Collection and processing of amniotic fluid
Donor consent, screening and infectious disease testing of amniotic fluid (AF) were previously described . The collection and processing of AF were also previously described . Prior to processing AF, several aliquots were removed and stored frozen at − 80 °C (i.e. pre-processed AF). Upon the completion of the final processing step, aliquots of pAF were removed and the samples were maintained at − 80 °C until the time of use.
Bacterial culture and preparation of inoculums
Clinical isolates of ESKAPE bacterial strains were purchased from ATCC (Manassas, VA): E. faecium ATCC® 51559™, S. aureus ATCC® 25923™, K. pneumoniae ATCC® 700603, A. baumannii ATCC® 49466™, P. aeruginosa ATCC® 15692™, and E. aerogenes ATCC® 49469™. All bacterial strains were cultured and maintained as instructed by ATCC®. Optimal culture media used are tryptic soy broth (TSB) for S. aureus, P. aeruginosa, E. aerogenes, A. baumannii, brain heart infusion (BHI) medium for E. faecium and nutrient broth for K. pneumoniae. Preparation of bacterial inoculum was performed as described previously . Briefly, bacteria were cultured in their defined optimal culture medium at 37 °C with shaking until optical density reached 0.2 to 0.6 at 600 nm (OD600). The number of colony forming units (CFUs) for each strain was estimated based on an OD600 = 1.0, which corresponds to 109 CFU/mL. To prepare the inoculum for antibacterial assays, the bacterial stocks were serially diluted with culture medium or pAF to approximately 1 × 103 CFU/mL of bacteria. For each experiment, the actual CFU of each inoculum was determined by preparing serial dilutions and then plating onto TSB agar plates (BD, Franklin Lakes, NJ).
Inhibition of bacterial growth by pAF
An inoculum of P. aeruginosa or S. aureus (1 × 103 CFU/mL) was added to pAF that had been prepared by diluting pAF in TSB. The cultures were incubated at 37 °C for 24 h. The growth of bacteria was monitored using an alamarBlue assay (ThermoFisher Scientifics; Waltham, MA) by following the manufacture’s protocol. The fluorescent intensity was measured using a TECAN Spark 10 M plate reader (TECAN, Morrisville NC) at Ex560 nm/Em590 nm. The growth of bacteria in the presence of pAF was normalized to the growth in the absence of pAF.
Quantification of bacterial growth in the presence of pAF
Bacterial growth was assessed as described with modifications . Each strain of bacteria (1 × 103 CFU/mL) was added to 1 mL of AF or 1 mL optimal culture medium. The cultures were incubated at 37 °C with shaking for 24 h. Serial dilutions were then prepared for each culture and plated onto TSB agar plates. CFUs were counted after overnight incubation at 37 °C. The antibacterial activity of pAF was expressed in log reductions, which was calculated as the log10CFU (Control) − log10CFU (pAF). Data from two independent experiments (n = 3 for each experiment) were pooled together to calculate the mean and standard deviation.
Quantitative Protein arrays were performed as previously described . Briefly, pAF from three maternal collections were sent to RayBiotech to simultaneously and quantitatively measure the concentration of 400 human cytokines using the Quantibody ® Human Cytokine Antibody Array 9000 (RayBiotech, In., Norcross, GA). Controls and serial dilutions of cytokine standards were prepared according to the manufacturer’s instructions and were added to chip wells. After processing the chips according to the manufacturer’s instructions, the chips were analyzed using the Quantibody® Q-Analyzer software (RayBiotech, Inc.). Proteins were classified according to their biological function by surveying the Human Protein Reference Database (http://www.hprd.org/index_html), Cytokines & Cells Online Pathfinder Encyclopedia (COPE) http://www.copewithcytokines.de/), GeneCards® (http://www.genecards.org/), and the biomedical literature in PubMed (http://www.ncbi.nlm.nih.gov/pubmed).
Detection of human lysozyme, cystatin C and lactoferrin in pAF using ELISA
The presence of lysozyme, cystatin C and lactoferrin from different lots of pAF was quantified using quantitative sandwich ELISA assays according to the manufacturer’s instructions (Abcam, Cambridge, MA): Lysozyme (Human Lysozyme ELISA kit ab108880), Lactoferrin (Human Lysozyme ELISA kit ab200015), and cystatin C (Human Lysozyme ELISA kit ab179883).
Selective depletion of lysozyme and lactoferrin from AF was accomplished by immunoprecipitation (IP) as described . Briefly, rabbit polyclonal anti-lysozyme antibody (ab2408) and anti-lactoferrin antibody (ab15811) were purchased from Abcam. Anti-lysozyme antibodies (15 μg/mL) and anti-lactoferrin antibodies (4 μg/mL) were added individually (IP) or together (Co-IP) to 1 mL of AF. As a control, the same volume of PBS was added to 1 mL of AF. The samples were mixed at 4 °C for overnight. A 50 µL slurry of Protein A/G agarose plus resin: sc-2003 (Santa Cruz Biotechnology; Dallas, TX) was washed twice with 1 mL of PBS and mixed with each pAF with or without antibodies for 4 h at 4 °C. Mixtures were then centrifuged at 2000 rpm for 3 min and resulting supernatants were transferred to individual bacterial culture tubes. Inoculums of 1 × 103 CFU of P. aeruginosa or S. aureus were added to each culture tube and incubated with shaking at 37 °C. After 24 h, CFUs for each culture were quantified by serial dilution plating as previously described .
Each independent experiment contained 3 or more biological repeat samples (n ≥ 3), and data is presented as the mean ± standard deviation. One-way ANOVA with a Tukey’s multiple comparisons test was performed to determine statistical significance. Differences were considered significant at a p value of < 0.05.