The importance of efflux pumps in bacterial antibiotic resistance

Abstract

Efflux pumps are transport proteins involved in the extrusion of toxic substrates (including virtually all classes of clinically relevant antibiotics) from within cells into the external environment. These proteins are found in both Gram-positive and -negative bacteria as well as in eukaryotic organisms.1 Pumps may be specific for one substrate or may transport a range of structurally dissimilar compounds (including antibiotics of multiple classes); such pumps can be associated with multiple drug resistance (MDR). In the prokaryotic kingdom there are five major families of efflux transporter:2 MF (major facilitator), MATE (multidrug and toxic efflux), RND (resistance-nodulation-division), SMR (small multidrug resistance) and ABC (ATP binding cassette). All these systems utilize the proton motive force as an energy source,3 apart from the ABC family, which utilizes ATP hydrolysis to drive the export of substrates. Recent advances in DNA technology and the advent of the genomic era have led to the identification of numerous new members of the above families, and the ubiquitous nature of efflux pumps is remarkable. Transporters that efflux multiple substrates, including antibiotics, have not evolved in response to the stresses of the antibiotic era. All bacterial genomes studied contain several different efflux pumps; this indicates their ancestral origins. It has been estimated that ∼5–10% of all bacterial genes are involved in transport and a large proportion of these encode efflux pumps.2,4 There is some debate as to the ‘normal’ physiological role of efflux transporters, as antibiotic susceptible as well as resistant bacteria carry and express these genes. In many cases, efflux pump genes are part of an operon, with a regulatory gene controlling expression. Increased expression is associated with resistance to the substrates, e.g. resistance to bile salts and some antibiotics in Escherichia coli is mediated by over-expression of acrAB.5 Although genes encoding efflux pumps can be found on plasmids, the carriage of efflux pump genes on the chromosome gives the bacterium an intrinsic mechanism that allows survival in a hostile environment (e.g. the presence of antibiotics), and so mutant bacteria that over-express efflux pump genes can be selected without the acquisition of new genetic material. It is probable that these pumps arose so that noxious substances could be transported out of the bacterium, allowing survival. Indeed it is now widely accepted that the ‘intrinsic resistance’ of Gram-negative bacteria to certain antibiotics relative to Gram-positive bacteria is a result of the activity of efflux systems.6 Efflux systems that contribute to antibiotic resistance have been described from a number of clinically important bacteria, including Campylobacter jejuni (CmeABC7,8), E. coli (AcrAB-TolC, AcrEF-TolC, EmrB, EmrD9), Pseudomonas aeruginosa (MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY-OprM9), Streptococcus pneumoniae (PmrA10), Salmonella typhimurium (AcrB11) and Staphylococcus aureus (NorA12). All of these systems efflux fluoroquinolones and the RND pumps (CmeB, AcrB and the Mex pumps) also export multiple antibiotics. Over-expression of efflux pumps can result from mutations within local repressor genes13–15 or may result from activation of a regulon regulated by a global transcriptional regulator such as MarA or SoxS of E. coli.16,17 The broad substrate range of efflux systems is of concern, as often over-expression of a pump will result in resistance to antibiotics of more than one class as well as some dyes, detergents and disinfectants (including some commonly used biocides). Cross-resistance is also a problem; exposure to any one agent that belongs to the substrate profile of a pump would favour over-expression of that pump and consequent cross-resistance to all other substrates of the pump. These may include clinically relevant antibiotics. An example of this is seen again with the mexAB system of P. aeruginosa; mutants that over-produce MexAB are less susceptible, if not fully resistant to a range of antibiotics (fluoroquinolones, β-lactams, chloramphenicol and trimethoprim) but also triclosan, a commonly used household biocide.18 The potential misuse of biocides and possible selection of bacteria cross-resistant to antibiotics has recently been debated in this journal and elsewhere.19–22 Over-expression of a multidrug resistance efflux pump alone often does not confer high-level, clinically significant resistance to antibiotics. However, such bacteria are better equipped to survive antibiotic pressure and develop further mutations in genes encoding the target sites of antibiotics.23 It has been shown that fluoroquinolone-resistant strains of E. coli are selected 1000-fold more readily from mar mutants than wild-type bacteria,24 and highly fluoroquinolone-resistant E. coli contain mutations in genes encoding the target topoisomerase enzymes and have reduced accumulation and increased efflux (p