In Escherichia coli the frequency of spontaneous single-step mutation to high levels of resistance to the newer 4-quinolone agent norfloxacin was confirmed to be over 300-fold lower than that to the older agent nalidixic acid. Serial passage on incremental concentrations of drug was necessary to produce mutants highly resistant to norfloxacin. Genetic analysis of one such highly resistant strain identified two mutations conferring drug resistance. One mutation, nfxA, mapped around 48 min on the E. coli genetic map and was shown to be an allele of gyrA by studies demonstrating an increased drug resistance of DNA gyrase reconstituted with the gyrase A subunit isolated from the mutant strain. These findings also identified the DNA gyrase A subunit as a target of norfloxacin. The second mutation, nfxB, mapped between 20 and 22 min and was associated with additional resistances to tetracycline, chloramphenicol, and cefoxitin and with decreases in outer membrane porin protein OmpF. The nfxA and nfxB mutations together accounted for most, but not all, of the norfloxacin resistance phenotype of this strain.The newer 4-quinolone antimicrobial agents such as norfloxacin (NFX) are structurally related to nalidixic acid (NAL) but have substantially increased potency and are broader in spectrum. The newer agents, therefore, show promise for use in treatment of a variety of bacterial infections. A potentially serious limitation to this usefulness, however, might be the development of bacterial resistance, a problem that occurred with NAL in certain settings (28).Resistance of bacteria to NAL and the 4-quinolones results from chromosomal mutations, but has not been found to be carried on plasmids or transposons (1). A target of NAL action is the essential bacterial enzyme DNA gyrase (9). The structure and functions of DNA gyrase have been the subject of several reviews (3, 9, 35). Mutations in the gene [gyrA (nalA)] encoding the A subunit of the enzyme DNA gyrase confer on the bacterium and the enzyme high-level resistance to NAL. In addition to two A subunits, this enzyme contains two B subunits, the products of the gyrB (cou) gene. Purified DNA gyrase has a variety of activities in vitro that are inhibited by NAL, including the introduction of negative superhelical twists into duplex circular DNA and the reversible interlocking of DNA circles like links in a chain. In the growing bacterium DNA gyrase is required for DNA replication, transcription of certain operons, DNA repair, and other processes, all of which are antagonized by NAL. The level of superhelical twisting of intracellular DNA is controlled at least in part by the activities of DNA gyrase and topoisomerase I, the product of the topA gene. Other genes in which mutations may cause altered levels of resistance to NAL include nalB (12), nalC (gyrB) (37), nalD (31, 37), icd (18), cya (18), and crp (17).Less is known about the mechanisms of resistance of bacteria to NFX and other newer 4-quinolones. The frequency with which bacteria spontaneously become resistant to h...

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 Vm. of 3 nmol min-' mg of protein-'. 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 fluoroquinolones are newly introduced broad-spectrum synthetic antimicrobial agents. They have greater activity against gram-positive and gram-negative bacteria than do the older quinolone analogs nalidixic acid and oxolinic acid (11, 28). Resistance to fluoroquinolones has been infrequent and limited to chromosomal mutations primarily affecting DNA gyrase and outer membrane permeability (28), with no reports of plasmid-mediated or transferable resistance. In Escherichia coli, the fluoroquinolones are thought to cross the outer membrane primarily through the OmpF porin (9), and indeed, fluoroquinolone-resistant mutants with a reduction or loss of OmpF have been isolated (1,10,12).In studying fluoroquinolone transport, we unexpectedly observed an endogenous active efflux for norfloxacin in plasmid-free susceptible E. coli. This is the second endogenous efflux for an antimicrobial agent seen in susceptible bacterial cells, the first being one described for the tetracyclines (18). In this study, we characterized norfloxacin efflux in wild-type strains and in an OmpF-deficient mutant. Our results suggest that the active efflux acts in concert with decreases in outer membrane permeability and could become a factor allowing for the emergence of strains resistant to the fluoroquinolones. MATERIALS AND METHODSBacterial strains. Plasmid-free E. coli K-12 strain AG100 (7); its ompF derivative LM218, constructed by P1 transduction (20) of ompF::TnS from strain MH450 (8); and E. coli ML strain with 0.5% Casamino Acids (Difco Laboratories, Detroit, Mich.), 0.2% glucose, and 0.001% B, (required by AG100) to an A530 of 0.75, washed in growth medium, and resuspended in medium to an A530 of 60. Spheroplasts were prepared from strain ML308-225 by using the lysozyme-EDTA method of Kaback (13). Norfloxacin uptake was assayed at 30°C upon addition at time zero of [3H]norfloxacin (74 mCi/mg; uniformly labeled in the piperazine ring; generously provided by Merck & Co.) to a final concentration of 0.125 ,uM, adjusted to a specific activity of 3.6 mCi/mg by additio...

Two genetic loci selected for norfloxacin (nfxB) and ciprofloxacin (cfxB) resistance were characterized. Both mutations have previously been shown to confer pleiotropic resistance to quinolones, chloramphenicol, and tetracycline and to decrease expression of porin outer-membrane protein OmpF. nfxB was shown to map at about 19 min and thus to be genetically distinct from ompF (21 min Chemother. 32:1187Chemother. 32: -1191Chemother. 32: , 1988), suggest a model for quinolone resistance by decreased permeation in which decreased diffusion through porin channels in the outer membrane interacts with a saturable drug efflux system at the inner membrane.Two mechanisms of resistance to quinolone antibacterial agents have been recently characterized: alteration of the target enzyme DNA gyrase and decreased drug accumulation. Drug resistance mutations in the gyrA and gyrB genes of DNA gyrase are specific to quinolones and have been identified in Escherichia coli (3, 12, 19, 21-23, 37, 39, 43, 47, 48), Pseudomonas aeruginosa (20,24,34,35), and Citrobacter freundii (2) strains exposed to nalidixic acid, pipemidic acid, norfloxacin, ciprofloxacin, and ofloxacin.Quinolone resistance caused by decreased drug accumulation is conferred by mutations in genes other than gyrA or gyrB. Several such genes have been characterized in E. coli

In this work, biosurfactant production by several yeast strains was evaluated using different culture media. The best results were obtained with the strain Wickerhamomyces anomalus CCMA 0358 growing in a culture medium containing glucose (1g/L) and olive oil (20g/L) as carbon sources. This strain produced 2.6g of biosurfactant per liter after 24h of growth. The crude biosurfactant reduced the surface tension of water to values around 31mN/m, and its critical micelle concentration was 0.9mg/mL. This biosurfactant was characterized through mass spectrometry (MS), and nuclear magnetic resonance (NMR) as a mixture of two different glycolipids, comprising a sugar moiety linked to one or three molecules of oleic acid. To the best of our knowledge, these biosurfactants are structurally different from those previously reported. Furthermore, the crude biosurfactant exhibited antimicrobial activity against several microorganisms, including the pathogens Candida albicans, Escherichia coli, Staphylococcus epidermidis and Streptococcus agalactiae, which opens the possibility for its use in several biomedical applications.

In this study, crude glycerol was used as a carbon source in the cultivation of wild yeasts, aiming for the production of microbial lipids and citric acid. Forty yeasts of different sources were tested concerning their growth in crude and commercial glycerol. Four yeasts (Lidnera saturnus UFLA CES-Y677, Yarrowia lipolytica UFLA CM-Y9.4, Rhodotorula glutinis NCYC 2439, and Cryptococcus curvatus NCYC 476) were then selected owing to their ability to grow in pure (OD600 2.133, 1.633, 2.055, and 2.049, respectively) and crude (OD600 2.354, 1.753, 2.316, and 2.281, respectively) glycerol (10%, 20%, and 30%). Y. lipolytica UFLA CM-Y9.4 was selected for its ability to maintain cell viability in concentrations of 30% of crude glycerol, and high glycerol intake (18.907 g/l). This yeast was submitted to lipid production in 30 g/l of crude glycerol, and therefore obtained 63.4% of microbial lipids. In the fatty acid profile, there was a predominance of stearic (C18:0) and palmitic (C16:0) acids in the concentrations of 87.64% and 74.67%, respectively. We also performed optimization of the parameters for the production of citric acid, which yielded a production of 0.19 g/l of citric acid in optimum conditions (38.4 g/l of crude glycerol, agitation of 184 rpm, and temperature of 30°C). Yarrowia lipolytica UFLA CM-Y9.4 presented good lipid production when in the concentration of 30 g/l of glycerol. These data may be used for production in large quantities for the application of industrial biodiesel.