Qassadi, Fatimah
(2024)
Treatment strategies to combat antimicrobial resistance in gastrointestinal infections.
PhD thesis, University of Nottingham.
Abstract
The growing threat of antimicrobial resistance (AMR) necessitates the exploration of antibacterial therapeutic strategies to treat bacterial infections. This thesis explores metabolic targets for conventional and emerging therapies aimed at gastrointestinal (GI) infections caused by Escherichia coli (E. coli) and Clostridioides difficile (C. difficile). The research evaluates plant-based natural compound such as berberine, conventional antibiotics like azithromycin, and microbiome-based therapies including bacteriophages, bacteriophages combined with azithromycin, faecal microbiota transplantation (FMT) and faecal filtrates derived from FMT donors.
Bacterial metabolic processes play a key role in cellular function and is closely associated with AMR mechanisms. Metabolomics has become an increasingly popular “omics” approach for understanding the underlying biological mechanisms related to AMR and for identifying important metabolic pathways and biomarkers. By characterising the metabolic profiles of bacteria, their responses to different therapeutics can be elucidated, facilitating the identification of previously unexplored therapeutic targets. This thesis demonstrates the use of Liquid chromatography-mass spectrometry (LC-MS) and Orbitrap secondary ion mass spectrometry (OrbiSIMS) to assess the effectiveness of these therapies and identify molecular targets and metabolic pathways related to treatment responses.
The study first examines the differential abundance of metabolites in pathogenic multidrug resistant (MDR) enteroaggregative E. coli (EAEC O104:H4) compared to non-pathogenic E. coli O157: H7 (B6914) strains. Mass spectrometry (MS)-based metabolomic analysis revealed significant differences in bacterial metabolomes. The results showed that the metabolome of pathogenic E. coli differs significantly from that of non-pathogenic E. coli. Several pathways with significant associations to bacterial resistance were identified, notably alterations in amino acid biosynthesis, tricarboxylic acid (TCA) cycle intermediates, and phosphatidylglycerol lipids, making them potential therapeutic targets.
Natural source-based antibacterial agents, including plant-derived compounds, are re-gaining interest due to their diverse mechanisms of action and potential against MDR pathogens. This thesis investigates the antimicrobial activity and the metabolic alterations induced by berberine in both pathogenic MDR EAEC O104:H4 and non-pathogenic E. coli O157: H7 strains. The metabolomic analyses revealed that berberine disrupts key metabolic pathways, including the TCA cycle and peptidoglycan biosynthesis, compromising bacterial membrane integrity. The metabolomics data further revealed a significant depletion of glutathione, a key antioxidant that neutralises reactive oxygen species (ROS) and maintains cellular redox balance. These disruptions were supported by ROS generation assays, resulting in significant oxidative stress. These metabolic pathways are essential for bacterial viability and could serve as metabolic targets for berberine’s antibacterial activity.
The investigation into azithromycin's mode of action revealed off-target related effects beyond its well-known role in inhibiting protein synthesis. This study uniquely employed a complementary metabolomics approach using both OrbiSIMS and LC-MS to evaluate azithromycin-induced metabolic changes in MDR pathogenic and non-pathogenic E. coli strains. Pathogenic strain exhibited specific alterations in the glutamate-glutamine cycle, while non-pathogenic strain showed changes in arginine biosynthesis. The integration of OrbiSIMS and LC-MS platforms provided comprehensive coverage of amino acids and lipid metabolites while validating the significant metabolic changes observed in OrbiSIMS data.
When combined with bacteriophage therapy (vB_EcoM_SVMS_56), azithromycin demonstrated a synergistic effect, particularly in disrupting branched-chain amino acids (BCAAs) biosynthesis and phospholipid metabolism. Membrane phospholipid alterations, observed through OrbiSIMS, suggest an adaptation mechanism to counteract azithromycin and phage-induced stress. The profiling of the bacterial samples with OrbiSIMS allowed for spatial distribution and mapping of the biomolecules in depth. The discriminative metabolites were spatially localised, showing that phospholipids were concentrated near the surface of the bacterial cells. These lipids are the major components of the membrane and are highly enriched at the surface of cells. BCAAs were detected throughout sample depth, emphasising the capability of OrbiSIMS for precise metabolic mapping.
This thesis also explores the potential of FMT for treating GI infections caused by MDR EAEC and toxigenic C. difficile. OrbiSIMS-based metabolomic analysis of faecal samples from patients with C. difficile infections (n =12; average age 63.8 years, 67% female), collected either before or 12-weeks after FMT, revealed significant changes in key metabolites, including those involved in glycosylphosphatidylinositols biosynthesis. Furthermore, the impact of faecal filtrates derived from FMT of healthy donors (n = 4; 2 males and 2 females; mean [Standard deviation (SD)] age, 33 [2.3] years) on the metabolomes of pathogenic bacterial strains, including EAEC (O104:H4 and O20:H19) and toxigenic C. difficile (VPI 10463 and CD630), was investigated. The metabolic changes induced by faecal filtrate were strain-specific, with significant alterations in glycerophospholipid metabolism and metabolites modulating pathogenicity and virulence such as isoleucine, proline and fumarate. OrbiSIMS enabled not only the detection but also the imaging of these essential metabolites, providing critical insights into their localisation and distribution.
The identification of metabolic signatures and biomarkers through MS-based metabolomic approaches could guide the development of more effective and targeted therapeutic strategies against GI infections and drug-resistant pathogens.
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