Changes and roles of intestinal fungal microbiota in coronary heart disease complicated with nonalcoholic fatty liver disease

Abstract

Background: Patients who suffered coronary heart disease (CHD) complicated with non-alcoholic fatty liver disease (NAFLD) were reported to have worse cardiac function and clinical outcomes than patients with CHD only. The mechanism was unclear. Previous study focused on the metabolism and showed it could be regulated by the microbiota. Few studies related to fungi. We aimed to investigate the characteristics of intestinal fungal microbiota in CHD patients complicated with NAFLD (CHD-NAFLD).

Methods: 72 People were recruited and equally divided into three groups, including CHD patients (without NAFLD), CHD-NAFLD patients, and healthy controls (HCs). Fecal samples were collected. The Illumina sequencing of the internal transcribed spacer 3-4 rRNA was applied.

Results: The BMI, uric acid and triglyceride in CHD-NAFLD patients increased compared with CHD patients. The abundance of Exophiala attenuata and Malassezia restricta in all CHD-NAFLD and CHD patients significantly reduced. The intestinal fungal microbiota in CHD-NAFLD patients showed an increase in the abundance of Preussia, Xylodon and Cladorrhinum, and a reduction in the abundance of Candida glabrata and Ganoderma. Among them, the abundance of Ganoderma was significantly lower than that in CHD patients. The ejection fraction was negatively correlated to the abundance of Xylodon. Uric acid was positively correlated with the abundance of Cladorrhinum and Preussia.

Conclusions: These changes of intestinal fungal microbiota in CHD-NAFLD patients may be important factors affecting the degree of metabolic disorder. But there are few reports on these fungi. More studies are needed to confirm the effects of these fungi on human.

Keywords: Non-alcoholic fatty liver disease; coronary heart disease; intestinal microbiota.

Figure 1

The diversity of the fecal fungal microbiota. A. The Shannon index in the overall CHD patients. B. The Chao1 index in the overall CHD patients. C. The PCoA analysis of the overall CHD patients. D. The Shannon index in CHD-NAFLD patients. E. The Chao1 index in CHD-NAFLD patients. F. The PCoA analysis of CHD-NAFLD patients. The “CHD+CHD-NAFLD” stood for the overall CHD patients. ns, not significant. 70 samples were used in each analysis. Kruskal-Wallis H test was used in the comparsion of Shannon index and Chao1 index. In the comparsion of PCoA analysis, adonis test was used.

Figure 2

The composition of the fungi at the phylum and genus level. A. The fungi at the phylum level in the overall CHD patients. B. The fungi at the genus level in the overall CHD patients. The top 13 genus in abundance was listed. C. The fungi at the phylum level in the CHD-NAFLD and CHD patients. D. The fungi at the genus level in the CHD-NAFLD and CHD patients. The top 13 genus in abundance was listed. 70 samples were used in each analysis.

Figure 3

The specific microbiota at the genus and species level. A. The specific fungal microbiota at the genus level. B. The specific fungal microbiota at the species level. The R3.5.1 with indicspecies package was used. Permutation test was performed. The shape of the graph represents the comparison in enrichment (circle) or depletion (triangle) between three groups. The size of the graph indicates the relative abundance. *P≤0.05; **P≤0.01; ***P≤0.001. 70 samples were used in analysis.

Figure 4

The comparison of fungal microbiota in CHD patients. A. The comparison of fungal microbiota between CHD patients and HCs at genus level. B. The comparison of fungal microbiota between CHD patients and HCs at species level. The t test and STAMP was used. 70 samples were used in each analysis.

Figure 5

The comparison of fungal microbiota in CHD-NAFLD patients. A. The comparison of fungal microbiota between CHD-NAFLD patients and HCs at genus level. B. The comparison of fungal microbiota between CHD-NAFLD patients and HCs at species level. C. The comparison of fungal microbiota between CHD-NAFLD patients and CHD patients using the Lefse. The t test and STAMP was used. 70 samples were used in analysis.

Figure 6

The correlation analysis between clinical indexes and fungal microbiota. A. The correlation analysis between clinical indexes and fungal microbiota at the genus level. B. The correlation analysis between clinical indexes and fungal microbiota at the species level. The Spearman’s correlation analysis was used. R 3.5.1 software with pheatmap package was use for visualization. *P≤0.05; **P≤0.01; ***P≤0.001. 70 samples were used in analysis.

Figure 7

Function annotation of fungal microbiota. The comparison of OTUs of function annotation in fungal microbiota between CHD patients, CHD-NAFLD patients and HCs. The software FunGuild was used. 70 samples were used in analysis.