Haemophilus influenzae isolates vary widely in their susceptibilities to the peptide deformylase inhibitor LBM415 (MIC range, 0.06 to 32 g/ml); however, on average, they are less susceptible than gram-positive organisms, such as Staphylococcus aureus and Streptococcus pneumoniae. Insertional inactivation of the H. influenzae acrB or tolC gene in strain NB65044 (Rd strain KW20) increased susceptibility to LBM415, confirming a role for the AcrAB-TolC pump in determining resistance. Consistent with this, sequencing of a PCR fragment generated with primers flanking the acrRA region from an LBM415-hypersusceptible H. influenzae clinical isolate revealed a genetic deletion of acrA. Inactivation of acrB or tolC in several clinical isolates with atypically reduced susceptibility to LBM415 (MIC of 16 g/ml or greater) significantly increased susceptibility, confirming that the pump is also a determinant of decreased susceptibility in these clinical isolates. Examination of acrR, encoding the putative repressor of pump gene expression, from several of these strains revealed mutations introducing frameshifts, stop codons, and amino acid changes relative to the published sequence, suggesting that loss of pump repression leads to decreased susceptibility. Supporting this, NB65044 acrR mutants selected by exposure to LBM415 at 8 g/ml had susceptibilities to LBM415 and other pump substrates comparable to the least sensitive clinical isolates and showed increased expression of pump genes.The continuing emergence and spread of cellular targetbased antibiotic resistance mechanisms is a serious and increasing threat to the effective treatment of microbial infections. There is an obvious need for the development of new compounds, directed at novel cellular functions, which will be active against current resistant strains. In general, target-based antibiotic development strategies are very effective for identifying potent and specific inhibitors of target proteins; however, historically there have been significant hurdles to the effective use of these inhibitors as broad-spectrum antibiotics. This has much to do with intrinsic resistance imparted by bacterial membrane impermeability and efflux. These issues are especially problematic in the case of gram-negative bacteria, where the outer membrane and efflux pumps have been shown to act synergistically to minimize intracellular accumulation of a variety of structurally unrelated compounds (reviewed in references 17, 18, and 19).There are five general groups of bacterial efflux pumps currently described: the major facilitator superfamily, small multidrug resistance family, the multidrug and toxic compound extrusion family, the ATP-binding cassette family, and the resistance-nodulation-division (RND) family (25). The RND family appears to have the broadest substrate range, and these pumps are therefore most generally relevant vis-a-vis drug resistance in gram-negative bacteria. Architecturally, they consist of an inner membrane proton-drug antiporter, an outer membrane channel, and a so...
