Frances Bolt (1), Nicole Strittmatter (1), Adam Burke (1), Monica Rebec (2), Tamás Karancsi (3), Daniel Simon (3), Tony Rickards (2), Zoltan Takats (1)
(1) Imperial College London, SW7 2AZ, London, United Kingdom (2) Department of Microbiology, Imperial College Healthcare NHS Trust (3) Waters Research Centre, HU 1033 Budapest, Hungary
The introduction of mass spectrometry for clinical microbiology laboratories has revolutionised work flows and provided timely and accurate clinical diagnoses. Rapid evaporative ionisation mass spectrometry (REIMS) has previously been show to allow the differentiation of fungi and bacterial species based upon their lipidome (Strittmatter et al., 2013; Strittmatter et al., 2014). In order to utilise this tool for clinical diagnostics and research we are creating a spectral library comprising over 50,000 isolates and 4000 species. A novel customised TECAN platform which incorporates automated colony imaging, colony picking and REIMS analysis has been developed to provide a reproducible system for high throughput workflows. REIMS on the customised TECAN EVO platform provides an ideal method for different applications including clinical diagnostics, lipidome and metabonomic analysis.
Prior to the introduction of mass spectrometry, most notably matrix-assisted laser desorption ionisation Time of Flight (MALDI-TOF), in clinical microbiology laboratories most bacterial identification was based upon biochemical characteristics including sugar utilisation and fermentation which was time consuming, variable and required expertise to perform. The advent of mass spectrometry for rapid microbial diagnostics has improved patient care through timely diagnosis and targeted treatments. MALDI-TOF is based upon the analysis of ionised proteins within the mass range of 2−20 kDa where over half are of ribosomal origin (Krause et al., 1999; Ryzhov et al., 2001). Whilst MALDI-TOF has reduced the time to identification of microbial colonies it requires the addition of a matrix to assist in their lysis and ionisation and, in some instances, additional extraction steps are necessary for accurate species classification.
Rapid evaporative ionisation mass spectrometry (REIMS) provides accurate speciation of bacteria and yeasts direct from colonies without the need for additional preparative steps (Strittmatter et al., 2013; Strittmatter et al., 2014). This technique allows the lipidomic profile of bacteria and fungi to be determined. After applying a radiofrequency electrical current to the micro-organism the resulting vapour, containing gas phase ions of metabolites and structural lipids, is channelled to a mass spectrometer for REIMS analysis. Analysis of the resulting lipidome enabled the robust species level identification of yeast and 28 clinically important bacteria (Strittmatter et al., 2014). Furthermore, it has shown promising results for strain level resolution and information on antimicrobial resistance. In order to utilise this technology for the identification of unknown microorganisms a spectral library comprising mass spectra is currently being developed to encompass the breadth of microbial taxonomy. To ensure the REIMS taxonomic designation is accurate 16S rRNA and MALDI-TOF mass spectrometry based speciation (Bruker Daltonics, Billerica, MA) will also be performed for each isolate. Notably, the REIMS process has been adapted for high throughput analysis using a novel TECAN EVO Freedom platform (Tecan, Männedorf, Switzerland) with an integrated Pickolo system (SciRobotics, Israel). This provides a single system for automated colony picking, REIMS analysis and molecular processing. Each of the 34 agar plates analysed during one run is visualised using the Pickolo image analysis and either the operator or the Pickolo software can select colonies for automated REIMS. Furthermore, down-stream molecular workflows are optimised by the automatic transfer of selected colonies to a 96 well plate which can then be used for nucleic acid extraction. This novel platform provides simple, accurate and reproducible REIMS with minimal user input.
Materials and Methods
Over 50,000 isolates, obtained from numerous research and clinical microbiology laboratories, will be analysed by REIMS to ensure the spectral library is diverse. Each isolate was cultured according to their specific growth requirements and identified to species level by 16S rRNA sequencing analysis and MALDI-TOF based mass spectrometry on the MALDI Biotyper (Bruker Daltonics, Billerica, MA). DNA was extracted using a QIAcube (QIAGEN, UK) according to manufacturer’s instructions and the 16S rRNA gene was amplified using the method described by Woo et al. (Woo et al., 2001) with the exception that all PCR reactions were conducted using the Hot StarTaq Master Mix Kit (Qiagen, UK). Although REIMS processing and analysis was based upon the methods described by Strittmater and colleagues (Strittmatter et al., 2014) this was automated using the TECAN EVO Freedom platform (Tecan, Männedorf, Switzerland) with an integrated Pickolo system (SciRobotics, Israel) as described above. Antimicrobial susceptibilities were determined according to Clinical and Laboratory Standards Institute guidelines (CLSI, Pennsylvania, US) and, where appropriate, carbapenemase production was assessed using the Xpert Carba-R assay (Cepheid, CA, US). The REIMS data was validated by ensuring the species designations were concordant with the 16S rRNA and MALDI-TOF data.
RESULTS AND CONCLUSIONS
Robust species level identification was achieved for bacteria and fungi using REIMS and was in concordance with data obtained by 16S rRNA and MALDI-TOF analysis. Preliminary data indicate that it can provide finer level taxonomic classifications for some bacteria and information on antimicrobial susceptibilities. A spectral library is currently being developed to ensure much of the breadth of microbial taxonomy is captured. The TECAN instrument, coupled with the Pickolo software, provides an ideal automated platform for high throughput investigations which minimises many user related variables. REIMS analysis offers many promising applications from clinical diagnostics to metabolomics and lipidomics.
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