Physiological Characterization of Metronidazole Resistance in Bacteroides fragilis
Bacteroides fragilis, an anaerobic Gram-negative bacterium, abundantly present as commensal in the human gut, can cause infections once it reaches the surrounding tissues or enters the bloodstream during surgery, trauma, or disease. Infections caused by B.fragilis are commonly treated with tigecycline, ampicillin, carbapenems, and metronidazole, unfortunately, because of the ability to quickly acquire the resistance, many antibiotics are rendered useless for the treatment. Currently, due to the relatively low resistance rate, carbapenems and metronidazole are the preferable choices for treatment. This thesis aims to use a wide range of biochemical and analytical methods, including oxygen scavenging measurements, Fourier-transformed infrared spectroscopy (FTIR), lipid profile analysis, and comparative mRNA profiling of B.fragilis Nim-positive and Nim-negative strains to enhance our understanding of Nim-mediated metronidazole resistance. Oxygen scavenging measurements aim to reveal the difference in the ability of Nim-positive/Nim-negative strains to remove oxygen and how this ability impacts metronidazole resistance. Additionally, lipid analysis will show changes cells undergo after during resistance to metronidazole (both Nim-positive/Nim-negative strains). RNAseq will be used to discover Nim-specific alterations in the gene expression profiles in B.fragilis with or without metronidazole resistance. Furthermore, the role of thioredoxin reductase (TrxR), an important enzyme involved in oxidative defense, will be studied relative to Nim-mediated metronidazole resistance.
This thesis project will improve our understanding of Nim-mediated metronidazole resistance as well as changes in physiology B.fragilis undergoes when expressing Nim-proteins.
Methods and Skills:
Comet assay; RT-PCR; RT-qPCR; PCR-based genotyping; cloning; recombinant protein expression; CRISPR/Cas9; library preparation; DNA barcoding; 1D/2D-SDS-PAGE; Western blot; MTT assay; proteomics: mass spectrometry (Orbitrap Q Exactive), sample fractionation, FASP, SPE, label free quantitation, TMT labeling, in-gel digestion
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