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The efflux pump AcrAB is important in the antibiotic resistance and virulence of several pathogenic bacteria. We report that deletion of the Yersinia pestis AcrAB-TolC homolog leads to increased susceptibility to diverse substrates, including, though unlike in Escherichia coli, the aminoglycosides. Neither is the Y. pestis pump affected by the efflux pump inhibitor phenylalanine-arginine beta-naphthylamide. In mouse plague models, pump deletion does not have a significant effect on tissue colonization.

ABSTRACTThe efflux pump AcrAB is important in the antibiotic resistance and virulence of several pathogenic bacteria. We report that deletion of the Yersinia pestisAcrAB-TolC homolog leads to increased susceptibility to diverse substrates, including, though unlike in Escherichia coli, the aminoglycosides. Neither is the Y. pestispump affected by the efflux pump inhibitor phenylalanine-arginine beta-naphthylamide. In mouse plague models, pump deletion does not have a significant effect on tissue colonization.

ABSTRACTThirteen spontaneous multiple-antibiotic-resistant (Mar) mutants of Escherichia coliAG100 were isolated on Luria-Bertani (LB) agar in the presence of tetracycline (4 μg/ml). The phenotype was linked to insertion sequence (IS) insertions in marRor acrRor unstable large tandem genomic amplifications which included acrABand which were bordered by IS3or IS5sequences. Five different lonmutations, not related to the Mar phenotype, were also found in 12 of the 13 mutants. Under specific selective conditions, most drug-resistant mutants appearing late on the selective plates evolved from a subpopulation of AG100 with lonmutations. That the lonlocus was involved in the evolution to low levels of multidrug resistance was supported by the following findings: (i) AG100 grown in LB broth had an important spontaneous subpopulation (about 3.7 × 10−4) of lon::IS186mutants, (ii) new lonmutants appeared during the selection on antibiotic-containing agar plates, (iii) lonmutants could slowly grow in the presence of low amounts (about 2× MIC of the wild type) of chloramphenicol or tetracycline, and (iv) a lonmutation conferred a mutator phenotype which increased IS transposition and genome rearrangements. The association between lonmutations and mutations causing the Mar phenotype was dependent on the medium (LB versus MacConkey medium) and the antibiotic used for the selection. A previously reported unstable amplifiable high-level resistance observed after the prolonged growth of Mar mutants in a low concentration of tetracycline or chloramphenicol can be explained by genomic amplification.

ABSTRACTThe genetic basis for fluoroquinolone resistance was examined in 30 high-level fluoroquinolone-resistant Escherichia coliclinical 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 soxSRNA in any of the clinical strains. The AcrA protein of the AcrAB multidrug efflux pump was overexpressed in 19 of 30 strains of E. colitested, 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 acrRgene reduced the level of AcrA in the mutants and partially restored antibiotic susceptibility 1.5- to 6-fold. This study shows that mutations in acrRare an additional genetic basis for fluoroquinolone resistance.

ABSTRACTThe mdt(A) gene, previously designatedmef214, from Lactococcus lactissubsp.lactisplasmid pK214 encodes a protein [Mdt(A) (multiple drug transporter)] with 12 putative transmembrane segments (TMS) that contain typical motifs conserved among the efflux proteins of the major facilitator superfamily. However, it also has two C-motifs (conserved in the fifth TMS of the antiporters) and a putative ATP-binding site. Expression of the cloned mdt(A) gene decreased susceptibility to macrolides, lincosamides, streptogramins, and tetracyclines in L. lactisand Escherichia coli, but not in Enterococcus faecalisor inStaphylococcus aureus. Glucose-dependent efflux of erythromycin and tetracycline was demonstrated in L. lactisand in E. coli.

ABSTRACTFluoroquinolone-resistant mutants, selected from a wild-typeEscherichia coliK-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 genegyrA(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 acrABlocus 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.

Escherichia colisensitivity to tetracycline involves transport and accumulation of the antibiotic within the cell by two different uptake systems: an initial rapid uptake, which occurs over the initial 6 min of contact of the cell with tetracycline, and a slower uptake system, which continues indefinitely and whose rate of uptake is 1/10 that of the rapid system. Only the slow uptake system is blocked by inhibitors of energy-driven systems; it appears to be particularly dependent upon energy from oxidative phosphorylation. Although both uptake systems lead to accumulation of intracellular tetracycline and contribute to the cell's sensitivity, the rapid uptake system appears to be the more important. While these studies confirm active transport of tetracycline into the cell, they demonstrate that a critical uptake system which appears insensitive to metabolic inhibitors occurs initially.

ABSTRACTThe contribution of regulatory genes to fluoroquinolone resistance was studied with clinical Escherichia colistrains bearing mutations in gyrAand parCand with different levels of fluoroquinolone resistance. Expression of marAand soxSwas evaluated by Northern blot analysis of isolates that demonstrated increased organic solvent tolerance, a phenotype that has been linked to overexpression of marA,soxS, and rob. Among 25 cyclohexane-tolerant strains detected by a screen for increased organic solvent tolerance (M. Oethinger, W. V. Kern, J. D. Goldman, and S. B. Levy, J. Antimicrob. Chemother. 41:111–114, 1998), we found 5 Mar mutants and 4 Sox mutants. A further Mar mutant was detected among 11 fluoroquinolone-resistant, cyclohexane-susceptible E. colistrains used as controls. Comparison of the marORsequences of clinical Mar mutants with that of E. coliK-12 (GenBank accession no. M96235) revealed point mutations in marRin all mutants which correlated with loss of repressor function as detected in a marO::lacZtranscriptional assay. We found four other amino acid changes in MarR that did not lead to loss of function. Two of these changes, present in 20 of the 35 sequenced marORfragments, identified a variant genotype of marOR. Isolates with the samegyrAand parCmutations showed increased fluoroquinolone resistance when the mutations were accompanied by overexpression of marAor soxS. These data support the hypothesis that high-level fluoroquinolone resistance involves mutations at several chromosomal loci, comprising structural and regulatory genes.

ABSTRACTA carbapenem-resistant clinical isolate of Escherichia coli, which lacked OmpF and OmpC porins, carried a marRmutation and expressed a functional yedS, a normally nontranslated gene. MarR and YedS are described here as having effects on the ability of this strain to resist carbapenems. Additionally, expression of YedS was regulated by the small RNA MicF in a MarA-dependent way. These findings illustrate how broadly bacteria can mutate within a selective clinical setting, in this case, resistance to carbapenems, by altering three porin genes and one regulatory gene.

ABSTRACTThree Mycobacterium smegmatismutants 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 inhAgene on a multicopy plasmid. Replacement of the mutant chromosomalinhAgenes with wild-type inhAeliminated resistance. These results suggest that M. smegmatisInhA, like its Escherichia colihomolog FabI, is a target for triclosan.