ABSTRACT: Resistance-nodulation-division (RND) efflux pumps confer multidrug resistance in Gram-negative bacteria and are critical for many physiological functions including virulence and biofilm formation. The common Escherichia coli laboratory strain, K-12 MG1655, has six recognized RND transporters (AcrB, AcrD, AcrF, CusA, MdtBC and MdtF). However, by studying >20,000 E. coli assemblies, we show that E. coli belonging to phylogroups B2, D, E, F and G, which are commonly associated with infection, possess an additional, seventh RND transporter, EefB. It is found in a five-gene operon, eefRABCD, which also encodes a TetR family transcription factor, a periplasmic adapter protein, an outer membrane factor and major facilitator superfamily pump. In contrast, E. c