Metronidazole availability during bacterial growth in microplates

The bacterial growth during exposition to antibiotics remains a central activity in the testing and understanding of the efficacy of antibiotics. Objectives: To evaluate metronidazole availability during bacterial growth in microplates using a HPLC method. Methods: Reference strains of human oral microorganisms Prevotella intermedia, Campylobacter rectus and Streptococcus sanguis were tested for their capacity to grow with metronidazole. Bacteria were initially grown on agar plates optimized for growth of the specific species. Plates were incubated for 7 days at 35ºC, in 80% N₂, 10% CO₂ and 10% H₂. Aliquots of these samples were used to inoculate eppendorf tubes containing 2% mycoplasma broth (BBL) and placed into a 96-well microtiter plate. For the antibacterial assays, metronidazole was dissolved to a final concentration ranging (0,1,2,4,8,16,32 and 64 µg/ml).The microplates were seeded with bacterial inoculum and the samples were then incubated and growth evaluated by microplate reader at 0, 27 and 68 h under anaerobic conditions by the increase 630 nm absorbance. For metronidazole analysis using HPLC method 1µL of each well was mixed with acetonitrile in phosphate buffer and centrifuged. The supernatant was separated, injected into the HPLC system and monitored by UV absorbance at 318 nm. Results: The area ratio(R) with a tinidazole internal standard (0.06 µg/mL) was linear over a concentration range of 0 to 64 µg/mL (R²=0.99). Metronidazole concentration no showed correlation with bacterial curves to P.intermedia (p=0.546) and C.rectus (p=0.635). The antibiotic inhibited bacterial growth (≥1 µg/ml) to both microorganisms with a metabolite being found by HPLC method. Metronidazole was inactive against S.sanguis and there was significant correlation between bacterial OD values and µg/mL levels in S.sanguis microplate (p=0.0036; Pearson r =0.884; 95%confidence interval 0.475 to 0.979). Conclusions: This data indicated that there is bacterial metabolic factors involved into metronidazole availability during bacterial growth.