Saudi Cultural Missions Theses & Dissertations
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Item Restricted Investigating The Role of AAL Cassette Proteins in Polysaccharide Biosynthesis in Burkholderia species(University of Leeds, 2024) Albreki, Raghad; Hemsworth, GlynIt is hypothesized that the Amino Acid Ligase (AAL) - Acyl Carrier Protein (ACP) complex, encoded within the AAL cassette in Burkholderia, is essential for the first stage of surface exopolysaccharide (EPS) production, which is a crucial component in the formation of bacterial biofilms and offers insights into their roles in both antibiotic resistance and biofilm formation. AAL: ACP1 and ACP2 proteins were produced separately in E. coli, purified, and modified using an ideal buffer system to test this theory. Subsequently, to verify modification and evaluate protein-protein interactions, the modified proteins were examined using native gel electrophoresis, mass spectrometry, and DTNB tests. In addition, the amino acid transfer activity was assessed using the AMP-Glo assay. ACP1 was successfully modified by the study, whereas only minor alterations were observed in ACP2. However, using native gel electrophoresis, no persistent interactions between AAL and the ACPs were found, and the AMP-Glo assay showed difficulties in identifying amino acid transfer, indicating the need for additional adjustments. To further comprehend AAL-ACP interactions, future research should concentrate on improving the modification processes and using more sensitive approaches. These developments could help create fresh tactics to combat Burkholderia species' biofilm growth and antibiotic resistance.15 0Item Restricted The Influence of Iron and Lignin on Molecules of the Gastrointestinal Microbiome and Its Effect on the Conjugation of Carbapenem Resistance Plasmids(University of Birmingham, 2022-09) Alharbi, Abdullah; Buckner, MichelleThe gastrointestinal microbiome has acted a reservoir for antibiotic resistance genes by harbouring favourable conditions for the the conjugative transfer of resistance plasmids. Resistance against carbapenems, the last resort β-lactam antibiotics, has been on the rise and is mainly mediated by the spread of carbapenemase-encoding plasmids by Klebsiella pneumoniae. Dietary intake of iron and iron chelators can modulate the metabolic and microbial profile of the gastrointestinal microbiome. This project focused on investigating the influence that molecules produced by the gastrointestinal microbiome might have on the conjugation of carbapenem resistance plasmids in K. pneumoniae in reaction to iron and lignin supplementation. A fluorescence reporter system was used to determine the effect of iron and lignin co-supplementation to an ex-vivo colonic supernatant on the conjugation of an endemic carbapenemase-encoding plasmid called pKp-QIL. This was followed by plating-based conjugation assays which measured the frequency of conjugation of a plasmid from clinical isolate called pCPE16_3 in an iron and lignin co-supplemented ex-vivo colonic supernatant. This project has revealed that lignin has no effect on the conjugation of resistance plasmids in iron- available gastrointestinal microbiome.17 0Item Restricted Responses of the Resident Oral Microbiome of Children to Amoxicillin Therapy(2022-11) Alshaye, Mona Mohammad; Do, ThuyBackground: The World Health Organisation considers antibiotic resistance an increasing global threat that creates a clinical and financial burden on healthcare systems worldwide. The British Prime Minister in July 2014 announced that antibiotic resistance is not a distant threat, with 25,000 people a year already dying from infections resistant to antibiotic drugs in Europe alone. Dentists account for 7 to 10% of all antibiotic prescriptions. Several paediatric dentistry guidelines recommend amoxicillin for the management of dental abscesses. Antibiotics undoubtedly impact our microbiome. The knowledge of the effect of antibiotic use on the oral microbiome is limited, especially in children. A better understanding of the effect of antibiotics and their disturbance to the oral microbiome is required. Aims: This longitudinal cohort study examines the hypothesis that the oral microbiome is altered in function and in composition through amoxicillin administration to children and the oral microbiome is a reservoir for antibiotic resistance genes (ARGs) from both oral and non-oral organisms. Method: A total of 14 children aged 4-11years old attending the Leeds Dental Hospital who were indicated for antibiotic prescription were recruited. Saliva samples and clinical information were collected from children before, two-week and three-month after amoxicillin therapy. Oral microbial profiles (taxonomic and functional) were obtained using a shotgun metagenomic and meta-transcriptomic approaches. Unique reads were annotated using blastp against the CARD database (e-value ≤ 1e-5) to characterise ARGs. Comparison analysis was used to evaluate the composition, function, and resistome of the oral microbiome at different timepoints. Results: There were great inter-individual differences in taxonomic, functional, and resistome profiles. The ubiquitous oral microbiome was identified at different taxonomy levels before and after antibiotic exposure and included bacterial, fungal, archaeal, and viral components. Streptococcus was found to be the most abundant (18.9%) and active (26.3%) genus in the oral microbiome and showed resilience, where it was depleted in the two-week timepoint and recovered after three months of amoxicillin therapy. The statistically significant changes in the composition of the oral microbiome occurred at the two-week timepoint at the phylum, genus, and species levels (q-value < 0.05). KEGG pathway annotations indicated high proportions of genes involved in carbohydrate and amino acid metabolisms (32,453 and 25,712 genes respectively) in all samples. The functional metagenomic and metatranscriptomic investigations of the KEGG and eggNOG analyses generally agreed. CAZy analyses revealed an enrichment of the six CAZy families in all the samples, with glycoside hydrolases being the most enriched family. The statistically significant changes in the function profile of the oral microbiome occurred at the two-week and three-month timepoints. The study identified 68 ARG types in the samples. A statistically significant increase in ARGs number after three months compared to the baseline was found. Additionally, thirty ARGs showed statistically significant abundance changes between timepoints, including the ARGs conferring resistance to amoxicillin (TEM-1, OXA-85, OprM, golS, and mtrE). The dominant antibiotic resistance mechanisms of the identified ARGs were antibiotic efflux (44.2%) followed by antibiotic inactivation (19.1%). Conclusion: Differences in the metagenome, metatranscriptome and resistome of children is showing following treatment with amoxicillin. The study indicates a substantial but incomplete recovery of the oral microbiome following three months of amoxicillin therapy.29 0