In recent years, the role of metabolomics has become increasingly more important in the advancement of many research fields including medical studies. Due to lack of metabolomics research in the area of infectious disease and the rise in antibiotic resistance, there is a need for further studies on the modes of antibiotic action and the mechanisms of resistance of pathogenic microorganisms at the metabolome level. This study aimed to investigate effects of DNA synthesis inhibitors on the metabolome of E. coli and to develop a workflow for discrimination between E. coli isolates down to the sub-species level using a variety of methods, which can inform the choice of analytical techniques in metabolomics research. A metabolomics-based approach was used to elucidate metabolic alterations in E. coli K-12 upon challenge with trimethoprim at two pH levels (5 and 7) which mimic human urine acidity. FT-IR spectroscopy was used as a preliminary experiment to produce bacterial fingerprints and GC-MS was applied to generate global metabolic profiles in each condition. At pH 7, as the drug molecules exhibited higher permeability, stronger direct effects of the antibiotic were observed, i.e. decreased levels of nucleotides. Trehalose, an osmoprotectant, was up-regulated in these stress conditions and this up-regulation was mirrored by a decrease in glucose levels. This also correlated with up-regulation of pyruvate-related products (e.g. alanine, citrate and malate). Other off-target related effects were observed such as alterations in the levels of various amino acids upon trimethoprim challenge. This study offered a wider view of drug action at pH levels similar to healthy human urine. A high throughput FT-IR spectroscopy method was developed to discriminate between pathogenic E. coli isolates based on sequence type. This method employed a Bioscreen as a micro-culture incubator instead of traditional sample preparation (shaking flasks), which can be labour intensive and time consuming. Excluding the washing step in the protocol enabled discrimination between isolates of different sequence types. Moreover, a reproducible workflow of lipid analysis based on LC-MS was developed and applied on four pathogenic isolates with different sequence type and susceptibility to ciprofloxacin. This workflow enabled detection of a wide range of lipid classes and determination of significant alterations in lipid levels related to susceptibility to ciprofloxacin. Stressed and control isolates were also analysed using the developed Bioscreen FT-IR approach to assess phenotypic fingerprint differences, which were in line with the LC-MS-ve class distribution. Further investigation by means of four analytical platforms (FT-IR, GC-MS, LC-MS-ve and LC-MS+ve) was applied on E. coli ST131 isolates characterised using classical microbiological tests (virulence factors and metabolic tests). Procrustes transformation was used to compare between the analytical methods and the microbiological tests in terms of their capacity to discriminate between the different isolates. As indicated above, the results from FT-IR and LC-MS-ve were comparable and in line with virulence tests, while GC-MS and metabolic tests were in agreement. Complementary information generated by different analytical techniques and microbiological tests may indicate the requirement for careful selection of the method of investigation and may suggest the need to continue using a combination of methods which are applied to study different features of bacterial physiology.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:617979 |
| Date | January 2014 |
| Creators | Alrabiah, Haitham Khalid M. |
| Contributors | Goodacre, Roy |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/advanced-metabolomics-for-the-discrimination-of-uropathogenic-escherichia-coli-and-their-response-to-antibiotics(1c78e191-8652-4ff9-9eae-b746ed1c9e0e).html |
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