Multidrug resistance pump AcrAB-TolC is required for high-level, Tet(A)-mediated tetracycline resistance in Escherichia coli

Abstract

Objectives: Starting from the observation that Escherichia coli tolC mutations severely reduced the high-level resistance to tetracycline afforded by Tn10- and plasmid-encoded Tet(A) pumps, we studied the mechanism of this susceptibility. Methods: The MIC of tetracycline for MC4100 tolC::Tn10 and several tolC mutants carrying the Tn10 in other sites on the chromosome (thr::Tn10) was determined. The effect of a tolC mutation on the level of expression of Tn10 tet(A) was examined by using a tet(A)::lacZ gene fusion. Influence of tolC mutations on tetracycline efflux and accumulation was quantified by spectrofluorometric assays. The contribution of the AcrAB multidrug efflux system to high-level tetracycline resistance was measured in a Tn10-carrying acrAB null mutant strain. Results: Tn10- and plasmid-encoded Tet(A) conferred 5- to 6-fold lower levels of tetracycline resistance in tolC mutants, as compared with control strain tolC⁺. Spectrofluorometric analyses showed that this resulted from a decrease in drug efflux in tolC mutants. Chlortetracycline resistance was also compromised by loss of TolC. Mutational loss of the AcrAB multidrug efflux transporter had the same effect as tolC mutations on tetracycline resistance. This indicated that tolC mutations act through inactivation of the AcrAB system. Conclusions: Our results are compatible with the hypothesis that the AcrAB pump is an important component in the development of high levels of resistance to tetracycline in E. coli, perhaps by working in combination with Tet(A).