Assessing the contributions of the multidrug efflux pump components acrE and acrA in mediating resistance to kanamycin in E. coli BW25113 : Steps towards the generation of acrA/acrE double mutants using CRISPR/Cas9 system.

SUMMARY Efflux pumps in Escherichia coli are responsible for their intrinsic resistance to antimicrobial compounds. AcrAB-TolC and AcrEF-TolC are structurally similar efflux pumps that have been proposed to be responsible for the export of kanamycin. AcrS is a repressor of acrAB and possibly acrEF. AcrS deletion was shown to increase the expression of acrE and confer an increase in kanamycin resistance. Therefore, it was suggested that AcrEF-TolC and possibly AcrAB-TolC were contributors to the development of kanamycin resistance. Since AcrAB-TolC is constitutively expressed in the cell and at higher levels than AcrEF-TolC, we proposed that it has a greater contribution to the resistant phenotype. Furthermore, the pumps have high structural similarity, so we hypothesized that the loss of one pump could be compensated by the other, thus a double mutant would exhibit a lowered resistance due to the loss of the two main pumps responsible for the efflux kanamycin. To test this hypothesis, we used the wild-type strain E. coli MG1655, an ΔacrAΔkan and a ΔacrEΔkan deletion mutant and attempted to construct a double mutant ΔacrAΔacrEΔkan. A minimum inhibitory concentration (MIC) assay comparing the single mutants and the wildtype strain resulted in a MIC of 8  μg/mL for all strains. This suggests that a single loss of acrA or acrE expression has no apparent effect in kanamycin resistance. In our attempt to generate a double mutant with CRISPR/Cas9 system, technical difficulties were faced in the cloning of the pCRISPR-SacB-gDNA. Therefore, this study also sought for and provided preliminary evidence in the characterization of the pCRISPR-SacB plasmid using restriction digest and partial genome sequencing.