Efficacy of ß-lactam and inhibitor combinations in a diffusion chamber model in rabbits

A. Georgopoulos*, A. Buxbaum and W. Graninger

University Clinic for Internal Medicine I, Clinical Department for Infectious Diseases and Chemotherapy, University of Vienna, Austria


    Abstract
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Using a diffusion chamber in rabbits, we evaluated therapy with the combination of ceftriaxone plus the ß-lactamase inhibitor tazobactam in comparison with ceftriaxone alone. One sensitive and one resistant strain of Escherichia coli, Enterobacter cloacae and Klebsiella pneumoniae were inoculated into one of the six diffusion chambers, implanted in the same animal. In order to simulate pharmacokinetics in humans, both substances were administered in decreasing doses. Ceftriaxone was given 0, 2, 4 and 6 h after infection in dosages of 45, 35, 25 and 15 mg/kg of body weight, while tazobactam was administered either in one dose at 0 h, or divided into two doses at 0 and 1 h or 0 and 4 h, or divided into three doses at 0, 1 and 4 h after infection. The ratio of ceftriaxone:tazobactam was fixed at 8:1. Ceftriaxone, in combination with tazobactam, given in one dose immediately after infection showed a significant reduction in bacterial count. All other combinations of ceftriaxone and tazobactam did not differ from ceftriaxone in monotherapy. Co-administration of the ß-lactamase inhibitor tazobactam significantly enhanced the activity of ceftriaxone against all three tested species.


    Introduction
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
The cephalosporins of the third generation are potent antibiotic substances in the treatment of life-threatening infections. In the last few years, however, an increase in resistance, especially among Enterobacteriaceae, has been reported, resulting from a continuous spread of broad-spectrum ß-lactamases. Therefore a combination of a cephalosporin and a ß-lactamase inhibitor is used in order to reactivate the antibiotic and to prevent the emergence of resistant bacteria. 1,2,3 One potential combination is ceftriaxone, a third-generation cephalosporin, and tazobactam, a penicillanic acid-sulphone derivative which is a potent, irreversible ß-lactamase inhibitor. 4,5,6,7,8,9 Several factors make it difficult to evaluate the efficacy of combinations of antimicrobial agents on the basis of clinical reports, including the severity of the infection, the immune status of the host, interactions with other drugs, and the criteria chosen for assessing success or failure. Therefore, experimental therapeutic effects in animal models can help in the interpretation of a drug's efficacy. 10,11

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.


    Material and methods
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Bacterial strains

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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Figure 1. Diffusion chamber with polyethylene ring closed by semipermeable membranes and two catheters.

 
Experimental infection of rabbits

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


    Results
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
The results of the in-vitro susceptibility tests before and after the experiments are summarized in the Table. There were no changes in the MICs after therapy: no development of resistance could be detected. When tazobactam was added to ceftriaxone, ceftriaxone-resistant strains regained their sensitivity.


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Table. MICs of ceftriaxone and tazobactam alone and in combination, before (b) and after (a) experiments
 
Killing curves for the tested strains are shown in Figures 2,3,4,5. Bacterial densities of E. coli, K. pneumoniae and E. cloacae within diffusion chambers of untreated controls reached c. 10 8 cfu/mL of diffusion chamber fluid 6- 8 h after infection. Figure 2 shows that ceftriaxone alone reduced colony counts of ceftriaxone-sensitive K. pneumoniae below the detection limit (5 x 10 2 cfu/mL of cage fluid) within 8 h. In the case of E. coli this effect could be observed in 12- 24 h (not shown). Tazobactam, when added in one dose immediately after infection, enhanced the potency of ceftriaxone against ceftriaxone-sensitive E. coli and K. pneumoniae: significant reductions in bacterial counts were seen 4- 6 h after infection. All other combinations of ceftriaxone and tazobactam did not differ from ceftriaxone in monotherapy. Moreover, good results with ceftriaxone in combination with tazobactam were obtained for ceftriaxone-resistant isolates of E. coli and K. pneumoniae (Figure 3): when tazobactam was added to ceftriaxone at 0 h, reduced bacterial counts (<10 3 cfu/mL) could be observed 6 h after infection. After this time point, however, strains recovered and reached >10 4 cfu/mL.



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Figure 2. Efficacy of ceftriaxone and tazobactam against K. pneumoniae 337. {blacklozenge}, Control; {blacksquare}, ceftriaxone; {blacktriangleup}, ceftriaxone + tazobactam (0 h); {circ}, ceftriaxone + tazobactam (0, 1 h); {square}, ceftriaxone + tazobactam (0, 4 h); •, ceftriaxone + tazobactam (0, 1, 4 h).

 


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Figure 3. Efficacy of ceftriaxone and tazobactam against K. pneumoniae 335. {blacklozenge}, Control; {triangleup}, ceftriaxone; {blacktriangleup}, ceftriaxone + tazobactam (0 h); {circ}, ceftriaxone + tazobactam (0, 1 h), {square}, ceftriaxone + tazobactam (0, 4 h); •, ceftriaxone + tazobactam (0, 1, 4 h).

 


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Figure 4. Efficacy of ceftriaxone and tazobactam against E. cloacae 2. {blacklozenge}, Control; {blacksquare}, ceftriaxone; {blacktriangleup}, ceftriaxone + tazobactam (0 h); {circ}, ceftriaxone + tazobactam (0, 1 h); {square}, ceftriaxone + tazobactam (0, 4 h); •, ceftriaxone + tazobactam (0, 1, 4 h).

 


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Figure 5. Efficacy of ceftriaxone and tazobactam against E. cloacae 538. {blacklozenge}, Control; {blacksquare}, ceftriaxone; {blacktriangleup}, ceftriaxone + tazobactam (0 h); {circ}, ceftriaxone + tazobactam (0, 1 h); {square}, ceftriaxone + tazobactam (0, 4 h); •, ceftriaxone + tazobactam (0, 1, 4 h).

 
When the Enterobacter strains were tested, ceftriaxone alone was ineffective against both ceftriaxone-sensitive (Figure 4) and ceftriaxone-resistant (Figure 5) isolates. Therapy with ceftriaxone in combination with tazobactam, given in one dose, led to significantly lower residual bacterial counts, compared with controls. The bacterial densities of both ceftriaxone-sensitive and, to a lesser degree, ceftriaxone-resistant strains were reduced.

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.


    Discussion
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
This paper describes in-vivo studies in a modified cage model in rabbits which allows evaluation of the activity of antimicrobial substances when cellular immune defence mechanisms are evaded. Different regimens of ceftriaxone alone and in combination with tazobactam were tested in a model using diffusion chambers in rabbits. In previous work we gradually fixed the experimental parameters we have used here: (i) clinical strains of K. pneumoniae,E. coli and E. cloacae sensitive and resistant to ceftriaxone; (ii) an inoculum that ensured constant and exponential kinetics of infection; (iii) dosage regimens that should allow concentrations in rabbit serum and tissue to be achieved similar to those found in humans (pharmacokinetic data from previous studies, unpublished).

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.


    Notes
 
* Correspondence address. University Clinic for Internal Medicine I, Clinical Department for Infectious Diseases and Chemotherapy, General Hospital of Vienna, Währinger Gürtel 18-20, A-1090 Vienna. Tel: +43-1-40400/5139; Fax: +43-1-40400/5167 Back


    References
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
1 . Allan, D. J. & Moellering, R. C. (1985). Management of infections caused by gram-negative bacilli: the role of antimicrobial combinations. Reviews of Infectious Diseases 7, Suppl. 4, 559–71.

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, 2070–4.[ISI][Medline]

3 . Chambers, H. F. & Fournier, M. A. (1993). Efficacy of cefoperazone in combination with sulbactam in experimental Staphylococcus aureus endocarditis in rabbits. Journal of Antimicrobial Chemotherapy 32, 453–8.[Abstract]

4 . Acar, J. F., Gutmann, L. & Kitzis, M. D. (1988). ß-Lactamases in clinical isolates. Spectrum implications of sulbactam/ampicillin. Drugs 35, Suppl. 7,,12–16.

5 . Appelbaum, P. C., Jacobs, M. R., Spangler, S. K. & Yamabe, S. (1986). Comparative activity of ß-lactamase inhibitors YTR 830, clavulanate, and sulbactam combined with ß-lactams against ß- lactamase producing anaerobes. Antimicrobial Agents and Chemotherapy 30, 789–91.[ISI][Medline]

6 . Aronoff, S. C., Jacobs, M. R., Labrozzi, P. H. & Yamabe, S. (1986). Synergy of amoxicillin combined with clavulanate and YTR 830 in experimental infection in mice. Journal of Antimicrobial Chemotherapy 18, 271–6.[Abstract]

7 . Gutmann, L., Kitzis, M. D., Yamabe, S. & Acar, J. F. (1986). Comparative evaluation of a new ß-lactamase inhibitor, YTR 830, combined with different ß-lactam antibiotics against bacteria harbouring known ß -lactamases. Antimicrobial Agents and Chemotherapy 29, 955–7.[ISI][Medline]

8 . Jacobs, M. R., Aronoff, S. C., Johenning, S. & Yamabe, S. (1986). Comparative activities of the ß-lactamase inhibitors YTR 830, clavulanate, and sulbactam combined with extended-spectrum penicillins against ticarcillin-resistant Enterobacteriaceae and pseudomonads. Journal of Antimicrobial Chemotherapy 18, 177–84.[Abstract]

9 . Mentec, H., Vallois, J. M., Bure, A., Saleh-Mghir, A., Jehl, F. & Carbon, C. (1992). Piperacillin, tazobactam, and gentamicin alone or combined in an endocarditis model of infection by a TEM-3 producing strain of Klebsiella pneumoniae or its susceptible variant. Antimicrobial Agents and Chemotherapy 36,1883 –9.[Abstract]

10 . Fournier, J. L., Ramisse, F., Jacolot, A. C., Szatanik, M., Petitjean, O. J., Alonso, J. M. et al. (1996). Assessment of two penicillins plus ß-lactamase inhibitors versus cefotaxime in treatment of murine Klebsiella pneumoniae infections. Antimicrobial Agents and Chemotherapy 40, 325–30.[Abstract]

11 . Scavizzi, M. R., Elbhar, A., Fenelon, J. P. & Bronner, F. D. (1993). Multi-dimensional analysis for interpreting antibiotic susceptibility data. Antimicrobial Agents and Chemotherapy 37, 929.[ISI][Medline]

12 . National Committee for Clinical Laboratory Standards. (1995). Performance Standards for Antimicrobial Susceptibility Testing— Fifth Informational Supplement: Approved Standard M100- S5. NCCLS, Villanova, PA.

13 . Mimoz, O., Jacolot, A., Padoin, C., Caillon, J., Louchahi, K., Tod, M.et al. (1997). Cefepime and amikacin synergy against a cefotaxime-susceptible strain of Enterobacter cloacae in vitro and in vivo. Journal of Antimicrobial Chemotherapy 39, 363–9.[Abstract]

14 . Fantin, B. & Carbon, C. (1992). In vivo synergism: contribution of animal models. Antimicrobial Agents and Chemotherapy 36, 907–12.[ISI][Medline]

15 . Nix, D. E., Goodwin, S. D., Peloquin, C. A., Rotella, D. L. & Schentag, J. J. (1991). Antibiotic tissue penetration and its relevance: models of tissue penetration and their meaning. Antimicrobial Agents and Chemotherapy 35, 1947–52.[ISI][Medline]

16 . Pechere, J. C. & Vladoianu, I. R. (1992). Development of resistance during ceftazidime and cefepime therapy in a murine peritonitis model. Journal of Antimicrobial Chemotherapy 29, 563–73.[Abstract]

17 . Kobasa, W. D. & Kaye, D. (1983). Aztreonam, cefoperazone, and gentamicin in the treatment of experimental Enterobacter aerogenes endocarditis in rabbits. Antimicrobial Agents and Chemotherapy 24, 321–4.[ISI][Medline]

18 . Levison, M. E. & Kobasa, W. D. (1984). Mezlocillin and ticarcillin alone and combined with gentamicin in the treatment of experimental Enterobacter aerogenes endocarditis. Antimicrobial Agents and Chemotherapy 25, 683–6.[ISI][Medline]

19 . Pefanis, A., Thauvin-Eliopoulos, C., Eliopoulos, G. M. & Moellering, R. C. (1993). Efficacy of ceftriaxone plus tazobactam in a rat model of intraabdominal abscess due to Bacteroides fragilis. Journal of Antimicrobial Chemotherapy 32, 307– 12.[Abstract]

20 . Leleu, G., Kitzis, M. D., Vallois, J. M., Gutmann, L. & Decazes, J. M. (1994). Different ratios of the piperacillin/tazobactam combination for the treatment of experimental meningitis due to Klebsiella pneumoniae producing the TEM-3 extended spectrum ß-lactamase. Antimicrobial Agents and Chemotherapy 38, 195–9.[Abstract]

21 . Cleeland, R. & Squires, E. (1984). Antimicrobial activity of ceftriaxone: a review. American Journal of Medicine 77, Suppl. 4C, 3 – 11.[ISI][Medline]

22 . Bartlett, J. G., Dezfulian, M. & Joiner, K. (1983). Relative efficacy and critical interval of antimicrobial agents in experimental infections involving Bacteroides fragilis. Archives of Surgery 118, 181–4.[Abstract]

23 . Pefanis, A., Thauvin-Eliopoulos, C., Holden, J., Eliopoulos, G. M., Ferraro, M. J. & Moellering, R. C. (1994). Activity of fleroxacin alone and in combination with clindamycin or metronidazole in experimental intra-abdominal abscess. Antimicrobial Agents and Chemotherapy 38, 252–5. ,[Abstract]

24 . Cagni, A., Chuard, C., Vaudaux, P. E., Schrenzel, J. & Lew, D. P. (1995). Comparison of sparfloxacin, temafloxacin, and ciprofloxacin for prophylaxis and treatment of experimental foreign-body infection by methicillin-resistant Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 39, 1655–60.[Abstract]

25 . Climo, M. W., Markowitz, S. M., Williams, D. S., Hale-Cooper, C. G. & Archer, G. L. (1997). Comparison of the in-vitro and in-vivo efficacy of FK037, vancomycin, imipenem and nafcillin against staphylococcal species. Journal of Antimicrobial Chemotherapy 40, 59–66.[Abstract]

26 . Chuard, C., Lucet, E. C., Rohner, P., Herrmann, M., Auckenthaler, R., Waldvogel, F. A. et al. (1991). Resistance of Staphylococcus aureus recovered from infected foreign body in vivo to killing by antimicrobials. Journal of Infectious Diseases 163, 1369–73.[ISI][Medline]

27 . Eliopoulos, G. M., Thauvin-Eliopoulos, C. & Moellering, R. C. (1992). Contribution of animal models in the search for effective therapy for endocarditis due to enterococci with high-level resistance to gentamicin. Clinical Infectious Diseases 15, 58–62.[ISI][Medline]

28 . Schaad, H. J., Chuard, C., Vaudaux, P., Waldvogel, F. A. & Lew, D. P. (1994). Teicoplanin alone or combined with rifampin compared with vancomycin for prophylaxis and treatment of experimental foreign body infection by methicillin-resistant Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 38, 1703–10.[Abstract]

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, 1053–7.[Abstract]

Received 3 August 1998; returned 19 October 1998; revised 3 November 1998; accepted 24 November 1998