New nalidixic acid resistance mutations related to deoxyribonucleic acid gyrase activity.

In Escherichia coli K-12 mutants which had a new nalidixic acid resistance mutation at about 82 min on the chromosome map, cell growth was resistant to or hypersusceptible to nalidixic acid, oxolinic acid, piromidic acid, pipemidic acid, and novobiocin. Deoxyribonucleic acid gyrase activity as tested by supercoiling of lambda phage deoxyribonucleic acid inside the mutants was similarly resistant or hypersusceptible to the compounds. The drug concentrations required for gyrase inhibition were much higher than those for cell growth inhibition but similar to those for inhibition of lambda phage multiplication. Transduction analysis with lambda phages carrying the chromosomal fragment of the tnaA-gyrB region suggested that one of the mutations, nal-31, was located on the gyrB gene.     

Synthesis and antimicrobial activity of N-alkyl and N-aryl piperazine derivatives

A series of substituted piperazine derivatives have been synthesized and tested for antimicrobial activity. The antibacterial activity was tested against Staphylococcus aureus (MTCCB 737), Pseudomonas aeruginosa (MTCCB 741), Streptomyces epidermidis (MTCCB 1824) and Escherichia coli (MTCCB 1652), and antifungal activity against Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger. All synthesized compounds showed significant activity against bacterial strains but were found to be less active against tested fungi. In vitro toxicity tests demonstrated that compounds 4d and 6a showed very less toxicity against human erythrocytes.     

Sensitivity to antibiotics of bacterial strains isolated from clinical specimens during the years 1985-1986

Sensitivity of 312 strains of staphylococci, 386 strains of streptococci and 1193 strains of aerobic gram-negative bacilli to the selected antibiotics was tested. These strains were isolated from the clinical material at the Clinical Hospital No. 1 in Warsaw within 1985-1986. Staphylococci were sensitive to pristinamycin, cefazolin, fusidic acid, oxacillin, and clindamycin. In 1986, a decrease in the number of strains sensitive to these antibiotics, except cefazolin, was seen. In case of streptococci the most active proved chloramphenicol and gentamicin but a significant decrease in the percentage of sensitive strains was also noted in 1986. The highest number of gram-negative bacilli was sensitive to amikacin, colistin, nalidixic acid, pipemidic acid, and gentamicin. In 1986, a decrease in the percentage of sensitive strains was noted. Amikacin and colistin were the most active against Pseudomonas spp. while amikacin and nalidixic and pipemidic acids--against Proteus spp. Comparison of the results with those obtained in 1981-1984 has shown that the sensitivity of staphylococci changed the most significantly and this change was unfavourable. Gentamicin and amikacin remained the most active against gram-negative bacilli while amikacin and colimycin against Pseudomonas spp. In case of anaerobes the majority of strains was sensitive to chloramphenicol, tetracycline and clindamycin. Metronidazole was active against high percentage of Clostridium spp. and all gram-negative bacilli while the percentage of gram-positive bacilli and cocci was sensitive to metronidazole.     

Effect of Nalidixic Acid and Hydroxyurea on Division Ability of Escherichia coli fil+ and lon− Strains

Short periods of incubation in medium containing nalidixic acid or hydroxyurea, followed by a return to normal growth conditions, induced filament formation in Escherichia coli B (fil(+)) and AB1899NM (lon(-)) but not in B/r (fil(-)) and AB1157 (lon(+)). These drugs reversibly stopped deoxyribonucleic acid (DNA) synthesis with little or no effect on ribonucleic acid (RNA) synthesis or mass increase. The initial imbalance caused by incubation in these drugs was the same for B and B/r as was macromolecular synthesis following a return to normal growth conditions. DNA degradation caused by nalidixic acid was measured and found to be the same for B and B/r. Hydroxyurea caused no DNA degradation in these two strains. Survival curves as determined under various conditions by colony formation suggested that the property of filament formation was responsible for the extrasensitivity of fil(+) and lon(-) strains to either nalidixic acid or hydroxyurea. E. coli B was more sensitive to either drug than was B/r or B(s-1). Pantoyl lactone or liquid holding treatment aided division and colony formation of nalidixic acid-treated B but had no effect on B/r. Likewise, the filament-former AB1899NM was more sensitive to nalidixic acid than was the non-filament-former AB1157. The sensitivity of B/r and B(s-1) to nalidixic acid was nearly the same except at longer times in nalidixic acid, when B(s-1) appeared more resistant. Even though nalidixic acid, hydroxyurea, and ultraviolet light may produce quite different molecular alterations in E. coli, they all cause a metabolic imbalance resulting in a lowered ratio of DNA to RNA and protein. We propose that it is this imbalance per se rather than any specific primary chemical or photochemical alterations which leads to filament formation by some genetically susceptible bacterial strains such as lon(-) and fil(+).     

Bactericidal effect of 5-azacytidine on Escherichia coli carrying EcoRII restriction-modification enzymes

5-Azacytidine was found to be bactericidal to Escherichia coli carrying plasmids specifying EcoRII restriction-modification systems, but not to the same strains lacking these plasmids. Of other base analogs tested, only 5(beta-D-ribofuranosyl)isocytidine had similar, although weaker, effects. Plasmids that had lost the EcoRII restriction-modification system did not confer sensitivity to 5-azacytidine. Mutants defective in the restriction function remained sensitive to the toxic effects of the drug; however, a mutant defective in the modification function lost most of the sensitivity to 5-azacytidine. For the bactericidal effect to be seen, the cells had to be growing; cells in the stationary phase of growth were not killed by the drug. The drug inhibited the methylase enzyme, and an inhibitor of the enzyme could be detected in vitro in extracts of cells that had been treated with 5-azacytidine. This nalidixic acid inhibited its formation. Coumermycin but not nalidixic acid antagonized the bactericidal effect of the drug; however, coumermycin was more effective in preventing the inhibition of the methylase by 5-azacytidine than was nalidixic acid.     

Genotoxic potential of quinolone antimicrobials in the in vitro comet assay and micronucleus test

The purpose of this study was to examine the genotoxicity of quinolone antimicrobials. We investigated the genotoxic potential of eight quinolones, namely nalidixic acid (NA), pipemidic acid (PPA), oxolinic acid (OA), piromidic acid (PA), enoxacin (ENX), ofloxacin (OFLX), norfloxacin (NFLX) and ciprofloxacin (CPFX), by the in vitro alkaline single-cell gel electrophoresis (comet) assay at pH>13. WTK-1 cells (mutant p53) were treated with each of the eight quinolones at 62.5-1000 microg/mL for 2, 4 and 20 h. NFLX and CPFX significantly induced DNA damage concentration-dependently after 4 and 20 h treatment, but this damage was recoverable. On the other hand, DNA was not damaged in the cells treated with six other quinolones. In the cells treated with NFLX and CPFX for 20 h, DNA migration was compared by the comet assay at pH 10, 12.1 and >13. The comet assay both at pH 12.1 and >13 showed increased DNA migration, but there was no positive response in the comet assay at pH 10. In the in vitro micronucleus (MN) test, WTK-1 cells were treated with each of four quinolones (NA, PPA, NFLX and CPFX) at 15.63-125 microg/mL for 20 h. NFLX significantly increased MNs in the cells, but no changes were noted in the cells treated with three other quinolones. These results suggest that NFLX and CPFX induced DNA single strand breaks (SSBs), and that NFLX-induced SSBs resulted in chromosome aberrations.     

Ecological Implications of an Improved Direct Viable Count Method for Aquatic Bacteria

The direct viable count method first described by Kogure et al. (Can. J. Microbiol. 25:415-420, 1979) was improved by using an antibiotic cocktail instead of nalidixic acid alone. We screened 100 marine isolates from two coastal areas for their sensitivities to five replication-inhibiting antibiotics, including four quinolones (nalidixic, piromidic, and pipemidic acids and ciprofloxacin) and one (beta)-lactam (cephalexin). It was shown that growth inhibition of all isolates cannot be readily achieved by using a single antibiotic. Inhibition was much more efficient when all the antibiotics were combined, making it possible to use this method with natural communities. In combination, the concentration of each antibiotic could be lowered and the incubation time could be increased without any growth. Under such conditions, it was shown that the fraction of substrate-responsive cells within natural marine communities is much greater (1 to 2 orders of magnitude) than those reported by traditional procedures. Furthermore, the new procedure made substrate-responsive cells more clearly distinguishable. These improvements resulted in an increased incubation time and were related to metabolic expression of slow-growing cells and/or to the recovery of starved cells. The increased fraction of viable cells within marine communities has ecological implications on the metabolic role of nonculturable cells.     

Inhibition of Ribonucleic Acid Synthesis by Nalidixic Acid in Escherichia coli

The effect of low concentrations of nalidixic acid on ribonucleic acid (RNA) synthesis in Escherichia coli was examined. It was observed that RNA synthesis in exponentially growing cells was not significantly affected, in harmony with previous studies. However, RNA synthesis was markedly depressed by nalidixic acid during starvation for an amino acid or during chloramphenicol treatment. This effect was not caused by increased killing or inhibition of nucleoside triphosphate synthesis by nalidixic acid. The pattern of radioactive uracil incorporation into transfer RNA or ribosomes was not changed by the drug. The sensitivity of RNA synthesis to nalidixic acid in the absence of protein production may be useful in probing the amino acid control of RNA synthesis.     

Effect of Rifamycins and Related Antibiotics on the Deoxyribonucleic Acid-Dependent Ribonucleic Acid Polymerase of Vaccinia Virus Particles

A number of compounds related to rifampin which act as expected in the Escherichia coli system have been tested for their ability to inhibit the vaccinia particle deoxyribonucleic acid-dependent ribonucleic acid (RNA) polymerase in vitro. Some compounds are inactive even at concentrations of 500 mug/ml, others are able to produce partial inhibition, and others strongly inhibit the enzyme activity at 150 mug/ml or less. The inhibition, where present, operates immediately but appears to be at least partially reversible. At least one compound which is without effect against bacterial RNA polymerase is a potent inhibitor of the viral RNA polymerase. As the enzyme activity of rifampin-resistant mutants of vaccinia virus is inhibited to the same extent as that of the wild type, the observed in vitro effect on vaccinia virus RNA polymerase is not identical with the in vivo effect specifically directed against a vaccinia-specified protein.     

The influence of nalidixic acid on Escherichia coli growth in milk

The high antibacterial activity of nalidixic acid against Escherichia coli, cultivated in raw and pasteurized milk has been shown. The low oxygen reduction potential had no influence on the antibacterial activity of this drug. The natural antibacterial agents in active milk from an inflamed udder have reduced the efficacy of nalidixic acid inhibition of the growth of E. coli.     

The interaction of trimethoprim and quinolones against Gram-negative fish pathogens

The effect of combination of trimethoprim with other non-sulphonamide antibacterial agents, in particular oxolinic acid and nalidixic acid, was evaluated against Gram-negative fish pathogens. The species included Aeromonas salmonicida, Yersinia ruckeri , some Vibrio spp. and Escherichia coli as a reference. The extent of synergy found by other workers with these substances against human Gram-negative bacteria was not apparent here. Some positive interaction between trimethoprim and oxolinic acid was found with Aer. salmonicida, Y. ruckeri and E. coli and between trimethoprim and nalidixic acid with Y. ruckeri in double disc diffusion tests but was not supported by fractional inhibitory concentration indices. The combinations were not effective in preventing emergence of resistance in passage on a drug gradient. Trimethoprim-resistant isolates of Aer. salmonicida were inhibited by low levels of oxolinic acid but the converse did not apply.     

Determination of piromidic acid residues in trout muscle tissue and in urine by liquid chromatography with post-column modification of pH and fluorimetric detection

A rapid high-performance liquid chromatographic method has been developed to determine piromidic acid in trout muscle tissue and in urine, in the presence of nalidixic, 7-hydroxymethylnalidixic, oxolinic and pipemidic acids and cinoxacin. A Nova-Pak C₁₈ column was used with acetonitrile–4·10⁻⁴ M oxalic acid (40:60, v/v) as the mobile phase. A post-column change of pH was made with NaOH. Fluorimetric detection at 456 nm (λ_ex 275 nm) was used. The instrumental detection limit was 5.91 ng/ml, based on height of peak. Pretreatment of the urine samples was not necessary and fish samples were extracted with sodium hydroxide solutions and cleaned by means of an extraction with chloroform. Detection limit was 147 ng/ml for urine and 5.91 ng/g for trout muscle. Good separation without interference from any other components was obtained. Recovery was better than 87% in urine and better than 72% in trout muscle tissue.     

Enumeration and characterization of bacteria in mineral water by improved direct viable count method

Fifteen strains from two emergent mineral waters were isolated and tentatively identified with API 20NE and BIOLOG GN systems. These strains were screened for their sensitivities to seven replication-inhibiting antibiotics of the (fluoro)quinolone group (nalidixic and pipemidic acid, flumequine, norfloxacin, ofloxacin, pefloxacin and ciprofloxacin). It was shown that the direct viable count (DVC) procedure could be improved by using certain antibiotic cocktails, which were active against the isolates. Geometric bacterial features were successfully determined with image analysis and adapted software (ICONIX, Perfect Image). Elongations were significant and allowed rapid discrimination of antibiotic inhibited and non-inhibited strains. Particular isolates in a mixed culture were characterized and enumerated after only 14 h exposure with the appropriate antibiotic cocktail. This method can also be applied to other communities, such as mixed cultures in bio-fermentors or in food with known microflora.     

DM423菌株诱变前后对吡哌酸的耐药性测定

为了研制一种对腹泻病人疗效快,又不造成菌群失调的药物。我们确定吡哌酸与DM423菌联合应用。首先把DM423菌株诱变成耐药菌株,经多次诱变筛选结果是,耐药量比原菌株提高了2000倍,耐药性相对稳定,耐药株生物学特性与原菌株相同。定名为DM423-1菌株。     

In-situ enumeration and probing of pyrene-degrading soil bacteria

Inferences about which microorganisms degrade polycyclic aromatic hydrocarbons in contaminated soils have largely been obtained using culture-based techniques, despite the low percentage of microorganisms in soil that are believed to be culturable. We used a substrate-responsive direct viable count method to identify and quantify potential polycyclic aromatic hydrocarbon-degrading bacteria in a soil containing petroleum wastes. Bacteria were extracted and their response to substrates determined in the presence of DNA gyrase inhibitors, which cause viable and active cells to elongate. When yeast extract, a widely used carbon source, was added as a growth substrate, together with nalidixic acid, piromidic acid and ciprofloxacin, a significant increase in elongated cells to 47%, 37% and 22%, respectively, was observed within 24 h. With pyrene as the main substrate, 10 mg L(-1) of nalidixic acid or piromidic acid caused 18-22% and 8-12%, respectively, of the cells to elongate within 24 h; whereas the effect of 0.5 mg L(-1) ciprofloxacin was not significant until 53 h later. Enlarged cells were identified and enumerated by fluorescent in situ hybridization, using Alpha-, Beta- and Gammaproteobacteria, and domain Bacteria-specific probes. The Bacteria-specific probe detected 35-71% of the total microorganisms detected by the DNA-binding dye 4,6-diamidino-2-phenylindole. Initially, 44%, 13% and 5% of the total bacteria in the soil extract were Alpha-, Beta- and Gammaproteobacteria, respectively. Without pyrene or a gyrase inhibitor, these subgroups decreased to 30% of the total population but were predominant with piromidic acid or unchanged with ciprofloxacin when pyrene was the main substrate. The proportion of elongated Alpha- and Betaproteobacteria (potential pyrene degraders) increased significantly (P<0.05). This approach links phylogenetic information with physiological function in situ without the conventional cultivation of bacteria and can be used to probe and enumerate degradative groups at even a finer level of discrimination.     

Glutathione transferase in bacteria: subunit composition and antigenic characterization

The presence of glutathione transferase (GST; EC 2.5.1.18) in Escherichia coli ATCC 25922, E. coli ATCC 25422, Proteus vulgaris ATCC 8427, Pseudomonas aeruginosa ATCC 27853, Klebsiella oxytoca CIP 666, K. oxytoca AF 101, Enterobacter cloacae CIP 6085, Serratia marcescens CIP 6755, and Proteus mirabilis AF 2924 was investigated. Using 1-chloro-2,4-dinitrobenzene as substrate, GST activity was found in the glutathione-(GSH-)affinity-purified fraction of all strains tested. SDS-PAGE analysis of GSH-affinity-purified enzyme indicated that the GSTs of all these bacteria are dimers of two identical subunits of Mr about 22,500. Rabbit antiserum directed against the major isoenzyme present in Proteus mirabilis AF 2924, Pm-GST-6.0, was used to investigate the antigenic properties of bacterial GSTs. Western blot analysis indicated that a GST antigenically identical to Pm-GST-6.0 is present in Enterobacter cloacae CIP 6085, Escherichia coli ATCC 25422 and Proteus vulgaris ATCC 8427, but absent in Escherichia coli ATCC 25922, Klebsiella oxytoca CIP 666, K. oxytoca AF 101 and Serratia marcescens CIP 6755. The presence of Pm-GST-6.0, but not mammalian GST, increased the MIC values of amikacin, ampicillin, cefotaxime, cephalothin and nalidixic acid for E. coli ATCC 25922. It is suggested that bacterial GST may represent a defense against the effects of antibiotics.     

Quinazolinone-based rhodanine-3-acetic acids as potent aldose reductase inhibitors: Synthesis,functional evaluation and molecular modeling study

A series of quinazolinone-based rhodanine-3-acetic acids was synthesized and tested for in vitro aldose reductase inhibitory activity. All the target compounds displayed nanomolar activity against the target enzyme. Compounds 3a, 3b, and 3e exhibited almost 3-fold higher activity as compared to the only marketed reference drug epalrestat. Structure-activity relationship studies indicated that bulky substituents at the 3-phenyl ring of the quinazolinone moiety are generally not tolerated in the active site of the enzyme. Insertion of a methoxy group on the central benzylidene ring was found to have a variable effect on ALR-2 activity depending on the nature of peripheral quinazolinone ring substituents. Removal of the acetic acid moiety led to inactive or weakly active target compounds. Docking and molecular dynamic simulations of the most active rhodanine-3-acetic acid derivatives were also carried out, to provide the basis for further structure-guided design of novel inhibitors.     

In Vitro Antioxidant Profile of Phenolic Acid Derivatives

Several caffeic acid esters isolated from propolis exhibit interesting antioxidant properties, but their in vivo use is compromised by hydrolysis of the ester bond in the gastrointestinal tract. Therefore, a series of caffeic acid amides were synthesized and their in vitro antioxidant profile was determined. A series of hydroxybenzoic acids, hydroxycinnamic acids, and the synthesized caffeic acid amides were tested for both their 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging and microsomal lipid peroxidation-inhibiting activity. Some of the highly active antioxidants were further tested by means of electron paramagnetic resonance for their hydroxyl radical scavenging activity. Since a promising antioxidant compound should show a lipid peroxidation-inhibiting activity at micromolar level and a low cytotoxicity, the cytotoxicity of the phenolic compounds was also studied. In all the assays used, the caffeic acid anilides and the caffeic acid dopamine amide showed an interesting antioxidant activity.     

Synthesis, in vitro and in vivo antimycobacterial activities of diclofenac acid hydrazones and amides

Various diclofenac acid hydrazones and amides were synthesized and evaluated for in vitro and in vivo antimycobacterial activities against Mycobacterium tuberculosis. Preliminary results indicated that most of the compounds demonstrated better in vitro antimycobacterial activity (MIC: 0.0383-7.53 microM) than diclofenac (MIC: 21.10 microM) and ciprofloxacin (MIC: 9.41 microM). Among the synthesized compounds, 1-cyclopropyl-6-fluoro-8-methoxy-7-[[N4-(2-(2-(2,6-dichlorophenylamino)phenyl)acetyl)-3-methyl]-N1-piperazinyl]-4-oxo-1,4-dihydro-3-quinoline carboxylic acid (5d) was found to be the most active compound in vitro with MIC of 0.0383 microM and was more potent than first line antitubercular drug isoniazid (MIC: 0.1822 microM). In the in vivo animal model 5d decreased the bacterial load in lung and spleen tissues with 2.42- and 3.66-log10 protections, respectively, at 25 mg/kg body weight.     

Determination of quinolones in plasma samples by capillary electrophoresis using solid-phase extraction

The potential of capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MEKC) have been investigated for the separation and quantitative determination of 10 quinolone antibiotics. The influence of different conditions, such as the buffer and pH of the electrolyte, the surfactant and the ion-pairing agents added to the electrolyte and the organic modifier were studied. A buffer consisting of 40 mM sodium tetraborate at pH 8.1 containing 10% (v/v) methanol was found to be a highly efficient electrophoretic system for separating lomefloxacin, enoxacin, norfloxacin, pipemidic acid, ofloxacin, piromidic acid, flumequine, oxolinic acid, cinoxacin and nalidixic acid. A solid-phase extraction method to remove the sample matrix (pig plasma samples) was developed on a C₁₈ cartridge using a mixture of methanol–water (70:30, v/v). The method is specific and reproducible and mean recoveries were in the range 94.0±4.2% and 123.3±4.1% for pig plasma samples over the range used. A linear relationship between concentration and peak area for each compound in pig plasma samples was obtained in the concentration range 5–20 mg l⁻¹ and detection limits were between 1.1 and 2.4 mg l⁻¹.