Treatment with Sub-inhibitory Kanamycin Induces Adaptive Resistance to Aminoglycoside Antibiotics via the AcrD Multidrug Efflux Pump in Escherichia coli K-12

Abstract

Research has shown that exposing Escherichia coli cells to sub-inhibitory concentrations of kanamycin induces adaptive resistance upon subsequent exposure to lethal levels of both structurally-similar and unrelated antibiotics. AcrD is an efflux pump that forms a complex with the outer membrane pore TolC and the periplasmic membrane fusion protein AcrA. Together, this complex works to export a variety of aminoglycosides and amphiphilic compounds from the cell. The involvement of AcrD in the observed adaptive resistance was assessed by comparing the induction of adaptive resistance upon exposure to kanamycin in wildtype and acrD -deficient Escherichia coli strains. Both strains were pretreated with subinhibitory concentrations of kanamycin and subsequently exposed to lethal concentrations of ampicillin, kanamycin, nalidixic acid, streptomycin, and tetracycline. Growth was monitored over 18 hours via optical density readings. Comparing the relative growth of the different treatments revealed that adaptive resistance was only induced in the wild-type strain towards kanamycin and streptomycin. This indicates that AcrD is necessary for the induced adaptive resistance and that it is specific to aminoglycosides. Additionally, molecular techniques were used to assess the level of expression of acrA, which was found to be upregulated in both the wild-type and acrD deletion strains following pretreatment with kanamycin. RNA was isolated from pretreated and control wild-type and acrD deletion cells, converted to cDNA, and analyzed using semi-quantitative PCR to analyze levels of acrA expression. These results are consistent with the hypothesis that AcrD is an inducible protein whose up regulation contributes to the previously observed adaptive resistance.