MSACL 2016 US Abstract

Using Rapid Evaporative Ionisation Mass Spectrometry (REIMS) to Assign Taxonomic Classifications to Microbial Isolates and Mixed Communities

Simon Cameron (Presenter)
Imperial College London

Bio: I joined Prof. Zoltan Takat's research group in 2015 at Imperial College London as a Research Associate in Microbial Populations and Metabonomics. I received my BSc (2011) and PhD (2015) from Aberystwyth University, Wales, UK. My research interests are in the broad field of microbial ecology with a focus on the human microbiome and its response to disease and stress. I employ microbial profiling techniques including 16S rRNA gene amplicon sequencing and metagenomics, and metabolome fingerprinting and profiling approaches. I currently work as part of the MicrobeID team at Imperial College London to apply rapid evaporative ionisation mass spectrometry (REIMS) to assign taxonomic and functional classification to microbial isolates and to the profiling of mixed microbial communities.

Authorship: SJS Cameron (1), F Bolt (1), A Burke (1), N Strittmatter (1), T Rickards (2), K Hardiman (1), P Inglese (1), A Abdolrasouli (2), M Rebec (2), T Karancsi (1), D Simon (1), KA Veselkov (1), Z Takats (1)
(1) Imperial College London, London, UK (2) Imperial College Healthcare NHS Trust, London, UK

Short Abstract Mass spectrometry has revolutionised clinical microbiology laboratories’ work flows, and decreased diagnosis times. Rapid evaporative ionisation mass spectrometry (REIMS) has previously been show to allow the differentiation of fungi and bacterial species based upon their lipidomic profiles. Work is currently underway to construct a REIMS mass spectra library of approximately 50,000 isolates from 4,000 species, using a high-throughput, semi-automated platform incorporating colony imaging and picking. This mass spectra library will be utilised in the identification of microbial isolates without the preparative steps required for MALDI-ToF. Furthermore, the effectiveness of using REIMS analysis to identify microbes from multiple species samples will be evaluated. This will be completed without the prior isolation of pure microbial cultures, thereby significantly reducing time to diagnosis.

Long Abstract

This presentation will cover the on-going work within the MicrobeID team at Imperial College London to assess the utility of using the REIMS platform to assign species-level taxonomic classification to microorganisms, and the creation of a REIMS mass spectra library. Additionally, preliminary work to utilise the REIMS platform in microbial community profiling will be presented and discussed.

INTRODUCTION: Prior to the introduction of mass spectrometry, most notably matrix-assisted laser desorption ionisation time of flight (MALDI-ToF), into clinical microbiology laboratories, the majority of identifications relied upon biochemical characterisations. These included sugar utilisation and fermentation; but were time consuming, variable, and required expertise to perform. The utilisation of mass spectrometry for rapid microbial identification has improved patient care through reduced diagnosis times, and targeted clinical interventions. MALDI-ToF based platforms are the most widely used in clinical diagnostic laboratories. Microbial identifications using this method rely on the characterisation of ionised proteins within the mass range of 2−20 kDa; where over 50% are of ribosomal origin [1,2]. Although, the introduction of MALDI-ToF has reduced the time to identification of microbial colonies, it requires the addition of a matrix to assist in ionisation and, in some instances, additional extraction steps are required for accurate species identification. Furthermore, in the majority of investigations, it is still reliant upon the isolation of pure microbial cultures before analysis can be carried out. This is frequently the major factor in determining the length of time required for microbial identification. MALDI-ToF based systems can also have difficulty in the species-level classification of some microorganisms, such as Salmonella sp., and in sub-species analysis.

REIMS has previously been demonstrated to provide accurate species-level classification of bacteria and yeasts direct from colonies; without the need for additional preparative steps [3,4]. In comparison to MALDI-ToF based platforms, REIMS utilises the lipidomic profile of bacteria and fungi to determine their species-level classification. The REIMS system works by applying a radiofrequency electrical current to a microbial colony to rapidly evaporative it. The resulting vapour, containing gas phase ions of metabolites and structural lipids, is channelled to a mass spectrometer for analysis. Previous work by members of this group has shown that this method of lipidomic analysis is able to provide robust, species-level identification of yeast and 28 clinically important bacteria [4].

In order to utilise this technology for the identification of unknown microorganisms, a spectral library comprising mass spectra is currently being developed; which will encompass the breadth of microbial taxonomy. To ensure the REIMS taxonomic designation is accurate, full-length 16S/18S rRNA gene and ITS region sequences and MALDI-ToF mass spectrometry based speciation using the Bruker Biotyper system (Bruker Daltonics, Billerica, MA) will be assigned to each mass spectra in our library. Recent work in our group has focussed on optimising the REIMS process for high-throughput analysis. This incorporates a novel TECAN EVO Freedom platform (Tecan, Männedorf, Switzerland) with an integrated Pickolo system (SciRobotics, Israel) which provides a single system for colony picking, REIMS analysis, and molecular processing.

Due to its analytical approach, REIMS may have further utility as a profiling tool for the microbial communities present within clinical and non-clinical samples. Currently, for the majority of situations, clinical diagnostic microbiology relies upon the isolation of pure microbial cultures before classification using MALDI-ToF, or other methods, can be attempted. Through the utilisation of our mass spectra database, it may be possible for REIMS to identify clinically relevant microbes from multi-species communities, without the time-consuming requirement of pre-analysis isolation.

MATERIALS AND METHODS: Construction of a mass spectra library of approximately 50,000 isolates across approximately 4,000 microbial species is currently underway. Isolates are collected from clinical diagnostic and non-clinical microbiology laboratories, and cultured according to their specific growth requirements. Each isolate is then subjected to MALDI-ToF analysis using the Bruker Biotyper system; with a score of 2.0 or above confirming species-level classification. In addition, genomic DNA is extracted from each isolate using a combined temperature and enzymatic degradation method. DNA is then subjected to PCR to amplify the sequence target (eg: 16/18S rRNA gene or ITS region) and then sequenced by an external provider using the Sanger method. Resulting sequences are used to interrogate established databases to assign species and strain-level taxonomic classifications. REIMS analysis is based upon a modified version of the methods detailed by Strittmater et al., [4], specifically the replacement of the bi-polar forceps with an automated TECAN EVO Freedom platform (Tecan, Männedorf, Switzerland) with automated Pickolo system (SciRobotics, Israel).

RESULTS AND DISCUSSION: Species-level identification has been achieved for bacteria and fungi using REIMS with classification in concordance with taxonomy assigned by 16S/18S rRNA gene sequencing and MALDI-ToF analysis. The mass spectra library is currently being developed to increase the breadth of microbial taxonomy covered, and to widen the utility of the REIMS system to non-clinical samples. The TECAN EVO Freedom instrument coupled with the Pickolo software provides an ideal automated platform for high throughput investigations; which minimises user related variables. The REIMS platform offers many promising applications. It may further revolutionise clinical microbiology diagnostics by allowing the identification of clinically-relevant microbes without the prior requirement to isolate pure microbial cultures.

References & Acknowledgements:

References:

[1] Krause, E., Wenschuh, H. & Jungblut, P. R. The Dominance of Arginine-Containing Peptides in MALDI-Derived Tryptic Mass Fingerprints of Proteins. Anal. Chem. 71, 4160–4165 (1999).

[2] Ryzhov, V. & Fenselau, C. Characterization of the Protein Subset Desorbed by MALDI from Whole Bacterial Cells. Anal. Chem. 73, 746–750 (2001).

[3] Strittmatter, N. et al. Analysis of Intact Bacteria using Rapid Evaporative Ionisation Mass Spectrometry. Chem. Commun. 49, 6188–90 (2013).

[4] Strittmatter, N. et al. Characterization and Identification of Clinically Relevant Microorganisms using Rapid Evaporative Ionization Mass Spectrometry. Anal. Chem. 86, 6555–62 (2014).

Acknowledgements:

This work is supported by research grants awarded to Prof. Zoltan Takats from the Waters Corporation and the UK Biotechnology and Biological Sciences Research Council (BBRSC).

Financial Disclosure

IP Royalty: no

Planning to mention or discuss specific products or technology of the company(ies) listed above:

yes