Abstract
Amniotic membrane (AM) is used to treat a range of ophthalmic indications but must be presented in a non-contaminated state. AM from elective caesarean sections contains natural microbial contamination, requiring removal during processing protocols. The aim of this study was to assess the ability of antibiotic decontamination of AM, during processing by innovative low-temperature vacuum-drying. Bioburden of caesarean section AM was assessed, and found to be present in low levels. Subsequently, the process for producing vacuum-dried AM (VDAM) was assessed for decontamination ability, by artificially loading with Staphylococcus epidermidis at different stages of processing. The protocol was highly efficient at removing bioburden introduced at any stage of processing, with antibiotic treatment and drying the most efficacious steps. The antibacterial activity of non-antibiotic treated AM compared to VDAM was evaluated using minimum inhibitory/biocidal concentrations (MIC/MBC), and disc diffusion assays against Meticillin-resistant Staphylococcus aureus, Meticillin-resistant S. epidermidis, Escherichia coli, Pseudomonas aeruginosa and Enterococcus faecalis. Antibacterial activity without antibiotic was low, confirmed by high MIC/MBC, and a no inhibition on agar lawns. However, VDAM with antibiotic demonstrated effective antibacterial capacity against all bacteria. Therefore, antibiotic decontamination is a reliable method for sterilisation of AM and the resultant antibiotic reservoir is effective against gram-positive and –negative bacteria.
Highlights
Treatment of ocular surface disease, ulcers and infections remains a challenge
We identify the level of microbiological contamination, or bioburden, of elective caesarean section (ECS) amniotic membrane (AM), and go on to assess the robustness of decontamination during our optimised, innovative protocol for preserving AM, using vacuum-drying and a broad spectrum antibiotic/ antifungal cocktail[18]
The natural floral contamination of fresh AM obtained by ECS in the Queens’s Medical Centre, Nottingham University Hospitals NHS trust, was shown to be considerably lower than literature reported from normal vaginal deliveries20,24,25. 40% of the tested AM showed no signs of bioburden contamination, similar to results previously reported (39%)[20]
Summary
Treatment of ocular surface disease, ulcers and infections remains a challenge. Severe bacterial ocular infections are commonly treated with corticosteroids and an intensive course of antibiotics[1,2]. Associated with low post-transplantation infection complications[15], cryopreservation has been shown to damage the amnion[16] which impairs the viability and proliferative capacity of epithelial cells[17], decrease levels of many bioactive substances including epidermal growth factors (EGF) and transforming growth factors (TGF), and is constrained by distribution difficulties[18]. Dried AM followed by radiation sterilisation has been shown to be degraded with reduced growth factor content[14] This effect on the stability and function of biological molecules such as growth factors, cytokines and structural proteins, affects the quality of the AM as a biological treatment for eye injury, and justifies the development of an appropriate approach for AM preservation.
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