University Clinic for Internal Medicine I, Clinical Department for Infectious Diseases and Chemotherapy, University of Vienna, Austria
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Abstract |
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Introduction |
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In the present study we used a diffusion chamber model in rabbits to compare the in-vivo efficacies of ceftriaxone alone and ceftriaxone plus tazobactam in various dosage schemes against cephalosporin-sensitive and cephalosporin-resistant isolates of Enterobacteriaceae.
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Material and methods |
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Three ceftriaxone-resistant isolates of Enterobacteriaceae (Escherichia coli G87, Klebsiella pneumoniae 335 and Enterobacter cloacae 538) and three ceftriaxone-sensitive isolates (E. coli G98, K. pneumoniae 337 and E. cloacae 2) were used in this study; all were clinical isolates. For preparation of the infective inoculum, the organisms were grown in brain- heart infusion broth for 24 h, and the suspensions divided into multiple aliquots, which were stored at -196°C in liquid nitrogen. On the day of infection, the bacterial inoculum was thawed and colony counts were performed.
Antimicrobial agents
Ceftriaxone and tazobactam were kindly supplied by Hoffmann-La-Roche (Vienna, Austria). Stock solutions of the antimicrobial drugs were prepared and stored at -196°C in liquid nitrogen.
In-vitro susceptibility testing
Ceftriaxone was tested alone and in combination with tazobactam. The ratio ceftriaxone:tazobactam was fixed at 8:1. The susceptibility of the test strains to the antibiotics used in this study was determined in Mueller- Hinton broth by a micro-dilution technique in accordance with NCCLS guidelines. 12 Testing was performed by using inoculum concentrations of 10 5 cfu/mL.
Implantation of diffusion chamber
Six diffusion chambers (consisting of a plastic ring, closed on both sides by a membrane with a pore width of 0.22 µm and two fixed catheters) were implanted intra-peritoneally in female chinchilla rabbits under aseptic conditions (Figure 1). Studies were started 5- 7 days after implantation and completed 3- 6 weeks afterwards. The rabbits were kept in wire cages at 22°C room temperature and fed with pellets and water.
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After aseptically aspirating the diffusion chamber fluid, the diffusion chamber was inoculated with a suspension (5 x 10 5 cfu/mL) of the test strain via the cathether.
Establishment of therapy
The rabbits were divided into six groups, each consisting of six animals (with six diffusion chambers). In the first group of experiments (group 1), ceftriaxone alone was administered iv at 0 h (= time of infection) and 2, 4 and 6 h afterwards at the indicated doses: 45, 35, 25 and 15 mg/kg of body weight. The second set of experiments (groups 2- 5) were performed with ceftriaxone and tazobactam in combination. Although the doses of ceftriaxone were the same as in group 1, the application scheme for tazobactam was varied: group 2: 15 mg/kg of body weight at 0 h; group 3: 10 mg/kg of body weight at 0 h and 5 mg/kg of body weight 1 h after infection; group 4: 10 mg/kg of body weight at 0 h and 2.5 mg/kg of body weight 1 and 4 h after infection; group 5: 10 mg/kg of body weight at 0 h and 5 mg/kg of body weight 4 h after infection. The ratio of ceftriaxone:tazobactam was fixed in all experiments at 8:1. Group 6 was not treated and was used as control.
Assessment of therapy
In each experiment at 1, 2, 4, 6, 8, 12 and 24 h after infection the diffusion chamber fluid was aspirated and colony counts were performed. Colony count reduction in the diffusion chamber fluid of the treated animals was evaluated in comparison with the control group, using the chi-squared test.
Resistance to antimicrobial agents
The tested strains of E. coli, K. pneumoniae and E. cloacae recovered from diffusion chamber fluid at the end of treatment were screened for changes in susceptibility to ceftriaxone and ceftriaxone plus tazobactam using the micro-dilution technique, described previously. 12
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Results |
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For all three tested species the addition of tazobactam significantly enhanced the activity of ceftriaxone; best results were achieved when tazobactam was added in one dose immediately after infection.
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Discussion |
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The results obtained here show that ceftriaxone-sensitive strains of K. pneumoniae and E. coli were eradicated by ceftriaxone alone in 8- 24 h. When added in one dose, tazobactam shortened the time needed for bacterial clearance to 4- 6 h. For ceftriaxone-resistant isolates good results were also obtained with the combination of ceftriaxone and tazobactam.
The bacterial killing curves of both ceftriaxone-sensitive and ceftriaxone-resistant E. cloacae isolates demonstrate no effect from ceftriaxone in monotherapy. Failure of monotherapy with cephalosporins has been reported in other animal models, especially when testing E. cloacae. The emergence of resistant clones during therapy was not observed in the present study. 13 A satisfactory explanation of in-vivo bacterial persistence despite single antibiotic treatment remains elusive. 14,15,16
Antibiotic combinations consisting of a ß-lactam antibiotic and a ß-lactamase inhibitor or a ß-lactam and an aminoglycoside have frequently produced an increased bactericidal effect in in-vivo experimental models of aerobic Gram-negative bacillary infections, which has generally paralleled an increased rate of killing in vitro. 17,18 Similar observations for E. cloacae have been noted in this study: when combined with tazobactam (given in one dose immediately after infection), ceftriaxone significantly lowered residual bacterial counts compared with controls. Concerning the dosage of the two drugs we, like other authors, 19 found a ceftriaxone:tazobactam ratio of 8:1 to be very effective. The experimental settings of various studies show that the dose ratio of ß-lactam and ß-lactamase inhibitor may be a vital influence in the effectiveness of these combinations. 9,10,20
Tazobactam, the ß-lactamase inhibitor used in these experiments, effectively inhibits ß-lactamases from a broad range of Gram-positive and Gram-negative bacteria and consequently substantially enhances the in-vitro potency of ß-lactam antibiotics, like ceftriaxone, which as single agents may have only limited activity. 19,21 This in-vitro enhanced activity may not occur in vivo when simultaneous dosing is employed of these two agents, whose pharmacokinetic profiles differ considerably. Our study demonstrates clearly a beneficial effect of tazobactam administration with ceftriaxone only when tazobactam was given in one dose immediately after infection. All other combinations did not differ from ceftriaxone in monotherapy. The disadvantages of delaying antimicrobial therapy have been noted before in several experimental models. 22,23 This effect may be attributed to the relative resistance of organisms in the stationary growth phase. Another explanation, well documented for staphylococci, is that the suppression of resistant subpopulations of bacterial strains is required for antibiotic effectiveness in in-vivo models of infections. 24,25
It can be concluded that the rational selection of therapeutic regimens of antimicrobial substances against a variety of bacterial agents based on in-vitro data alone is still problematic. 26,27,28,29 These data further emphasize the value of performing experiments in animals for the evaluation of dosage regimens of antibiotics and the parameters influencing their in-vivo activities. Appropriate dose regimens of various ß-lactam antibiotic and tazobactam combinations may deserve further comparative studies in experimental infections. However, despite differences in pharmacokinetic profiles, the combination of ceftriaxone and tazobactam appears to be a promising regimen for the treatment of infections due to members of the family Enterobacteriaceae.
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Notes |
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References |
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2 . Caron, F., Gutmann, L., Bure, A., Pangon, B., Vallois, J. M., Pechinot, A. et al. (1990). Ceftriaxone- sulbactam combination in rabbit endocarditis caused by a strain of Klebsiella pneumoniae producing extended-broad-spectrum-TEM-3-ß-lactamase. Antimicrobial Agents and Chemotherapy 34, 20704.[ISI][Medline]
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29 . Thauvin-Eliopoulos, C., Tripodi, M. F., Moellering, R. C. & Eliopoulos, G. M. (1997). Efficacies of piperacillin- tazobactam and cefepime in rats with experimental intra-abdominal abscesses due to an extended-spectrum ß-lactamase-producing strain of Klebsiella pneumoniae. Antimicrobial Agents and Chemotherapy 41, 10537.[Abstract]
Received 3 August 1998; returned 19 October 1998; revised 3 November 1998; accepted 24 November 1998