Interplay between Efflux Pumps May Provide Either Additive or Multiplicative Effects on Drug Resistance

Abstract

The effects of simultaneous expression of several efflux pumps on antibiotic resistance were investigated in Escherichia coli and Pseudomonas aeruginosa. Several combinations of efflux pumps have been studied: (i) simultaneous expression of a single-component efflux pump, which exports antibiotics into the periplasm, in combination with a multicomponent efflux pump that accomplishes efflux directly into the external medium; (ii) simultaneous expression of two single-component pumps; and (iii) simultaneous expression of two multicomponent pumps. It was found that when efflux pumps of different structural types were combined in the same cell (the first case), the observed antibiotic resistance was much higher than that conferred by each of the pumps expressed singly. Simultaneous expression of pairs of single-component or multicomponent efflux pumps (the second and third cases) did not produce strong increases in antibiotic resistance.Efflux of antibiotics out of cells is broadly recognized as a major component of bacterial resistance to many classes of antibiotics (26,28). This efflux occurs due to the activity of membrane transporter proteins, the so-called drug efflux pumps. Some efflux pumps selectively extrude specific antibiotics, while others, referred to as multidrug resistance (MDR) pumps, expel various structurally diverse antibiotics. While antibiotic-specific efflux pumps are usually encoded on transmissible plasmids and transposons, genes encoding many MDR pumps are normal constituents of bacterial chromosomes. Efflux pumps occur as either single-component or multicomponent systems. In gram-negative bacteria, single-component efflux pumps extrude their substrates into the periplasmic space (40). Examples of such single-component efflux pumps include the transposon-encoded tetracycline-and chloramphenicol-specific pumps, TetA and CmlA, respectively (2, 38), and the MDR pump MdfA, encoded in the chromosome of Escherichia coli (6). Multicomponent efflux pumps (which are found exclusively in gram-negative bacteria) traverse both inner and outer membranes. Examples include the MDR pumps AcrAB-TolC (19) and MexAB-OprM (34) from E. coli and Pseudomonas aeruginosa, respectively. Each pump contains a transporter located in the cytoplasmic membrane (as exemplified by AcrB or MexB), an outer membrane channel (TolC or OprM), and a periplasmic linker protein (AcrA or MexA), which is thought to bring into contact the other two components (42). This structural organization allows extrusion of substrates directly into the external medium, bypassing the periplasm and the outer membrane (27). The outer membrane of gram-negative bacteria serves as an efficient permeability barrier for both hydrophobic and hydrophilic antibiotics (29). Therefore, when antibiotics are extruded directly into the external medium, two independent mechanisms, efflux and low uptake through this permeability barrier, contribute to decreased intracellular accumulation of antibiotics (26).A single bacterial cell may contain multiple efflux pumps ...

A coordinated network of transporters with overlapping specificities provides a robust survival strategy

“…Indeed, the findings from our experiments on ethidium transport support the model proposed here (Fig. 5) and in previous work (26) that imply that if drug removal from the periplasmic space is not fast enough, the drug will quickly leak back into the cytoplasm, and the single-component transporter cannot cope with the load.…”

“…The results described here and elsewhere (10,26) provide strong evidence that AcrAB-TolC removes drugs only from the periplasm (Fig. 5).…”

“…The interactions between the MDTs demonstrated here and in previous studies (10,26) imply the development of a very robust survival mechanism provided by overlapping specificities of several MDTs. Along with the transporters, the cell response includes such components as drug-degrading enzymes, and this response provides the cell with a survival strategy to cope with resistance determinants present in the environment.…”

“…Simultaneous expression of a single-component efflux pump that exports antibiotics into the periplasm and a multicomponent efflux pump that accomplishes efflux directly into the external medium has been reported to have a synergistic effect on drug resistance (10,26). No synergistic effects were detected in strains with multiple deletions from the same family, either MFS or SMR (4), or in a mdfA and norE double-null strain (10).…”

A PhoPQ-Regulated ABC Transporter System Exports Tetracycline in Pseudomonas aeruginosa

“…Overexpression of genes encoding ␤-lactam and efflux pump can be triggered through regulatory genes (9). Interestingly, it has been demonstrated that the interplay between chromosomal ␤-lactamase and efflux systems or between efflux pumps also plays an important role in intrinsic resistance to antibiotics in P. aeruginosa (10,11). Systemic analysis of the resistome of P. aeruginosa is needed, which will provide an opportunity for understanding the complex phenomenon of intrinsic antibiotic resistance from a broader, global point of view.…”