Tissue decontamination is a challenge for tissue banks. However, the standardization and validation of specific decontamination procedures and antibiotic cocktails have rarely been pursued, consequently a wide range of antibiotic formulations, temperatures and exposure times are currently adopted for this purpose (Heng et al. 2013; De By et al. 2012; Germain et al. 2010).
Taking the assessments reported by Pitt et al. (2014), on the efficacy of different antibiotic cocktails at 4 and 22 °C against a wide panel of bacterial strains, we designed 4 different antibiotic cocktails. After a preliminary screening we focused on two of them which were tested against a panel of bacterial species commonly isolated from allograft tissues and compared them to the antibiotic cocktail currently in use in our facility at FBTV (cocktail Z). We did not include any anti-mycotic drugs, since in our procedure the presence of fungi in the pre-decontamination analysis is an exclusion criterion for tissue. For the same reason our bacterial panels did not include Streptococcus pneumoniae, Streptococcus pyogenes, Serratia marcescens, Meningococcus or Pseudomonas aeruginosa strains or strains belonging to the genus Clostridium or Mycobacterium.
The efficacy of the cocktails was tested both at 4 °C for 24 or 48 h and at 22 °C for 6 h. To prevent drug carry-over interfering with the results, the bacteria were rinsed in a drug-free medium before viability counts. Two of the new cocktails (C and D) killed a broad spectrum of bacteria even at 4 °C and were clearly more effective than cocktail Z. Specifically, after incubation for 48 h at 4 °C, 61 and 67 % of the strains showed a kill rate higher than 95 % when incubated with cocktail C or D, respectively, compared to 44 % of the strains reaching the same kill rate when incubated with cocktail Z. This effect was probably due to the broader spectrum of action of the antibiotics contained in the new cocktails compared to cocktail Z (Table 2). Cocktails C and D, both contained vancomycin and gentamicin, plus meropenem (cocktail C) or ciprofloxacin (cocktail D). The superiority of cocktails C and D over cocktail Z mainly lies in their increased bactericidal activity against Streptococcus and Staphylococcus strains (Fig. 1), which are the most commonly detected genera in allografts. In particular, cocktail D showed a kill rate higher than >90 % activity against all tested strains of Streptococcus and Staphylococcus.
Treatment at a higher temperature (such as room temperature, 22 °C) for shorter times would increase decontamination efficacy (in particular allowing higher depletion of more resistant species including Enterecoccus faecalis) and reduce processing times, however this procedure might not be applicable to all tissues due to their shorter survival at this temperature. It would be interesting in the future to measure tissue vitality at this temperature by performing specific tests, and to assess the efficacy of short decontamination procedures using shorter incubation times.
It is important to point out that susceptibility towards antibiotics varies within species, so the panel of clinical and reference isolates used in this work cannot cover all of the possible susceptibility spectra of bacterial contaminants found in the field. One drawback of our work was that our killing experiments were performed in liquid medium, in different conditions compared to those usually experienced by the bacteria during allograft decontamination. To alleviate this drawback, the medium was added with 0.5 % foetal calf serum to increase the concentration of proteins, which are obviously highly represented in tissues and can bind with antibiotics.
With regard to the above mentioned issue, we will shortly be analysing the microbiological results after one year with the new cocktail to verify its efficacy in allograft decontamination compared to cocktail Z.
It is also worth noting that our experiments were performed starting with cultures that had a bacterial concentration of 105–106 cells/ml, a much higher load than that found in allograft tissues, since a drug’s kill rate is usually inversely proportional to the bacterial load (i.e. inoculum effect; Udekwu et al. 2009); the bactericidal activity shown in Figs. 1 and 4 might be underestimated with respect to that obtained during allograft decontamination.
In conclusion, in this work we characterized two antibiotic formulations that displayed several advantages validating our initial hypothesis that a cocktail including gentamicin, ciprofloxacin or meropenem would be more efficient than the one currently in use at FBTV (cocktail Z): (1) both were found to be more potent both at 4 and at 22 °C; (2) both contained three antibiotics instead of four, reducing the cost of decontamination; and (3) both contained antibiotics which are more readily available than lyncomicin and polymyxin B (present in cocktail Z), as they are commonly used in clinical practice. In addition, in Italy polymixin B is no longer available as an antibiotic for systemic use, but only for topical use due to its toxicity.