Awareness of the problem of antimicrobial resistance (AMR) has escalated and drug-resistant infections are named among the most urgent problems facing clinicians today. Our experiments here identify a transporter interactome and portray its essential function in acquisition of antimicrobial resistance. By exposing E. coli cells to consecutive increasing concentrations of the fluoroquinolone norfloxacin we generated in the laboratory highly resistant strains that carry multiple mutations, most of them identical to those identified in clinical isolates. With this experimental paradigm, we show that the MDTs function in a coordinated mode to provide an essential first-line defense mechanism, preventing the drug reaching lethal concentrations, until a number of stable efficient alterations occur that allow survival. Single-component efflux transporters remove the toxic compounds from the cytoplasm to the periplasmic space where TolC-dependent transporters expel them from the cell. We postulate a close interaction between the two types of transporters to prevent rapid leak of the hydrophobic substrates back into the cell. The findings change the prevalent concept that in Gram-negative bacteria a single multidrug transporter, AcrAB-TolC type, is responsible for the resistance. The concept of a functional interactome, the process of identification of its members, the elucidation of the nature of the interactions and its role in cell physiology will change the existing paradigms in the field. We anticipate that our work will have an impact on the present strategy searching for inhibitors of AcrAB-TolC as adjuvants of existing antibiotics and provide novel targets for this urgent undertaking.

Multidrug Transporters (MDTs) are major contributors to the acquisition and maintenance of Antimicrobial Resistance (AMR), a growing public health threat of broad concern. Despite the large number of MDTs, the overwhelming majority of the studies performed thus far in Gram-negative bacteria emphasize the supremacy of the AcrAB-TolC complex. To unveil the potential role of other MDTs we studied the behavior of a null AcrB Escherichia coli strain when challenged with chloramphenicol, a bacteriostatic antibiotic. We found that such a strain developed an extremely high-level of resistance to chloramphenicol, cross resistance to quinolones and erythromycin and displayed high levels of expression of the single component MFS transporter MdfA and multiple TolC-dependent transporters. The results suggest that the high versatility of the whole ensemble of transporters, the bacterial Effluxome, is an essential part of a strategy of survival in everchanging, at times noxious, environments. The concept of a functional Effluxome presents an alternative to the existing paradigms in the field and provides novel targets for the search for inhibitors of transporters as adjuvants of existing antibiotics.

Antibiotic resistance is one of the biggest public health challenges of our time. Here we present a novel approach to recognizing molecular mechanisms essential for maintaining high-level clinically relevant antibiotic resistance. To identify essential genes in this context, we subjected Escherichia coli EV18, a strain highly resistant to quinolones, to random transposon mutagenesis. This strain’s unique advantage is that the screen is performed at very high concentrations of the antibiotic, non-permissive for most strains. The transposon’s insertion affected the transcription of five genes required for the maintenance of resistance in EV18. Three of these genes (YihO, YhdP, and WaaY) have not been previously identified as essential for high-level antimicrobial resistance (AMR). Our work provides a new perspective to identify and characterize novel players crucial for maintaining AMR in E. coli.