Development of a homologous gene replacement approach to study ciprofloxacin resistance in clinical Escherichia coli isolates
AuthorValla, Sanne Ekroll
Urinary tract infections are common amongst infectious diseases in humans, and often these infections are caused by uropathogenic Escherichia coli. Effective antimicrobial treatment of infections are medical achievements that should not be taken for granted, as antimicrobial resistance has developed and causes treatments to become ineffective. Further, resistance traits can be spread between unrelated bacteria through mechanisms such as conjugation, where plasmids (mobile genetic elements) are potential carriers of multidrug resistance traits. Treatment strategies outside the production of novel antimicrobial drugs are being investigated. Collateral sensitivity is an example of a treatment strategy that specifically targets resistant bacteria. By gaining antimicrobial resistance to an initial drug, susceptibility towards other antimicrobials can increase due to the initial resistance. The aim of this project was to develop a homologous gene replacement approach to introduce or repair defined mutations known to cause ciprofloxacin resistance in Escherichia coli. This would enable investigation of the effects specific resistance-causing mutations have on collateral susceptibility changes in different strain backgrounds. Methods of traditional cloning by ligation, as well as the more modern isothermal cloning method, were used to build constructs that would replace the original genomic target by homologous gene replacement. While we were able to build gene constructs with defined mutations by isothermal cloning, moving these constructs into integrative plasmids for homologous gene replacement proved challenging. However, one construct was ligated into an integrative plasmid, but has yet to be transferred by conjugation to the clinical isolate of interest. Ultimately, we were able to design and optimize several cloning approaches to introduce or repair mutations, but further work is necessary to enable the efficient use of homologous gene replacement in the future.
PublisherUiT Norges arktiske universitet
UiT The Arctic University of Norway
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