Laboratoire d'Antibiologie Bactériennes, Institut de Bactériologie, Faculté de Médecine Hôpitaux Universitaires de Strasbourg, 3 rue Koeberlé, F-67000 Strasbourg, France
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Abstract |
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Introduction |
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Since then, the percentage of strains exhibiting resistance mediated by production of ß-lactamases has steadily increased,1519 and resulted in the development of more stable antibiotics with regard to those enzymes.
Co-amoxiclav, consisting of a ß-lactam antibiotic, amoxicillin, combined with a ß-lactamase inhibitor, clavulanic acid, was first used in clinical practice in the UK in the 1980s.18 In that type of presentation, the active substances act synergically. Cooper et al.20 and Yogev et al.21 have shown, in vitro and in vivo, that the bactericidal activity of co-amoxiclav depends on the concentrations of the two active substances.
One of the problems with co-amoxiclav is that it is required to be used three times per day, which lowers patient compliance. Thus, a formulation allowing twice daily usage would be welcome. The aim of the present study was to investigate the ex vivo bactericidal activity of human serum sampled in patients having received co-amoxiclav 1.125 g po (amoxicillin 1 g + clavulanic acid 0.125 g) in a bd oral regimen for a pharmacokinetic study. This bactericidal activity was evaluated up to 12 h after drug intake against a strain of H. influenzae producing a TEM-type penicillinase.
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Materials and methods |
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The strain of H. influenzae was isolated at the Institute of Bacteriology (University Hospital) of Strasbourg from the sputum of a patient hospitalized in the intensive care unit. The strain produces a TEM-type penicillinase.
In vitro susceptibility
The MICs of co-amoxiclav and amoxicillin alone were determined by broth macrodilution in MuellerHinton medium supplemented with factors V and X, as per the reference method.22 The bacterial inoculum was prepared from a 5 h culture in the exponential growth phase and diluted to obtain concentrations from 106 to 107 cfu/mL. The minimum bactericidal concentration (MBC) was determined by counting the viable bacteria in the tubes with no visible growth. MBC was defined as the lowest concentration leaving only 0.01% survivors after 24 h of exposure at 37°C under an atmosphere enriched with 5% CO2.
Serum specimens
Blood samples were obtained from 12 patients included in a pharmacokinetic study performed over the 12 h period post-co-amoxiclav administration (amoxicillin 1 g + clavulanic acid 0.125 g). The sera were pooled, by time point and in equal volumes, in order to obtain a set of samples representative of the mean concentration curve and a sufficiently large volume to implement all the kinetic determinations to document bactericidal potency. The mean serum concentrations determined and sampling time points are shown in Table 1.
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Timekill curves
Of the 12 pharmacokinetic time points scheduled in the protocol, eight were included in the ex vivo bactericidal study: these were at 0.5, 0.75, 1, 1.5, 2.5, 5, 8 and 12 h. Each experimental time point, reflecting the pool of 12 volunteers, was serially diluted from 1:2 to 1:256 in MuellerHinton medium supplemented with factors V and X + 5% albumin bovine fraction V (Euromedex, Souffelweyersheim, France). The kinetics of bactericidal activities were determined on each of the dilutions,25 following addition of an initial bacterial inoculum of 106107 cfu/mL. Bacterial counts were conducted after 1, 3 and 6 h of serum exposure, by subculturing 50 µL of each dilution on chocolate agar plates, after incubation at 37°C under an atmosphere enriched with 5% CO2 for 2448 h. For each experiment, an antibiotic-free growth control was run.
Pharmacodynamic parameters
For each test serum dilution, an index of surviving bacteria (ISB) was calculated, as described by Garraffo & Drugeon:26
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where AUC is the area under the curve, determined by the trapezoidal method.
The values thus obtained enabled the relationship between ISB and log concentration for each dilution to be plotted and the following parameters determined.27
LEC, lowest effective concentration.
Theoretically equivalent to the first antibiotic concentration inducing a decrease of at least 1% in the value of the ISB. The LEC was determined from the linear regression plot of log serum antibiotic concentration versus ISB. In practice, it is more reasonable, given the limited sensitivity of the methods, to define the LEC as equivalent to an ISB of 80%.
MEC, maximum effective concentration.
Theoretically equivalent to the first antibiotic concentration reducing the ISB to 0%. The MEC was evaluated graphically, by calculating the intercept of the linear regression plot with the x-axis (antibiotic concentration). In practice, it is more realistic to use a rounded value equivalent to the antibiotic concentration that reduces the ISB to 20%.
MBAP, maximum bactericidal activity period.
Defined as the time over which the antibiotic concentrations in vivo are greater than or equal to the MEC.
TDBA, theoretical duration of bactericidal activity in vivo.
Estimated by plotting the LEC value on the mean pharmacokinetic profile for all the subjects receiving the same dose of antibiotic. The TDBA is equivalent to the period over which serum antibiotic concentrations are greater than the LEC.
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Results |
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The curves showing the ex vivo bactericidal kinetics of co-amoxiclav 1.125 g against the ß-lactamase-producing H. influenzae strain under study are shown in Figures 1 and 2.
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The ISBs at 3 and 6 h clearly show the absence of a relationship between ISB and amoxicillin concentrations (data not shown). After 3 h of bacterial exposure to the antibiotic, a large number of serum dilutions had not yet achieved maximum effect (ISB 20%). The maximum effect was obtained after 6 h exposure. The potency of amoxicillin (in the presence of clavulanic acid), with the exception of rare serum dilutions, was at a maximum from the outset, even for low concentrations. The ISB was of the order of 1020%.
These results clearly reflect the markedly time-dependent effect of amoxicillin (in the presence of clavulanic acid) against the penicillinase-producing H. influenzae strain used in the study. The pharmacodynamic parameters are shown in Table 2, and represented in Figure 3
according to the mean serum concentration time curves of amoxicillin and clavulanic acid.
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Discussion |
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Conclusion
The results generated by this study enable us to consider that the co-amoxiclav 1.125 g formulation is able to exert a potent and lasting bactericidal effect on the H. influenzae strain resistant to amoxicillin through penicillinase production. Moreover, that bactericidal activity was maintained over 12 h. The above data indicate that the use of the ß-lactam plus ß-lactamase inhibitor combination in a bd regimen may be effective
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Notes |
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References |
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2 . Simonet, M., Gehanno, P., Ichou, F. & Véron, M. (1990). Etude bactériologique de la sinusite aiguë de l'adulte. Médecine et Maladies Infectieuses 20, 916.
3 . François, M., Bingen, E., Margo, J. N., Sichel, J. Y., Lambert, N. & Narcy, P. (1988). Etude bactériologique de l'otite moyenne aiguë en pratique hospitalière et en pratique libérale. Archives Françaises de Pédiatrie 45, 4716.
4 . Veyssier, P. (1997). Infections chez le sujet agé. La Presse Médicale 26, 1.
5 . Swift, C. G. (1994). Pharmacokinetics and prescribing in the elderly. Journal of Antimicrobial Chemotherapy 34, Suppl. A, 2532.[ISI][Medline]
6 . Gunn, B. A., Woodall, J. B., James, J. F. & Thornsberry, C. (1974). Ampicillin resistant Haemophilus influenzae meningitis. Lancet ii, 845.
7 . Thomas, W. J., McReynolds, J. W., Mack, C. R. & Bailey, D. W. (1974). Ampicillin resistant Haemophilus influenzae meningitis. Lancet i, 313.
8 . Khan, W., Ross, S., Rodriguez, W., Controni, G. & Saz, A. K. (1974). Haemophilus influenzae resistant to ampicillin. A report of two cases. Journal of the American Medical Association 229, 228301.
9 . Mackintosh, T. F. & Dadswell, J. V. (1974). Ampicillin resistant Haemophilus influenzae meningitis. Lancet ii, 1034.
10 . Williams, J. D., Kattan, S. & Cavanagh, P. (1974). Penicillinase production by Haemophilus influenzae. Lancet i, 103.
11 . Ried, A. J., Simpson, I. N., Harper, P. B. & Amyes, S. G. B. (1987). Ampicillin resistance in Haemophilus influenzae: identification of resistance mechanisms. Journal of Antimicrobial Chemotherapy 20, 64556.[Abstract]
12 . Juteau, J. M. & Levesque, R. C. (1990). Sequence analysis and evolutionary perspectives of ROB-1 ß-lactamase. Antimicrobial Agents and Chemotherapy 34, 13549.[ISI][Medline]
13 . Scriver, S. R., Walmsley, S. L., Kau, C. L., Hoban, D. J., Brunton, J., McGeer, A. et al. (1994). Determination of antimicrobial susceptibilities of Canadian isolates of Haemophilus influenzae and characterization of their ß-lactamases. Antimicrobial Agents and Chemotherapy 38, 167880.[Abstract]
14 . Rubin, L. G., Yolken, R. H., Medeiros, A. A. & Moxon, E. R. (1981). Ampicillin treatment failure of apparently ß-lactamasenegative Haemophilus influenzae type B meningitis due to novel ß-lactamase. Lancet i, 1008.
15 . Powell, M., Koutsia-Carouzou, C., Voutsinas, D., Seymour, A. & Williams, J. D. (1987). Resistance of clinical isolates of Haemophilus influenzae in the United Kingdom 1986. British Medical Journal 295, 1769.[ISI][Medline]
16 . Doern, G. V., Jorgensen, J. H., Thornsberry, C., Preston, D. A., Tubert, T., Redding, J. S. et al. (1988). National collaborative study of the prevalence of antimicrobial resistance among clinical isolates of Haemophilus influenzae. Antimicrobial Agents and Chemotherapy 32, 1805.[ISI][Medline]
17 . Machka, K., Braveny, I., Dabernat, H., Dornbusch, K., Van Dyck, E., Kayser, F. H. et al. (1988). Distribution and resistance patterns of Haemophilus influenzae: a European cooperative study. European Journal of Clinical Microbiology and Infectious Disease 7, 1424.[ISI][Medline]
18 . Neu, H. C., Wilson, A. P. R. & Grüneberg, R. N. (1993). Amoxicillin/clavulanic acid a review of its efficacy in over 38 500 patients from 1979 to 1992. Journal of Chemotherapy 5, 6793.[ISI][Medline]
19 . Doern, G. V., Brueggemann, A. B., Pierce, G., Holley, H. P., Jr & Rauch, A. (1997). Antibiotic resistance among clinical isolates of Haemophilus influenzae in the United States in 1994 and 1995 and detection of ß-lactamase-positive strains resistant to amoxicillin-clavulanate: results of a national multicenter surveillance study. Antimicrobial Agents and Chemotherapy 41, 2947.
20 . Cooper, C. E., Slocombe, B. & White, A. R. (1990). Effect of low concentrations of clavulanic acid on the in-vitro activity of amoxicillin against ß-lactamase-producing Branhamella catarrhalis and Haemophilus influenzae. Journal of Antimicrobial Chemotherapy 26, 37180.[Abstract]
21 . Yogev, R., Melik, C. & Kabat, W. J. (1981). In vitro and in vivo synergism between amoxicillin and clavulanic acid against ampicillin-resistant Haemophilus influenzae type b. Antimicrobial Agents and Chemotherapy 19, 9936.[ISI][Medline]
22 . Courvalin, P., Drugeon, H., Flandrois, J. P. & Goldstein F. (1990). Détermination de la concentration minimale inhibitrice et de la concentration minimale bactéricide en milieu liquide. In Bactéricidie, Aspects Théoriques et Thérapeutiques pp. 3356. Maloine, Paris.
23 . Foulstone, M. & Reading, C. (1982). Assay of amoxicillin and clavulanic acid, the components of amoxicillinclavulanate, in biological fluids with high-performance liquid chromatography. Antimicrobial Agents and Chemotherapy 22, 75362.[ISI][Medline]
24 . Jehl, F., Monteil, H. & Brogard, J. M. (1987). Détermination directe de l'acide clavulanique dans les liquides biologiques par HPLC. Pathologie Biologie 35, 7026.[ISI][Medline]
25 . Courvalin, P., Drugeon, H., Flandrois, J. P. & Goldstein, F. (1990). Pouvoir bactériostatique et bactéricide d'un liquide biologique. In Bactéricidie, Aspects Théoriques et Thérapeutiques pp. 34750. Maloine, Paris.
26 . Garraffo, R. & Drugeon, H. B. (1992). Détermination de la posologie optimale d'un antibiotique chez l'homme à l'aide d'un modèle pharmacodynamique in vivo/ex vivo. In Méthodes Nouvelles en Pharmacologie Clinique Pédiatrique. Springer Verlag.
27 . Jehl, F., Kamili, N., Elkhaïli, H. & Monteil, H. (1997). Pharmacodynamie in vitro de l'amoxicilline et bactéricidie ex vivo après 1g per os sur S. pneumoniae résistant à la pénicilline. Médecine et Maladies Infectieuses 27, Spécial, 4557.[ISI]
28 . Cooper, C. E., Slocombe, B. & White, A. R. (1990). Effect of four concentrations of clavulanic acid on the in-vitro activity of amoxy-cillin against ß-lactamase-producing Branhamella catarrhalis and Haemophilus influenzae. Journal of Antimicrobial Chemotherapy, 26, 37180.[Abstract]
29 . Moitie, D., Simonet, M. & Veron, M. (1989). Comparaison de l'activité in vitro des associations amoxicilline + acide clavulanique et ampicilline + sulbactam sur cinquante souches de H. influenzae productrices d'une bêta-lactamase. Pathologie Biologie 37, 3903.[ISI][Medline]
30 . Murbach, V., Dhoyen, N., Linger, L., Monteil, H. & Jehl, F. (1999). Mise en évidence in vitro d'un véritable effet post-inhibiteur de bêta-lactamases (EPIBLA) de l'acide clavulanique sur Klebsiella pneumoniae et Haemophilus influenzae. Pathologie Biologie 47, 4628.[ISI][Medline]
31 . Gaillot, O. & Simonet, M. (1994). Activité comparée de bêtalactamines orales sur cinquante souches d'Haemophilus influenzae productrices d'une bêta-lactamase, en fonction de l'inoculum bactérien. Pathologie Biologie 42, 3757.[ISI][Medline]
Received 5 February 2001; returned 29 May 2001; revised 4 July 2001; accepted 21 July 2001