproducibility of Matrix- ¤ Current address: Institute for Medical Microbiology, Virology and Hygiene, University Medical Center Assisted Laser Desorption Ionization-Time of Flight Hamburg-Eppendorf, Hamburg, Germany Mass Spectrometry (MALDI-TOF MS) Based * [email protected] Typing: Employment of Bioinformatics in a Multicenter Study. PLoS ONE 11(10): e0164260. doi:10.1371/journal.pone.0164260 Abstract Editor: Frederique Lisacek, Swiss Institute of Bioinformatics, SWITZERLAND Background Received: May 19, 2016 The technical, biological, and inter-center reproducibility of matrix-assisted laser desorption Accepted: September 22, 2016 ionization-time of flight mass spectrometry (MALDI TOF MS) typing data has not yet been Published: October 31, 2016 explored. The aim of this study is to compare typing data from multiple centers employing Copyright: © 2016 Oberle et al. This is an open bioinformatics using bacterial strains from two past outbreaks and non-related strains. access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and Material/Methods reproduction in any medium, provided the original Participants received twelve extended spectrum betalactamase-producing E. coli isolates author and source are credited. and followed the same standard operating procedure (SOP) including a full-protein extrac- Data Availability Statement: A link to the raw data tion protocol. All laboratories provided visually read spectra via flexAnalysis (Bruker, Ger- is in the manuscript. The data can be analysed with many). Raw data from each laboratory allowed calculating the technical and biological various (free) softwareÐin our publication we have used a commercial software (Bionumerics), reproducibility between centers using BioNumerics (Applied Maths NV, Belgium). however, even for this software a free trial version is available, so that everybody could reproduce our Results data. Technical and biological reproducibility ranged between 96.8±99.4% and 47.6±94.4%, Funding: Support was provided by the Swiss National Research Foundation: PZ00P3_154709; respectively. The inter-center reproducibility showed a comparable clustering among identi- [www.snf.ch]. cal isolates. Principal component analysis indicated a higher tendency to cluster within the PLOS ONE | DOI:10.1371/journal.pone.0164260 October 31, 2016 1 / 13 MALDI-TOF MS Typing and Bioinformatics Competing Interests: The authors have declared same center. Therefore, we used a discriminant analysis, which completely separated the that no competing interests exist. clusters. Next, we defined a reference center and performed a statistical analysis to identify specific peaks to identify the outbreak clusters. Finally, we used a classifier algorithm and a linear support vector machine on the determined peaks as classifier. A validation showed that within the set of the reference center, the identification of the cluster was 100% correct with a large contrast between the score with the correct cluster and the next best scoring cluster. Conclusions Based on the sufficient technical and biological reproducibility of MALDI-TOF MS based spectra, detection of specific clusters is possible from spectra obtained from different cen- ters. However, we believe that a shared SOP and a bioinformatics approach are required to make the analysis robust and reliable. Introduction Matrix-assisted laser desorption/ionization-timeof flight mass-spectrometry (MALDI-TOF MS) is commonly used for the rapid identification of bacterial species via characterization of highly specific protein mass-spectra profiles[1,2]. For microbiological routine diagnostics, the MALDI-TOF MS technology focuses on a mass-range between 2 to 20kDa, covering mainly ribosomal proteins harbouring a high diversity between species[3–5]. In addition, this technol- ogy can also be used for typing purposes at a sub-species level. Most publications focused on an improved resolution of species identification within a bacterial complex or group e.g. Bacillus cereus group[6,7], Burkholderia cepacia complex[8,9], or Mycobacterium abscessus complex [10,11]. In addition, several reports have indicated that MALDI-TOF MS based typing may be used for rapid infection control investigations including a wide range of bacterial species[12– 20]. The technology allows the comparison of mass-spectrometryspectra data with a potential resolution down to a single amino acid difference between outbreak strains and non-related isolates. The range and frequency of shifts in the mass-spectra peak profiles can be used to determine the relatedness between single isolates within an outbreak. We have recently estab- lished a standard operating procedure to type extended spectrum β-lactamse (ESBL)-produc- ing Escherichia coli and could show a highly similar dendrogram compared to pulsed field gel electrophoresis[15]. However, in none of these studies the overall technical, biological, and inter-center repro- ducibility of a same set of bacteria has been addressed. This would be a crucial step to validate the robustness of MALDI-TOF MS based typing. An investigation in its robustness would allow further implementation of this new and rapid typing method. It would also allow estab- lishing a protocol to prepare the samples for sub-typing. In addition, interpretation of complex MALDI-TOF spectra in the context of typing application may be challenging [21]. We hypothesize, that (i) the variance at a sub-species level may be determined by using MALDI-TOF MS, and (ii) the results are dependent on high quality profiles including a techni- cal and biological reproducibility, followed by a bioinformatics work-up. Therefore, we aimed to assess the technical, biological and inter-center reproducibility of MALDI-TOF MS based typing in a blinded multicenter study including six microbiology diagnostic laboratories exam- ining two nosocomial outbreaks and two non-outbreak related isolates of ESBL-producing E. coli. PLOS ONE | DOI:10.1371/journal.pone.0164260 October 31, 2016 2 / 13 MALDI-TOF MS Typing and Bioinformatics Materials and Methods Bacterial isolates. We included twelve isolates of ESBL-producing E. coli collected of two hospi- tal-related outbreak clusters (six and four each) and two non-outbreak related isolates from the University Hospital Basel and the Cantonal Hospital Aarau. The non-outbreak related isoaltes were from urinary tract isolates of the University Hospital Basel. The relatedness of the isolates was previously analysed with pulsed field gel electrophoresis (S1 Fig). These isolates were sent to the six participating diagnostic microbiology laboratories (see affiliations). The centers were blinded for the outbreak and non-outbreak related isolates. In one center, two technicians inde- pendently performed MALDI-TOF MS typing, resulting in two data sets from the same center (referred to as center 5A and 5B). All participating centers used the Microflex MALDI-TOF MS system (Bruker Daltonics, Bremen, Germany). Technicians had low or no experience in MALDI-TOF MS based typing. Therefore, we used a previously published standard operating procedure (SOP) to provide a protocol guiding, step by step, through the whole procedure [15]. All participants were blinded regarding the relatedness of the isolates. All peak profile raw data from each center can be downloaded here: https://figshare.com/articles/ AllRawSpectraEgliPlosOne_zip/3749454 SOP. Each center received a SOP for the exact handly of the samples. The SOP was explained by a common instructor. Briefly, all bacterial isolates were stored at -80°C, thawed and sub-culti- vated prior to sending to the participating laboratories. Each participating laboratory re-culti- vated the isolates at standard conditions on a blood agar plate in an aerobic atmosphere at 37°C for 18–20h. All twelve isolates had to be sub-cultivated simultaneously to ensure the same age among the colonies to control the senescence-associatedchanges in the mass peak spectrum. Twenty ul of bacterial colony material (corresponds to a full 1μL loop) was used for ethanol-for- mic acid (70%) protein extraction [15]. For each protein extraction, four separate spectra were recorded (quadruplicates) using the FlexControl software (Bruker Daltonics, Bremen, Germany) to assess technical reproducibility.
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