Initial Rate Of Killing Research Articles

In Vitro Anaerobic Pharmacokinetic/Pharmacodynamic Model to Simulate the Bactericidal Activity of Levornidazole Against Bacteroides fragilis

PurposeThis study was designed to correlate the pharmacokinetic/pharmacodynamic (PK/PD) parameters with PD indices of levornidazole against Bacteroides fragilis and to calculate the PK/PD target value for levornidazole to attain its expected maximal bactericidal effect using an in vitro anaerobic dynamic PK/PD model. MethodsAn anaerobic dynamic PK/PD model was developed in vitro. The scheme for PK modeling was designed according to the PK parameters of levornidazole in the human body. The device of 2-compartment PK/PD model was constructed by using digital control of flow rate to simulate 4 regimens of single-dose intravenous infusion of levornidazole to determine the bactericidal activity of levornidazole against the 3 strains of B fragilis within 72 hours. PD parameters such as reduction of colony count within 24 hours (∆Log24h), area under bactericidal curve (AUBC), and 2-hour initial killing rate (IKR) were calculated and correlated with PK/PD parameters. Sigmoid Emax model of levornidazole was established to calculate PK/PD target values to attain corresponding PD effect. FindingsPK and PD validation proved the stability of the model in simulating levornidazole against B fragilis and the precision and accuracy in the results of PK modeling. Cmax and AUC0–24h found only –1.46% and –6.72% differences from the values in vivo. Our study found that ∆Log24h, AUBC, and IKR were more correlated with AUC0–24h/MIC and Cmax/MIC than with %T>MIC. According to ∆Log24h, the PK/PD target values of AUC0–24h/MIC, Cmax/MIC, and %T>MIC of levornidazole against B fragilis were 157.6%, 14.1%, and 56.4%, respectively. ImplicationsOur findings are useful for optimizing the clinical dosing regimen of levornidazole sodium chloride injection to attain maximal bactericidal effect.

Modified Pharmacokinetic/Pharmacodynamic model for electrically activated silver-titanium implant system

Silver-based systems activated by low intensity direct current continue to be investigated as an alternative antimicrobial for infection prophylaxis and treatment. However there has been limited research on the quantitative characterization of the antimicrobial efficacy of such systems. The objective of this study was to develop a semi-mechanistic pharmacokinetic/pharmacodynamic (PK/PD) model providing the quantitative relationship between the critical system parameters and the degree of antimicrobial efficacy. First, time-kill curves were experimentally established for a strain of Staphylococcus aureus in a nutrientrich fluid environment over 48 hours. Based on these curves, a modified PK/PD model was developed with two components: a growing silver-susceptible bacterial population and a depreciating bactericidal process. The test of goodness-of-fit showed that the model was robust and had good predictability (<TEX>$R^2</TEX><TEX>></TEX><TEX>0.7$</TEX>). The model demonstrated that the current intensity was positively correlated to the initial killing rate and the bactericidal fatigue rate of the system while the anode surface area was negatively correlated to the fatigue rate. The model also allowed the determination of the effective range of these two parameters within which the system has significant antimicrobial efficacy. In conclusion, the modified PK/PD model successfully described bacterial growth and killing kinetics when the bacteria were exposed to the electrically activated silver-titanium implant system. This modeling approach as well as the model itself can also potentially contribute to the development of optimal design strategies for other similar antimicrobial systems.

Neutrophils from Cystic Fibrosis Patients Are Defective in Killing of Phagocytosed Pseudomonas aeruginosa.

Cystic fibrosis (CF), the most common genetic disease in Caucasians, is caused by mutations of the gene encoding the CF transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel. CF has long been recognized as an epithelial disease whose most severe complications often occur in the lung. The clinical manifestations include persistent bacterial infection, prominent neutrophil infiltration and small airway obstruction. Even though dramatic advances have been made towards understanding of CF pathogenesis, the link between the CFTR chloride channel defect and a clinical defect in bacterial eradication has not been fully established. Our published data demonstrated that CFTR is expressed in human neutrophils and their phagolysosomes. CF neutrophils are defective in the chlorination of phagocytosed Pseudomonas aeruginosa (PAO1), indicating defective intraphagolysosomal hypochlorous acid (HOCl) production. In the current report, we assessed the bacterial killing abilities of neutrophils from CF and normal individuals. Percoll-purified peripheral blood neutrophils were incubated with opsonized PAO1 at a ratio of 1:1 or 1:50. To define the role of chloride in the killing process, two different Ringer's buffers with either 0 mM chloride or 135 mM chloride were exploited. At various time points (0, 15, 30 and 60 minutes) after incubation of neutrophils with bacteria, samples were aliquoted to assay for viable bacteria. After correction for bacterial growth over the experimental period, the bacterial viability at each time point relative to that of the initial value was obtained. Two-way ANOVA tests indicated that CF neutrophils had a significantly lower initial rate of killing of PAO1 than that in normal controls. This defect is more pronounced under the condition of high bacterial load and low extracellular chloride. Surprisingly, the low extracellular chloride significantly affected neutrophil-mediated bacterial killing even for the normal neutrophils, suggesting the dependence of this ion in the killing process. Our data provide evidence to suggest the potential role of CFTR in supplying intraphagolysosomal chloride to produce hypochlorous acid (HOCl), an oxidant essential for the killing of HOCl-sensitive bacteria.

Disinfection characteristics of waterborne pathogenic protozoaGiardia lamblia

Giardia lamblia is a parasitic protozoa which is transmitted in the form of a cyst through untreated water and also treated drinking water. Since its presence in water has led to frequent outbreaks of giardiasis and death in many countries, the removal and disinfection of this protozoan cyst from the water supply are of great concern for public health. This study examined the disinfection characteristics ofG. lamblia cysts isolated from a Korean patient with giardiasis. When using sodium hypochlorite includdig 5 or 10 ppm chlorine, the killing rate was initially rapid however, the disinfection slowed down and a Blog reduction could not be achieved even after 2 h. The disinfection effectiveness was also reduced at a lower temperature, thereby implying that the risk of a giardiasis outbreak will be higher in the winter season. A CT (concentration time) curve was constructed based on the results with sodium hypochlorite for use in designing and predicting disinfection performance. The organic chlorination disinfectant SDIC (sodium dichloroisocyanurate) produced a lower pH and a much higher residual effect than sodium hypochlorote. The disinfection of cysts by SDIC continued steadily throughout 2 h of contact, although the initial killing rate was lower than that with sodium hypochlorite.

Killing of Staphylococcus aureus by tumor necrosis factor-alpha-activated neutrophils. The role of serum opsonins, integrin receptors, respiratory burst, and degranulation.

We have examined the effects of TNF priming on the killing of Staphylococcus aureus by human neutrophils. In the absence of serum opsonins, neutrophils failed to kill S. aureus, and TNF priming did not induce the cells to become bactericidal. Normal human serum, containing complement activity, promoted the killing of the bacteria by neutrophils. Pretreatment of neutrophils for 30 min with TNF significantly enhanced their bactericidal activity. The effects of TNF on neutrophil bactericidal activity was dependent on serum concentration and the degree of enhancement induced increased up to a concentration of 1%. The kinetics of bacterial killing showed that TNF-only enhanced the initial rate of killing, over the first 30 min. Little killing of bacteria occurred in the presence of complement-inactivated serum, and TNF did not stimulate this killing. These results suggest that TNF enhances the neutrophil complement-dependent killing of S. aureus. TNF increased the expression of CR3 (CD11b/CD18) and CR4 (P150, 95; CD11c/CD18) adhesion receptors but not LFA-1 (CD11a/CD18); and mAb against the alpha-chain of either CR3 or CR4 but not LFA-1 prevented the enhancing effects of TNF on the neutrophil bactericidal activity.

Serum Bactericidal Titres After Cefoperazone With and Without Sulbactam

11 healthy volunteers were enrolled in a crossover ex vivo study comparing cefoperazone (2g) with the combination of cefoperazone (2g) + sulbactam (1g). The antibiotics were given by intravenous infusion over 30 minutes. Blood samples were obtained 30 minutes and 12 hours after the end of infusion for the measurement of serum concentrations, bactericidal titres and killing rates. Two parameters were derived from the killing curves: the initial rate of killing and the relative bioactivity (area under the time-kill curve as a percentage of the growth control curve). 60 test organisms were studied including oxacillin-susceptible and -resistant Staphylococcus aureus, Escherichia coli, Enterobacter cloacae, Serratia marcescens, Bacteroides fragilis and Acinetobacter baumanii. Mean serum concentrations of cefoperazone were 131.6 and < 2 mg/L at 30 minutes and 12 hours, respectively, after the end of infusion. Synergy between cefoperazone and sulbactam was observed in two-thirds of the strains by the checkerboard method, including oxacillin-resistant S. aureus. Using the serum bactericidal titres, synergy occurred mainly against A. baumanii and B. fragilis, but restricted to the senjm obtained at 30 minutes. Using the killing curve method in serum, synergy on both the relative bioactivity and killing rate was observed on the serum obtained at 30 minutes, but not at 12 hours.

Serum Bactericidal Titres after Cefoperazone and Ceftazidime With and Without Amikacin

We enrolled 14 human volunteers in a crossover ex vivo study comparing 6 regimens including amikacin (0.5g), ceftazidime (2g), cefoperazone (2 and 4g) and the combination of each cephalosporin (2g) with amikacin. The antibiotics were given by intravenous infusion over 30 minutes. Blood samples were obtained 1, 4, 6 and 12 hours after the end of infusion to measure serum concentrations, bactericidal titres and killing rates. Two parameters were derived from the killing curves: the initial rate of killing and the relative bioactivity (area under the time-kill curve as a percentage of the control growth curve). The elimination half-life of cefoperazone was decreased by concomitant administration of amikacin, resulting in higher trough levels. Although synergy was seen in the checkerboard method in half of the strains, serum bactericidal titres unusually showed synergy which was more frequent with cefoperazone + amikacin than with cefhzidirne + amikacin. For both cephalosporins, the relative bioactivity, but not the killing rate, was dependent on concentration. Ceftazidime had a higher intrinsic activity despite the higher serum concentrations obtained with cefoperazone. The addition of amikacin conferred concentration dependency of the killing rate to the combination. None of the regimens tested provided enough antimicrobial activity to support once daily administration.

Hypoxia-selective antitumor agents. 3. Relationships between structure and cytotoxicity against cultured tumor cells for substituted N,N-bis(2-chloroethyl)anilines

A series of aniline mustards with a wide range of electron-donating and -withdrawing substituents in the 3- and 4-positions has been synthesized and evaluated for cytotoxicity in cell culture to examine the potential of using nitro group deactivated nitrogen mustards for the design of novel hypoxia-selective anticancer drugs (Denny, W. A.; Wilson, W. R. J. Med. Chem. 1986, 29, 879). Hydrolytic half-lives in tissue culture media, determined by bioassay against a cell line (UV4) defective in the repair of DNA interstrand cross-links showed the expected dependence on the Hammett electronic parameter, sigma, varying from 0.13 h for the 4-amino analogue to greater than 100 h for analogues with strongly electron-withdrawing substituents. Cytotoxic potencies in aerobic UV4 cultures showed a similar dependence on sigma. This dependence predicted that the 4-nitroaniline mustard would be 7200-fold less potent than its potential six-electron reduction product, the 4-amino compound, in growth inhibition assays using a 1-h drug exposure. The measured differential was much lower (225-fold) because of the instability of the latter compound, but a differential of 17,500-fold was observed in the initial rate of killing by using a clonogenic assay. The potential for formation of reactive mustards by reduction to the amine or hydroxylamine was demonstrated by the 4-nitroso compound, which had an aerobic toxicity similar to that of the amine. Although these features confirmed the original rationale, the 3-nitro- and 4-nitroaniline mustards had only minimal hypoxic selectivity against UV cells. Toxicity to hypoxic cells appears to be limited by the low reduction potentials of these compounds and consequent lack of enzymatic nitroreduction. However, this study has demonstrated that nitro groups can be used to latentiate aromatic nitrogen mustards and indicates that examples with higher reduction potentials could provide useful hypoxia-selective therapeutic agents.

In-vitro evaluation of the antifungal activity of amphotericin B entrapped into liposomes during storage for one year

The stability of the antifungal activity of amphotericin B entrapped in small sonicated liposomes (ampholiposomes) was studied in vitro over a one-year period. Preparations of ampholiposomes stored at -20 degrees C or at 4 degrees C were compared monthly with freshly-prepared ampholiposomes and a commercial preparation of amphotericin B-deoxycholate (Fungizone; Squibb) by a killing curve method with Candida albicans. The bioactivity of the four preparations, each containing 1.5 or 2 mg/l of amphotericin B, was measured as the initial rate of killing and the 'relative bioactivity'. Relative bioactivity was calculated as the percentage reduction of the area under the growth curve compared with control growth. Storage of ampholiposomes for one year did not decrease their antifungal activity. Storage of ampholiposomes containing 1.5 mg/l amphotericin B for one year at -20 degrees C, but not at 4 degrees C, gave a significant increase in relative bioactivity and killing rate in comparison with freshly-prepared ampholiposomes. This was probably due to modifications in the spatial configuration of phospholipids and amphotericin B. The persisting antifungal activity of ampholiposomes stored for one year should allow the preparation of large batches to perform comparative clinical studies.

In-vitro activity of SCH39304 in comparison with amphotericin B and flaconazole

The in-vitro activity of SCH39304, a new imidazole, was compared with that of amphotericin B using two media (Sabouraud dextrose and tissue culture (TC) medium) with or without 25% human serum. At an inoculum of 1.5 X 10(4) cfu/ml, the IC95% for amphotericin B ranged from 0.08 to 0.3 mg/l irrespective of the medium. At the same inoculum, the IC95% of the two imidazoles ranged from 0.3 to greater than 80 mg/l. The inoculum effect (from 1.5 X 10(4) to 1.5 X 10(5) cfu/ml) was less than 1 dilution for the three antifungal agents. Serum increased the inhibitory activity (IC80%) of SCH39304 and fluconazole but not of amphotericin B. The IC80%, IC95% and minimal fungicidal concentration (MFC) for amphotericin B usually ranged within two dilutions. Using kill curve methodology, the initial killing rate (0-4 h) of amphotericin B correlated with concentration (P less than 0.001), and was faster in TC medium than in Sabouraud dextrose (P less than 0.0006). SCH39304 and fluconazole did not produce any significant killing over the range of concentrations tested (0.5-20 mg/l). SCH39304 was similar to fluconazole.

Bactericidal kinetics of various dosages of fleroxacin simulated in bacterial cultures.

From in-vitro data, recommendations for dosing with fleroxacin are presented. Serum pharmacokinetics of 250, 400, 500, 800, 1000 and 1500 mg once daily dosages were simulated in bacterial cultures. The bactericidal kinetics of clinical isolates of Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus with MICs for fleroxacin similar to MIC90 or above were investigated. Bacterial populations of all strains with MICs equal to or below 2 mg/l were reduced by at least 99% by a once daily dosage of 400 mg of fleroxacin. 500 mg once per day was high enough to induce a two log reduction of P. aeruginosa MIC 4 mg/l. At a 250 mg dosing mutants with MICs four times above the MICs of the initial strains were selected. The increased concentrations of fleroxacin after multiple dosing enhanced bactericidal activity. Once daily dosing increased the initial rate of killing but reduced the extent of inactivation in comparison with twice daily dosing of the same total amount. From our in-vitro investigation a once daily dosage of 400 mg of fleroxacin should be effective against causative organisms with an MIC of up to 2 mg/l, both in the rate and extent of killing and to minimize the risk for selection of resistant mutants.

The relative efficacies of tobramycin and ciprofloxacin against Pseudomonas aeruginosa in vitro and in normal and granulocytopenic mice.

Ciprofloxacin was studied in vitro and in an experimental thigh infection model in mice to evaluate its efficacy against Pseudomonas aeruginosa in comparison with that of tobramycin. The in vivo experiments were carried out in normal mice as well as mice rendered granulocytopenic by irradiation. The MIC of ciprofloxacin for two Pseudomonas strains was 0.125 mg/l and 0.25 mg/l and that of tobramycin 0.25 mg/l and 4.0 mg/l, respectively. In vitro short-term growth experiments revealed that as far as initial killing rate is concerned, ciprofloxacin was 2.20 times as potent as tobramycin against the first strain and 45.4 times as potent against the second strain. The in vivo experiments were performed by injecting the micro-organism into the thigh muscle and counting colony forming units (CFUs) after several hours of exposure to the antibiotics. The results for irradiated mice indicate that ciprofloxacin was 2.0 times as potent as tobramycin against the first strain and 37.8 times as potent against the second strain, when related to dosage. For normal mice these values were 2.0 and 16.0, respectively, which is more than would be expected from the in vitro experiments because the mean plasma concentrations of tobramycin were about four times higher than those of ciprofloxacin.

The relationship between luminol chemiluminescence and killing of staphylococcus aureus by neutrophil granulocytes.

Luminol chemiluminescence induced by phagocytosis of bacteria was studied in a system consisting of polymorphonuclear granulocytes (PMN), serum, luminol and Staphylococcus aureus. To evaluate the quantitative relationship between luminol chemiluminescence and the bactericidal process time courses for both variables were compared. It was found that initial rate of increase of chemiluminescence and initial rate of killing of bacteria were well correlated whereas the correlation was poorer for later stages of the process. When the rate of the bactericidal process was varied by changing concentrations of bacteria and PMN, directly proportional variations of initial rates of increase of chemiluminescence were observed. This is interpreted as reflecting an accumulation of oxidizing radicals as the result of a phagocytosis dependent gradual activation of the NADPH oxidase system, leading to luminol oxidation and/or killing of bacteria. However, by thermal inactivation of PMNs, chemiluminescence could be diminished whereas killing remained essentially unaffected, showing that these two processes could be uncoupled. Also, addition of erythrocytes to the PMN suspension was associated with decreased chemiluminescence and lysis of erythrocytes with an increased chemiluminescence, emphasizing the importance of proper control of the components of the leucocyte test suspension.

Role of rabbit lysozyme in in vitro serum and plasma serum bactericidal reactions against Bacillus subtilis.

The antibacterial activity of purified rabbit lysozyme was kinetically investigated at concentrations comparable to those in normal rabbit serum and plasma serum. The bactericidal capability, lysozyme content, and electrophoretic composition of "purified beta-lysin," fractionated from normal rabbit serum, were also examined. In contrast to the extensive antibacterial activity of dilute normal rabbit serum observed in vitro, rabbit lysozyme was only weakly bactericidal for Bacillus subtilis. Inhibition of lysozyme enzymatic and bactericidal activities in normal rabbit serum by antilysozyme immunoglobulin G slightly reduced the initial rate of killing. The addition of neutralizing antibody or histamine (another lysozyme inhibitor) to partially purified bactericidal serum fractions had no effect on killing kinetics. Increasing the ionic strength of reaction mixtures containing normal serum or partially purified bactericidal fractions to levels which completely inhibited lysozyme activity resulted in stimulation of their respective killing kinetics. The addition of inhibitors to normal rabbit plasma serum completely eliminated its bactericidal activity. With regard to the killing of B. subtilis by rabbit and human blood fractions, these analyses clearly demonstrated that (i) although lysozyme is not a significant antibacterial component of normal rabbit serum, it represents the principal factor in normal rabbit plasma serum; (ii) different primary bactericidal mechanisms which are not detectable by singlepoint analyses operate in the sera of different species; and (iii) purified beta-lysin isolated from normal rabbit serum by the classical procedure is a heterogenous mixture of components.

The effects of colony differences and olfactory bulb lesions on muricide in rats

A series of four experiments was performed to study the effects of colony differences in rats either with or without olfactory bulb damage. Olfactory bulb damage was found to have the opposite effect upon muricide in rats from two different colonies, with OB damage causing an increase from a low initial rate in one colony and a decrease from a high rate in the other. In addition, one experiment dealt with the possibility that the high initial rate of killing in one colony was due to deprivation during shipping. The results did not provide conclusive evidence for an artifact. The overall results were discussed in terms of generalizing from studies employing rats from different colonies, and suggestions for improving generality were made.