Antimicrobial Peptides Secreted From Human Cryopreserved Viable Amniotic Membrane Contribute to its Antibacterial Activity

Yong Mao,Matthew Moorman,Tyler Hoffman,Joachim Kohn,Anya Singh-Varma,Alla Danilkovitch,Yi Duan-Arnold

doi:10.1038/s41598-017-13310-6

Abstract

Chronic wounds remain a large problem in the field of medicine and are often associated with risk of infection and amputation. Recently, a commercially available human cryopreserved viable amniotic membrane (hCVAM) has been shown to effectively promote wound closure and reduce wound-related infections. A sprevious study indicates that hCVAM can inhibit the growth of bacteria associated with chronic wounds. In the present study, we investigated the mechanism of hCVAM antimicrobial activity. Our data demonstrate that antimicrobial activities against common pathogens in chronic wounds such as P.aeruginosa, S.aureus and Methicillin-resistant S.aureus (MRSA) are mediated via the secretion of soluble factors by viable cells in hCVAM and that these factors are proteins in nature. Further, we show that genes for antimicrobial peptides (AMPs) including human beta-defensins (HBDs) are expressed by hCVAM and that expression levels positively correlate with antimicrobial activity of hCVAM. At the protein level, our data indicate that HBD2 and HBD3 are secreted by hCVAM and directly contribute to its activity against P. aeruginosa. These data provide evidence that soluble factors including AMPs are hCVAM antimicrobial agents and are consistent with a role for AMPs in mediating antimicrobial properties of the membrane.

Highlights

Fresh human placental membranes have been used in treating wounds for more than 100 years[3,4]
Methicillin-resistant S.aureus (MRSA) growth was more sensitive to human cryopreserved viable amniotic membrane (hCVAM) condition medium than MSSA, suggesting that secreted soluble factors play a larger role in hCVAM activity of methicillin-resistant forms of S. aureus than methicillin-sensitive forms
We focused on P. aeruginosa, one of the most common pathogens in chronic wounds, and two S. aureus strains, MRSA and MSSA

Summary

Introduction

Fresh human placental membranes have been used in treating wounds for more than 100 years[3,4]. In vitro studies demonstrated a significant reduction in the growth of ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa or Enterobacter aerogenes) pathogens in the presence of hCVAM, suggesting that hCVAM retains intrinisic antimicrobial properties of the amnion[7]. These properties likely contribute to the positive outcomes observed with hCVAM in clinical studies. Previous studies have demonstrated that endogenous viable cells in fresh amnion and hCVAM are the source of cytokines and immune modulators which mediate a number of functional properties of the membrane[5,16,17,18]. Results demonstrate that HBDs secreted by endogenous cells mediate hCVAM antimicrobial activity against P. aeruginosa

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