关键词: 肠道蠕虫, 肠道菌群, 16S rRNA, 代谢组学

Abstract:

Objective To reveal the effect of mild asymptomatic hookworm infection on intestinal microflora and metabolome in middle-aged and the elderly people, to provide reference information for study on interaction of helminth-intestinal microflora. Methods In October to November 2022, from the parasite infection survey in residents at the surveillance site for soil-transmitted nematodes infection in Qimen County and Huangshan District of Huangshan City, Anhui Province, the hookworm egg positives detected by modified Kato-Katz thick smear method were assigned in infection group, and the hookworm egg negatives in control group. Fresh fecal samples from the examinees were collected for extraction of intestinal microflora DNA, of which 16S rRNA gene sequence was amplified by PCR and sequenced. The abundance-based coverage estimator (ACE), PD-wholetree, Chao index and Shannon index were calculated. The principal coordinate analysis (PCoA) and β diversity analysis of the unweighted pair group method with arithmetic mean (UPGMA) hierarchical clustering were used to compare the difference in the distribution of intestinal microflora between the infection group and the control group, to screen the indicator flora by indicator value analysis. The metabolites in the fecal samples were extracted with methanol-water extraction method for metabolome analysis by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) for non-targeted sequencing. Orthogonal partial least squares discriminant analysis (OPLS-DA) was used to establish a model for screening differential metabolites between the infection group and the control group, and generate the volcano plot. The differential metabolites were screened based on P-values, variable weight value, and difference multiple. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to search the metabolic pathways of related differential metabolites. Enrichment analysis of differential metabolites was performed, and the degree of linear correlation between two metabolites was analyzed by Pearson correlation analysis. Results A total of 22 subjects were studied, including 11 subjects of positive fecal eggs and 11 subjects of negative control. The average age of the infected group was 72.2 years old, and all of them presented mild asymptomatic hookworm infection. The average age of the control group was 53.1 years old. A total of 1 755 649 16S rRNA sequences were obtained and 1 245 amplicon sequence variants were clustered after quality control and assembly. A total of 389 species from 15 phyla, 22 classes, 58 orders, 94 families, and 191 genera were identified. The results of the diversity index analysis showed that the ACE, PD whole tree, Chao and Shannon indexes of intestinal flora diversity in the infection group were 188.768, 16.533, 192.667 and 5.195, respectively, whereas the control group were 167.829, 15.294, 157.371 and 4.898, respectively. No significant difference was seen between the two groups （t = 1.266, 0.952, 1.266, 0.962, P > 0.05）. The PCoA analysis demonstrated that the intestinal microbiota of the infected group and the control group overlapped to varying degrees but could still be divided into two distinct taxa. Additionally, the UPGMA hierarchical clustering analysis exhibited that both the infection group and the control were clustered in the same group first, and then clustered with the other group, and only one case overlapped. Multivariate statistical analysis showed seven flora dominating in the infection group, including Prevotella, Feacalibacterium, Klebsiella variicola, Parasutteralla, Coprococcus, Clostridium UCG-014 and Enterobacterium; while in the control group, only B. vulgatus was abundant. The results of indicator value analysis revealed the bacteria with the largest indicator value that affect the intestinal environment in the infection group, including Prevotella, Klebsiella, Bacteroides, Dialister, Enterobacterium, Coprococcus, UCG-002 and Faecalibacterium in the infection group; while in the control group, Clade_Ⅰa, Clade_Ⅲ, Blautia, Alistipes, Lachnoclostridium, Bilophila, Turicibacter and Muribaculaceae were dominant. The OPLS-DA model could effectively distinguish the metabolites in fecal samples of the infection group and the control. A total of 400 different metabolites were identified in the two groups, of which 156 were significantly increased and 244 decreased. The KEGG enrichment analysis indicated that the differential fecal metabolites were mainly enriched in protein digestion and absorption (P = 0.000) and central carbon metabolism (P = 0.000). Positive correlations were noted in 15 pairs metabolites i.e. between delta2-THA and (22E)-3 beta-hydroxy-5 alpha-chola-7,22-dien-oic acid (r = 0.935, P < 0.01), and negative correlations were seen in 9 pairs metabolites i.e. between lysoPE and aminovaleric acid betaine (r = -0.500, P < 0.05). Conclusion Asymptomatic hookworm infection in middle-aged and elderly people in rural area of Huangshan City may affect the composition and metabolism of intestinal microflora, which may create a symbiotic and colonization environment for probiotics

Key words: Helminth infection, Gut microbiota, 16S rRNA gene, Metabolome