The antimicrobial resistance (AMR) crisis, where new antibiotic discovery is not keeping pace with the emergence of resistant pathogens, is driven by mobile genetic elements (MGEs). MGEs can autonomously transfer between bacteria, along with AMR genes. The widespread use of antibiotics in the clinic, in agriculture, and animal husbandry, has accelerated the MGE-mediated transfer of AMR genes in the environment. However, despite playing such an important role in the AMR crisis, the dynamics and mechanisms behind the transmission of genes are poorly understood. Furthermore, which natural and man-made compounds inhibit or promote their movement in these environments is unknown.
One method to combat the rise in AMR is to identify small molecules as probes to understand the molecular basis of transmission and apply this information to prevent MGE-mediated resistance dissemination. Since conjugation is the main mechanism for AMR gene transfer, targeting MGEs that use conjugation, such as conjugative plasmids (e.g. Tn1549) and conjugative transposons (e.g. F plasmid), has the potential to prevent the emergence of multi-drug resistant pathogens. In this work, a high throughput assay modeled after Tn1549 excision was screened against a library of known bioactive compounds to find modulators of the integrase and excisionase activity. Several fluoroquinolone antibiotics including ciprofloxacin were identified as dose-dependent inhibitors of excision, which acted by changing supercoiling levels in the cell. Ciprofloxacin enhanced conjugation frequency of Tn1549 at sub-MIC concentrations relative to an untreated control and inhibited conjugation frequency at higher concentrations. A second project was focused on a high throughput conjugation assay based on the separation of the lux operon between a donor and recipient cell, such that only transconjugants produce luminescence to reflect active gene transfer. This work furthers our understanding of the development of assays to target MGEs and screening for inhibitors of their movement. / Thesis / Master of Science (MSc) / Antibiotics are small molecules that cure bacterial infections. However, their efficacy is fading as a result of the ability of mobile genetic elements (MGEs) to spread antimicrobial resistance genes between bacteria. Conjugative plasmids (CPs) and conjugative transposons (CTns) are two of the major types of MGEs that contribute to the dissemination of antimicrobial resistance in pathogens. The goal of this research is to search for inhibitors of CTns and CPs in order to prevent the emergence of multi-drug resistant bacteria. High throughput assays were designed to model both a CTn (Tn1549) and a CP (F plasmid) to find small molecules targeting their movement. A screen of the Tn1549 excision assay identified fluoroquinolone antibiotics that inhibit excision in a dose-dependent manner and indirectly inhibit the integrase used to excise the CTn. Ciprofloxacin, a fluoroquinolone, inhibited the conjugation frequency of Tn1549. Future work will focus on identifying new inhibitors of these MGEs and their characterization.
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/24805 |
| Date | January 2019 |
| Creators | Hansen, Drew M. |
| Contributors | Wright, Gerard D., Chemical Biology |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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