Processed human amniotic fluid retains its antibacterial activity

Abstract

Background: Human amniotic fluid (AF) contains numerous nutrients, trophic factors and defense proteins that provide a nurturing and protective environment for fetal development. Based on reports that AF has antibacterial, anti-inflammatory and regenerative properties, we designed a novel method to process AF for use in clinical care.

Methods: Six randomly selected lots of processed AF (pAF) were examined to determine whether they retained their antibacterial activity against a panel of wound-associated pathogens E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, and E. aerogenes (ESKAPE). To identify proteins in pAF that might be responsible for its antibacterial activity, three different lots of pAF were analyzed with quantitative cytokine arrays that consisted of 400 unique human proteins. One protein identified by microarrays, lactoferrin, and a second prominent antibacterial protein that was not identified by microarrays, lysozyme, were examined by depletion experiments to determine their contribution to the antibacterial activity of pAF.

Results: All six lots of pAF exhibited antibacterial activity against ESKAPE microorganisms, especially against the pathogens predominately found in chronic wounds (i.e. S. aureus and P. aeruginosa). Thirty-one of the peptides on the microarray were annotated as having antibacterial activity and 26 of these were detected in pAF. Cystatin C and lactoferrin were among the most highly expressed antibacterial proteins in pAF. Cystatin C and lactoferrin were confirmed by ELISA to be present in pAF along with lysozyme. Immunoprecipitation of lactoferrin and lysozyme reduced, but did not abolish the antibacterial activities of pAF.

Conclusion: Our data demonstrate that pAF maintains antibacterial activity via the preservation of antibacterial proteins against a broad spectrum of wound-associated pathogens.

Keywords: Amniotic fluid; Antibacterial; ESKAPE; Immunoprecipitation.

Fig. 1

Relative antibacterial activity of AF before and after processing. The growth of P. aeruginosa (a) or S. aureus (b) in tryptic soy broth without AF or in TSB with different concentrations of AF were quantified using alamarBlue assay. The percent of growth reduction with AF/pAF is calculated as: fluorescent intensity of culture with AF/fluorescent intensity of control (no AF) × 100. Data are presented as mean ± SD (n = 3)

Fig. 2

Antibacterial activity of processed AF. The growth of P. aeruginosa (a) or S. aureus (b) in different lots of pAF, PBS or were quantified by serial dilution and CFU counting after 24 h culture. Data are presented as mean ± SD for each lot (n = 3). Statistical analysis was performed to compare each lot of pAF with Control (Ctrl). **p < 0.01 and ***p < 0.005

Fig. 3

Proteins tested in pAF with known antibacterial activity. Quantibody^® Human Cytokine Antibody Arrays 9000 were used to examine the presence or absence of 400 different peptides in pAF. Of the 400 peptides that were tested (i.e. three lots other than six lots examined for antibacterial activity), an average of 304 ± 20 proteins (n = 3) were positively identified in pAF. Thirty-one of the 400 proteins that were tested were known as antibacterial peptides. Of the 31 antibacterial proteins that were tested, 26 were present in pAF. Among the detectable antibacterial peptides, 17 of them are also classified as chemokines (black bars) and 9 of them are not chemokines (white bars)

Fig. 4

The presence of antibacterial proteins in multiple lots of AF. The levels of lysozyme (a), cystatin C (b) or lactoferrin (c) in different lots of AF were quantified using ELISA as described in Materials and methods. Data are presented as mean ± SD (n = 4)

Fig. 5

Immunoprecipitation (IP) of lysozyme and lactoferrin reduced the antibacterial activity of pAF. The growth of P. aeruginosa (a) or S. aureus (b) in pAF after IP or Co-IP of lysozyme and lactoferrin or after a mock IP was quantified. Data are presented as mean ± SD (n = 3), *p < 0.05, **p < 0.01