Identification of specific metabolites in mycolactone producing mycobacteria and Buruli ulcer infection: diagnostic biomarkers through metabolomic

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Buruli ulcer is a severe, slow progressing skin infection which destroys tissue and is caused by bacteria found in the environment called Mycobacterium ulcerans. The disease is classified as a neglected tropical disease despite being described in over 30 countries including Ghana, Togo, Benin, Côte d'Ivoire, Cameroon, Mexico, Papua New Guinea, South East Asia and Australia, and mostly affects individuals in rural communities living in infected areas. The World Health Organisation's Global Buruli Ulcer Initiative has identified the development of simple diagnostic tools as one of the priority areas in the control of the disease. Metabolomics is a growing field in analytical science and biology which seeks to identify novel biomarkers that can distinguish between diseased and non-diseased individuals. In a pilot study, funded under the Cambridge-Africa Partnership for Research Excellence (CAPREx), we sought to determine the best types of sample for the identification of metabolites from the bacteria that can be found only in Buruli ulcer patients. We collected tissue biopsy, swabs and fine needle aspirates from 28 Buruli ulcer confirmed patents and 21 patients with tropical ulcers that were not Buruli ulcer. The samples were pre-processed at the Department of Biochemistry, Cell and Molecular Biology at the University of Ghana, to extract the required components for gas chromatography-mass spectrometry (GC-MS) at the University of Cambridge. We identified tissue biopsy and fine needle aspirates as the best specimen for the identification of metabolites in preference to lesion swabs. Interestingly, metabolites identified in both groups of patients included cadaverine, putrescine, pinitol, palmitate, naphthalene, chlopyrifos and oxaspiro. The former two metabolites are interesting because they classify all the samples as containing degenerating tissue. The fatty acid palmitate is a common metabolite present in human tissue. Pinitol and the later three identifies metabolites are chemical residues that may have been present in the treatment poultice used as unorthodox remedies by the patients. In this proposal, we are seeking to further this pilot study, building on our observations and collecting the tissue biopsies and fine needle aspirates from a larger patient sample group to allow us to perform a more detailed analysis of the compounds found within the bacteria and also within infected tissue. We also intend to expand the scope of the biological samples from affected individuals by including urine samples in our analysis which may provide a more convenient biofluid to sample. To gain more insight into the novelty of the biomarkers we identify, we will include another phase of experimentation which involves the characterization of the compounds found in M. ulcerans and other similar bacteria strains that infect people in Ghana including M. pseudishottsii, M. liflandii and M. marinum DL. Data generated from this project will be important in the identification of new diagnostics and also give us insight to the biology of the mycobacterium during infection which may lead to better prospects for future treatments.