Bacterial Dissemination to the Brain in Sepsis

Abstract

Rationale: Sepsis causes brain dysfunction and neuroinflammation. It is unknown whether neuroinflammation in sepsis is initiated by dissemination of bacteria to the brain and sustained by persistent infection, or whether neuroinflammation is a sterile process resulting solely from circulating inflammatory mediators.

Objectives: To determine if gut bacteria translocate to the brain during sepsis, and are associated with neuroinflammation.

Methods: Murine sepsis was induced using cecal ligation and puncture, and sepsis survivor mice were compared with sham and unoperated control animals. Brain tissue of patients who died of sepsis was compared with patients who died of noninfectious causes. Bacterial taxa were characterized by 16S ribosomal RNA gene sequencing in both murine and human brain specimens; compared among sepsis and nonsepsis groups; and correlated with levels of S100A8, a marker of neuroinflammation using permutational multivariate ANOVA.

Measurements and main results: Viable gut-associated bacteria were enriched in the brains of mice 5 days after surviving abdominal sepsis (P < 0.01), and undetectable by 14 days. The community structure of brain-associated bacteria correlated with severity of neuroinflammation (P < 0.001). Furthermore, bacterial taxa detected in brains of humans who die of sepsis were distinct from those who died of noninfectious causes (P < 0.001) and correlated with S100A8/A9 expression (P < 0.05).

Conclusions: Although bacterial translocation is associated with acute neuroinflammation in murine sepsis, bacterial translocation did not result in chronic cerebral infection. Postmortem analysis of patients who die of sepsis suggests a role for bacteria in acute brain dysfunction in sepsis. Further work is needed to determine if modifying gut-associated bacterial communities modulates brain dysfunction after sepsis.

Keywords: S100A8; cecal ligation and puncture; inflammation; microbiome; sepsis.

Figure 1.

Multiple bacterial taxa are detectable in the brain during early sepsis. Abdominal sepsis was induced in mice by cecal ligation and puncture, and bacterial DNA detected in homogenized brain tissue was studied after 24 hours. (A) Brain bacterial communities 24 hours after operation are distinct from unoperated control subjects in both sham-operated and septic animals (permutational multivariate ANOVA, P = 0.03), and all three groups are distinct by principal component analysis (P < 0.001 for all pairwise comparisons). (B) Multiple bacterial taxa are detected in brain homogenates 24 hours after induction of sepsis, without clear predominance of a single taxon. These taxa are generally not represented in the sequences recovered from either sham-operated or unoperated animals. Gray bars indicate background signal detected in brains of unoperated mice. Data presented are means ± SEM. CLP = cecal ligation and puncture; Otu = operational taxanomic unit; PC = principal component.

Figure 2.

Brain microbial communities evolve after cecal ligation and puncture. Sepsis was induced by cecal ligation and puncture, and bacterial DNA detected in homogenized brain tissue was studied in surviving mice and compared with sham-operated, antibiotic-treated, and untreated control animals after 5 days. (A) Brain bacterial communities 5 days after induction of sepsis are distinct from those isolated from all control groups (permutational multivariate ANOVA, P < 0.002). (B) By 5 days post–cecal ligation and puncture, the detected microbial taxa are dominated by Bacteroidales OTU008, which is not detected in control groups. (C) Bacteroidales OTU008 dominates the bacterial community 5 days after sepsis (**P < 0.01) and returns to background levels by 14 days. Gray bars indicate background signal detected in brains of unoperated mice. Values presented as means ± SEM. Statistical significance was determined by Kruskall-Wallis one-way ANOVA with Dunn multiple comparison test (C). OTU = operational taxanomic unit; PC = principal component.

Figure 3.

Experimental sepsis results in translocation of live aerobic and anaerobic bacteria to the brain. (A) Five days after cecal ligation and puncture, Enterococcus faecalis was isolated from the brains of five of six postsepsis mice but was undetectable in the brains of mice in all control groups (P = 0.002). (B) OTU008 corresponds to a live bacterium, not carryover of nucleic acids. PCR specific to OTU008 shows the presence of bacterial growth in brain homogenates in anoxic conditions after sepsis but fails to detect OTU008 signal in raw brain homogenates or in unoperated control animals. Values presented as means ± SEM. Statistical significance was determined by Kruskall-Wallis one-way ANOVA with Dunn multiple comparison test (A). OTU = operational taxanomic unit.

Figure 4.

Neuroinflammation is associated with altered brain microbiota in patients with sepsis. Bacterial DNA was isolated and sequenced from postmortem brain specimens in patients who died of sepsis or a noninfectious illness. (A) Heterogeneous bacterial taxa are detected in brains of patients with and without sepsis. Model-based analysis of multivariate abundance identified Haemophilus, Neisseria, and Moraxella spp. as differentially abundant in sepsis and nonsepsis brain communities. A single brain specimen was enriched with Enterobactericiae sp. (OTU0017) and two were enriched with Bacteroides sp. (OTU0014). Pink boxes indicate upper airway–associated taxa, and brown boxes indicate gut-associated taxa. (B) Bacterial communities detected in cerebral cortex specimens from patients with sepsis were distinct from those of patients who died of noninfectious illness (P < 0.001). S100A8/A9 (S100 calcium binding protein A8 and A9 heterodimer) concentration is correlated with differences in bacterial taxa (P < 0.05). The area of each circle is proportional to the level of S100A8/A9 protein. Statistical significance was determined by Mann-Whitney U test (A) and permutational multivariate ANOVA (adonis) (B). OTU = operational taxanomic unit; PC = principal component.

Figure 5.

Neuroinflammation is enhanced in postsepsis brains and correlated with changes in brain microbiota in experimental sepsis. S100A8 (S100 calcium binding protein A8) expression was correlated both with OTU008 abundance (A, permutational multivariate ANOVA [adonis], P < 0.001) and overall microbial community structure 5 days after operations (B, permutational multivariate ANOVA [adonis], P ≤ 0.001). (B) Relative expression of S100A8 is proportional to marker size. In addition to increased tissue damage–associated molecular pattern expression, sepsis survivor mice exhibit increased reactivity by microglia and brain tissue macrophages. (C) Immunfluorescence of motor cortex show markedly increased size and density of Iba1⁺ microglial cell processes. (D) Immunoreactivity for Iba1 is increased in cecal ligation and puncture survivors on Day 5 post–cecal ligation and puncture in both area CA1 and overlying motor cortex (Mann-Whitney U test, P = 0.035 in both areas). *P < 0.05. (E) Microglia/macrophages were isolated by flow cytometry as a CX3CR1⁺CCR2⁻ cell population. (F) When stimulated ex vivo with LPS, CX3CR1⁺CCR2⁻ cells isolated from sepsis survivor mice 5 days post–cecal ligation and puncture produce increased tumor necrosis factor-α compared with sham-operated (ANOVA, P < 0.0001; Sidak post hoc, P = 0.0002) or antibiotic-treated mice (Sidak post hoc, P < 0.0001). ***P < 0.001. Values are presented as means ± SEM. A.U. = arbitrary units; CCR2 = C-C motif chemokine receptor 2; CLP = cecal ligation and puncture; CX3CR1 = C-X3-C motif chemokine receptor 1; GFP = green fluorescent protein; Iba1 = ionized calcium binding adapter molecule 1; OTU = operational taxanomic unit; PC = principal component; RFP = red fluorescent protein; S1008A8/A9 = S100 calcium binding protein A8 and A9 heterodimer; TNF = tumor necrosis factor.