Background: The aim of this study was to validate the microbiological testing of cornea organ culture medium according to the European Pharmacopoeia (chapter 2.6.27.) using RESEP (ALCHIMIA) a new medical device for the removal of antibiotics and blood culture bottles with the automatic BACTEC system (Becton Dickinson, Franklin Lakes, New Jersey, USA).
Methods: For the validation 10-100 colony forming units of Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Bacillus subtilis, Aspergillus niger, Clostridium sporogenes, Enterobacter cloacae and Staphylococcus epidermidis (ATCC 12228) were inoculated in 9 ml of cornea organ culture medium (MEM with 2% fetal australian calf serum and Streptomycin (130 µg/ml), Penicillin (60 µg/ml) and Amphotericin B (2,5 µg/ml); Merck, Biochrom GmbH, Germany). The medium was treated with RESEP for 20 minutes, inoculated in BACTEC™ Plus Aerobic/F and BACTEC™ Plus Anaerobic/F blood culture bottles and incubated until a positive reading was shown or for at least 14 days at 36 ±1°C. Medium control samples, without RESEP treatment, containing the same microorganisms, were inoculated in BACTEC vials directly. Microbial growth was controlled by direct inoculation of BACTEC flasks (growth control). The decrease of antibiotics from the medium by RESEP was determined by high performance liquid chromatography (HPLC).
Results: After 20 minutes of treatment at room temperature with RESEP (confirmed by HPLC) the antibiotics were completely eliminated . In samples treated with RESEP, the growth of all microorganisms could be detected within 3 days of incubation in the BACTEC system and showed no significant delay compared to the growth controls. In contrast, untreated medium samples showed no reproducibility of Staphylococcus aureus, Enterobacter cloacae, Candida albicans and Bacillus subtilis.
Conclusions: The microbiological testing of cornea organ culture medium with the automatic BACTEC blood culture system using RESEP for the removal of antibiotics has been successfully validated according to the European Pharmacopoeia (Chapter 2.6.27.)