Intraphagocytic Bactericidal Activity of Bacterial DNA Gyrase Inhibitors against &lt;i&gt;Serratia marcescens&lt;/i&gt;

A fluorometric assay, based on the natural fluorescence of the quinolone nucleus, was used to determine the uptake of ofloxacin by human polymorphonuclear leukocytes. The ratio of cellular concentration to extracellular concentration (CIE) at 20 min and 37°C was 7.2, using an extracellular concentration of 5 ,ug/ml. Uptake was rapid and was not affected by pH (5 to 9), but required elevated environmental temperature and cell viability. The metabolic inhibitors sodium fluoride and sodium cyanide significantly decreased the uptake of ofloxacin. The penetration of ofloxacin was not affected by the presence of glucose or adenosine, but was decreased by L-amino acids (lysine, leucine, and glycine). These results suggest that ofloxacin could be transported via an amino acid transport system and that the fluorometric assay is a useful method for determining the intracellular penetration of fluoroquinolones, avoiding the use of radiolabeled antimicrobial agents.The new quinolones have been shown to be very active against different intracellular pathogens such as mycobacteria and Legionella pneumophila (4, 5). These antimicrobial agents are able to penetrate into phagocytes and remain active intracellularly (2,3,9,12 In this study a fluorometric assay, based on the natural fluorescence of the quinolone nucleus, was used to evaluate the uptake of ofloxacin by human polymorphonuclear leukocytes (PMN). The possible mechanism involved in the membrane transport of this antimicrobial agent is also evaluated.MATERIALS AND METHODS Isolation of PMN. PMN were recovered from heparinized venous blood of healthy donors and were purified by previously described methods (10). PMN preparations were 97% pure. Final cell suspensions were adjusted to 5 x 106 PMN/ml in Hanks balanced salt solution. The PMN were 95% viable by trypan blue exclusion.Ofloxacin uptake by human PMN. A previously described fluorometric assay to measure quinolone uptake by bacterial cells was adapted to measure ofloxacin uptake by human PMN (1). Ofloxacin was kindly supplied by Hoescht AG, Frankfurt, Federal Republic of Germany. In these experiments 2.5 x 106 PMN were incubated in 0.5 ml of Hanks balanced salt solution containing clinically relevant concentrations of ofloxacin (2 and 5 ,ug/ml). After different incubation times at 37°C, cells were separated from extracellular * Corresponding author.solution by centrifugation through a water-impermeable silicone-oil barrier in a microcentrifuge tube (7). The entire cell pellet, obtained by cutting off the portion of the microcentrifuge tube containing the pellet, was placed in 2 ml of 0.1 M glycine-HCl buffer (pH 3.0) and agitated vigorously in a vortex shaker. Incubation for 2 h at room temperature was sufficient to release fully PMN-associated antibiotic. The samples were centrifuged for 5 min at 5,600 x g, and the amount of antibiotic was determined by fluorescence emission of the supernatant with a fluorescence spectrophotometer (SFM 25; Kontron Instruments, Zurich, Switzerland). The fluorescence excitation and e...

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Fluorometric measurement of ofloxacin uptake by human polymorphonuclear leukocytes

Pascual

Garcı́a

Perea

1989

“…Some of these effects have been observed on leukocytes. It has been found that ciprofloxacin penetrates leukocytes and reaches intracellular levels with bactericidal activity (4,20). The incorporation of [ 3 H]thymidine into T lymphocytes incubated with phytohemagglutinin was increased in the presence of fluoroquinolones, but the progression of mitogen-stimulated lymphocytes through the S and G 2 /M stages of the cell cycle and the secretion of immunoglobulins (Ig's) by pokeweed mitogen-stimulated B lymphocytes were inhibited (7).…”

mentioning

confidence: 99%

Modification of immune response in mice by ciprofloxacin

Jiménez-Valera

Sampedro

Moreno

et al. 1995

Some studies have suggested that the addition of ciprofloxacin to in vitro cultures of mitogen-stimulated lymphocytes exerts inhibitory effects on cell cycle progression and immunoglobulin (Ig) secretion. We tested the effects of this drug on some immunity parameters in BALB/c mice. Mice treated intraperitoneally with ciprofloxacin (10 mg/kg of body weight per day) for 3 consecutive days and immunized with sheep erythrocytes 24 h after the last injection showed significant suppression of hemolytic IgG-forming cells, whereas the response of IgM-forming cells remained unchanged. When treatment lasted 7 days the response of antibodyforming cells was not modified. When the 3-day treatment was started at 24 h after immunization with sheep erythrocytes, the response of IgM-forming cells was increased, whereas the response of IgG-forming cells was suppressed. Delayed-type hypersensitivity to sheep erythrocytes was significatively suppressed in animals that received the 3-day treatment with ciprofloxacin and were immunized subcutaneously 24 h after the last injection. In vitro proliferation of lymphocytes from ciprofloxacin-treated mice in response to either lipopolysaccharide or concanavalin A was also suppressed. Leukopenia and an increase in the level of granulocytemacrophage colony-forming cells in bone marrow were also observed in ciprofloxacin-treated mice. These results, together with those from other reports, suggest that modification of the biological responses by ciprofloxacin is a complex phenomenon that may be influenced by several factors.The knowledge of possible influences of antibiotics on the immune response seems to be of great importance for the clinical approach to the process of therapy. This is specially interesting when antibiotics are given to immunosuppressed patients. The capacity to modify the immune response to unrelated antigens has been observed with numerous antibiotics, including ␤-lactams (8, 19), aminoglycosides (19), tetracyclines (10), rifamycins (2), and others.The fluoroquinolones are antimicrobial agents with a broad antimicrobial spectrum (21). The primary target of fluoroquinolones is the bacterial DNA gyrase (topoisomerase II) (21). However, some inhibitory effects of these agents on the eucaryotic enzymes involved with DNA replication have been observed (9). A number of quinolones have been shown to possess genotoxicity in vitro, and this has been considered a possible explanation for some of the abnormal eucaryotic cellular responses obtained after treatment with these agents (9). Some of these effects have been observed on leukocytes. It has been found that ciprofloxacin penetrates leukocytes and reaches intracellular levels with bactericidal activity (4,20). The incorporation of [ 3 H]thymidine into T lymphocytes incubated with phytohemagglutinin was increased in the presence of fluoroquinolones, but the progression of mitogen-stimulated lymphocytes through the S and G 2 /M stages of the cell cycle and the secretion of immunoglobulins (Ig's) by pokeweed mitogen-stimulated B ...

“…In the past decade, pipemidic acid (4) has been found to possess higher antibacterial activity against Pseudomonas aeruginosa, Escherichia coli, Proteus vulgaris, Klebsiella pneumoniae, and Enterobacter and Citrobacter organisms (5,(12)(13)(14) than structurally similar agents, such as piromidic (11) and nalidixic acid (3), and thus has been applied to chemotherapy of parenchymatous urinary tract infections. The known chronic and acute toxicities of pipemidic acid are rather low (6)(7)(8)(9); however, numerous pharmacokinetic studies have been conducted to determine the most effective concentrations in serum for clinical use.…”

mentioning

confidence: 99%

Determination of pipemidic acid in plasma by normal-phase high-pressure liquid chromatography

Ito¹,

Inoue²,

Morikawa³

et al. 1985

An improved high-pressure liquid chromatography procedure for determining the concentration of pipemidic acid in human plasma was developed, which uses a normal-phase silica gel column and aqueous mobile solvent system similar to that used in reverse-phase chromatography. Precolumn derivatization of pipemidic acid was achieved by a methylation reaction with boron trifluoride in methanol after extraction from plasma with chloroform containing 4% ethanol. The percent recovery of pipemidic acid was 88.3 7.7, the variation of which became negligible when quinacrine was used as an internal standard for the determination. High-pressure liquid chromatography analysis was performed by conventional silica gel and mobile solvent mixtures containing 3% of 0.14% HCl04 solution, 19% methanol, and 78% chloroform. The UV detector was set at 265 nm. The detection limit of pipemidic acid methyl ester was as low as 10 ng of the injection amounts or 0.5 ,ug of the plasma per ml, with 0.01 absorbance units (full scale) and a signal-to-noise ratio of 3.In the past decade, pipemidic acid (4) has been found to possess higher antibacterial activity against Pseudomonas aeruginosa, Escherichia coli, Proteus vulgaris, Klebsiella pneumoniae, and Enterobacter and Citrobacter organisms (5, 12-14) than structurally similar agents, such as piromidic (11) and nalidixic acid (3), and thus has been applied to chemotherapy of parenchymatous urinary tract infections. The known chronic and acute toxicities of pipemidic acid are rather low (6-9); however, numerous pharmacokinetic studies have been conducted to determine the most effective concentrations in serum for clinical use. In 1975, Japanese workers (10) recommended a biological assay technique that uses the thin-layer cup plate of E. coli Kp strain as an indicator organism. More recently, French workers (M. Le Duff, D. Gibassier, P. A. Sado, and R. Le Verge, Abstr. C. R. Congr. Eur. Biopharm. Pharmacokinet. 1st, series 2, p. [508][509][510][511][512][513][514][515] 1981) have reported a high-pressure liquid chromatography procedure for determining the concentration of pipemidic acid in rabbit plasma. Since pipemidic acid has a very strong basic moiety in its molecular structure, conventional chromatographic procedures would not be adequate for pharmacological studies. This paper presents a new chromatographic technique which uses a silica gel (stationary phase) and reversed mobile-phase solvents for accurately determining pipemidic acid content in human plasma.MATERIALS AND METHODS Extraction and precolumn derivatization. A 2-ml plasma specimen was spiked with 20 ,ug of quinacrine per 20 ,ul of methanol solution, and the resulting mixture was acidified to ca. pH 4.5 with 100 ,ul of 2 N hydrochloric acid. Neutral and acidic contaminants were eliminated by washing the mixture with 2 ml of chloroform in a 10-ml test tube with a vortex mixer. The organic layer was eliminated, and the aqueous layer was diluted with 1 ml of distilled water. The aqueous mixture was adjusted at ca. pH 7.0 with 2 N so...