Deletion of the major Escherichia coli multidrug transporter AcrB reveals transporter plasticity and redundancy in bacterial cells

Abstract

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.

Why most transporter mutations that cause antibiotic resistance are to efflux pumps rather than to import transporters

“…A recent experimental survey of several hundred gene knockouts in E. coli , using fluorescent probes as antibiotic surrogates showed that dozens of such efflux transporters could be active and thereby contribute to lowering the steady-state uptake 47 . There is also considerable redundancy and plasticity 75 . Thus, as expected from metabolic control analysis, while there is little effect of single-gene knockouts on fluxes 76 , there can be potentially very large effects on the concentrations of intermediary metabolites 77, 78 or, as in our model, the intracellular concentration of an antibiotic of interest,…”

Acidification of Cytoplasm in Escherichia coli Provides a Strategy to Cope with Stress and Facilitates Development of Antibiotic Resistance

“…EV18 and EVC are highly resistant strains developed in our laboratory and have collected a considerable number of mutations that support resistance to NF and chloramphenicol, respectively 23,24 . The results thus far show that regardless of the antibiotic exposure during evolution (NF or chloramphenicol), the resistant strains have a lower pH i , most likely due to the activation of the mar response.…”

“…EV18 and EVC are highly resistant strains that originated from E. coli BW25113 cells subjected to an in vitro lab evolution process. They carry multiple mutations that accumulated sequentially to confer high-level resistance, two to three orders of magnitude higher than the wild type strains 23,24 . Per previous findings, the wild-type cells maintain their cytoplasmic pH within a range of pH 7.4-7.7 over an external pH range of 6.0-8.1 [13][14][15] .…”

Mapping the Role of AcrAB-TolC Efflux Pumps in the Evolution of Antibiotic Resistance Reveals Near-MIC Treatments Facilitate Resistance Acquisition

“…Adaptive evolution studies have begun exploring how certain antibiotic pressures influence the evolution of resistance. For instance, studies using a “morbidostat”—a continuous culture device that dynamically adjusts antibiotic concentrations to inhibitory levels—have found numerous targets that can be readily mutated to promote resistance ( 9 – 11 ), as well as identifying how drug switching can limit the evolution of resistance ( 12 ). While these studies have provided pivotal insights for this field, the morbidostat design causes antibiotic concentrations to rise to levels that exceed clinically relevant concentrations due to toxicity for patients ( 13 ).…”