The RND efflux pump EefABC is highly conserved within lineages of E. coli commonly associated with infection.

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. ImportanceEfflux pumps are molecular machines that export molecules out of bacterial cells. The efflux pumps belonging to the RND family are particularly important as they export antibiotics out of Gram-negative bacterial cells, contributing to antibiotic resistance. The important human pathogen, E. coli, has been previously reported to have six RND pumps. However, we show that phylogroups of E. coli commonly associated with infection encode a seventh RND pump, EefABC which is highly conserved, suggesting an important biological function. While the function of EefABC in E. coli remains to be resolved, it does not seem to transport antimicrobial compounds. These findings are important because they reveal a new RND pump, potentially involved in virulence and survival in the host, that could represent a new therapeutic target. Additionally, it again shows that laboratory type strains of common bacterial pathogens are not representative of those that are infection causing.