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VOL. 75 (2), 217-231, 2009 (CU)II IN VIVO INTERACTION WITH CEFOTAXIME

The susceptibility tests were as follows: One loop of each of the
precultured strains was spread on to the whole surface of agar-plates.
Immediately after the inoculation paper discs soaked with 20 ìL of
the assays solutions were placed on to the agar media. The plates
were kept in the fridge for 30 minutes to allow the liquid diffusion
through out the agar media while the bacterial growth was arrested.
Finally the plates were cultured for one day at 37 ºC and the
microbicide effect of the solutions were measured by the size of the
diameter of the growth inhibition zone (GIZ) surrounding each disc.

The assayed liquids were: free metal, free cefotaxime and the
complex metal-antibiotic.

Standard solutions of free cefotaxime was prepared based on the
Minimal Inhibitory Concentration (MIC) reported in literature (9),
being the solutions 5 times over the MIC of each species: for S.
aureus spp aureus and E. coli, the MICs were 3.3 µM and 0.12 µM
respectively. In the assays with B. subtilis, since it was assumed to
be unsensitive to the antibiotic, the mother solution was as in S.
aureus spp aureus.

The highest concentrations of free metal assayed dissolved in
water wee 5 times the maximum amount of Cu(II) found by us in
poisoned rats (10). The source of Cu(II) was obtained from the
acetate monohydrate salt at 44 µM.

To measure the activity of the complex antibiotic-metal was
necessary one previous procedure to isolate the antibiotic bounded
to copper. Free antibiotic and the metal salt were dissolved together
at saturation, 0.4 M for both components, at room temperature to
promote the complexation reaction. The complex was separated from
the residual contamination by chromatophy in a HPLC column
exactly as the conditions as we identified the free cefotaxime in rats.
The chromatografied buffer containing the complex was collected in
1.5 ml eppendorfs.

The resulting complex was dissolved in a methanol buffer. To
prevent the methanol toxicity, the alcohol was removed by
dissecation in a vacuum trap. The final power debris was weighted,
resuspended in water at saturation, homogenized and pooled. The
dry amount of the complex antibiotic-Cu was 90 mg. We assumed all
the dry weight to be the complex since the phosphate coeluted from
the isolation buffer was negligible (5 µg).

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