Journal of Antimicrobial Chemotherapy (2001) 48, 501-506
© 2001 The British Society for Antimicrobial Chemotherapy
Ex vivo 12 h bactericidal activity of oral co-amoxiclav (1.125 g) against ß-lactamase-producing Haemophilus influenzae
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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The aim of the study was to evaluate the in vitro/ex vivo bactericidal activity of a new coamoxiclav single-dose sachet formulation (1 g amoxicillin + 0.125 g clavulanic acid) against a ß-lactamase-producing strain of Haemophilus influenzae. The evaluation covered the 12 h period after antibiotic administration. Serum specimens from the 12 healthy volunteers included in the pharmacokinetic study were pooled by time point and in equal volumes. Eight of 12 pharmacokinetic sampling time points were included in the study. At time points 0.5, 0.75, 1, 1.5, 2.5, 5, 8 and 12 h post-dosing, the kinetics of bactericidal activity were determined for each of the serial dilutions. Each specimen was serially diluted from 1:2 to 1:256. The index of surviving bacteria (ISB) was subsequently determined for each pharmacokinetic time point. For all the serum samples, bactericidal activity was fast (3–6 h), marked (3–6 log10 reduction in the initial inoculum) and sustained over the 12 h between-dosing interval. The results obtained also confirmed that the potency of the amoxicillin plus clavulanic acid combination was time dependent against the species under study and that the time interval over which the concentrations were greater than the MIC (t > MIC) was 100% for the strain under study. The data thus generated constitute an interesting prerequisite with a view to using co-amoxiclav 1.125 g in a bd oral regimen.
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Introduction |
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Haemophilus influenzae is among the most frequently encountered bacteria responsible for community-acquired upper and lower respiratory tract infections, such as chronic bronchitis1 and pneumonia, acute sinusitis2 and acute otitis media,3 in both adults and children. In adults, advanced age plays a very important role due to the progressive weakening of the immune defences, the rapid course of the infection4 and changes in pharmacokinetic and pharmacodynamic parameters.5 Prior to 1970, H. influenzae was considered susceptible to ampicillin and amoxicillin. In 1974, Gunn et al.6 isolated the first resistant strain of H. influenzae type B in the United States. In 1974, Thomas et al.7 also isolated a resistant strain. Various studies have shown that the resistance mechanism developed by those strains involve the production of enzymes8–10 able to inactivate ß-lactams, previously considered the first-line treatment for H. influenzae infections. In the majority of cases, the enzyme responsible for resistance is a plasmid penicillinase of type TEM-1.11 Subsequently, other teams discovered a second enzyme of the ROB-1 type.12–14
Since then, the percentage of strains exhibiting resistance mediated by production of ß-lactamases has steadily increased,15–19 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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Microorganism
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 Mueller–Hinton 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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The active substances in co-amoxiclav were assayed by high-performance liquid chromatography (HPLC), using the protocol reported previously.23,24
Time–kill 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 Mueller–Hinton 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 106–107 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 24–48 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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For the H. influenzae strain tested, MICs/MBCs were 8/>64 mg/L for amoxicillin and 0.25/4 mg/L for co-amoxiclav.
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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, 1:2; 
, 1:4; 
, 1:8; 
, 1:16; 
, 1:32; 
, 1:64; 
, 1:128; 
, 1:256.
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For all the time point samples (0.5–12 h), the bactericidal activity was marked (3–6 log10) and rapid at the 1:2 and 1:4 dilutions. At 0.5 and 0.75 h (Figure 1a and b
), the level of bactericidal activity for the 1:2, 1:4 and 1:8 dilutions led to a 6 log10 decrease in 3 h. The inoculum reduction observed at time points 1, 1.5, 2.5, 5, 8 and 12 h was between 3 and 5 log10 for the 1:2 and 1:4 dilutions. 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 10–20%.
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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The absence of correlation between concentration and ISB means that little importance can be attributed to the pharmacodynamic parameters determined (Table 2
), except t > MIC. However, the ISBs at 3 and 6 h show that the LEC and MEC are very low, and also show that for an MEC evaluated at c. 0.50 mg/L, there is an MBAP of the order of 10 h (83.3% of the bd interval). It will also be observed that t > MIC is 12 h (100% of the bd interval). The bactericidal activity of co-amoxiclav 1.125 g is thus intense throughout the interval between the two daily intakes of the antibiotic. Although the trough clavulanic acid concentrations become very low (100 ng/mL), or even below the limit of detection (50 ng/mL) (Table 1), between 10 and 12 h post-intake, amoxicillin maintains its bactericidal potency against the penicillinase-producing H. influenzae strain. This confirms the excellent inhibitory potency of clavulanic acid with respect to the enzymes produced by H. influenzae. In this context, Cooper et al.28 and Moitie et al.29 have already demonstrated in vitro that at concentrations of the order of 0.1 mg/L, ß-lactamase-producing H. influenzae again becomes susceptible to amoxicillin. In this study, the value was present, on the plot, up to 8 h post-dosing, and thus largely explains the good bactericidal activity of amoxicillin. For the 12 h sample, the clavulanic acid concentration was <0.05 mg/L. Up to concentrations of 0.01 mg/L, clavulanic acid is known to inhibit the ß-lactamases produced by Moraxella catarrhalis.20 A concentration of that order of magnitude could explain the activity of amoxicillin against our strain of H. influenzae in the 12 h specimen. It should be noted that we cannot be dealing with a post-inhibitory effect of clavulanic acid,30 since the bacterium was freshly placed in contact with the sera. However, the delay in resuming growth subsequent to pre-exposure to coamoxiclav may occur in vivo and contribute to the prolongation of the duration of action of co-amoxiclav on our strain. In addition, in all probability, even in the presence of a large bacterial inoculum31 at the site of infection, the inhibitory effect on ß-lactamases is largely ensured by clavulanic acid at the concentrations determined between 0 and 12 h. 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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* Corresponding author. Tel: +33-3-9024-3790; Fax: +33-3-8825-1113; E-mail: francois.jehl{at}medecine.u-strasbg.fr
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References |
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Received 5 February 2001; returned 29 May 2001; revised 4 July 2001; accepted 21 July 2001
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