[Differential analysis of intestinal flora in patients with hepatic blastomycosis based on second-generation sequencing].

Abstract

Exploring the variability of the intestinal flora of patients with hepatic blastocysticercosis and searching for members of the intestinal microflora that may play a role in the disease process by means of macro-genome sequencing technology. A case-control study was used to include fecal samples from patients with hepatic vesicular schistosomiasis admitted to Qinghai Provincial People's Hospital between October 2023 and January 2024 and individuals attending health checkups. The experimental group (AE group) consisted of 10 patients with liver vesicular schistosomiasis and the control group (NC group) consisted of 9 individuals attending health checkups. Macrogenomic sequencing was performed on these two groups of samples using the Illumina Novaseq 6000 sequencing platform, using fastp (v0.20.1) to remove junctions, and bbmap (v38.93-0) to remove the hosted sequences, followed by sequence splicing using MEGAHIT (v1.2.9), and then using prodigal (v2.6.3) to The spliced scaffold was subjected to ORF prediction and translated into amino acid sequences, followed by the construction of a non-redundant gene set using MMSeqs2 (v13.45111), and finally compared with the non-redundant gene set using salmon (v1.8.0). Species were annotated by the non-redundant database, species abundance was calculated in each sample, and the two sets were tested using Wilcoxon rank sum test. Finally, the differences in intestinal flora between the two groups were statistically analyzed using linear discriminant analysis, and the correlation between the differential intestinal flora and clinical indicators was analyzed using redundancy analysis (RDA). The results showed that the effective data volume of each sample was distributed from 10.41 to 12.46 G. The number of ORFs in the de-redundantly constructed gene catalogue (non-redundant gene set) was 4 951 408, and the annotation rate of the non-redundant genes was 97.97% when compared with the NR database. The ages of the study subjects in the two groups were (44.78±4.58) years in the NC group and (42.90±10.44) years in the AE group, and the difference was not statistically significant (t=0.530, P=0.476). The two groups were matched for body mass index (BMI) (t=2.368, P=0.142), gender (χ²=0.200, P=0.655), and dietary habits. There was no statistically significant difference in alpha diversity in the AE group (ACE index, t=0.942; chao1 index, t=0.947; shannon index, t=0.813, the simpson's index, t=0.613, P>0.05), while beta diversity analysis showed significant differences in the overall structure of the two communities (Stress=0.054 5). A total of 120 species were annotated at the phylum level, of which two differed. While 1 736 species were annotated at the genus level, 69 were different, and 309 were different at the species level. The AE group ranked the top 6 in terms of abundance of Anaplasma, Escherichiaceae, Clostridium, Alternaria, Ruminalia, and Treponema spp. at the genus level; whereas, Segatella, Prevotella, E. faecalis, Rossella, and beneficial rod-shaped bacteria were more abundant in the NC group. There were differences in the abundance and diversity of intestinal flora between the two groups, and the structure of community composition was significantly different. Statistical results by linear discriminant analysis (LDA) showed that LDA scores >2 in the NC group included beneficial bacillus spp. and E. faecalis spp. in young infants, etc. LDA scores >2 in the AE group at the mid-species level included Clostridium polterococcus, unknown microorganisms in the genus Clostridium intestinalis, Hathaway's Henkett's bacillus, and Clostridium oryzae in the genus Clostridium refractory to culture and small Clostridium spp. in the AE group. Clostridium intestinalis. The RDA results showed a negative correlation between beneficial rod genera and liver function indices, and a positive correlation between Clostridium intestinalis genera and liver function indices. In conclusion, patients with hepatic blastomycosis have altered intestinal flora abundance and diversity, with significant structural changes in community composition and differences in several genera, including Mycobacterium anisopliae and Clostridium intestinalis, and imbalances in the intestinal flora may affect hepatic function by influencing intestinal metabolites and may have an impact on the development of hepatic blastomycosis, a finding that warrants further in-depth study.