Validation of standardized industrial antibiotic cocktails

Abstract: The aim of the present study was to evaluate the performances of the industrial antibiotic cocktail BASE.128 as compared to the solutions currently used by a Cardiovascular Tissue Bank. Cardiovascular tissues (n=6), including heart valves and blood vessels, were procured from HB and NHB donors and processed by the Cardiovascular Tissue Bank.

Tissues were divided into two equal halves and processed in parallel using BASE.128RED (AL.CHI.MI.A. S.r.l) at 4°C for 24h (valves) and 72h (vessels) or an antimycotic free antibiotic solution (Tissue Bank prepared) at 4°C for 24h (valves) and 72h (vessels) for decontamination, and BASE (AL.CHI.MI.A. S.r.l) with 10% CRYO.ON (AL.CHI.MI.A. S.r.l) and 2% human albumin or RPMI1640 with 10% DMSO and 2% human albumin (Tissue Bank prepared) for cryopreservation. Before and after decontamination, two bacteriological tests were performed on liquid and tissue samples by the hospital microbiology service using BACT-ALERT and/or Thioglycollate and TSB medium. The frozen tissues and liquid samples were transported to the AL.CHI.MI.A.’s lab and sterility test was performed according to the EP, after thawing and elimination of the residual antibiotics, which was assessed by agar diffusion test. Contaminants present in the samples positive to the sterility test were identified by ribosomal DNA sequencing.

The results of the bacteriological analysis performed by the hospital bacteriological service showed that all tissues from NHB donors were initially contaminated by one or more bacterial spp. (Staphylococcus spp., Streptococcus spp., Bacillus spp., Enterococcus spp., Corynebacterium spp., C. Albicans, E. Coli). After tissue decontamination with the bank prepared solution, one tissue resulted still contaminated by C. Albicans and E. Coli contamination of one cryopreservation medium was detected. Conversely, the decontamination with BASE.128RED eliminated all contaminants from all investigated tissues and liquids.
Agar diffusion test showed the presence of antibiotic residues in all decontaminated tissues and confirmed the elimination of residues in liquid and tissue samples submitted to sterility testing. The sterility test showed a significant incidence of false negative in tissue samples treated with the bank solution. Ribosomal DNA sequencing identification mostly demonstrated the presence of environmental contaminants. No false negative was detected in tissues decontaminated with BASE.128RED.
We demonstrated that the presence of residual antibiotics in tissue and liquid samples undergoing bacteriological analysis leads to false negative results. The BASE.128RED medical device decontaminates effectively the human cardiovascular tissues. Validation of the decontamination processes and microbiological methods is necessary to warrant the safety of allografts.