In vitro inhibition of bacterial DNA gyrase by cinodine, a glycocinnamoylspermidine antibiotic.

Cinodine, a broad-spectrum glycocinnamoylspermidine antibiotic, binds to DNA and irreversibly inhibits bacterial and phase DNA synthesis. Cinodine was found to inhibit the activity of Micrococcus luteus DNA gyrase in vitro, but it did not inhibit the activities of two other DNA-binding enzymes, namely, topoisomerase I and BamHI. Although we cannot yet conclude that DNA gyrase is an intracellular target of the drug, in vitro inhibition of the enzyme by cinodine appears to be specific.     

Inhibition of bacterial isoprenoid synthesis by fosmidomycin, a phosphonic acid-containing antibiotic

The mechanism of action of fosmidomycin (FR-31564), a phosphonic acid containing antibiotic, was examined against Escherichia coli, Micrococcus luteus and other bacteria. It converted growing Esch. coli cells into spheroplasts or swollen cells, but did not inhibit the enzymatic reactions of cell wall synthesis in ether-treated cells. Unlike bicyclomycin, phenethyl alcohol and mitomycin C, it did not reduce the amount of envelope lipoprotein, phospholipids, or DNA, but did reduce the amount of menaquinones and ubiquinones in growing Esch. coli cells. It also inhibited the biosynthesis of both carotenoids and menaquinones in M. luteus cells, suggesting that inhibition of the biosynthesis of a common precursor of these isoprenoids (possibly farnesyl pyrophosphate) might be the main site of its antibacterial activity.     

Quantification of the carbapenem antibiotic ertapenem in human plasma by a validated liquid chromatography-mass spectrometry method

BACKGROUND: Ertapenem (Invanz) is a newly developed carbapenem beta-lactam antibiotic. LC-MS is the method of choice for therapeutic drug monitoring (TDM) of a variety of drugs including antibiotics. No validated LC-MS method for ertapenem quantification is described in the literature so far. METHODS: A rapid and robust LC-MS quantification method for ertapenem was developed and validated for clinical routine application in plasma samples. After immediate stabilisation with MES buffer (pH 6.5), samples were prepared for LC-MS analysis using simple protein precipitation. LC-MS coupling was realised by the use of a Phenomenex Synergi 4micro Polar-RP A80 Mercury LC column (10 x 2.0 mm) in combination with a Single-MS (Agilent 1100 LC-MSD SL) operating in negative selected ion monitoring (SIM) detection mode with ceftazidime as internal standard for adequate selective and sensitive analysis. RESULTS: LC-MS method validation by means of determination of limit of detection (LOD 0.1 microg mL-1), lower limit of quantification (LLOQ 1 microg mL-1), linearity (0.1-50 microg mL-1), recovery (> 90%), intra- and inter-day precision (RSD < 10%), accuracy (> 90%), inter-subject variability (< 10% at LLOQ), drug stability in plasma (> 3 months) and in post-extracted samples (> 99% for 24 h), and matrix effects (process efficiency > 90%) showed excellent performance parameters considering Guidance for Industry - Bioanalytical Method Validation. CONCLUSION: This method is perfectly appropriate for routine quantification of ertapenem and possibly other polar carbapenem beta-lactam antibiotics.     

In-vitro antibiotic inactivation by mammalian cell and killed bacterial preparations

Inactivation of a range of antibiotics acting at different points in the metabolism of the bacterial cell was detected by estimating the MIC and MBC in the presence of liver and other tissue preparations. High temperature treatment and sonication of liver cells increased their ability to inactivate antibiotic action. This treatment would have almost completely destroyed enzyme activity, which was, therefore, not thought likely to be the cause of the phenomenon. The loss of antibiotic activity may be related to "protein binding" and a dialysis experiment showed that penicillin binding with liver homogenate was very much greater than with human albumin. It may be that increased disruption of tissue cells by physical methods exposes more active binding sites which reduces the bioavailability of antibiotics. Some degree of binding specificity was indicated in experiments in which DNA was shown to block antibiotics acting primarily on DNA--related synthesis and RNA blocked antibiotics acting on RNA--related metabolism. Suggestions are made for the cause of failure of antibiotic treatment in certain clinical situations.     

In vivo and in vitro studies of antibiotic release from and bacterial growth inhibition by antibiotic-impregnated polymethylmethacrylate hip spacers

The antimicrobial properties and the elution characteristics of gentamicin-vancomycin-loaded hip spacers were studied in vivo and in vitro. Vancomycin elution was greater than gentamicin elution. The antibiotic concentrations in vivo were less than those in vitro. Not dependent on implantation duration, growth inhibition by spacers in vitro was observed for 2 weeks. The reason for protracted wound healing cannot be insufficient antibiotic release.     

The rapid evaluation of bacterial growth and antibiotic susceptibility in blood cultures by selected ion flow tube mass spectrometry

We have measured the production of volatile organic compounds (VOCs) by selected ion flow tube mass spectrometry (SIFT-MS) in the headspaces of conventional BacT/ALERT blood culture bottles (Biomerieux, Durham, NC) artificially infected with 5 bacterial strains. Uninfected blood samples were inoculated with Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli, and Neisseria meningitidis. Growth and species identification were determined at 6 h by measuring a panel of 9 VOC products. Two species, E. coli and S. aureus, were also incubated in the presence of gentamicin or flucloxacillin, respectively, above or below their demonstrated MIC. The concentration-dependent antibiotic susceptibility of both strains was demonstrated by the inhibition of VOC production at 22 h (P < .05). These results suggest incorporating SIFT-MS detection of microbial VOCs as a sensitive method for bacterial detection, identification, and determination of antibiotic susceptibility in a conventional blood culture system.     

Effect of storage and changes in bacterial growth phase and antibiotic concentrations on antimicrobial tolerance in Staphylococcus aureus.

A new bacteriocin produced by Aerococcus viridans was purified. Bacteria grown on liquid medium synthesized a bacteriocin, viridicin, which can be extracted from the cells by treatment with 0.86 M NaCl solution. Viridicin production was not induced by ultraviolet irradiation or by treatment with mitomycin C. The bacteriocin was purified by ultrafiltration, gel filtration, and preparative polyacrylamide disc gel electrophoresis. The molecular weight was between 100,000 and 120,000. The purified viridicin was homogeneous on polyacrylamide gel electrophoresis. The viridicin was composed of protein, carbohydrate, and lipid.     

In vitro mechanism of inhibition of bacterial cell growth by allicin.

Diallyl thiosulfinate (allicin) is the agent found in garlic which is responsible for the antibacterial and antifungal activity of extracts of this plant. The effect of bacteriostatic concentrations of allicin (0.2 to 0.5 mM) on the growth of Salmonella typhimurium revealed a pattern of inhibition characterized by: (i) a lag of approximately 15 min between addition of allicin and onset of inhibition, (ii) a transitory inhibition phase whose duration was proportional to allicin concentration and inversely proportional to culture density, (iii) a resumed growth phase which showed a lower rate of growth than in uninhibited controls, and (iv) an entry into stationary phase at a lower culture density. Whereas DNA and protein syntheses showed a delayed and partial inhibition by allicin, inhibition of RNA synthesis was immediate and total, suggesting that this is the primary target of allicin action.     

Chemotherapy drug potency assessment method of ovarian cancer cells by digital holography microscopy

Drug potency assessment plays a crucial role in cancer chemotherapy. The selection of appropriate chemotherapy drugs can reduce the impact on the patient''s physical condition and achieve a better therapeutic effect. Various methods have been used to achieve in vitro drug susceptibility assays, but there are few studies on calculating morphology and texture parameters quantitatively based on phase imaging for drug potency assessment. In this study, digital holography microscopy was used to get phase imaging of ovarian cancer cells after adding three different drugs, namely, Cisplatin, Adriamycin, and 5-fluorouracil. Based on the reconstructed phase imaging, four parameters of ovarian cancer cells changed with time, such as the average height, projected area, cluster shade, and entropy, were calculated. And the half-inhibitory concentration of cells under the effect of different drugs was calculated according to these four parameters. The half-inhibitory concentration, which can directly reflect the drug potency, is associated with the morphological and texture features extracted from phase images by numerical fitting. So, a new method for calculating the half-inhibitory concentration was proposed. The result shows that the morphological and texture feature parameters can be used to evaluate the sensitivity of ovarian cancer cells to different drugs by fitting the half-inhibitory concentration numerically. And the result provides a new idea for drug potency assessment methods before chemotherapy for ovarian cancer.     

Effect of a bacterial lipopolysaccharide on biliary excretion of a beta-lactam antibiotic, cefoperazone, in rats.

Klebsiella pneumoniae O3 lipopolysaccharide (LPS) has been found to dramatically modify the pharmacokinetics of the beta-lactam antibiotic cefazolin in rats. This study investigated the effect of LPS on the biliary excretion of the beta-lactam antibiotic cefoperazone (CPZ) in rats. CPZ is known to be actively secreted into the bile by a carrier-mediated transport system. LPS (250 micrograms/kg of body weight) was infused for 20 to 30 min 2 h before an intravenous administration of CPZ (20 mg/kg). The pharmacokinetic parameters of CPZ were estimated by a noncompartment model. LPS induced a significant decrease in the systemic clearance (by approximately 50%) and an increase in the mean residence time of CPZ. Significant decreases were also seen in the bile flow rate and in the biliary recovery of unchanged CPZ in the LPS-treated rats. LPS tended to increase the proportion of urinary excretion of CPZ. LPS significantly decreased the biliary clearance (by approximately 55%) and renal clearance (by approximately 35%) of CPZ. However, no changes in the volume of distribution at steady state for CPZ were observed between the treatment groups. Our findings suggest that LPS induces changes in the pharmacokinetics of CPZ as a result of changes occurring in the biliary secretory system.     

The influence of fatty acids on serum thyroxine determination by competitive protein-binding radioassay.

Thyroxine concentration, determined by competitive protein-binding radioassay (T4(D)), increased in serum stored at 20 degrees C and 37 degrees C. The increase correlated well with the increase in nonesterified fatty acids (NEFA). In vitro addition of NEFA to serum and in vivo increase in NEFA induced by post-prandial heparin injection resulted in a proportional increase in T4(D) values. Protein-bound iodine and thyroxine concentration, as determined by radioimmunoassay, were not influenced by storage and changes in NEFA concentration. NEFA interfere with T4(D), leading to elevated results.     

Enhancement of antibiotic susceptibility and suppression of Mycobacterium avium complex growth by poloxamer 331.

The resistance of Mycobacterium avium complex (MAC) to antibiotics is thought to be enhanced by its outer glycolipid layer, which protects the organisms from antibiotics and host defense mechanisms. We hypothesized that surfactants which disrupt the lipid barrier might be of therapeutic value. We evaluated the ability of 10 poloxamer surfactants to inhibit the growth of MAC organisms and to potentiate antimycobacterial drug activity in broth culture using a radiometric assay. Very large, small, or hydrophilic poloxamers had little or no effect. However, certain hydrophobic poloxamers, especially P331, retarded the growth of most isolates of MAC and produced a synergistic effect with rifampin. The MIC of rifampin required to inhibit the growth of MAC was reduced from a mean of 14.6 micrograms/ml (range, 4 to > 32 micrograms/ml) to 1.4 micrograms/ml (range, < 1.125 to 4 micrograms/ml) by 1.0 mg of P331 per ml (P < 0.01). Enhancement of antibiotic susceptibility was observed with concentrations of poloxamer as low as 10 micrograms/ml. These studies suggest that P331 might be useful in increasing the effectiveness of antibiotic therapy of MAC infections.     

Mutant prevention concentration as a measure of antibiotic potency: studies with clinical isolates of Mycobacterium tuberculosis

The mutant prevention concentration (MPC) of a C-8-methoxy fluoroquinolone exhibited a narrow distribution for 14 genetically diverse clinical isolates of Mycobacterium tuberculosis, indicating that results from single-isolate studies are likely to be representative. When one isolate was challenged with a variety of antituberculosis agents, C-8-methoxy fluoroquinolones were exceptional in having MPCs below the maximum concentration attained in serum by use of commonly recommended doses.     

Limits of the estimate of serum folate-binding protein by radioassay

Various factors that influence the measurement of serum folic acid-binding protein (FABP) by radioassay were examined. Varying the protein concentration and the composition of the charcoal suspension can increase or reduce the value of the blank i.e. the estimate of FABP. Moreover, it was found that the value of the FABP depends on the quantity of folic acid in the incubation medium. Reproducible results can only be obtained with rigorously defined conditions of concentration of the different reactants.     

Quantification of antiangiogenic and antivascular drug activity by kinetic analysis of DCE-MRI data

Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) can be used to quantify the response of tumors to vascular targeting agents. Tumor response is frequently assessed using mathematical models that describe the distribution of contrast agents over time as a function of fundamental characteristics of vascular physiology. Generally, mathematical models of biological systems are abstractions that attempt to retain fundamental physiologic characteristics, thereby allowing for multiple potential modeling approaches and structures. Various DCE-MRI modeling techniques are discussed in this article.     

Differentiation-stimulating potency of differentiated HL60 cells after drug treatment

Differentiation therapy in the treatment of leukemia is often hampered by limitations on using certain pharmaceutical regents or on the required doses due to various reasons, such as drug-resistance and retinoic acid syndrome. To circumvent these problems, a strategy might be developed on the basis of the ability of drug-differentiated cells to stimulate differentiation in leukemia cells. Using the promyelocytic leukemia cell line HL60 as a cell model, we assessed the differentiation-stimulating potency of differentiated granulocytes and monocytes/macrophages after treatments with all-trans retinoic acid (ATRA) and 12-O-tetradecanoylphorbol-13-acetate (TPA), respectively. ATRA- and TPA-differentiated cells were able to stimulate differentiation in fresh HL60 cells, accompanied by inhibition on cell growth to various extents. The differentiated cells of the second generation, especially those originated from TPA treatment, were as potent as the drugs themselves in stimulating differentiation in fresh HL60 cells. On the basis of “differentiation induced by differentiated cells”, we explored the feasibility of ex vivo therapy.