Abstract: Our previous studies indicated that the use of antibiotic cocktails during tissue processing can lead to an antibiotic carry-over effect, which in turn can generate false negative results in microbiological analysis.

Purpose: To validate a single-use device for elimination of antibiotics from liquid samples undergoing microbiological analysis to prevent interference with microbial growth.

Methods: The efficacy of the ResEP device was tested on 5 ml of BASE.128 (AL.CHI.MI.A.S.r.l.) and determined by HPLC after 5, 10, 20, 30 min. The recovery of S. Aureus, P. Aeruginosa, C. Albicans, B. Subtilis, A. Niger and C. Sporogenes was determined by dilution plating of inoculants containing 1-10, 10-100 and 100-1000 CFU, 20 min after treatment.

The device was further tested by three cardiovascular and skin Italian banks. Standard bacteriological tests were performed on transport, rinsing and cryopreservation liquids (BACT-ALERT Biomerieux or thioglycollate/TSB media) of twenty tissue samples decontaminated with BASE.128; additional bacteriological tests were performed on liquid samples using ResEP device, after antibiotic removal. The presence of antibiotic residues in all samples undergoing the microbiological analyses was evaluated by HPLC.

Results: The HPLC analysis of the samples treated with ResEP device showed complete antibiotic removal from the BASE.128 within 20 min of treatment. A total bacterial recovery was obtained for all investigated inoculants.
HPLC analysis on processing liquids showed that important antibiotic residues were present in rinsing and cryopreservation liquids, after decontamination of skin and cardiovascular tissues. The ResEP device removed efficiently antibiotic residues from all liquid samples prior to microbiological analysis allowing the detection of 10 % of false negative results.

Conclusion: The ResEP device was validated for easy and quick removal of antibiotic residues from liquid samples undergoing microbiological analysis, showing complete microbial recovery and high specificity for BASE.128 medium and allowing the detection of false negative results in microbiological analyses.

Abstract: Elimination of residual antibiotics from allograft/human tissues intended for transplantation after decontamination is paramount to ensure tissue quality and safety. We monitored the fate of antibiotics during the tissue processing phases after decontamination of pig heart valves in an antibiotic cocktail containing Vancomycin, Cefotaxime and Amphotericin B.

Introduction: Decontamination is a crucial step in allograft processing. Tissue banks have established their own decontamination procedures using different solutions, depending on tissue, as well as different time and temperature conditions, which often require a complex validation process.

Purpose: To investigate the decontamination efficacy of the industrially manufactured medical device BASE.128 both in vitro and in the presence of artificially contaminated tissues.

Methods: In vitro time-kill studies: BASE.128, containing three different antibiotics, was inoculated with 108 CFU/device of reference microorganisms according to Pharmacopoeia: Staphylococcus Aureus, Candida Albicans, Pseudomonas Aeruginosa, and an additional bacterial strain: methicillin-resistant Staphylococcus Epidermidis. Media were incubated at 4°C, 22°C and 37°C. Microbial content was quantified after 3h, 6h, 12h, and 24h of incubation at all three temperatures by membrane filtration sterility test.

Contaminated tissues: Sterile pig heart valve, bone and skin were superficially contaminated with 106 – 105 CFU/tissue of the selected microorganisms. Tissues were then immersed in BASE.128 for 6h at 37°C or 24h at 4°C. After decontamination, tissue samples were transferred to Trypton Soy Broth containing a mixture of antibiotic-neutralizing resins. Samples were stirred for 1 h for an effective antibiotic neutralization and then incubated at 37°C for 14 days for microbiological analysis. Elimination of antibiotics was checked by agar diffusion test, immediately after antibiotic neutralization. Tissue decontamination was examined both qualitatively by visual inspection of media for turbidity for up to 14 days and quantitatively by plating sample media on Petri dishes.

Results: In vitro studies showed that a minimum decontamination efficacy of 6, 5 and 4 logs was achieved after 12 h incubation in BASE.128 at 37°C, 22°C and 4°C, respectively. Time-kill curves of the investigated microorganisms showed that the decrease in incubation temperature delays the decontamination effects and reduces it of 1 log at 4°C.
Artificial contamination of heart valve, bone or skin, and subsequent decontamination in BASE.128 showed that the presence of the tissue does not affect the decontamination efficacy of BASE.128. Based on our qualitative and quantitative results, an average decrease of 5 logs was observed for all investigated microorganisms in all examined tissues at both decontamination conditions. The agar diffusion test showed the absence of bacterial growth inhibition, thus indicating the absence of residual antibiotic activity.

Conclusions: Our results show that the medical device BASE.128 eliminates effectively all the investigated microorganisms from tissues with high decontamination efficacy. The time-kill studies demonstrate the relevance of time and temperature for tissue decontamination. Conversely, the presence of tissue does not affect the decontamination efficacy of BASE.128. In conclusion, BASE.128 is a validated decontamination medium for different tissues, which fits the standard decontaminating procedures of Tissue Banks.