Biochemical and Structural Basis of Antimicrobial Resistance in Clinical Isolates of Extended-Spectrum Beta-Lactamase-Producing Enterobacteriaceae from Nigerian Tertiary Hospitals

Extended-spectrum beta-lactamase-producing Enterobacteriaceae represent a critical antimicrobial resistance threat in Nigerian tertiary hospitals, and understanding their biochemical and structural mechanisms of resistance is essential for developing rational clinical countermeasures and novel enzyme inhibitors. This dissertation characterises the biochemical and structural basis of antimicrobial resistance in clinical ESBL-producing Enterobacteriaceae from five Nigerian tertiary hospitals across the South West, South East, North Central, and North West zones. Four hundred and twenty clinical Enterobacteriaceae isolates, predominantly Escherichia coli and Klebsiella pneumoniae, were collected from blood, urine, wound, and sputum cultures. ESBL production was confirmed by phenotypic tests and genotypically by multiplex PCR targeting CTX-M, TEM, SHV, and OXA genes. Whole-genome sequencing of sixty representative isolates by Illumina short-read sequencing characterised the full resistome including beta-lactamase variant alleles, plasmid-mediated resistance genes, and chromosomal mutations. Crystal structures of the three most prevalent CTX-M variant enzymes were solved at high resolution by X-ray crystallography using synchrotron data, enabling structural comparison with published CTX-M-15 and CTX-M-27 reference structures. Inhibitor kinetics of novel beta-lactamase inhibitor candidates including avibactam, relebactam, and two synthetic compounds were determined by steady-state enzyme kinetic analysis. Nigerian ESBL isolates show a predominance of CTX-M-15, consistent with global trends, but novel CTX-M variant alleles with altered inhibitor sensitivity profiles were identified in four isolates. Structural analysis reveals extended active site loop configurations in Nigerian variants that explain differential inhibitor binding. Keywords: ESBL, antimicrobial resistance, beta-lactamase, structural biochemistry, Nigerian hospitals.