A Study On Antimicrobial Properties Of Human Amniotic Homogenate Supernatant In Vitro

Background and Aims Amnion is a biological material which is cheap and easy to acquire. Pure human amnion has low antigenicity and amnion transplantation has been a focus in ocular surface operation. The main effects of amnion on clinical application include accelerating epithelization, maintaining normal epithelium type, decreasing inflammatory response and mitigating vascularization. The antimicrobial property of amniotic fluid has been well proved, but the antimicrobial property of amnion is still argued and reported seldom up to now.Antimicrobial peptides are a class of small molecule peptides which are produced by biological defense system to against exogenous pathogens. Meanwhile, they are also important defense substances of natural immune system. Antimicrobial peptides have a widely distribution and can be found in nearly all kind of biological species. There have been thousands of antimicrobial peptides being extracted since they were first discovered in 1980s. Human-derived antimicrobial peptides, which consist of human beta defensin (HBD), cathecidins and histatin, mainly exist in neutrophils and epithelial cells of skin and mucous membranes. For the problem of antibiotic resistance is getting more and more serious now, the research of antimicrobial peptides has become a hotspot.Recently, King et al. reported that HBD1-3 expression were founded in placenta, fetal membrane, placenta tophoblast, amniotic epithelial and decidua. At the same time, IL-1βinduced HBD2 mRNA expression in primary cultured trophoblast in vitro. Stock et al also demonstrated there were HBD1-3 mRNA and protein expressions in primary cultured amniotic epithelial cells. After treatment with 10ng/ml IL-1β, HBD2 mRNA expression showed bimodal increases, and there were 51 times and 67 times increases after treatment for 6h and 12h, respectively. Furthermore, HBD2 protein expression significant increased as well after treatment for 24h. The author believed that amnion could produce a powerful antibacterial substance to prevent infection in pregnancy, and IL-1β, pregnancy-associated inflammatory cytokines, induced HBD2 expression.Based on the above founding, we think that human amnion should have a stronger role to resist pathogenic microorganisms. In present study, we are investigating the antimicrobial properties of human amniotic homogenate supernatant (HAHS) to supply theoretical supports for a better use of human amnion in clinic ophthalmology,explore a new convenient and effective strategy treating with ocular infectious diseases.Methods1. Fresh amniotic membranes were obtained from healthy parturients under sterile condition using the standard and method, discribed by Talmi et al.,then we maked acellular amnion. Using Staphylococcus aureus ATCC25923 ,we tested the antibacterial activities of fresh amnion and acellular amnion both when the endepidermis faced to superior and inferior using the K-B method.2. Preparing fresh human amniotic homogenate supernatant, using Bicinchoninic acid (BCA) method to measure the protein concentration of it,we tested antimicrobial spectrum of fresh HAHS also by K-B method.There are 3 standard strains, 4 clinically isolated sensitive strains, 4 clinically isolated multi-drug-resistant strains, 3 clinically isolated strains of fungi as tested bacterium.3. The HAHS's antimicrobial activity was compared with ten normally used antibiotics in eyes by Broth Microdilution Method and colony-counting method,using staphylococcus aureus ATCC25923 and E. coli ATCC25922 bacteria as a test.4. The impacts of temperature, pH values and the preservation time on antibacterial activity of HAHS were researched by comparing the decreased range of absorbance value D (600) when equal quantity test bacterium react with HAHS influenced by factors at different levels for 1h。Staphylococcus aureus ATCC25923 was used as test bacteria.5. We made transmission electron microscope specimens,and observed the possible antimicrobial target of HAHS by TEM.Results1. We used S. aureus ATCC25923 to test the antibacterial activities of fresh amnion and acellular amnion. The results showed the medium attached with acellular amnion had no bacteriostatic ring, and the amnion epithelium was proved to remove completely by HE staining. However, there were significant bacteriostatic ring around fresh human amnion. HE staining showed the epithelium was complete.2. Kirby-Bauer method was used to test the antimicrobial spectrum of HAHS. The results indicated that HAHS had a marked antibacterial role for most selected bacteria including S. aureus ATCC25923, E. coli. ATCC25922, P. aeruginosa ATCC27853, Steptococcus Pneumoniae, StapHylococcus epidermidis, StapHylococcus haemolyticus, Proteus mirabilis, Enterococcus faecalis, Blastomyces albicans, Fusarium solani and Aspergillus fumigatus. However, HAHS did not have significant antibacterial effect on Enterobacter cloacae, E. coli and Methicillin-resistant StapHylococcus aureus (MRSA).3. Compared by molality, in the S. aureus ATCC25923 group:MIC value of HAHS was much smaller than chloramphenicol, was equal with clindamycin and was greater than the other three antibiotics, while MBC value of HAHS was not greater than that of any one of five antibiotics; In the E. coli. ATCC25922 group: MIC value of HAHS was less than those of sulfacetamide sodium and cefuroxime sodium, was equal with that of tobramycin and was greater than those of gentamycin and norfloxacin. But, MBC value of HAHS was not greater than those seleted five antibiotics.4. HAHS had a good bactericidal action on S. aureus ATCC25923 when pH value was 7(△D(600)=0.00338). Meanwhile, HAHS had the same effect on ATCC25923 at the other four pH values (pH values were 5, 6, 8 and 9 respectively). The antibacterial activities of HAHS at five pH values were not significant different. At 37℃, HAHS had a good bactericidal action on S. aureus ATCC25923(△D(600)=0.00295). Furthermore, when the temperatures were -20℃,4℃,20℃and 60℃respectively, the bactericidal action of HAHS had no marked change than that of 37℃. However, treatment with HAHS at 80℃, its bactericidal action decreased significantly than those of the other five temperatures(△D(600)=0.00152,P<0.05), but HAHS still had bactericidal effect to some extent.We also observed the changes of HAHS antibacterial activity when HAHS was stored for 1 day, 3 day, 7day, 14 day, 28 day and 56 day at 4℃and 20℃, respectively. The results proved that HAHS antibacterial activity did not has significant change with the extension of storage time at the two temperatures. After storage for 56 day, HAHS could still kill S. aureus ATCC25923 effectively,△D (600) values of HAHS at 4℃and 20℃were 0.00297 and 0.00281, respectively.5. Observed under TEM: Normal E. coli was integrity, smooth and had splitting phenomenon usually. After treatment with HAHS for 1h, the shape of E.coli was shrinkage, the thickness and length became different, the surface was rough and it was difficult to observe splitting phenomenon of E.coli. Meanwhile, HAHS led to rupture of E. coli membrane with release of cellular contents.As to S. aureus, normal bacteria was ball type and full, the membrane was integrity and proliferation was active. After treatment with HAHS for 1h, the shape of staphylococcus aureus was irregular, the surface was rough and the splitting phenomenon could not be found. In addition, we also observed cell wall of bacteria was destroyed seriously and protoplast released.Conclusions1. Fresh amnion and HAHS both had significant antibacterial role, while acellular amnion had no effect on bacteria. The results showed antibacterial substances of amnion mainly existed in epithelial cells, so we considered fresh amnion should be used for reconstructing ocular surface in clinical operation.2. HAHS had broad antimicrobial spectrum. It had antibacterial effect on G+, G- , fungi and even multiple drug-resistant bacteria which usually induced ocular infection.3. Antibacterial capacity of fresh HAHS was greater than those of chloramphenicol, sulfonamides and cefuroxime, was equal with those of clindamycin and tobramycin. Moreover, bactericidal capacity of HAHS was no less than those of ten selected antibiotics.The values of MBC and MIC were equal for HAHS,this result illustrated HAHS's antibacterial effect was kill bacteria directly.4.HAHS was steady in nature and easy to save under clinical conditions,it can be an effective and conveninent treatment for ocular surface infectious diseases.5.The antibacterial effect of HAHS was exerted through plasma membrane of bacterium.