Isolation of a Staphylococcus aureus beta-lactamase-dicloxacillin complex and kinetic studies on the reactivation of the enzyme.

Exposure of the beta-lactamase from Staphylococcus aureus to the slowly reacting substrates cloxacillin or dicloxacillin results in time-dependent inactivation of the enzyme. Methods for the rapid separation of a beta-lactamase-dicloxacillin complex from excess inhibitor, using centrifuged columns of Sephadex G-25 or DEAE-Sephadex G-25, are described. The enzyme-dicloxacillin complex releases active enzyme, with specific activity identical to that of untreated enzyme, after storage at pH 7.5 at 15 degrees C. Full reactivation was accompanied by the release of 0.8 eq of hydrolyzed dicloxacillin. The complex is stable for up to 40 h when stored at pH 3 at 4 degrees C. The reactivation process, which occurs with first-order kinetics at 15 degrees C and pH values between 4 and 8, displays a pH dependence with apparent pKa's of 4.6 and 8.5, and a limiting value of the reactivation rate constant of 0.022 min-1. Deviation from first-order kinetics at pH 9 is consistent with a competing irreversible inactivation of the enzyme at that pH. This behavior differs substantially from that of the similarly inactivated beta-lactamase I from Bacillus cereus, whose rate of reactivation is independent of pH, but which undergoes irreversible denaturation at acidic pH [A. L. Fink, K. M. Behner, and A. K. Tan (1987) Biochemistry 26, 4248-4258]. Addition of hydroxylamine to the S. aureus beta-lactamase-dicloxacillin, complex stimulates the rate of reactivation by a maximum of 35%. This effect is hyperbolically dependent on the concentration of hydroxylamine with half-maximal stimulation at 2.8 mM. The Km for ampicillin hydrolysis catalyzed by the partially reactivated enzyme is identical to that measured for catalysis by the untreated enzyme. We discuss our observations in relation to models for the transient inhibition process.

Effect of antibiotic protein binding on the killing rate of Staphylococcus aureus and on the paradoxical phenomenon.

The killing kinetics of Staphylococcus aureus, exposed to various concentrations of dicloxacillin in broth and in broth with 40 g/l human albumin was studied. When the free concentrations of dicloxacillin were identical in the two media, no difference in killing capacity could be demonstrated at concentrations above MIC, indicating that only the free antibiotic fraction is antibacterially active. However, at concentrations identical to the MIC, a better bactericidal effect in the medium containing albumin was found. In experiments where equal total concentrations were compared in the two media, an increasing bactericidal effect in the medium containing albumin could be demonstrated at concentrations between 10-100 X MIC. The most probable explanation for this was a prominent paradoxical effect with increasing antibiotic concentrations on the killing rate of S. aureus in broth. This effect was neutralized in the presence of albumin due to the lower free antibiotic concentration in this medium.

On the formation of a dicloxacillin-p-hydroxymercuribenzoate suicide complex mediated by beta-lactamase I from Bacillus cereus.

p-Hydroxymercuribenzoate is a non-competitive inhibitor of beta-lactamase I from Bacillus cereus and also, after preliminary preincubation, an inactivator of the enzyme. Submitted to the simultaneous action of PCMB plus dicloxacillin, the enzyme completely loses its activity. Extensive dialysis can restore the enzymatic activity only if preincubation had been carried out with either PCMB or dicloxacillin but not if both inhibitors had been simultaneously present. Mercaptoethanol protects the enzyme from the action of PCMB, but not from the severe inactivation caused by dicloxacillin-PCMB mixtures. All these data suggest the formation of a complex between PCMB and the acyl-enzyme intermediate generated upon hydrolysis of the beta-lactam bond of dicloxacillin.

Comparative study of the effects of some inducers with or without protein binding properties on bioavailability of isoxazolylpenicillins in rats.

The effect of various inducers with or without protein binding properties on serum levels and half life of Oxacillin, Cloxacillin and Dicloxacillin was studied. A total of 102 male rats classified in 3 "categories" according to the administered penicillin with 6 groups of rats in each of them were used. Each group was pretreated for 15 days with the following inducers: phenobarbital, diphenylhydantoin, diazepam, chlorpromazine and phenylbutazone. The control groups received saline. The d-glucaric acid concentration in the urine prior to and after the administration of inducers and the liver weight were taken as enzyme induction indices. The results showed a decrease of serum levels and half life of three penicillins with a negative correlation between urine d-glucaric acid and serum penicillin levels. Phenobarbital, diphenylhydantoin and chlorpromazine affected the 3 penicillins in the following statistically significant order: oxacillin, dicloxacillin, cloxacillin. Diazepam affected: cloxacillin, dicloxacillin, oxacillin, and phenylbutazone: dicloxacillin, cloxacillin and oxacillin. However all drugs finally produced a uniform effect on all 3 penicillins in the following decreasing order: phenobarbital (r = -0.910), diphenylhydantoin (r = -0.864), phenylbutazone (r = -0.851), chlorpromazine (r = -0.842) and diazepam (r = -0.821). For all inducers, the effect was most significant for oxacillin (r = -0.869), second most significant for dicloxacillin (r = -0.811) and finally for cloxacillin (r = -0.778). The results suggested an interaction of isoxazolylpenicillins and the above drugs.

Pharmacokinetics of dicloxacillin in serum and aortic wall during aneurysmal surgery.

Twenty-one patients undergoing elective surgery for abdominal aortic aneurysm were randomly assigned to receive 2 grams of dicloxacillin as an intravenous infusion either at the induction of anaesthesia, 3 hours or approximately 6 hours before surgery. Samples from serum and aortic tissue were simultaneously obtained so that antibiotic levels could be compared as a function of time. High concentrations of dicloxacillin in serum and aortic tissue were present already shortly after infusion and active levels were maintained in aortic tissue for approximately six hours. The kinetics of dicloxacillin was similar in serum and aortic tissue. These results support the need for antibiotic administration shortly before surgery to ensure adequate tissue levels of antibiotics.

Single-dose pharmacokinetics of dicloxacillin in healthy subjects of young and old age.

Dicloxacillin was administered as 2 g single intravenous doses to healthy young and old subjects of both sexes aged 19-32 years and 65-76 years, respectively, and the pharmacokinetics were studied. Peak serum concentrations were higher in young than in elderly subjects and in each age group in females than in males. The elimination rate was similar in all groups and the only striking pharmacokinetic differences observed were that the urinary recovery of active dicloxacillin was higher in young subjects and that the non-renal clearance was higher in elderly volunteers. The findings were interpreted to be due to a systemic metabolism of dicloxacillin, compensating for a reduced renal elimination in the elderly subjects. In all subjects dicloxacillin was well tolerated. No increases of serum creatinine were observed.

A multicenter comparison of related pharmacologic features of cephalexin and dicloxacillin given for two months to young children with cystic fibrosis.

Twenty-one cystic fibrosis patients under 3 years of age were enrolled in an open multicenter study to assess the feasibility of the study design and to compare selected pharmacologic features of cephalexin or dicloxacillin administered orally for 2 months. Patient tolerance and compliance were significantly less for dicloxacillin (p less than .01 and p less than .001, respectively). Superficial Candida infections were more common in the cephalexin group (p = 0.02), however increased stool frequency and nonspecific diaper rashes were more prevalent in patients receiving dicloxacillin (p less than .05). Staphylococcus aureus was isolated from respiratory secretions after 2 months from two dicloxacillin and no cephalexin patients. Areas under the curve and peak serum concentrations were higher for cephalexin (p less than .05 and p = .02), but antistaphylococcal activity in serum was higher for dicloxacillin (p less than .05) due to a lower mean MIC compared to cephalexin. Deep pharyngeal plus routine throat culture yielded more pathogens than either method alone. Express mail and central processing of respiratory specimens was efficient for most organisms, however there was some loss of Streptococcus pneumoniae and Haemophilus influenzae. Cephalexin was associated with better patient acceptance and compliance despite higher rates of superficial fungal infections as compared to dicloxacillin. Cephalexin, routine bacteriologic throat swabs processed locally or centrally, mail-in urine compliance assessment and a multicenter design are feasible components for a long-term prospective evaluation of antibiotic prophylaxis in patients with cystic fibrosis.

Laboratory evaluation of the serum dilution test in serious staphylococcal infection.

Four test media were studied to determine performance characteristics of serum dilution tests used to monitor antimicrobial therapy during serious Staphylococcus aureus infection being treated with highly protein-bound antibiotics. Serum inhibitory titers and serum bactericidal titers obtained with Mueller-Hinton broth supplemented with calcium and magnesium were 3- to 16-fold higher than titers obtained with whole human serum buffered with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). In cation-supplemented Mueller-Hinton containing 5% albumin or in cation-supplemented Mueller-Hinton combined with an equal volume of human serum, titers were 2- to 5-fold higher than in whole human serum buffered with HEPES. Clinical or animal studies are needed to establish whether the higher titers observed with patient serum containing highly protein-bound drugs diluted in low protein-content media would foster inadequate dosage regiments. In the meantime, both infectious disease clinicians and microbiologists should be aware of this potential pitfall.

Effects of mezlocillin on the pharmacokinetics of oxacillin and dicloxacillin.

The pharmacokinetics of oxacillin and dicloxacillin, with and without the coadministration of mezlocillin, were studied in 16 patients with normal or severely impaired renal function. After a single administration to patients in terminal renal failure a decrease in total oxacillin clearance from 279 to 159 ml/min/1.73 m2 and total dicloxacillin clearance from 249 to 43 ml/min/1.73 m2 was observed. After coadministration of mezlocillin total oxacillin clearance fell by 38% in all patients irrespective of their renal function. This was caused by a reduction in renal and non-renal elimination rather than by a change in the volume of distribution. With the exception of a minor reduction in renal excretion, the pharmacokinetics of dicloxacillin showed no significant changes. In terminal renal failure the oxacillin dose needs to be reduced by half only when combined with mezlocillin. For dicloxacillin, however, the same dosage adjustment is required irrespective of whether administration is single or combined.

Comparison of oral penicillinase-resistant penicillins: contrasts between agents and assays.

We observed the comparative serum levels and mean peak serum antistaphylococcal activity in eight fasting adults who received 500 mg each of dicloxacillin, cloxacillin, oxacillin, and nafcillin. Dicloxacillin achieved higher and more prolonged serum levels and greater peak serum antistaphylococcal titers than the other drugs studied. The higher degree of protein binding of dicloxacillin was reflected in a greater disparity between the peak antistaphylococcal activity observed when dilutions were done in serum compared to broth. The lesser protein-bound penicillins showed less disparity, but this effect was offset by the higher serum levels obtained by dicloxacillin. The higher protein binding of dicloxacillin did not prevent its having equal or superior antistaphylococcal activity in serum when the drugs were given in equal doses.

Effect of free fatty acids on protein binding of antimicrobial agents.

The effect of free fatty acids (FFAs) on the binding of antibiotics to human serum proteins was studied by addition of palmitic acid to albumin solutions and normal pooled serum. FFA reduced the binding of dicloxacillin, cefamandole, and sulfamethoxazole at molar ratios of FFA to albumin of greater than 2.0. In contrast, FFA enhanced the binding of benzylpenicillin, cephalothin, and cefoxitin at physiologic molar ratios of FFA to albumin. Elevated levels of FFAs induced in vivo by heparin produced similar results with dicloxacillin and benzylpenicillin. Addition of FFA to serum, but not to albumin, reduced the binding of clindamycin and trimethoprim. That the binding of these drugs was much lower in albumin than in serum suggested the presence in serum of another antibiotic-binding protein and that FFAs can modulate binding to this protein. Binding of chloramphenicol was unaffected by FFAs. The possible clinical significance of the changes in protein binding due to FFAs is discussed.

Tissue distribution of beta-lactam antibiotics. Experimental studies in rabbits.

Tissue distribution of ampicillin, dicloxacillin and cefuroxime was studied in rabbits. Different methods allowing frequent samples to be obtained from the same animal were used. Tissue fluid was obtained from subcutaneously implanted steel net cages and by implantation of thin cotton threads under the muscle fascia. A microtechnique was developed for determination of antibiotic concentrations in small muscle samples. The concentration time course in serum and tissue was followed after a single intravenous or intramuscular injection of the antibiotic. In tissue cage fluid, levels of ampicillin and dicloxacillin were low as compared to the serum concentrations. Also, maximum levels occurred later than in serum and elimination was slower. In contrast, rapid peak levels were achieved in muscle tissue with all three investigated antibiotics and the muscle concentrations declined parallel to the serum levels. The results also indicated that beta-lactam antibiotics are not homogeneously distributed in muscle tissue but mainly confined to the extracellular fluid volume. Whole tissue levels could therefore underestimate the actual antibiotic concentrations in the tissue fluid where the bacteria causing soft tissue infections are most likely to be found. Serum levels, however, seemed to be a good indicator for the concentration time course in tissue fluid.

Penetration of ampicillin and dicloxacillin into tissue cage fluid in rabbits: relation to serum and tissue protein binding.

The pharmacokinetics of ampicillin and dicloxacillin following intravenous injection was studied in a tissue cage model in rabbits. Serum protein binding was 98.6% for dicloxacillin and 46.7% for ampicillin, and there were no major differences between the degree of binding in tissue case fluid. The highly bound dicloxacillin gave lower but more sustained levels in tissue cage fluid than ampicillin, and showed a very long elimination half-life. The findings suggest that the flow of albumin into the interstitial space could be of importance for the tissue penetration of highly protein bound antibiotics. The results also indicate dose-dependent pharmacokinetics for dicloxacillin.

[Clinical study of sodium dicloxacillin for intravenous administration in heart surgery patients]. / Klinicheskoe izuchenie diklosatsillina natrievoi soli dlia vnutrivennogo vvedeniia u kardiokhirurgicheskikh bol'nykh.

Sodium dicloxacillin for intravenous administration manufactured in the USSR was studied clinically in 21 cardiosurgical patients. The patients were operated with the use of artificial circulation. 16 patients received the drug for counteracting purulent inflammatory complications during the postoperative period. 5 septic patients were treated with dicloxacillin with curative purposes. The antibiotic proved to be highly effective in treatment and prevention of postoperative purulent complications in patients subjected to open-heart operations. 80 per cent of the Gram-stained isolates were sensitive to dicloxacillin. No pronounced complications with respect to the liver were observed in the patients treated with dicloxacillin.

Influence of sulfaethidole on the human pharmacokinetics of dicloxacillin.

In a five-patient crossover study, serum levels of dicloxacillin after intravenous administration of dicloxacillin in the absence and presence of sulfaethiodole were measured. Significantly greater serum concentrations of dicloxacillin were noted when dicloxacillin was administered with sulfaethidole. Pharmacokinetic evaluation of the data suggests that the higher serum concentrations were primarily the result of changes in the extravascular distribution for dicloxacillin in the presence of sulfaethidole. Although examination of the distribution rate constants for dicloxacillin in a two-compartment open model would suggest a lowering of serum concentrations, the experimental data clearly indicate that the serum and tissue compartment dicloxacillin concentrations increased in the presence of sulfaethiodle, indicating that protein binding in the central as well as extravascular compartments could be affected by sulfaethidole.

[Clinical evaluation of use of combinations of beta-lactamines in anti-infective therapy]. / Contributo clinico all'impiego, in terapia antinfettiva, delle associazioni di beta-lattamine.

Clinical studies were undertaken in our Department of Medical Clinic about the therapeutic effects of two associated beta-lactamines in various respiratory infections. 20 patients affected by acute and chronic bronchitis were treated with the following standard protocol: cefadroxil (500 mg) plus dicloxacillin (250 mg) were given simultaneously four times daily for 8 days. For the clinical evaluation of the therapeutic index of drugs, the following parameters were checked in all patients on admission and repeated at the end of treatment: subjective symptoms (dyspnea, cough), fever, WBC count, sedimentation rate, antistreptolysin titre, chest film and the functional indices of hepatic, renal, hemopoietic tissues. To study the pharmacokinetics we measured the amounts of drugs in the body fluids; plasma levels and urinary concentration were determined 6 hours after the first administration of drugs. Our observations show a synergism of action between the cefadroxil and dicloxacillin; the efficacy of this treatment is confirmed in the majority of the patients: 95% of patients affected by acute respiratory infections show a significant decrease in symptoms and 55% a complete recovery.