Buzzi Marina, Guarino Anna, Gatto Claudio, Manara Sabrina, Dainese Luca, Polvani Gianluca, Tóthová Jana D'Amato
Cardiovascular Tissue Bank of Emilia-Romagna, Azienda Ospedaliero-Universitaria Policlinico S. Orsola-Malpighi, Bologna, Italy.
Cardiovascular Tissue Bank of Lombardia, Centro Cardiologico Monzino, Milan, Italy.
PLoS One. 2014 Nov 14;9(11):e112679. doi: 10.1371/journal.pone.0112679. eCollection 2014.
We investigated the presence of antibiotics in cryopreserved cardiovascular tissues and cryopreservation media, after tissue decontamination with antibiotic cocktails, and the impact of antibiotic residues on standard tissue bank microbiological analyses. Sixteen cardiovascular tissues were decontaminated with bank-prepared cocktails and cryopreserved by two different tissue banks according to their standard operating procedures. Before and after decontamination, samples underwent microbiological analysis by standard tissue bank methods. Cryopreserved samples were tested again with and without the removal of antibiotic residues using a RESEP tube, after thawing. Presence of antibiotics in tissue homogenates and processing liquids was determined by a modified agar diffusion test. All cryopreserved tissue homogenates and cryopreservation media induced important inhibition zones on both Staphylococcus aureus- and Pseudomonas aeruginosa-seeded plates, immediately after thawing and at the end of the sterility test. The RESEP tube treatment markedly reduced or totally eliminated the antimicrobial activity of tested tissues and media. Based on standard tissue bank analysis, 50% of tissues were found positive for bacteria and/or fungi, before decontamination and 2 out of 16 tested samples (13%) still contained microorganisms after decontamination. After thawing, none of the 16 cryopreserved samples resulted positive with direct inoculum method. When the same samples were tested after removal of antibiotic residues, 8 out of 16 (50%) were contaminated. Antibiotic residues present in tissue allografts and processing liquids after decontamination may mask microbial contamination during microbiological analysis performed with standard tissue bank methods, thus resulting in false negatives.
我们研究了在用抗生素鸡尾酒对组织进行去污处理后，冷冻保存的心血管组织和冷冻保存介质中抗生素的存在情况，以及抗生素残留对标准组织库微生物分析的影响。16个心血管组织用组织库制备的鸡尾酒进行去污处理，并由两个不同的组织库按照其标准操作程序进行冷冻保存。在去污前后，样本按照标准组织库方法进行微生物分析。解冻后，使用RESEP管对冷冻保存的样本在去除和未去除抗生素残留的情况下再次进行检测。通过改良的琼脂扩散试验测定组织匀浆和处理液中抗生素的存在情况。所有冷冻保存的组织匀浆和冷冻保存介质在解冻后立即以及无菌试验结束时，在接种金黄色葡萄球菌和铜绿假单胞菌的平板上均诱导出明显的抑菌圈。RESEP管处理显著降低或完全消除了受试组织和介质的抗菌活性。根据标准组织库分析，50%的组织在去污前被发现细菌和/或真菌呈阳性，16个受试样本中有2个（13%）在去污后仍含有微生物。解冻后，16个冷冻保存的样本采用直接接种法检测均未呈阳性。当去除抗生素残留后对相同样本进行检测时，16个中有8个（50%）被污染。去污后同种异体组织移植物和处理液中存在的抗生素残留可能会在使用标准组织库方法进行微生物分析时掩盖微生物污染，从而导致假阴性结果。
