The chicken gut metagenome and the modulatory effects of plant-derived benzylisoquinoline alkaloids

Abstract

Background: Sub-therapeutic antibiotics are widely used as growth promoters in the poultry industry; however, the resulting antibiotic resistance threatens public health. A plant-derived growth promoter, Macleaya cordata extract (MCE), with effective ingredients of benzylisoquinoline alkaloids, is a potential alternative to antibiotic growth promoters. Altered intestinal microbiota play important roles in growth promotion, but the underlying mechanism remains unknown.

Results: We generated 1.64 terabases of metagenomic data from 495 chicken intestinal digesta samples and constructed a comprehensive chicken gut microbial gene catalog (9.04 million genes), which is also the first gene catalog of an animal's gut microbiome that covers all intestinal compartments. Then, we identified the distinctive characteristics and temporal changes in the foregut and hindgut microbiota. Next, we assessed the impact of MCE on chickens and gut microbiota. Chickens fed with MCE had improved growth performance, and major microbial changes were confined to the foregut, with the predominant role of Lactobacillus being enhanced, and the amino acids, vitamins, and secondary bile acids biosynthesis pathways being upregulated, but lacked the accumulation of antibiotic-resistance genes. In comparison, treatment with chlortetracycline similarly enriched some biosynthesis pathways of nutrients in the foregut microbiota, but elicited an increase in antibiotic-producing bacteria and antibiotic-resistance genes.

Conclusion: The reference gene catalog of the chicken gut microbiome is an important supplement to animal gut metagenomes. Metagenomic analysis provides insights into the growth-promoting mechanism of MCE, and underscored the importance of utilizing safe and effective growth promoters.

Keywords: Antibiotic; Benzylisoquinoline alkaloid; Chicken; Chlortetracycline; Growth promoter; Gut metagenome; Microbiome.

Fig. 1

Chicken gut microbial gene catalog. a Diagram of chicken intestinal tract. The microbial densities in the foregut and hindgut were labeled. b Rarefaction curves of detected genes from the whole set of 495 samples (Total) and from subgroups of LY, AA, and Distribution. A total of 9.04 million non-redundant genes were detected, and the rarefaction curve including all samples approaches saturation at the end of sampling. The gene number of a specific number of samples was calculated after random samplings repeated 100 times with replacement, and the median was plotted. c Venn diagram of gut microbial genes shared between the chicken, human, and pig catalogs. The criteria for shared genes were sequence identity > 95% and overlap > 90% of the shorter gene. d Taxonomic annotation of the chicken gut gene catalog at the superkingdom and phylum levels. e Venn diagram of KEGG orthologous groups (KOs) present in and shared by chicken, human, and pig catalogs. f Comparison of KEGG functional profiles (relative gene abundance summarized into KEGG functional categories and genes without functional annotations were excluded) of gut microbiome among chickens, humans, and pigs. Asterisks denote Wilcoxon rank-sum test result (P < 0.005)

Fig. 2

Comparison of gut microbiome in different intestinal compartments of chickens. a Microbial diversity (Shannon index) at gene, genus, OG, and KO levels. Box plots show median ± interquartile range (IQR) and 1.5 IQR ranges (whiskers), with outliers denoted by dots. b The non-metric multidimensional scaling (NMDS) plot based on Bray-Curtis dissimilarities at species level. An obvious difference was observed between the foregut (duodenum, jejunum, and ileum) and hindgut (cecum and colorectum). c Differences in microbial functions between the foregut and hindgut based on KEGG functional categories (Wilcoxon rank-sum test, Storey’s methods for multiple tests adjustment). Chicken gut microbial co-occurrence network analysis based on core genus (average relative abundance > 0.1%) d in the foregut and e hindgut. Solid line: Spearman’s rank correlation coefficient > 0.30; dash line: Spearman’s rank correlation coefficient < − 0.30. The size of nodes was proportional to the relative abundance of genera

Fig. 3

Differences in the chicken intestinal microbiome at different ages. a The NMDS plot of microbial communities in the foregut at different ages. The analysis was based on Bray-Curtis dissimilarities at the species level, and samples were grouped according to the ages. b Microbial diversity (Shannon index) in the foregut at gene, genus, OG, and KO levels. Box plots show median ± interquartile range (IQR) and 1.5 IQR ranges (whiskers), with outliers denoted by dots. The relative abundance changes in major c phyla and d genera at different ages in both the foregut and hindgut. The area of the circles represents the relative abundance of each phylum and genus. e Relative abundance of KEGG metabolic pathways of the microbiome in the foregut at different ages

Fig. 4

Differences in microbial changes after CTC and MCE treatment. a The NMDS plot of microbial communities in CTC and MCE groups, based on Bray-Curtis dissimilarities at the species level. The obvious difference was in the foregut. b The average relative abundances of genera increased by MCE or CTC in the foregut. Kitasatospora and Streptomyces were significantly (P < 0.05) increased by CTC. c The average relative abundance of ARGs was increased (P < 0.1) by CTC in the foregut. d The heatmap of KEGG metabolic pathways significantly altered by CTC or MCE in the foregut (18 samples for each group, including 9 samples from AA chickens and 9 samples from LY chickens). The relative abundance of each pathway was colored according to the row z-score ((value – row mean)/row standard deviation). Red, black, and white rectangles at the right side of the heatmap represent significant increase (P < 0.05), significant decrease (P < 0.05), and no significant change (P > 0.05) compared to the BLANK, respectively. The Kruskal-Wallis test (Storey’s methods for adjustment) was followed by a post-hoc Wilcoxon rank-sum test

Fig. 5

The putative mechanisms of growth promotion by altering the foregut microbiota through CTC and MCE treatment. (Left) The antibiotic CTC as an exogenous pressure interfered with gut microbial competition and increased the Kitasatospora and Streptomyces, which are multi-antibiotic-resistant bacteria and antibiotic producers. The induced multi-antibiotics and antibiotic synergist (clavulanic acid) amplify the antimicrobial effects. Additionally, CTC enhanced microbial synthesis pathways of nutrients and secondary bile acids in the host. (Right) MCE increased Lactobacillus to benefit the host in many aspects, such as producing vitamins and generating lactate for anaerobic bacteria to produce butyrate, an anti-inflammatory compound and energy source for the intestine. Some bacteria were competitively inhibited by Lactobacillus. Additionally, MCE promoted the synthesis pathways of amino acids, vitamins, and secondary bile acids to provide nutrition for the host