The multi-kingdom microbiome catalog of the chicken gastrointestinal tract

Abstract

Chicken is an important food animal worldwide and plays an important role in human life by providing meat and eggs. Despite recent significant advances in gut microbiome studies, a comprehensive study of chicken gut bacterial, archaeal, and viral genomes remains unavailable. In this study, we constructed a chicken multi-kingdom microbiome catalog (CMKMC), including 18,201 bacterial, 225 archaeal, and 33,411 viral genomes, and annotated over 6,076,006 protein-coding genes by integrating 135 chicken gut metagenomes and publicly available metagenome-assembled genomes (MAGs) from ten countries. We found that 812 and 240 MAGs in our dataset were putative novel species and genera, respectively, far beyond what was previously reported. The newly unclassified MAGs were predominant in Phyla Firmicutes_A (n = 263), followed by Firmicutes (n = 126), Bacteroidota (n = 121), and Proteobacteria (n = 87). Most of the classified species-level viral operational taxonomic units belong to Caudovirales. Approximately, 63.24 % of chicken gut viromes are predicted to infect two or more hosts, including complete circular viruses. Moreover, we found that diverse auxiliary metabolic genes and antibiotic resistance genes were carried by viruses. Together, our CMKMC provides the largest integrated MAGs and viral genomes from the chicken gut to date, functional insights into the chicken gastrointestinal tract microbiota, and paves the way for microbial interventions for better chicken health and productivity.

Keywords: Antibiotic resistance gene; Archaeome; Chicken; Metagenome‑assembled genomes; Microbiome; Virome.

Fig. 1

Pipeline for the construction of the chicken multi-kingdom microbiome catalog, including bacterial, archaeal, and viral genomes. A) Overview of the overall strategy and datasets employed for CMKMC. Metagenomic sequencing data from the samples spanning origin, geography, breed, and farming system as well as 12,339 chicken MAGs from 799 metagenome data were integrated and used to construct the CMKMC. The reconstructed microbial genomes were clustered to strain- and species-level genome bins at 99 % and 95 % of the ANI, respectively. A total of 6,087 novel MAGs (1,883 nonredundant MAGs) divided into medium-quality MAGs (≥50 % completeness and ≤ 10 % contamination) and high-quality MAGs (>90 % completeness and < 5 % contamination) were reconstructed. SGBs containing at least one reference genome in the GTDB were considered kSGB, and the SGBs without reference genomes were considered uSGBs. B) Quality of the 18,201 bacterial genomes in the CMKMC. C) Quality of the 225 archaeal genomes in the CMKMC. D) Quality of the 33,411 viral genomes in the CMKMC. E) Composition of the genomes in the CMKMC. For CMKMC MAGs, the quality criteria are selected as near complete: > 90 % completeness and ≤ 5 % contamination according to CheckM and at least 18 tRNA, high-quality: > 90 % completeness and < 5 % contamination, and medium-quality: ≥ 50 % completeness and ≤ 10 % contamination. For viral genomes, the quality was evaluated using VIBRANT. Abbreviations: CMKMC, chicken multi-kingdom microbiome catalog; MAGs, metagenome-assembled genomes; SGBs, species-level genome bins; ANI, average nucleotide identity; VFs, virulence factors; VFDB, virulence factor database; kSGB, known SGBs; uSGBs, unknown SGBs; GTDB, Genome Taxonomy Database LPM, live poultry markets; vOTU, viral operational taxonomic units.

Fig. 2

Taxonomic annotation and phylogenetic tree of the CMKMC genomes. A) Taxonomic classification of 18,426 MAGs at different levels. B) The number of SGBs in each phylum. C) The number of newly generated SGBs in each phylum. D) The percentage of uSGBs in each phylum. Classification rates of newly generated SGB (bacterial and archaeal MAGs) in GTDB in each phylum. E) The phylogenetic relationship between the 3,050 bacterial and 21 archaeal MAGs in the CMKMC and their taxonomic classification according to GTDB–Tk. The annotations from inside to outside represent annotations at the species-level (different colors represent different phyla), newly generated (in blue), unclassified family (in brown), unclassified genus (in dark blue), and unclassified species (in purple). The three phyla are from Archaea and the others belong to bacteria. Abbreviations: SGBs, species-level genome bins; kSGB, known SGBs; uSGBs, unknown SGBs; MAGs, metagenome-assembled genomes; CMKMC, chicken multi-kingdom microbiome catalog; GTDB, Genome Taxonomy Database.

Fig. 3

Taxonomic classifications of the 33,411 viral genomes. A) Virus taxonomy for DNA VCs. Virus taxonomy is genome-based. Most of the vOTUs (91.76%) were assigned to the known virus. B)–E) phylum (B), class (C), family (D), and genus (E) level taxonomy and virus proportion in the chicken virome database. Most of the vOTUs (99.76%) classified into existing classes were under the order Caudovirales. Detailed taxonomic assignments of individual vOTUs are presented in Supplementary Table 9. Genus level taxonomy and proportion of the 1,636 vOTUs that could be assigned to existing genera or families using vConTACT2. Abbreviations: VCs, viral clusters; vOTUs, viral operational taxonomic units; DNA, deoxyribonucleic acid.

Fig. 4

Functional annotation of the CMKMC genomes. A) Overview of ARGs identified in the CMKMC genomes. B) The diversity of the AMR gene family. C) The antibiotic resistance mechanisms of the detected ARGs. D) Overview of beta-lactamase ARGs identified in the CMKMC genomes. E) The top ten most abundant plasmids found in the CMKMC genomes. F) The top 15 most abundant VFs found in the CMKMC genomes. G) The prevalence of the 409 CAZyme subclasses found in the CMKMC genomes. H) The top 20 most abundant CAZyme subclasses found in the CMKMC genomes. I) Distribution of ARGs, plasmids, and VFs in the CMKMC genomes. Abbreviations: CMKMC, chicken multi-kingdom microbiome catalog; ARGs, antibiotic resistance genes; AMR, antimicrobial resistance; VFs, virulence factors; CAZyme, carbohydrate-active enzyme; AA, auxiliary activities; CBM, carbohydrate-binding module; CE, carbohydrate esterase; GH, glycoside hydrolase; GT, glycosyl transferase; PL, polysaccharide lyase.

Fig. 5

Host prediction of the CMKMC virome. A) The prevalence of the CMKMC virome lifestyle. B) Virus diversity as a function of the number of predicted hosts. The viruses could be divided into specialist (number of hosts = 1) and generalist (number of hosts > 1). C) Virus distributions as a function of their number of predicted hosts. D) Overview of known viral hosts grouped at the kingdom level. E) Distribution of putative viral hosts grouped at the phylum level. F) Distribution of the viruses as a function of the taxonomic classification of their hosts in top 20 genera. Abbreviations: CMKMC, chicken multi-kingdom microbiome catalog.

Fig. 6

AMGs and ARGs carried by chicken gut viruses. A) Number of genes annotated by KEGG, Pfam, and VOG in the CMKMC virome. B) A bar plot showing the AMG categories identified in the rumen virome. See Supplementary Table 15 for the detailed AMGs, full annotation of the final AMG-carrying contigs, and AMG functional category annotation. C) The metabolism categories of the detected AMGs. D) Overview of the metabolism pathways of the detected AMGs. E) The number of ARG-carrying viruses and their ARG classes identified in the vial contigs. F) The top 14 most abundant ARGs found in the CMKMC virome. Abbreviations: AMGs, auxiliary metabolic genes; ARGs, antibiotic resistance genes; KEGG, Kyoto Encyclopedia of Genes and Genomes; VOG, virus orthologous group; CMKMC, chicken multi-kingdom microbiome catalog; Pfam, protein family; AMR, antimicrobial resistance; MDR, multidrug resistance; MLS, macrolide-lincosamide-streptogramin.