Laura M. McMurry

Triclosan targets lipid synthesis

Triclosan is a broad-spectrum antibacterial and antifungal agent,, which acts by previously undetermined mechanisms, that is used in products such as antiseptic soaps, toothpastes, fabrics and plastics. Here we show that triclosan blocks lipid synthesis in Escherichia coli, and that mutations in, or overexpression of, the gene fabI (which encodes enoyl reductase, involved in fatty acid synthesis) prevents this blockage. This is, to our knowledge, the first evidence that triclosan acts on a specific bacterial target, rather than as a nonspecific ‘biocide’.

Cross-resistance to fluoroquinolones in multiple-antibiotic-resistant (Mar) Escherichia coli selected by tetracycline or chloramphenicol: decreased drug accumulation associated with membrane changes in addition to OmpF reduction.

Chromosomal multiple-antibiotic-resistant (Mar) mutants of Escherichia coli, selected on agar containing low concentrations of tetracycline or chloramphenicol, were 6- to 18-fold less susceptible to the fluoroquinolones than were their wild-type E. coli K-12 or E. coli C parental strains. The frequency of emergence of such mutants was at least 1,000-fold higher than that of those selected by the fluoroquinolone norfloxacin directly. When Mar mutants, but not wild-type cells, were plated on norfloxacin, mutants resistant to high levels of norfloxacin (2 micrograms/ml) appeared at a relatively high (approximately 10(-7] frequency. In addition to decreased amounts of OmpF, Mar mutants had other outer membrane protein changes and were four- to eightfold less susceptible to fluoroquinolones than was an ompF::Tn5 mutant lacking only OmpF. Accumulation of [3H]norfloxacin was more than threefold lower in the Mar mutants than in wild-type cells and twofold lower than in the OmpF-deficient derivative. These differences were not attributable to a change in the endogenous active efflux system for norfloxacin in E. coli. Norfloxacin-induced inhibition of DNA synthesis was threefold lower in intact cells of a Mar mutant than in susceptible cells, but this difference was not seen in toluene-permeabilized cells. Insertion of Tn5 into marA (min 34.05 on the chromosome) led to a return of the wild-type patterns of norfloxacin accumulation, fluoroquinolone and other antimicrobial agent susceptibilities, and outer membrane protein profile, including partial restoration of OmpF. These findings together suggest that marA-dependent fluoroquinolone resistance is linked to decreased cell permeability, only part of which can be accounted for by the reduction in OmpF. Once mutated to marA, cells can achieve high levels of quinolone resistance at a relatively high frequency. Images

Ineffectiveness of Topoisomerase Mutations in Mediating Clinically Significant Fluoroquinolone Resistance in Escherichia coli in the Absence of the AcrAB Efflux Pump

ABSTRACT Fluoroquinolone-resistant mutants, selected from a wild-typeEscherichia 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 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 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.

Endogenous active efflux of norfloxacin in susceptible Escherichia coli.

Escherichia coli was shown to have an energy-dependent reduced uptake of the fluoroquinolone antimicrobial agent norfloxacin. Studies of everted inner membrane vesicles suggested that this reduced accumulation involved a carrier-mediated norfloxacin active efflux generated by proton motive force with an apparent Km of 0.2 mM and a Vmax of 3 nmol min-1 mg of protein-1. Other hydrophilic, but not hydrophobic, quinolones competed with norfloxacin for transport. Porin (OmpF)-deficient E. coli cells were twofold less susceptible to norfloxacin and showed twice as much energy-dependent reduction in drug uptake. However, active efflux assayed in everted vesicles from the OmpF strain was unchanged compared with that in the parental strain. These findings suggest that in the OmpF mutant decreased outer membrane permeability, combined with active efflux across the inner membrane, in some manner results in decreased steady-state uptake of norfloxacin and lowered drug susceptibility.

The Clostridium perfringens Tet P determinant comprises two overlapping genes: tetA(P), which mediates active tetracycline efflux, and tetB(P), which is related to the ribosomal protection family of tetracycline‐resistance determinants

The complete nucleotide sequence and mechanism of action of the tetracycline‐resistance determinant Tet P, from Clostridium perfringens has been determined. Analysis of the 4.4 kb of sequence data revealed the presence of two open reading frames, designated as tetA(P) and tetB(P), The tetA(P) gene appears to encode a 420 amino acid protein (molecular weight 46079) with twelve transmembrane domains. This gene was shown to be responsible for the active efflux of tetracycline from resistant ceils. Although there was some amino acid sequence similarity between the putative TetA(P) protein and other tetracycline efflux proteins, analysis suggested that TetA(P) represented a different type of efflux protein. The tetB(P) gene would encode a putative 652 amino acid protein (molecular weight 72639) with significant sequence similarity to Tet(M)‐like cytoplasmic proteins that specify a ribosomal‐protection tetracycline‐resistance mechanism. In both C. perfringens and Escherichia coli. tetB(P) encoded low‐level resistance to tetracycline and minocycline whereas tetA(P) only conferred tetracycline resistance. The tetA(P) and tetB(P) genes appeared to be linked in an operon, which represented a novel genetic arrangement for tetracycline‐resistance determinants. It is proposed that tetB(P) evolved from the conjugative transfer into C. perfringens of a fer (M)‐like gene from another bacterium.

Detection of an inducible membrane protein associated with R-factor-mediated tetracycline resistance.

Summary New synthesis of an R-factor-specific protein occurs in E. coli minicells containing tetracycline-resistant R factors when they are incubated in the presence of tetracycline. This protein is sometimes detected in small amounts in tetracycline-resistant minicells in the absence of the drug, but it is not detectable in minicells containing R factors which lack tetracycline resistance. The protein has a molecular weight of about 50,000 in sodium dodecyl sulfate (as determined by polyacrylamide gel electrophoresis) and is found preferentially in the minicell membrane.

Active efflux of chloramphenicol in susceptible Escherichia coli strains and in multiple-antibiotic-resistant (Mar) mutants.

The multiple-antibiotic resistance (mar) locus (min 34) regulates a resistance to chloramphenicol in Escherichia coli that does not involve acetyltransferase. Transport studies showed that wild-type cells had an apparent endogenous active efflux of chloramphenicol which depended on the proton motive force. This efflux was not altered by a 39-kb chromosomal deletion which included the mar locus. Nevertheless, mutations at the mar locus led to a stronger net chloramphenicol efflux. Therefore, a gene encoding the putative efflux system cannot be at the mar locus but may be positively influenced by that locus.

A new tetracycline resistance determinant, Tet H, from Pasteurella multocida specifying active efflux of tetracycline.

The tetracycline resistance determinant on plasmid pVM111 from an avian strain of Pasteurella multocida mediates tetracycline resistance by a regulated active efflux mechanism. DNA coding for the determinant did not hybridize at high stringency with DNA representing a group of common tetracycline resistance determinants. The DNA sequence, however; revealed a structural gene and a repressor gene which had significant (37 to 64%) sequence similarities with previously described classes of efflux-type tetracycline resistance genes from members of the family Enterobacteriaceae. The new determinant has been assigned to class H.

Energy-dependent efflux mediated by class L (tetL) tetracycline resistance determinant from streptococci.

The class L (TetL) tetracycline resistance determinant from streptococci specified resistance and an energy-dependent decreased accumulation of tetracycline in both Streptococcus faecalis and Escherichia coli. Using E. coli, we showed that the reduced uptake resulted from active efflux. The streptococcal class M determinant, known to render the protein synthesis machinery of S. faecalis resistant to tetracycline inhibition, did not alter tetracycline transport in either host.

Two Transport Systems for Tetracycline in Sensitive Escherichia coli: Critical Role for an Initial Rapid Uptake System Insensitive to Energy Inhibitors

Escherichia coli sensitivity 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 cells 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.