Abstract Background We evaluated the feasibility of plasma 16S rRNA gene amplicon sequencing in sepsis in order to determine if clinically interpretable profiles of bacterial DNA and composition are present in plasma. We tested whether these profiles differ across validated, clinical sepsis subtypes. Methods We performed bacterial DNA quantification using ddPCR amplification of the 16S rRNA gene and taxonomic identification using Illumina MiSeq 16S rRNA gene amplicon sequencing on plasma samples from 50 adults with sepsis (two specimens per patient: coordinator-collected and remnant; total n = 100). Extensive negative controls (blank wells, buffer, isolation controls, sterile water) and positive mock communities were sequenced in parallel. Plasma specimens were compared to negative controls using permutation testing. Among coordinator-collected samples, we compared previously validated, prognostically divergent sepsis subtypes (α, β, γ, δ) using Shannon diversity, measured ddPCR 16S copies/mL, and relative abundance of representative bacterial phyla (Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria). Results The cohort had a mean age of 54.4±16.8 years, 64.2% were men, and the mean SOFA score was 3.0±1.9. Plasma specimens contained significantly higher bacterial DNA compared with no-template controls (Mann-Whitney, p = 0.0012). Community composition was distinct between plasma and negative controls (PERMANOVA, p 0.001), indicating that the plasma contained distinct bacterial signature compared to background contamination. Ordination showed Enterobacteriaceae dominated the control signal, whereas Lactobacillus and Akkermansia represented plausible host-derived taxa. Subtype sample sizes were α (15), β (16), γ (6), and δ (13). Shannon diversity was uniformly low and similar across subtypes (Kruskal-Wallis, p = 0.92). Genus-level β-diversity revealed no global subtype effect (PERMANOVA, p = 0.82). ddPCR 16S copies/mL overlapped across subtypes, and major phyla (Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria) were similar. Conclusions Plasma 16S rRNA gene amplicon sequencing is feasible and reveals a circulating bacterial DNA signal distinct from contamination controls. We found no robust differences in diversity, bacterial burden, or overall composition across clinical sepsis subtypes, though interpretation is limited by small sample size. These findings support the feasibility of characterizing bacterial DNA in sepsis and motivate larger, powered studies to test endotype-specific hypotheses. This abstract is funded by: NIH
Chowdhury et al. (Fri,) studied this question.