Dysbiosis of Gut Microbiota and Short-Chain Fatty Acids in Encephalitis: A Chinese Pilot Study

Abstract

Background: Encephalitis, the inflammation of the brain, may be caused by an infection or an autoimmune reaction. However, few researches were focused on the gut microbiome characteristics in encephalitis patients.

Methods: A prospective observational study was conducted in an academic hospital in Guangzhou from February 2017 to February 2018. Patients with encephalitis were recruited. Fecal and serum samples were collected at admission. Healthy volunteers were enrolled from a community. Disease severity scores were recorded by specialized physicians, including Glasgow Coma Scale (GCS), Sequential Organ Failure Assessment (SOFA), and Acute Physiology and Chronic Health Evaluation-II (APACHE-II). 16S rRNA sequence was performed to analyze the gut microbiome, then the α-diversities and β-diversities were estimated. Short-chain fatty acids (SCFAs) were extracted from fecal samples and determined by gas chromatography-mass spectrometry. Serum D-lactate (D-LA), intestinal fatty acid-binding protein (iFABP), lipopolysaccharide (LPS), and lipopolysaccharide-binding protein (LBP) were measured by enzyme-linked immunosorbent assay (ELISA). The associations among microbial indexes and clinical parameters were evaluated by Spearman correlation analysis.

Results: In total, twenty-eight patients were recruited for analysis (median age 46 years; 82.1% male; median GCS 6.5; median SOFA 6.5; median APACHE-II 14.5). Twenty-eight age- and sex-matched healthy subjects were selected as controls. The β-diversities between patients and healthy subjects were significantly different. The α-diversities did not show significant differences between these two groups. In the patient group, the abundances of Bacteroidetes, Proteobacteria, and Bacilli were significantly enriched. Accordingly, fecal SCFA levels were decreased in the patient group, whereas serum D-LA, iFABP, LPS, and LBP levels were increased compared with those in healthy subjects. Correlation analyses showed that disease severity had positive correlations with Proteobacteria and Akkermansia but negative correlations with Firmicutes, Clostridia, and Ruminococcaceae abundances. The cerebrospinal fluid albumin-to-serum albumin ratio (CSAR) was positively related to the α-diversity but negatively correlated with the fecal butyrate concentration.

Conclusion: Gut microbiota disruption was observed in encephalitis patients, which manifested as pathogen dominance and health-promoting commensal depletion. Disease severity and brain damage may have associations with the gut microbiota or its metabolites. The causal relationship should be further explored in future studies.

Keywords: dysbiosis; encephalitis; gut microbiome; intestinal barrier; short-chain fatty acids.

FIGURE 1

The gut microbiota composition of encephalitis patients was significantly different from that of healthy subjects. (A,B) The β-diversity in the ENC and CON groups was calculated by the Bray-Curtis distance (A) and unweighted UniFrac distance (B) and is shown in the PCoA plot (Adonis test, Bray-Curtis distance, R² = 0.090, p < 0.001; unweighted UniFrac distance, R² = 0.124, p < 0.001). Each point represents the composition of the intestinal microbiota of one participant. (C,D) The α-diversity of the microbiota, presented as the Shannon index (C) and PD-whole tree index (D), was calculated from samples from encephalitis patients and healthy subjects (Mann-Whitney U test, Shannon index, p = 0.098; PD-whole tree index, p = 0.350). The boxplots display the 95% CIs, and the points lying outside the whiskers are referred to as outliers. (E,F) Average relative abundances of the predominant bacterial taxa at the phylum (E) and family (F) levels in the ENC and CON groups. (G) Cladogram based on LEfSe results of the CON and ENC groups. The red points represent the increased taxa in ENC group, while the blue points represent the increased taxa in CON group. ENC, patients with encephalitis; CON, healthy subjects serving as controls.

FIGURE 2

Associations of gut microbial taxa with clinical indexes. Heat map of Spearman’s rank correlation coefficient among 17 clinical indexes and 26 taxa with abundances higher than 0.1%. n = 28; ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; Spearman’s rank correlation. WBC, white blood cell count; NEU, neutrophil count; RBC, red blood cell count; HGB, hemoglobin; PLT, platelet count; ALT, alanine aminotransferase; Tbil, total bilirubin; TP, total protein; ALB, albumin; Cl, serum chlorine, K, serum potassium; BUN, blood urea nitrogen; SCr, serum creatinine; CRP, C-reactive protein; PCT, procalcitonin; DD, D-dimer; BNP, brain natriuretic peptide; NSE, neuron-specific enolase; S100B, S100 calcium-binding protein B; CSAR, cerebrospinal fluid albumin-to-serum albumin ratio; GCS, Glasgow Coma Scale; SOFA, Sequential Organ Failure Assessment; APACHE-II, Acute Physiology and Chronic Health Evaluation-II; IOS, length of stay in the ICU.

FIGURE 3

Decreased intestinal microbiota diversity in encephalitis patients is not associated with survival in an exploratory setting. Based on the α-diversities in healthy subjects, the patient cohort was split into two groups: α-diversity < Median and α-diversity > Median, for which a 120-day Kaplan–Meier survival plot is shown. Twenty-eight encephalitis patients were divided based on the median of Shannon index [A, Log Rank p = 0.619, HR = 0.7917, 95%CI = (0.3055, 2.052)], PD-whole tree index [B, Log Rank p = 0.264, HR = 0.5835, 95%CI = (0.2246, 1.516)], observed species [C, Log Rank p = 0.056, HR = 0.4035, 95%CI = (0.1543, 1.055)], Chao1 index (D, Log Rank p = 0.859, HR = 0.9182, 95%CI = (0.3548, 2.376)], successively. M, median. Numbers below the curve were patients at risk per group.

FIGURE 4

Comparison of six fecal SCFA levels between encephalitis patients and healthy controls. (A) acetate; (B) propionate; (C) butyrate; (D) isobutyrate; (E) valerate; (F) isovalerate. *p < 0.05; **p < 0.01; ***p < 0.001; Mann-Whitney U test. ENC, patients with encephalitis; CON, healthy subjects serving as controls.

FIGURE 5

Comparison of intestinal integrity biomarkers between encephalitis patients and healthy controls. (A) D-lactate; (B) iFABP; (C) LPS; (D) LBP. ***p < 0.001; Mann-Whitney U test. iFABP, intestinal fatty acid-binding protein; LPS, lipopolysaccharide; LBP, lipopolysaccharide-binding protein; ENC, patients with encephalitis; CON, healthy subjects serving as controls.