Antibiotic resistance: era of the multidrug pump

Sir, Over the past years, concerns have heightened over the escalating numbers of pathogenic microorganisms isolated that are resistant to multiple antibiotics (Berkelman et al. 1994, Science264: 368–370). This phenomenon poses major problems in the treatment of patients with hospital- or community-acquired infections caused by bacteria, yeasts, fungi, or parasitic organisms. Drug expulsion, mediated by membrane-associated drug efflux pumps, is one of the ingenious mechanisms that microorganisms use to evade the toxic effects of antibiotics. Particularly intriguing are the so-called multidrug transporters, which have specificity for compounds with very different chemical structures and cellular targets. Here, we report that a single ATP-dependent multidrug transporter of Lactococcus lactis, a bacterium used in dairy fermentations, confers resistance to a record of eight classes of clinically relevant broad-spectrum antibiotics. Although most microbial multidrug efflux systems known to date mediate drug–proton exchange (Paulsen et al. 1996, Microbiol Rev60: 575–608), the lactococcal multidrug transporter LmrA uses the free energy of ATP-hydrolysis to drive the export of toxic compounds from the inner leaflet of the cytoplasmic membrane (Bolhuis et al. 1996, EMBO J15: 4239–4245). LmrA, a 590-amino-acid integral membrane protein, is a member of the ATP-binding Cassette (ABC) Superfamily (Higgins, 1992, Annu Rev Cell Biol8: 67–113; van Veen et al. 1996, Proc Natl Acad Sci USA93: 10668–10672). LmrA shares significant sequence identity with the hop resistance protein HorA of the beer-spoilage bacterium Lactobacillus brevis, and with ABC transporters in pathogenic microorganisms (van Veen and Konings, 1998, Biochim Biophys Acta1365: 31–36). Furthermore, LmrA is a structural and functional homologue of the human multidrug transporter P-glycoprotein (van Veen et al. 1998, Nature391: 291–295). To address the role of LmrA in antibiotic resistance, LmrA was expressed in Escherichia coli strain CS1562, which is hypersensitive to drugs due to a deficiency of the TolC porin. The difference in in vivo antibiotic susceptibility between cells harbouring the lmrA containing plasmid pGKLmrA (van Veen et al. 1996, Proc Natl Acad Sci USA93: 10668–10672) or control plasmid pGK13 was studied in liquid cultures. LmrA expression resulted in an increased resistance to aminoglycosides, lincosamides, macrolides, quinolones, streptogramins, tetracyclines and chloramphenicol (Table 1). The resistance to trimethoprim and the glycopeptide vancomycin was unaltered. Surprisingly, LmrA expression also increased the resistance to certain β-lactam antibiotics. Because the targets of β-lactam antibiotics are extracellular transpeptidase enzymes involved in the biosynthesis of the cell wall, the role of multidrug transporters in β-lactam resistance is not immediately evident. However, our results and those obtained for the secondary MexAB (Li et al. 1995, Antimicrob Agents Chemother39: 1948–53) and AcrAB (Okuso et al. 1996, J Bacteriol178: 306–308) efflux systems demonstrate that the expression of multidrug transporters can result in a significant resistance to lipophilic β-lactam antibiotics. LmrA expression does not enhance the resistance to the more hydrophilic β-lactams such as meropenem, suggesting that insertion of β-lactam antibiotics into the cytoplasmic membrane is a requirement for LmrA-mediated drug resistance. Given the exceptionally broad antibiotic specificity of LmrA, the possible transfer of the lmrA gene to other bacteria in food or the digestive tract, and the presence of endogenous lmrA-like genes in pathogenic microorganisms, ABC-type multidrug transporters are a serious threat to the efficacy of valuable antibiotics. With few new drugs in the pipeline, prevention and circumvention of multidrug resistance are medical priorities. Knowledge of the molecular basis of antibiotic recognition and transport by multidrug transporters, which may allow the rational development of new drugs able to poison these proteins or bypass their efflux mechanism, is therefore urgently required.