Characterising Commensal and Pathogenic Staphylococcal Interactions with Neonatal and Adult Blood

Peripheral blood samples (0.45 ml) were collected from 8 preterm infants (30–32 weeks gestational age [GA]), 8 term infants (37–41 weeks) GA, and 8 adults (18–25 years). Samples were challenged ex vivo with a defined dose of approximately 1x10^7 colony-forming units (CFU) of either Staphylococcus epidermidis WT 1457 or Staphylococcus aureus ATCC 29523 for 90 minutes. An additional aliquot (0.1 ml) from each participant's blood was retained as an unstimulated baseline control. Following the bacterial challenge, a previously optimized RNA extraction protocol was employed to isolate total RNA from leukocytes and bacteria in all samples (n = 72). Ribosomal RNA was depleted from the purified RNA, which was subsequently sequenced using 150 bp paired-end reads on a NovaSeq 6000 platform (Illumina). Sequencing reads were aligned to host and bacterial reference genomes in silico, and bioinformatic analyses were performed to investigate host- and pathogen-specific transcriptional changes associated with early infection in this sepsis model.