Award details

Development of a rapid bacterial identification method based on direct mass spectrometric metabolic profiling

Reference	BB/L020858/1
Principal Investigator / Supervisor	Professor Zoltan Takats
Co-Investigators / Co-Supervisors	Professor Julian Marchesi, Dr Kirill Veselkov
Institution	Imperial College London
Department	Surgery and Cancer
Funding type	Research
Value (£)	485,889
Status	Completed
Type	Research Grant
Start date	01/08/2014
End date	30/09/2018
Duration	50 months

Abstract

The proposed research is aimed at the development of a novel microbial identification and community analysis method. The method is based on the rapid evaporation of bacterial cells or matrix containing bacterial cells using high frequency alternating electric current followed by mass spectrometric analysis of electrically charged molecular species. The method - termed Rapid Evaporative Ionization Mass Spectrometry (REIMS) - is expected to yield spectral information featuring metabolic species and polar lipids. Since lipid synthesis pathways are conservative and specific to a given species, REIMS analysis of bacterial cells allows species-level identification, as it is already suggested by preliminary data. The REIMS data will be complemented by more traditional LC-MS data giving detailed information on the detected species. The proposed work comprises the development of corresponding analytical instrumentation followed by large scale data collection. Analysis of 150 bacterial strains (playing important role in food-borne diseases) is planned following culturing under a wide range of different conditions. The collected data will serve as a learning set for developing multivariate statistical machine learning algorithms in order to perform identification of unknown strains. Statistical models will also be utilized to understand the metabolic differences between different strains, species or families and correlate these with genetic differences. Community analysis methods based on REIMS and LC-MS will also be developed. Analytical and biological interferences will be studied using the two different mass spectrometric approaches and data processing approaches will be developed to compensate for these and achieve absolute quantification of different species in mixed communities. Effects of food and biofluid matrices will also be studied and the method will further be developed for the culturing free, quantitative detection of bacteria in various natural matrices.

Summary

The project is aimed at the development of new tools for the identifications of various microorganisms including bacteria causing a wide range of diseases from common cold to bloodstream infections. Knowing exactly which type of bacterium is involved in a disease is very important, since the choice of appropriate medication largely depends on it. Likewise, in case of public health or food safety, the correct classification of bacterial contaminations helps with the identification of their source and elimination of the contamination. Currently, samples containing bacterial cells are collected and sent to laboratories. Microbiologists grow the bacteria in Petri-dishes containing special nutrients. Based on the types of nutrients the bacteria can use and the results of multiple chemical tests, the bacterium is tentatively identified. If proper classification is necessary, nucleic acids are extracted from the bacterial cells, and their base-pair sequence is determined, which helps in the unambiguous identification. All of these processes are time consuming, which considerably delays the efficient intervention both in case of infectious diseases and in case of a waterborne disease outbreak. The purpose of the proposed research is to develop an alternative, much faster technique for the identification of bacteria. Mass spectrometry is an analytical technique capable of the measurement of the weight of molecules, and also the selective detection of hundreds of different molecules at the same time. We plan to use this well-established technique for looking at some special building blocks of bacterial cells. One novel aspect of the research is that mass spectrometers are not used in the traditional way, including a lengthy preparation of bacterial cells prior to analysis, but the cells are simply heated up, and electrically charged molecules formed on the boiling of cells are analysed using mass spectrometry. The idea of rapid mass spectrometric analysis by using simplyheat has already been applied in case of surgery, where cancer tissue is identified in a similar way. In course of the proposed project we plan the adopt this technology (Rapid Evaporative Ionization Mass spectrometry; REIMS) for the analysis of bacteria grown in the laboratory and also for the direct analysis of liquid samples (ranging from pond water to blood) containing bacterial agents. We plan to build a large library of the spectroscopic fingerprints of the bacteria, which will be used as a training set for computer based search algorithms. The method, the database and the algorithm together will enable the unambiguous identification of bacteria in considerably shorter timeframe than the current routine. Furthermore, the proposed research can potentially lead to an approach, where bacteria are directly identified in their natural environment (e.g. in urine for a urinary infection) without growing them in the laboratory for several hours or days.

Impact Summary

The proposed research is aimed at the development of new chemotaxonomic bacterial identification methods and corresponding extensions suitable for the analysis of microbial communities. The main advantages of the envisioned methods include the possibility of real-time analysis, culturing-free analysis, improved quantification of bacterial species in communities and extended amount of information on the actual phenotype of the species. This latter point is important when the question is not aimed at the taxonomical classification of the bacterial strains, but rather at their biochemical function. Beneficiaries The primary beneficiary of the research is the Mass Spectrometry Division of Waters Corporation (Manchester, UK), utilizing the developed technologies in the form of products marketed for bacterial identification for various customer groups. In this sense, the following groups can directly benefit from the expected results of proposed project: - Microbiologist researchers - Clinical microbiologists - Biotechnology professionals/companies - Food safety authorities/food industry - Pharma industry Indirectly the entire society can benefit from these developments by receiving quicker and more accurate medical care in case of infectious diseases, by consuming safer food (including drinking water) items containing harmful bacteria at lower probability, having access to new pharmaceuticals produced by more reliable biotechnological processes and living in a cleaner environment, just to mention a few of the broader impacts.

Committee	Research Committee C (Genes, development and STEM approaches to biology)
Research Topics	Microbial Food Safety, Microbiology, Technology and Methods Development
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	Industrial Partnership Award (IPA)

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