Quantification of Plasma Inflammatory Markers

Cryopreserved plasma samples were used for quantification of inflammatory cytokines, chemokines and biomarkers using the V-PLEX Human Biomarker 40-Plex kit (MesoScale Diagnostics). Cell-free DNA levels were quantified using the Quanti-iT PicoGreen kit (Invitrogen) according to the manufacturer’s instructions and quantification of MPO–DNA complexes in plasma was performed as previously described^{55 (link)}. Differential abundance of 40-plex plasma inflammatory biomarkers was performed on the log₂-transformed concentration values using the limma package v.3.48.3 in R, as previously described^{67 (link)}. Dimensionality reduction and visualization of the differences in plasma inflammatory mediators between patients with and without microbiota Enterobacteriaceae enrichment was performed using NMDS of mediator concentrations using the Vegan package v.2.6 in R. Figures showing log₂FC of individual mediators between ICU patients with or without Enterobacteriaceae enrichment were calculated on absolute concentration using the limma package v.3.48.3 and plotted with ggplot2 . No clinical covariables were significantly associated with inflammatory mediator landscape composition (Supplementary Table 16).