Stuart B. Levy

The genetic basis for fluoroquinolone resistance was examined in 30 high-level fluoroquinolone-resistant Escherichia coli clinical isolates from Beijing, China. Each strain also demonstrated resistance to a variety of other antibiotics. PCR sequence analysis of the quinolone resistance-determining region of the topoisomerase genes ( gyrA/B, parC ) revealed three to five mutations known to be associated with fluoroquinolone resistance. Western blot analysis failed to demonstrate overexpression of MarA, and Northern blot analysis did not detect overexpression of soxS RNA in any of the clinical strains. The AcrA protein of the AcrAB multidrug efflux pump was overexpressed in 19 of 30 strains of E. coli tested, and all 19 strains were tolerant to organic solvents. PCR amplification of the complete acrR (regulator/repressor) gene of eight isolates revealed amino acid changes in four isolates, a 9-bp deletion in another, and a 22-bp duplication in a sixth strain. Complementation with a plasmid-borne wild-type acrR gene reduced the level of AcrA in the mutants and partially restored antibiotic susceptibility 1.5- to 6-fold. This study shows that mutations in acrR are an additional genetic basis for fluoroquinolone resistance.

In Escherichia coli , the MarA protein controls expression of multiple chromosomal genes affecting resistance to antibiotics and other environmental hazards. For a more-complete characterization of the mar regulon, duplicate macroarrays containing 4,290 open reading frames of the E. coli genome were hybridized to radiolabeled cDNA populations derived from mar -deleted and mar -expressing E. coli . Strains constitutively expressing MarA showed altered expression of more than 60 chromosomal genes: 76% showed increased expression and 24% showed decreased expression. Although some of the genes were already known to be MarA regulated, the majority were newly determined and belonged to a variety of functional groups. Some of the genes identified have been associated with iron transport and metabolism; other genes were previously known to be part of the soxRS regulon. Northern blot analysis of selected genes confirmed the results obtained with the macroarrays. The findings reveal that the mar locus mediates a global stress response involving one of the largest networks of genes described.

Fluoroquinolone-resistant mutants, selected from a wild-type Escherichia coli K-12 strain and its Mar mutant by exposure to increasing levels of ofloxacin on solid medium, were analyzed by Northern (RNA) blot analysis, sequencing, and radiolabelled ciprofloxacin accumulation studies. Mutations in the target gene gyrA (DNA gyrase), the regulatory gene marR , and additional, as yet unidentified genes (genes that probably affect efflux mediated by the multidrug efflux pump AcrAB) all contributed to fluoroquinolone resistance. Inactivation of the acrAB locus made all strains, including those with target gene mutations, hypersusceptible to fluoroquinolones and certain other unrelated drugs. These studies indicate that, in the absence of the AcrAB pump, gyrase mutations fail to produce clinically relevant levels of fluoroquinolone resistance.

MarR negatively regulates expression of the multiple antibiotic resistance operon ( marRAB ) in Escherichia coli . In this study, it was demonstrated that sodium salicylate, plumbagin, 2,4-dinitrophenol, and menadione–inducers of the marRAB operon in whole cells–all interfered with the repressor activity of MarR in vitro. It is proposed that these compounds can interact directly with MarR to affect its repressor activity.

Active efflux is a useful strategy by which bacteria evade growth inhibition by antibiotics. Certain semisynthetic tetracycline (TC) analogs, substituted at the 13th carbon at C-6 on ring C of the TC molecule, blocked TC efflux as revealed in everted membrane vesicles from class B TC-resistant (Tc ^r ) Escherichia coli (M. L. Nelson, B. H. Park, J. S. Andrews, V. A. Georgian, R. C. Thomas, and S. B. Levy, J. Med. Chem. 36:370–377, 1993). A representative C-13-substituted analog, 13-cyclopentylthio-5-OH-TC (13-CPTC), was shown to competitively inhibit TC translocation by the Tet(B) protein, blocking the uptake of TC into vesicles and therefore the efflux of TC from whole cells. Against Tc ^r E. coli , 13-CPTC, when used in combination with doxycycline, produced synergistic inhibition of growth. 13-CPTC was shown to increase the uptake of [ ³ H]TC into the resistant cells. 13-CPTC alone was a potent growth inhibitor against TC-susceptible (Tc ^s ) and Tc ^r Staphylococcus aureus and enterococci specifying class K or class L efflux-dependent TC resistance mechanisms or, unexpectedly, the class M ribosomal protection mechanism. These findings indicate that derivatives of TC, identified by their ability to block the Tet(B) efflux protein, can restore TC activity against Tc ^r bacteria bearing either of the two known resistance mechanisms. Blocking drug efflux and increasing intracellular drug concentrations constitute an effective approach to reversing TC resistance and may be generally applicable to other antibiotics rendered ineffective by efflux proteins.

Letters of the English alphabet have heretofore been used to name tetracycline resistance determinants. Since all 26 letters have now been used, a nomenclature employing numerals is recommended for future determinants, and one laboratory has offered to coordinate the assignment of numerals.

MarR negatively regulates expression of the multiple antibiotic resistance ( mar ) locus in Escherichia coli . Superrepressor mutants, generated in order to study regions of MarR required for function, exhibited altered inducer recognition properties in whole cells and increased DNA binding to marO in vitro. Mutations occurred in three areas of the relatively small MarR protein (144 amino acids). It is surmised that superrepression results from increased DNA binding activities of these mutant proteins.

Three Mycobacterium smegmatis mutants selected for resistance to triclosan each had a different mutation in InhA, an enoyl reductase involved in fatty acid synthesis. Two expressed some isoniazid resistance. A mutation originally selected on isoniazid also mediated triclosan resistance, as did the wild-type inhA gene on a multicopy plasmid. Replacement of the mutant chromosomal inhA genes with wild-type inhA eliminated resistance. These results suggest that M. smegmatis InhA, like its Escherichia coli homolog FabI, is a target for triclosan.

To understand resistance, imagine being a bacterium in a world bombarded with antimicrobials. Living in a human, you would face antibiotics being taken for routine infections and for non-threatening conditions like acne. As a susceptible strain you have to acquire a survival mechanism. This is not too difficult, as your resistant counterparts, though less common, are very …