A reporter gene system for the identification and characterisation of multiple antibiotic resistance (mar) in E. coli associated with altered expression of the AcrAB-TolC drug efflux pump

Abstract number: 1134_03_47

Matthiessen N., Heisig P.

Objectives:  

The increasing prevalence of bacterial resistance to antibiotics is a worldwide problem for the therapy of infectious diseases. Among the three basic mechanism leading to antibiotic resistance, i.e. alteration of the target, inactivation of the drug , and reduced accumulation of the drug at the target site, the latter has become the most important resistance-mechanism due to the following reasons: (i) it is the most abundant mechanism detectable in both Gram-negative and Gram-positive bacteria, (ii) it often mediates multi drug resistance (mdr) to a broad range of -- unrelated -- drug classes, (iii) it can favor the acquisition of additional mechanisms of resistance. Most frequently mdr is achieved by one of several mutations resulting in the deregulation of mdr efflux pump expression. The major multidrug efflux pump in E.coli AcrAB-TolC is a constitutively expressed tripartite complex consisting of a RND-type transporter AcrB, a membrane fusion protein AcrA and an outer membrane channel TolC. The expression of acrAB is regulated by a local repressor AcrR and known global regulator systems MarRAB, SoxRS and Rob. Since any mutation inactivating AcrR or a global regulator, like MarR may result in acrAB overexpression, detection of a resistance mutation requires extensive sequencing and additional susceptibility tests using different antibiotics.

Methods:  

Thus, a reporter gene system has been developed that senses alterations in the expression of the AcrAB-TolC efflux pump caused by induction and/or mutation/deletion of the local and/or global regulators. Briefly, the luciferase gene luc of the firefly Photinus pyralis as a reporter gene was fused to the promotor pacrAB by a modified PCR technique (SOEing) and inserted into the plasmid pBR322.

Results:  

Firefly luciferase as a reporter is advantageous due to the high sensitivity with no background activity, wide range of applicability, ease of use and cost efficiency. Alterations in the expression of acrAB either due to the presence of inductors or mutations in marR could be detected as increased luciferase activities by this reporter gene system.

Conclusions:  

Thus, this newly developed reporter gene system can be used to identify acrR mutants and to quantify alterations of the expression of the acrAB operon. Moreover, it is a useful tool to study under different environmental conditions the expression and to screen for inductors or inhibitors of the AcrAB-TolC efflux pump.