Sulbactam efficacy in experimental models caused by susceptible and intermediate Acinetobacter baumannii strains

María-Jesús Rodríguez-Hernándeza,*, Lucila Cuberosb, Cristina Pichardoa, Francisco J. Caballeroa, Ignacio Morenoa, M. Enrique Jiménez-Mejíasa, Andrés García-Curielb and Jerónimo Pachóna

a Service of Infectious Diseases and b Service of Microbiology, University Hospital Virgen del Rocio, Avenida Manuel Siurot s/n, 41013 Sevilla, Spain


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Sulbactam and imipenem were compared in an experimental pneumonia model in immunocompetent mice, using a susceptible strain of Acinetobacter baumannii, and in an experimental endocarditis model in rabbits, using an intermediately susceptible strain. In the former, sulbactam was as efficacious as imipenem in terms of survival, sterility of lungs and in the bacterial clearance from lungs and blood, provided that the t > MIC for sulbactam (1.84 h) was similar to that for imipenem (2.01 h). In the endocarditis model, imipenem (t > MIC, 2.12 h) was more efficacious than sulbactam (t > MIC, 1.17 h) in bacterial clearance from vegetations. These results show the efficacy of sulbactam in infections caused by susceptible strains of A. baumannii, with an MIC up to 4 mg/L, provided that doses reach a t > MIC similar to that of imipenem. The activity of sulbactam was time dependent.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Sulbactam is an antimicrobial that is frequently active in vitro against multiresistant strains of Acinetobacter baumannii and is an alternative to treatment with imipenem.1,2 Sulbactam has shown in vivo activity in models of systemic infection and pneumonia caused by Acinetobacter in neutropenic mice.3,4 The efficacy of sulbactam/ampicillin in different infections caused by susceptible strains of A. baumannii in open clinical studies has been reported.2,5,6 However, neither clinical randomized studies nor discriminative experimental models in immunocompetent animals have investigated the efficacy of sulbactam compared with imipenem in severe A. baumannii infections.

We compared the in vitro and in vivo activity of sulbactam with that of imipenem in experimental A. baumannii pneumonia and endocarditis models, using strains susceptible and intermediately susceptible to both antimicrobials.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In vitro studies

Two A. baumannii strains, both isolated from blood cultures, were used. Strain 731015 (susceptible to sulbactam and imipenem) was used in the pneumonia model and strain 333538 (intermediate to sulbactam and imipenem) in the endocarditis model.

Imipenem was obtained from Merck, Sharp & Dohme (Madrid, Spain) and sulbactam from Pfizer (Karlsruhe, Germany). MICs were measured by a tube dilution method.7 The MIC and MBC, and the susceptibility and resistance criteria were defined according to those of the National Committee for Clinical Laboratory Standards (NCCLS).7

Bactericidal activity against A. baumannii was evaluated by time–kill curves.8 For strain 731015, concentrations of sulbactam equivalent to 1 x, 2 x and 4 x MIC were used, and for the strain 333538, the concentration was equivalent to the Cmax in rabbits. Bacterial growth was counted at 0, 2, 4, 8 and 24 h after incubation at 37°C. An antibiotic was considered bactericidal when a 3 log10 decrease in cfu/mL was reached.8

In vivo studies

Experimental pneumonia model.
Immunocompetent C57BL/6 female mice weighing 14–16 g (BK Universal Ltd, Barcelona, Spain), as in a previously described model,9 were used, with an inoculum of 108 cfu/mL of A. baumannii 731015. Four hours after inoculation, sulbactam was administered for 72 h in two different doses: 140 mg/kg/day, im and td (n = 15), and 240 mg/kg/day, im td (n = 15). Groups of uninfected mice received the same doses for 72 h (non-infected treated groups). Surviving mice were killed 4 h after the last dose, and blood and lung samples processed for qualitative and quantitative culture, respectively, as described previously.9

Experimental endocarditis model.
New Zealand rabbits weighing 2–3 kg (BK Universal Ltd) were employed in a rabbit model of experimental endocarditis, as described by Perlman & Freedman10 and modified by Durack & Beeson.11 Four days after inserting the intracardiac catheter in the left ventricle, the rabbits were injected with 1 mL inoculum, containing 108 cfu A. baumannii 333538, through an ear vein. Blood samples were taken 24 h after the inoculation to confirm onset of endocarditis.

Thirty-two rabbits were inoculated and then divided into three groups. The control group (n = 12) did not receive antimicrobials. The other two groups received imipenem, 180 mg/kg/day, im td (n = 11), or sulbactam, 90 mg/kg/day, im td (n = 9), for 24 h. Groups of uninfected rabbits (n = 6) were given each antibiotic for 24 h (non-infected treated groups).

Surviving rabbits were killed 12 h after the last dose. Blood samples were taken immediately before killing and processed quantitatively. The heart was extracted, and the valve vegetations were dissected aseptically, weighed and homogenized before quantitative cultures were performed. If cultures were sterile they were assigned the value of log10 corresponding to the sensitivity level of the method (1 cfu).

Drug pharmacokinetics

Plasma drug levels were determined after the administration of single intramuscular doses. In the pneumonia model, doses of 35 and 60 mg/kg of sulbactam were used. After 10, 15, 30, 60, 90, 120 and 150 min, blood was taken as described previously.9 In the endocarditis model, doses of 60 mg/kg for imipenem and 30 mg/kg for sulbactam were used. After 10, 15, 30, 60, 120, 240 and 480 min, blood was taken from a marginal ear vein, using five rabbits for each time-point and antibiotic.

Plasma concentrations were measured using a bioassay method. The maximum concentration (Cmax, mg/L), the area under the concentration–time curve (AUC, mg•h/L), the terminal half-life (t1/2, h), the Cmax:MIC ratios and the t > MIC (h) were determined.

Statistical analysis

The two-tailed Fisher's test, the test of homogeneity of variances and the post hoc tests (Dunnet and Tukey test) were used. A P value <0.05 was considered significant. In the pneumonia model, the results of sulbactam were compared with those of controls and imipenem-treated groups as described previously.9


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The MIC and MBC of sulbactam for A. baumannii 731015 were 4 and 32 mg/L, respectively. MICs and MBCs for A. baumannii 333538 were 8 and 8 mg/L for imipenem, and 8 and 16 mg/L for sulbactam, respectively. In time–kill curves, sulbactam showed bactericidal activity after 4 h for a concentration of 4 x MIC against A. baumannii 731015. For A. baumannii 333538, neither imipenem nor sulbactam showed bactericidal activity.

The pharmacokinetic/pharmacodynamic parameters of each drug in mice and rabbits are shown in Table IGo.


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Table I. Antibiotic pharmacokinetic/pharmacodynamic parameters in mice and rabbits
 
Pneumonia model

Therapeutic efficacy.
The survival rate was 0% in the control group. The survival rates were 67% for animals treated with sulbactam 35 mg/kg and 87% for sulbactam 60 mg/kg. Both treatments improved survival compared with the control group (P < 0.01), with no difference between groups nor with the imipenem group. Survival in the non-infected treated groups was 100%.

There were no differences in lung sterility among the treatment groups (Table IIGo). Both sulbactam groups showed higher lung clearance of A. baumannii than the control group (P < 0.05). Lung bacterial clearance was similar for the imipenem and sulbactam 60 mg/kg groups; however, both showed higher lung clearance than sulbactam 35 mg/kg (P < 0.05).


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Table II. Effect of antibiotic therapy on the clearance of A. baumannii from lungs and blood (pneumonia model)
 
In the blood cultures, both sulbactam groups were significantly different from the control group (P < 0.05). There were no differences between both sulbactam groups and the imipenem group (Table IIGo).

Endocarditis model

Characterization of experimental endocarditis by A. baumannii.
A. baumannii was isolated in all blood cultures obtained 24 h after inoculation (mean ± S.D., 3.79 ± 0.6 cfu/mL). One of the 12 rabbits died (91.7% survival). Blood cultures taken from the 11 surviving rabbits before they were killed were positive (2.74 ± 0.97 cfu/mL) and A. baumannii was isolated from all valves (6.32 ± 1.81 cfu/g of vegetation).

Therapeutic efficacy.
The survival rate was 100% in both treatment groups and non-infected treated groups. There were no differences with the control group.

Both treatments increased the sterility of valves with respect to the controls. Imipenem showed a significant difference when compared with the control group in the bacterial clearance from vegetations (P < 0.05). There was no difference in the sterility of blood cultures between the treatment and the control groups. However, both imipenem and sulbactam increased the bacterial clearance from blood with respect to the control group (P < 0.05) (Table IIIGo).


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Table III. Effect of antibiotic therapy on the clearance of A. baumannii from valves and blood (endocarditis model)
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the in vitro study, sulbactam showed bactericidal activity in time–kill curves against the susceptible strain at 4 x MIC. Imipenem was bactericidal against this strain at 1 x MIC.9 In other studies, sulbactam showed bacteriostatic or bactericidal activity against susceptible strains of A. baumannii, regardless of the concentration used in relation to the MIC.35,12 Neither imipenem nor sulbactam showed bactericidal in vitro activity against the intermediate A. baumannii strain, which was similar to the results of Wolff et al.4

In the pneumonia model, sulbactam (140 mg/kg/day) improved the survival and the sterilization of lungs and blood compared with the controls. However, the results were significantly worse than those obtained with imipenem9 using a similar dose (120 mg/kg/day). When the dose of sulbactam was increased (240 mg/kg/day), reaching a t > MIC similar to that of imipenem9 (1.84 versus 2.01 h), sulbactam was as efficacious as imipenem, suggesting time-dependent activity for sulbactam. The t > MIC is the most predictive pharmacokinetic/pharmacodynamic parameter of the in vivo efficacy of ß-lactams in animal models.13 Wolff et al.,4 in a model of pneumonia in neutropenic mice using a strain susceptible to sulbactam (MIC 0.5 mg/L), showed that this antimicrobial was as efficacious as imipenem, with t > MIC for sulbactam >3 h.

To study the in vivo activity of sulbactam and imipenem against A. baumannii intermediately susceptible to both antimicrobials, an experimental endocarditis model was used. Both antimicrobials diminished the bacterial concentration in blood after 24 h of treatment. However, imipenem was better than sulbactam in the clearance of bacteria from vegetations, reflecting the higher t > MIC of imipenem (2.12 h) compared with sulbactam (1.17 h). These results support the pneumonia model conclusions, in the sense that sulbactam required a t > MIC similar to that for imipenem for the treatment of infections caused by A. baumannii.

In summary, the present study shows that sulbactam is as effective as imipenem in experimental pneumonia caused by A. baumanni, in immunocompetent mice, provided that dosages reached a similar t > MIC. These results support the efficacy of sulbactam, as reported in open clinical studies2,5,6 and in a pneumonia model in neutropenic mice.4


    Acknowledgments
 
Part of the information in this paper was presented at the Eighth Congress of the Spanish Society of Infectious Diseases and Clnical Microbiology (SEIMC), Palma de Mallorca, Spain, May 1998 (Abstract A-16) and in the Ninth European Congress of Clinical Microbiology and Infectious Diseases, Berlin, Germany, March 1999 (Abstracts P954 and P955). This study was supported by a research grant (FIS 95/1629) from the Fondo de Investigación Sanitaria.


    Notes
 
* Corresponding author. Tel: +34-955-01-23-76; Fax: +34-955-01-23-77; E-mail: jpachon{at}hvr.sas.cica.es Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Kuah, B. G., Kumarasinghe, G., Doran, J. & Chang, H. R. (1994). Antimicrobial susceptibilities of clinical isolates of Acinetobacter baumannii from Singapore. Antimicrobial Agents and Chemotherapy 38, 2502–3.[Abstract]

2 . Urban, C., Go, E., Mariano, N., Berger, B. J., Avraham, I., Rubin, D. et al. (1993). Effect of sulbactam on infections caused by imipenem-resistant Acinetobacter calcoaceticus biotype anitratus. Journal of Infectious Diseases 167, 448–51.[ISI][Medline]

3 . Obana, Y. & Nishino, T. (1990). In-vitro and in-vivo activities of sulbactam and YTR830H against Acinetobacter calcoaceticus. Journal of Antimicrobial Chemotherapy 26, 677–82.[Abstract]

4 . Wolff, M., Joly-Guillou, M. L., Farinotti, R. & Carbon, C. (1999). In vivo efficacies of combinations of ß-lactams, ß-lactamase inhibitors, and rifampin against Acinetobacter baumannii in a mouse pneumonia model. Antimicrobial Agents and Chemotherapy 43, 1406–11.[Abstract/Free Full Text]

5 . Corbella, X., Ariza, J., Ardanuy, C., Vuelta, M., Tubau, F., Sora, M. et al. (1998). Efficacy of sulbactam alone and in combination with ampicillin in nosocomial infections caused by multiresistant Acinetobacter baumannii. Journal of Antimicrobial Chemotherapy 42, 793–802.[Abstract]

6 . Jiménez-Mejías, M. E., Pachón, J., Becerril, B., Palomino-Nicás, J., Rodríguez-Cobacho, A. & Revuelta, M. (1997). Treatment of multidrug-resistant Acinetobacter baumannii meningitis with ampicillin/sulbactam. Clinical Infectious Diseases 24, 932–5.[ISI][Medline]

7 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fifth Edition: Approved Standard M7-A5. NCCLS, Wayne, PA.

8 . National Committee for Clinical Laboratory Standards. (1992). Methods for Determining Bactericidal Activity of Antimicrobial Agents—Tentative Guideline 771 E. NCCLS, Villanova, PA.

9 . Rodríguez-Hernández, M. J., Pachón, J., Pichardo, C., Cuberos, L., Ibáñez-Martínez, J., García-Curiel, A. et al. (2000). Imipenem, doxycycline and amikacin in monotherapy and in combination in Acinetobacter baumannii experimental pneumonia. Journal of Antimicrobial Chemotherapy 45, 493–501.[Abstract/Free Full Text]

10 . Perlman, B. B. & Freedman, L. R. (1971). Experimental endocarditis. II. Staphylococcal infection of the aortic valve following placement of a polyethene catheter in the left side of the heart. Yale Journal of Biology and Medicine 44, 206–13.[ISI][Medline]

11 . Durack, D. T. & Beeson, P. B. (1971). Experimental bacterial endocarditis. I. Colonization of a sterile vegetation. British Journal of Experimental Pathology 53, 44–9.[ISI]

12 . Joly-Guillou, M. L., Decré, D., Herrman, J. L., Bourdelier, E. & Bergogne-Bérézin, E. (1995). Bactericidal in-vitro activity of ß- lactams and ß-lactamase inhibitors, alone or associated, against clinical strains of A. baumannii: effect of combination with aminoglycosides. Journal of Antimicrobial Chemotherapy 36, 619–29.[Abstract]

13 . Craig, W. A. (1998). Pharmacokinetic/pharmacodynamic parameters: rationales for antibacterial dosing of mice and men. Clinical Infectious Diseases 26, 1–12.[ISI][Medline]

Received 4 August 2000; returned 8 October 2000; revised 27 November 2000; accepted 10 December 2000