Pugh, H.; Darby, E.; Burgess, L.; Colclough, A.; Meosa-John, A.-R.; Dunn, S.; Connor, C.; Perry, E.; McNally, A.; Bavro, V.; Blair, J. M. A.

Tripartite resistance-nodulation-division (RND) efflux pumps confer multidrug resistance (MDR) in Gram-negative bacteria and are critical for many physiological functions including virulence and biofilm formation. The common laboratory strain of E. coli, K-12 MG1655 has six recognised RND transporters participating in tripartite pump formation (AcrB, AcrD, AcrF, CusA, MdtBC, and MdtF). However, by studying >20,000 E. coli genomes 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. coli from phylogroups A, B1 and C, generally containing environmental and commensal strains, do not encode the operon and instead encode an uncharacterised ORF, ycjD. In phylogroups where the eefRABCD operon is present it was very highly conserved. In fact, conservation levels were comparable to that of the major E. coli RND efflux system AcrAB-TolC, suggesting a critical biological function. Protein modelling shows that this pump is highly divergent from endogenous E. coli RND systems with unique structural features, while showing similarities to efflux systems found in Pseudomonas aeruginosa. However, unlike other major RND efflux systems, EefABC does not appear to transport antimicrobials and instead may be important for infection or survival in the host environment.