Rahman, MA; (2017) Detection and characterisation of integrons, gene cassettes and cassette-located antibiotic resistance genes in the human oral metagenome. Doctoral thesis , UCL (University College London).

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Abstract

Antibiotic resistance is a serious threat to public health. Horizontal gene transfer (HGT) plays a critical role in the dissemination of antibiotic resistance, however, knowledge about the source of the resistance determinants and their mobile vectors is still limited. Integrons are natural gene capture and expression systems and are known for their role in dissemination of antibiotic resistance genes (ARGs) in clinically important pathogens. The human oral cavity is a reservoir of ARGs many of which were found on plasmids and transposons. However, the association of these ARGs with integrons have not been investigated before. In this study, a PCR-based metagenomic approach was used to investigate the presence of integrons carrying ARGs in the oral cavity of healthy human individuals from the UK (n=11) and Bangladesh (n=10). PCR primers targeting the mobile integrons (class 1, 2 and 3) as well as chromosomal integrons of Treponema were used to amplify integrons and associated array of gene cassettes (GCs) in the oral metagenome. By analysing the libraries of PCR amplicons, a large pool of GCs including the cassettes located at the first position of an integron were identified and most of them were found to be novel. The cassettes were predicted to carry open-reading frames encoding proteins of a diverse range of functions including antibiotic resistance, competence, plasmid stability and adaptation to stress. Two novel variants of D-alanine-D-alanine ligase (ddl) were identified and located in the first position within an integron for the first time. It was found that expression of ddls increase the resistance of the surrogate host (Escherichia coli) to D-cycloserine (D-cycloserine), an antibiotic used to treat multi-drug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis. A SNP at c.777 of the ddl variants was found to be responsible for alteration of minimum inhibitory concentration (MIC) of D-cycloserine. Cloning and sequencing of upstream sequence showed that the putative host of the Ddl encoding integron is likely to be a strain of Treponema denticola, one of the causative agents of periodontitis. The Ddl proteins encoded by the cassette genes were expressed and purified and their specific ligase activity was confirmed. The predicted 3D structures were also determined using I-TASSER tools and the generated 3D models were used to test the hypothesis that plant-based flavonoids could play an important role in the evolution ddls as integron GCs. It was found that the flavonoids namely quercetin and apigenin can bind to both ATP and D-alanine binding sites of the Ddls. This study shows that a PCR-based metagenomic approach can recover novel GCs including functional genes that confer resistance to clinically important antibiotics. The evidence for HGT of integron GCs and a hypothesis for evolution of ddls within integrons is also presented.

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