CLAVULANIC ACID - EFFECTIVE CONCENTRATIONS, ASSAY-METHODS AND TISSUE PENETRATION

Some bacterial betalactamases are inhibited by very low concentrations of clavulanic acid : (in mug/ml) Moraxella catarrhalis 0.01-0.5; Haemophilus influenzae 0.06-0.5; Bacteroides-Fusobacterium spp 0.5-2; Staphylococcus aureus 0.05-0.5; E. coli (TEM enzyme) 2-4, etc. In vitro, within a given betalactamase-producing genus, the percentage of R strains that become susceptible increases proportionally to the concentration of clavulanic acid combined with ticarcillin (TIC) or amoxycillin (AMX). With some genera, slight increases in the clavulanic acid concentration (in absolute values) considerably increase the number of strains that become susceptible. It is thus important to know the concentrations of clavulanic acid obtained in various sites of infection. During investigations of the tissue penetration of clavulanic acid, it is essential to use assay methods that are reliable in terms of accuracy, precision (in the quality-control sense) and the detection limit. Reliability must extend not only to the assay itself, but also to the sample preparation method. In practice, tissue homogenization - an essential step in the extraction of clavulanic acid - is carried out with no special precautions to limit heating. As a result, clavulanic acid is partially destroyed, and this leads to an underestimation of the concentration. This is also true for most other antibiotics. The risk of underevaluating the clavulanic acid concentration is increased during the assay procedure itself when a microbiological method is used. Indeed, incubation at 37-degrees-C for 18 h leads to marked degradation of clavulanic acid which, like most betalactam antibiotics, is unstable in vitro. Further errors are inherent in the principle on which microbiological assays are based : they consist of measuring the inhibition of bacterial growth by the antibiotic present in the sample, and there are several uncontrollable sources of error. With regard to enzyme inhibitors, the problem is even more acute, since the method involves measuring the inhibition of growth by the betalactam of a bacterium that produces a betalactamase, thanks to the neutralization of the latter by the inhibitor present in the biological sample. There are even more uncontrollable errors inherent in this process. Given these two sources of error - partial degradation of the antibiotic and the imprecision of microbiological assays - published clavulanic acid concentrations should be interpreted with care. Indeed, they are almost certainly lower than the real values, and even a difference of a few tenths of mug/ml can have a major impact on the proportion of R strains that become susceptible. By way on an example, Cooper et al (6) reported that 65 % of betalactamase + H. influenzae strains became susceptible in vitro to amoxycillin when 0.06 mug/ml of clavulanic acid was added, compared to 91 % when 0.12 mug/ml was added.