1 Laboratoire de Bactériologie-Virologie, Centre HospitaloUniversitaire de Nîmes, Groupe HospitaloUniversitaire de Carémeau, Place du Professeur Robert Debré, 30029 Nîmes Cedex 09; 2 Laboratoire de Bactériologie, Faculté de Médecine, 28 Place Henri Dunant, 63001 Clermont-Ferrand Cedex; 3 Laboratoire Universitaire dAntibiologie, Faculté de Médecine, Avenue Kennedy, 30900 Nîmes; 4 Laboratoire dAntibiologie, Faculté de Médecine, 1 rue Gaston Veil, 44035 Nantes, France
Received 7 October 2003; returned 3 December 2003; revised 13 January 2004; accepted 13 January 2004
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods: PAE and PLIE were studied for ESBL-producing strains of Escherichia coli and Klebsiella pneumoniae. Two ATCC ß-lactamase-negative strains of E. coli and K. pneumoniae were used as controls. The MICs of a ceftazidimesulbactam combination were determined with a fixed concentration of sulbactam (8 mg/L). The organisms were exposed to the antibiotics at twice the MIC for 2 h before removal of the antibiotics by filtration of the culture. Bacteria on the filter were resuspended in drug-free medium to determine the PAE and in medium containing ceftazidime, at the same concentration as originally present, to determine the PLIE.
Results: The PAE of ceftazidime was similar for bacteria producing the same ESBL except for E. coli producing CTX-M-1. PLIE values varied according to the type of ß-lactamase but similar results were observed for the strains producing the same ESBLs. PLIEs were longer than PAEs and were longer when the MICs of ceftazidime were lower.
Conclusions: To the best of our knowledge, we describe here for the first time an in vitro PLIE for a ceftazidimesulbactam combination on different bacteria producing different ESBLs. These findings indicate that suicide inhibitors may be used in combination with third-generation cephalosporins.
Keywords: ESBLs, PAE, PLIE, ceftazidimesulbactam, cephalosporins, suicide inhibitors
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ceftazidime pentahydrate was kindly supplied as laboratory reference material by GlaxoSmithKline (Durham, UK). Sulbactam sodium was kindly supplied as laboratory reference material by Pfizer Inc. (Groton, CT, USA).
Organisms
Nine strains of bacteria were used in this work. Two strains (one strain of Escherichia coli, one strain of Klebsiella pneumoniae) were ATCC strains that did not produce ß-lactamases. Seven were ESBL-producing strains (four strains of E. coli of which one was wild and the other three transconjugants, and three wild strains of K. pneumoniae). The susceptibility of these organisms was determined using a standard method.7 The MICs of the ceftazidimesulbactam combination were determined for a fixed concentration of sulbactam (8 mg/L, the standard concentration recommended for an antibiogram).8,9
Inocula
For all the strains, 40 µL bacterial culture incubated at 37°C for 18 h was diluted in MuellerHinton broth (Oxoid, Basingstoke, UK) to obtain a starting inoculum between 105 and 107 cfu/mL.
PAE and PLIE determinations
As previously described by Thorburn et al.,4 the organisms were exposed to the antibiotics at twice the MIC in MuellerHinton broth for 2 h before removal of the antibiotics by filtering the culture through a 0.25 µm pore size nylon filter (Corning Costar, New York, USA). The untreated control culture was also filtered. Two washes with 10 mL of a pre-warmed, drug-free test medium followed filtration. The bacteria on the filter were resuspended in 20 mL of a pre-warmed drug-free medium to determine the PAE and 20 mL of pre-warmed test medium containing ceftazidime, at the same concentration as originally present, to determine the PLIE. All the cultures were incubated at 37°C in an environmental orbital shaker.
Viable bacterial counts
The number of live bacteria was determined by taking samples every hour during the 2 h of pre-exposure to antibiotics, immediately after filtering, up to 9 h after exposure, and finally at 24 h. Samples were diluted 10-fold serially in a sterile, drug-free MuellerHinton broth, and 100 µL drops of each were plated on nutrient agar. The number of cfu was determined after 24 h of incubation.
The PAE was calculated as TC, where T was the time for the number of viable organisms in the test culture to increase by 1 log10 above the number observed immediately after filtration, and C was the time for the number of viable organisms in the untreated control culture to increase by 1 log10 above the number observed immediately after filtration.
The PLIE was calculated as TC, where T was the time for the number of viable organisms in the test culture to increase by 1 log10 above the number observed immediately after filtration, and C was the time for the number of viable organisms in the untreated control culture containing ceftazidime after filtration to increase by 1 log10 above the number observed immediately after filtration.
The PAE and PLIE were assayed three times. Means and standard deviations (S.D.) for all the experimental data were calculated.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The MICs of ceftazidime and the ceftazidimesulbactam combination for the test organisms are shown in Table 1.
|
PAE and PLIE values for each strain are shown in Table 1. Example curves are shown in Figure 1.
|
PLIE values varied according to the type of ß-lactamase. Similar results were observed for the strains which produced the same ESBLs (Table 1). The PLIEs were longer than the PAEs and were longer when the MICs of ceftazidime were lower.
For the E. coli strain TrMEN-1 which produces CTX-M-1, the PAE (0.3 h) turned out to be much shorter and the PLIE (5 h) longer than for the other strains of E. coli. These results seemed logical owing to the lower ceftazidime MICs for this strain.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The PAEs of ceftazidime, with or without sulbactam, on strains that did not produce ESBLs, were non-existent or negative; this is in agreement with data already published.10 The PAEs on strains producing ESBLs proved to be homogeneous depending on which strain was being studied: 0.80.9 h for K. pneumoniae, 0.50.7 h for the E. coli except for the strain producing CTX-M-1. For this strain, the MICs of ceftazidime were the lowest (32 mg/L). The greater susceptibility of this strain would explain the lower PAE. In all cases, the PAEs were less than 1 h long, and this short time span is consistent with data in the literature.4
The concentration of sulbactam used in this study was 8 mg/L, corresponding to the standard dose used for carrying out the antibiogram. For each strain, the concentration of ceftazidime corresponded to twice the MICs of the combination (as recommended by Thorburn et al.4). Most of the concentrations of ceftazidime used were consistent with the serum concentrations measured in vivo during discontinued intravenous perfusions of 1 or 2 g every 8 h or as a continuous perfusion of 100 mg/kg per 24 h.1113
The PLIEs varied according to the type of ß-lactamase. The ceftazidime plus sulbactam combination tested on K. pneumoniae CF1104 and E. coli CF1124 producing TEM-24 gave PLIEs of 0.7 and 0.8 h, respectively. For K. pneumoniae KP2102 and E. coli CF1004 producing SHV-4, the PLIEs were 2.2 and 2.5 h, respectively. Similar results were obtained with TEM-3 (Table 1). The PLIE therefore seems more dependent on the product of the concentrations of the ceftazidime plus sulbactam combination than the concentration of sulbactam alone in the culture medium. We noted that the shortest PLIE was obtained with bacteria for which the MIC of ceftazidime was the highest: K. pneumoniae CF1104 and E. coli CF1004. This would seem to be in relation to the bacteria producing the most ß-lactamases. These data are in agreement with findings by Thorburn et al. for K. pneumoniae and E. coli.4
The cephalosporin and suicide inhibitor combination has already been assessed in vitro and in vivo on bacteria producing ESBLs.1422 The interest in using sulbactam as a suicide inhibitor lies in the fact that it is available independently from ß-lactams. As early as 1985, Jones et al. had successfully tested in vitro a combination of a third-generation cephalosporin, cefoperazone, and sulbactam on ß-lactamase-producing Enterobacteriaceae.14 This combination, later used in man for intra-abdominal infections, fever in neutropenic cancer patients, lower respiratory infections and complicated urinary tract infections, has proved its efficacy.1522 Fantin et al. demonstrated the efficacy of a ceftriaxone plus sulbactam combination against E. coli producing SHV-2 in a rabbit endocarditis model.23 Other authors showed results for this combination in the same experimental model, with the infection from Pseudomonas aeruginosa.24 By combining sulbactam with ceftazidime or cefotaxime, the activity of these third generation cephalosporins could be improved in vitro against various strains (E. coli, E. cloacae, C. freundii, Acinetobacter spp.) producing ESBLs.25 However, the combination of ceftriaxone plus sulbactam used in the rabbit model for infectious endocarditis with K. pneumoniae producing TEM-3 did not satisfactorily sterilize the valvular vegetations. The authors cited a high level of ß-lactamase in the vegetations as being the cause of this failure.26 This would therefore confirm the need to maintain sufficient residual concentrations of the two components for as long as possible within the seat of the infection. Assessing the PLIE for such a combination could help to optimize this relationship between pharmacokinetics and pharmacodynamics.
We described, in this study, an in vitro PLIE associated with long PAE and PLIE of a ceftazidimesulbactam combination on different bacteria producing different ESBLs. This interesting finding merits further work particularly using animal experimental models. Such work could lead to new associations of suicide inhibitors with third- generation cephalosporins as well as new ways of administering them, in order to circumvent the ever increasing problem of bacterial resistance linked to ß-lactamases.
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Belaaouaj, A., Lapoumeroulie, C., Canica, M. M. et al. (1994). Nucleotide sequences of the genes coding for the TEM-like ß-lactamases IRT-1 and IRT-2 (formerly called TRI-1 and TRI-2). FEMS Microbiology Letters 120, 7580.[CrossRef][ISI][Medline]
3
.
Bradford, P. A. (2001). Extended-spectrum ß-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clinical Microbiology Reviews 14, 93351.
4 . Thorburn, C. E., Molesworth, S. J., Sutherland, R. et al. (1996). Postantibiotic and post-ß-lactamase inhibitor effects of amoxicillin plus clavulanate. Antimicrobial Agents and Chemotherapy 40, 2796801.[Abstract]
5 . Aguilar, L., Martin, M., Balcabao, I. P. et al. (1997). In vitro assessment of the effect of clavulanic acid at concentrations achieved in human serum on the bactericidal activity of amoxicillin at physiological concentrations against Staphylococcus aureus: implications for dosage regimens. Antimicrobial Agents and Chemotherapy 41, 14035.[Abstract]
6 . Murbach, V., Dhoyen, N., Linger, L. et al. (2001). Evidence for a true post-ß-lactamase-inhibitor effect of clavulanic acid against Klebsiella pneumoniae and Haemophilus influenzae. Clinical Microbiology and Infection 7, 6615.[CrossRef][ISI][Medline]
7 . National Committee for Clinical Laboratory Standards. (2003). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow AerobicallyThird Edition: Approved Standard M7-A6. NCCLS, Wayne, PA, USA.
8 . Soussy, C. J. (2003). Recommandations du Comité de lAntibiogramme de la Société Française de Microbiologie. [Online.] http://www.sfm.asso.fr (16 December 2003, date last accessed).
9 . Noguchi, J. K. & Gill, M. A. (1988). Sulbactam: a ß-lactamase inhibitor. Clinical Pharmacology 7, 3751.
10 . Craig, W. A. & Gudmundsson, S. (1996). The post-antibiotic effect. In Antibiotics in Laboratory Medicine, 4th edn (Lorian, V., Ed.), pp. 296329. Williams and Wilkins, Baltimore, MD, USA.
11 . Nicolau, D. P., Lacy, M. K., McNabb, J. C. et al. (1999). Pharmacokinetics of continuous and intermittent ceftazidime in intensive care unit patients with nosocomial pneumonia. Infectious Diseases in Clinical Practice 8, 459.[ISI]
12 . Sommers, D. K., Walters, L., Van Wyk, M. et al. (1983). Pharmacokinetics of ceftazidime in male and female volunteers. Antimicrobial Agents and Chemotherapy 23, 8926.[ISI][Medline]
13 . Vinks, A. A. T. M. M., Touw, D. J., Heijerman, G. M. et al. (1994). Pharmacokinetics of ceftazidime in adult cystic fibrosis patients during continuous infusion and ambulatory treatment at home. Therapeutic Drug Monitoring 16, 3418.[ISI][Medline]
14 . Jones, R. N., Barry, A. L., Thornsberry, C. et al. (1985). The cefoperazonesulbactam combination. In vitro qualities including ß-lactamase stability, antimicrobial activity, and interpretative criteria for disk diffusion tests. American Journal of Clinical Pathology 84, 496504.[ISI][Medline]
15 . Bodey, G. P., Abi-Said, D., Rolston, K. et al. (1996). Imipenem or cefoperazonesulbactam combined with vancomycin for therapy of presumed or proven infection in neutropenic cancer patients. European Journal of Clinical Microbiology and Infectious Diseases 15, 62534.[ISI][Medline]
16 . Bodey, G. P., Elting, L. S., Narro, J. et al. (1993). An open trial of cefoperazone plus sulbactam for the treatment of fever in cancer patients. Journal of Antimicrobial Chemotherapy 32, 14152.[Abstract]
17 . Greenberg, R. N., Cayavec, P., Danko, L. S. et al. (1994). Comparison of cefoperazone plus sulbactam with clindamycin plus gentamicin as treatment for intra-abdominal infections. Journal of Antimicrobial Chemotherapy 34, 391401.[Abstract]
18 . Jauregui, L. E., Appelbaum, P. C., Fabian, T. C. et al. (1990). A randomized clinical study of cefoperazone and sulbactam versus gentamicin and clindamycin in the treatment of intra-abdominal infections. Journal of Antimicrobial Chemotherapy 25, 42333.[Abstract]
19 . Koga, H., Tomono, K., Hirakata, Y. et al. (1996). Clinical evaluation of sulbactam/cefoperazone for lower respiratory tract infections. Correlation between the efficacy of sulbactam/cefoperazone and ß-lactamase. Japanese Journal of Antibiotics 49, 8007.[Medline]
20 . Li, J. T., Lu, Y., Hou, J. et al. (1997). Sulbactam/cefoperazone versus cefotaxime for the treatment of moderate-to-severe bacterial infections: results of a randomized, controlled clinical trial. Clinical Infectious Diseases 24, 498505.[ISI][Medline]
21 . Nomura, S., Hanaki, H. & Nagayama, A. (1997). Tazobactampiperacillin compared with sulbactamampicillin, clavulanic acidticarcillin, sulbactamcefoperazone, and piperacillin for activity against ß-lactamase-producing bacteria isolated from patients with complicated urinary tract infections. Journal of Chemotherapy 9, 8994.[ISI][Medline]
22 . Winston, D. J., Bartoni, K., Bruckner, D. A. et al. (1998). Randomized comparison of sulbactam/cefoperazone with imipenem as empirical monotherapy for febrile granulocytopenic patients. Clinical Infectious Diseases 26, 57683.[ISI][Medline]
23 . Fantin, B., Pangon, B., Potel, G. et al. (1990). Activity of sulbactam in combination with ceftriaxone in vitro and in experimental endocarditis caused by Escherichia coli producing SHV-2-like ß-lactamase. Antimicrobial Agents and Chemotherapy 34, 5816.[ISI][Medline]
24 . Xiong, Y. Q., Caillon, J., Zhou, X. Y. et al. (1995). Treatment of experimental rabbit infective endocarditis due to a multidrug-resistant Pseudomonas aeruginosa with high-dose ceftazidime alone and combined with amikacin or sulbactam or both. Journal of Antimicrobial Chemotherapy 35, 697706.[ISI][Medline]
25 . Zhang, Y. L. & Li, J. T. (2001). The in vitro activity of sulbactam combined with third generation cephalosporins against third generation cephalosporin-resistant bacteria. International Journal of Antimicrobial Agents 17, 1436.[CrossRef][ISI][Medline]
26 . Caron, F., Gutmann, L., Bure, A. et al. (1990). Ceftriaxonesulbactam 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]
|