Endemic and epidemic acinetobacter species in a university hospital: an 8-year survey.

The prevalence of the currently known Acinetobacter species and related trends of antimicrobial resistance in a Dutch university hospital were studied. Between 1999 and 2006, Acinetobacter isolates from clinical samples were collected prospectively. Isolates were analyzed by amplified fragment length polymorphism fingerprinting. For species identification, a profile similarity cutoff level of 50% was used, and for strain identification, a cutoff level of 90% was used. Susceptibility for antimicrobial agents was tested by disk diffusion by following the CLSI guideline. The incidences of Acinetobacter isolates ranged from 1.7 to 3.7 per 10,000 patients per year, without a trend of increase, during the study years. Twenty different species were distinguished. Acinetobacter baumannii (27%) and Acinetobacter genomic species (gen. sp.) 3 (26%) were the most prevalent. Other species seen relatively frequently were Acinetobacter lwoffii (11%), Acinetobacter ursingii (4%), Acinetobacter johnsonii (4%), and Acinetobacter junii (3%). One large cluster of A. baumannii, involving 31 patients, and 16 smaller clusters of various species, involving in total 39 patients, with at most 5 patients in 1 cluster, occurred. Overall, 37% of the A. baumannii isolates were fully susceptible to the tested antibiotics. There was a borderline significant (P = 0.059) trend of decreasing susceptibility. A. baumannii was the Acinetobacter species causing the largest burden of multiple-antibiotic resistance and transmissions in the hospital.

Inhibition of hBD-3, but not hBD-1 and hBD-2, mRNA expression by corticosteroids.

Intensive use of corticosteroids may be accompanied by increased susceptibility to infections; hence, we investigated the effects of dexamethasone on the expression of antimicrobial peptides, termed human beta-defensins (hBD), by cultured bronchial epithelial cells and mononuclear phagocytes. The results revealed that dexamethasone inhibited the (stimulated) expression of mRNA for hBD-3, but not hBD-1 and hBD-2 by these epithelial cells. Dexamethasone did not affect the (stimulated) mRNA expression of hBD-1 and hBD-2 by mononuclear phagocytes, whereas these cells did not express hBD-3 mRNA.

Intracellular activity of trovafloxacin against Staphylococcus aureus.

The effect of trovafloxacin on Staphylococcus aureus ingested by human granulocytes or monocytes was compared with that on S. aureus in cell-free medium. Maximum growth inhibition (E(R,max)) by the antibiotic was 0.530 log10/h for S. aureus within granulocytes, 0.912 log10/h for S. aureus within monocytes, and 1.830-1.916 log10/h for S. aureus in medium. EC50, the concentration at which 50% of the maximum growth inhibition is achieved, did not differ significantly under the conditions investigated. After inhibition of intracellular killing by granulocytes with sodium fluoride, the intracellular antibacterial activity of trovafloxacin was still less than that in medium. A 3.4 times higher concentration was needed to achieve the same effect on phagocytosed S. aureus as in cell-free medium. Trovafloxacin binds more strongly to granulocytes than to monocytes, the respective cellular concentrations being 10 and four times higher than that in medium. In conclusion, the activity of trovafloxacin against S. aureus ingested by human granulocytes or monocytes is less than that against S. aureus in cell-free medium and is not related to the cell-associated concentration. Intracellular conditions are not favourable for the antibacterial activity of trovafloxacin.

Pharmacokinetic and pharmacodynamic models of the antistaphylococcal effects of meropenem and cloxacillin in vitro and in experimental infection.

The efficacies of meropenem (MPM) and cloxacillin (CLC) against two Staphylococcus aureus strains were established in vitro. A pharmacodynamic model equation, based on the concept that the killing rate depends on concentration and time, was fitted to the numbers of CFU. The parameters of the equation are maximum killing rate, time point of maximum killing, and 50% effective concentration (EC50). The EC50s for the two strains were 0.047 and 0.040 mg/liter, respectively, for MPM and 0.105 and 0.121 mg/liter, respectively, for CLC. Calculated values of the parameters were used to predict the numbers of CFU at exponentially decreasing concentrations in vitro as well as in an experimental infection model. The prediction for in vitro conditions gave a satisfactory fit (R2, between 0.862 and 0.894). In vivo the numbers were predicted with the assumption that killing rate in vivo is proportional to that in vitro (R2, between 0.731 and 0.973). The proportionality factor ranged between 0.23 and 0.42; this variation was due mainly to covariation with growth rates in control animals, without other significant differences between antibiotics or strains.

The renal clearance of cefuroxime and ceftazidime and the effect of probenecid on their tubular excretion.

1. The renal tubular excretion of cefuroxime and ceftazidime in relation to the coadministration of probenecid was investigated in eight and two healthy subjects, respectively. 2. Cefuroxime or ceftazidime were administered by i.v. infusion and 1 g probenecid was administered orally after steady state plasma concentrations of the cephalosporin were reached. 3. In a second session the same antibiotic was administered at increasing infusion rates such that three different levels of plasma drug concentration were achieved. 4. The renal clearance of antibiotic was calculated based upon unbound plasma concentration, and tubular clearance was estimated by subtracting inulin clearance from the renal clearance of the antibiotic. 5. Non-linear regression analysis was used to estimate parameters describing the saturability of tubular excretion and the effect of probenecid inhibition, i.e. EC50 and Rtub,max, could be established for cefuroxime: EC50 was 248 (s.d. 130) mg l-1 and Rtub,max was 1.852 (s.d. 0.577) mg h-1. Tubular excretion of ceftazidime was practically zero. The EC50 of probenecid for inhibition of the tubular excretion of cefuroxime was 0.80 (s.d. 0.31) mg l-1. 6. The results indicate that in the therapeutic plasma concentration range of cefuroxime its renal clearance is not saturated. Probenecid at therapeutic doses will block tubular excretion of cefuroxime almost completely.

The passage of cloxacillin into cerebrospinal fluid in the absence of meningitis.

1. Eleven patients undergoing lumbar discectomy received cloxacillin by continuous i.v. infusion, starting before the operation. During the operation several blood samples and one CSF sample were taken. 2. Mean rate constants describing the passive transfer of drug from plasma to CSF (kp) and the largely active transfer in the opposite direction (kCSF) were estimated. 3. In some subjects the CSF albumin quotient, defined as the ratio between the albumin concentration in CSF and in plasma times 1000, was slightly elevated (up to 23) which caused a significant increase in the value of kp. 4. The estimate of mean kp for healthy individuals was 0.065 h-1, which corresponds to a half-life of 10 h. The estimate of mean kCSF was 2.10 h-1. This predicts a steady-state CSF drug concentration which is 3% of the unbound plasma drug concentration. 5. There was a significant lag between the time courses of plasma and CSF drug concentrations, presumably reflecting the time for drug to move from the choroid plexus to the lumbar sampling site. 6. Four other patients received cloxacillin for prophylactic or therapeutic reasons by continuous i.v. infusion. In three of those patients the albumin quotient was normal or slightly elevated and the steady-state CCSF/Cu ratio was similar to the predicted normal value. 7. These findings indicate that eradicating staphylococci from CSF in cases of meningitis with a low degree of inflammation may be difficult.

Comparison of the antibacterial efficacies of ampicillin and ciprofloxacin against experimental infections with Listeria monocytogenes in hydrocortisone-treated mice.

The efficacies of ciprofloxacin and ampicillin against Listeria monocytogenes in an immunosuppressed mouse model of listeriosis were compared. Immunosuppression was achieved by administration of 2.5 mg of hydrocortisone acetate daily. Both ciprofloxacin and ampicillin were effective in reducing the number of viable L. monocytogenes cells in the liver and spleen. After treatment with 100 mg of ampicillin per kg of body weight every 6 h for 3 days, virtually no L. monocytogenes could be recovered from the livers and spleens of the mice. In contrast, after treatment with 100 mg of ciprofloxacin per kg every 6 h for 3 days, a geometric mean of 5 x 10(4) CFU of L. monocytogenes was recovered from the spleens and 1 x 10(5) CFU was recovered from the livers of the mice. Results of the study show that the antibacterial efficacy of ampicillin is far superior to that of ciprofloxacin in our animal model of listeriosis.

Comparison of the antibacterial effects of cefepime and ceftazidime against Escherichia coli in vitro and in vivo.

The efficacies of cefepime and ceftazidime in an experimental Escherichia coli infection in granulocytopenic mice were related to their in vitro activities and their pharmacokinetic profiles. Cefepime had a higher intrinsic activity in vitro than ceftazidime, and it had a different pharmacokinetic profile, resulting in higher peak concentrations in plasma and a longer elimination half-life. To predict the antibacterial efficacy in vivo on the basis of in vitro activity and pharmacokinetics, we applied a mathematical model in which the in vitro effect is expressed as the difference in growth rate between control cultures and cultures grown in the presence of the antibiotic (ER), whereas the in vivo effect is given by the difference in the number of CFU between controls and antibiotic-treated animals (EN). The integral of ER over time, called ERt, was calculated by using in vivo concentrations. A significant linear relationship was found between EN and ERt for different doses at various times up to 4 h after administration, although the slope of this relationship was slightly but significantly less for cefepime (0.44) than for ceftazidime (0.59).

Diffusion of cloxacillin into synovial tissue.

After a 30 min i.v. infusion of 1 g cloxacillin, the concentrations of this antibiotic were measured in plasma and synovial tissue samples from 11 patients undergoing total hip replacement. Assuming passive distribution between plasma and tissue the rate constants of distribution were estimated. The mean half-life of distribution was 22 min. The concentration of free drug in synovial tissue was estimated to be 77% of the total tissue concentration. The maximum tissue drug concentration after an i.v. bolus dose is predicted to occur at about 37 min.

Modulation of the intestinal flora of mice by treatment with aztreonam and tigemonam.

Oral and parenteral administration of aztreonam and oral administration of tigemonam to conventional mice caused a decrease in the number of aerobic gram-negative rods in the feces. Oral treatment with high doses of aztreonam (greater than or equal to 25 mg/kg/day) and tigemonam (100 mg/kg/day) adversely influenced colonization resistance, whereas oral treatment with lower doses of the monobactams or parenteral treatment with aztreonam did not.

Comparison of the effects of aztreonam and tigemonam against Escherichia coli and Klebsiella pneumoniae in vitro and in vivo.

A study was performed to investigate the pharmacodynamics of aztreonam and tigemonam against Escherichia coli and Klebsiella pneumoniae in vitro and in vivo. The in vitro concentration-effect relationships were determined in short-term growth experiments. The in vivo dose-effect relationships were determined in an experimental thigh muscle infection in irradiated mice. In this model, E. coli was injected into one thigh muscle and K. pneumoniae was injected into the other. Throughout these experiments aztreonam was administered subcutaneously and tigemonam was administered orally. For analysis of the antibacterial pharmacodynamics, the following parameters were determined: the maximum effect as a parameter for efficacy, the 50% effective concentration (or dose) as a parameter for potency, and the slope of the concentration-effect relationship. To assess the relationship between the concentration of the antibiotic and the antibacterial effect in vivo, the pharmacokinetics of the two drugs in the plasma of mice were determined as well. The maximum in vitro and in vivo effects of aztreonam and tigemonam against both bacteria did not differ substantially. However, both drugs killed E. coli more effectively than K. pneumoniae, indicating that the maximum in vitro effect of these drugs against E. coli was higher than that against K. pneumoniae. The maximum in vivo effect of both drugs against E. coli was similar to that against K. pneumoniae. Furthermore, in vitro aztreonam was about twice as potent as tigemonam, but in vivo the reverse was the case. These findings were explained by pharmacokinetic differences between subcutaneously administered aztreonam and orally administered tigemonam, because concentrations of tigemonam in plasma remained at microbiologically active concentrations longer than those of aztreonam did.

Comparative study of the effects of four cephalosporins against Escherichia coli in vitro and in vivo.

A thigh muscle infection induced with Escherichia coli in irradiated mice was used as a model to compare the in vivo pharmacodynamics of the antibacterial effect of four cephalosporins (i.e., cefepime, ceftriaxone, ceftazidime, and cefoperazone) with the in vitro antibacterial pharmacodynamics of these drugs. The following in vitro pharmacodynamic parameters were determined: the maximum effect as a measure for efficacy, the 50% effective concentration as a parameter for potency, and the slope of the concentration-effect relationship. For analysis of the in vivo antibacterial pharmacodynamics, the same parameters were applied for the dose instead of the concentration. For the detection of a relationship between concentration and antibacterial effect in vivo, we determined the pharmacokinetics of the four cephalosporins in the plasma of mice. The results showed that, in general, there is a direct relationship between the in vivo and in vitro pharmacodynamics of these cephalosporins. The maximum effects of cefepime, ceftazidime, and cefoperazone were approximately similar in vivo and in vitro. The sequence of potency of these drugs was, in descending order, cefepime, ceftazidime, and cefoperazone. Ceftriaxone differed from the other three cephalosporins in that it displayed unexpected in vivo pharmacodynamics. Ceftriaxone was just as efficacious as the other three in vitro, but its maximum effect in vivo was much lower. This relatively low maximum effect of ceftriaxone in vivo was not explained by the pharmacokinetic characteristics of the drug. From the present results it can be concluded that the in vitro efficacy of cephalosporins does not necessarily have a predictive value for the in vivo efficacy.

Antibacterial activity of four cephalosporins in an experimental infection in relation to in vitro effect and pharmacokinetics.

The in vitro activity of four cephalosporins was compared with their effects in an experimental thigh infection (cefuroxime and cefamandole against Escherichia coli and cefamandole, ceftriaxone, and ceftazidime against Klebsiella pneumoniae) in granulocytopenic mice. The effect in vitro (ER) was defined as the difference between the growth rate without antibiotic and the growth rate at the steepest part of a 3-h growth curve in the presence of an antibiotic. The relation between concentration and ER was described with the Hill equation. Using pharmacokinetic parameters of the plasma concentrations in vivo and those of the Hill equation the corresponding time course of ER was calculated and by integration with respect to time (0tERdt), an estimate was obtained of the effect on bacteria. For all four antibiotics this estimate was significantly correlated with the actual values of the effect in vivo (EN), defined as the difference in numbers of bacteria between controls and antibiotic-treated animals at 4 h.

Interactions of methotrexate and cyclophosphamide with the pharmacokinetics of 5-fluorouracil in an animal model.

The interaction of methotrexate and/or cyclophosphamide with the pharmacokinetics of 5-fluorouracil (5-FU) was studied in tumor-bearing WAG/Rij rats. Four groups were formed including treatment with single-agent 5-FU (eight rats); 5-FU plus methotrexate (11 rats); 5-FU plus cyclophosphamide (12 rats); and 5-FU, cyclophosphamide, and methotrexate (13 rats). The area-under-the-plasma-concentration/time curve, total-body clearance, elimination half-life, mean residence time, and steady-state volume of distribution were computed and compared. The mean residence time and elimination half-life of 5-FU increased when methotrexate was included in the combination. The increase was significant (P less than 0.05) for 5-FU, cyclophosphamide, and methotrexate versus 5-FU and cyclophosphamide.

Pharmacokinetic interactions of cyclophosphamide and 5-fluorouracil with methotrexate in an animal model.

In tumor-bearing WAG/Rij rats the interaction of cyclophosphamide and/or 5-fluorouracil (5-FU) with methotrexate as manifested at the pharmacokinetic level was studied. Four groups were formed of at least ten animals. The control group, which received single-agent methotrexate, was compared with groups that received methotrexate plus cyclophosphamide, methotrexate plus 5-FU, and methotrexate plus cyclophosphamide plus 5-FU. There appeared to be an increase of 40% in the clearance of methotrexate by the triple combination. Cyclophosphamide especially diminished the terminal part of the concentration-time curve of methotrexate.

A gas chromatographic assay for the determination of 5,6-dihydrofluorouracil and 5-fluorouracil in human plasma.

A gas chromatographic assay for the determination of 5-fluorouracil (5-FU) and 5,6-dihydrofluorouracil (FDHU) is described. The selectivity and sensitivity of the method allows the determination of both 5-FU and FDHU in 200 microliters of plasma. Diphenylsuccinimide and chlorouracil were used as external and internal standard, respectively. The assay including the extraction shows a good linearity in the range 0-5000 ng/ml plasma for 5-FU as well as for FDHU. 5-FU and FDHU plasma concentrations of a number of patients with breast cancer treated with 5-FU were determined in order to demonstrate the usefulness of the method.