BaeR, EvgA and CpxR Differentially Regulate the Expression of acrD in Escherichia coli K-12 but Increased acrD Transcription Alone Does Not Demonstrate a Substantial Increase in Adaptive Resistance Against Kanamycin

Abstract

Escherichia coli K-12 possesses the transmembrane AcrAD-TolC multidrug efflux complex. This pump is involved in the efflux of a variety of antibiotics, as well as amphipathic compounds. Studies suggest that the pretreatment of E. coli K-12 with kanamycin resulted in adaptive resistance against kanamycin with an increased level of acrD expression. In this study, kanamycin-induced adaptive resistance was further explored. Single deletion strains of evgA, cpxR, and baeR are known regulators of acrD and were tested for their roles in acrD transcriptional regulation and associated kanamycin resistance. Minimum inhibitory concentration assays compared the growth of E. coli K-12 before and after exposure to sub-inhibitory levels of kanamycin, which tested for differences in the induction of adaptive resistance found in these transcriptional regulator deletion strains. acrD transcription was analyzed using quantitative PCR, which detected an increased level of acrD in baeR, cpxR, and evgA knockout strains by a factor of 4, 5.5, and 6 respectively. No direct correlation was found between pretreatment with sub-inhibitory levels of kanamycin and development of adaptive resistance, as the minimum inhibitory concentrations of pretreated strains were equivalent to the corresponding untreated strains. This is indicative of an absence, or very limited gain of adaptive resistance after pretreatment in these mutant strains. Since an increase in acrD transcription alone did not effectively induce adaptive resistance an increase in the expression of all components of the AcrAD- TolC efflux pump may be necessary before substantial kanamycin resistance can be gained.