Characterizing the gut phageome and phage-borne antimicrobial resistance genes in pigs

Abstract

Background: Mammalian intestine harbors a mass of phages that play important roles in maintaining gut microbial ecosystem and host health. Pig has become a common model for biomedical research and provides a large amount of meat for human consumption. However, the knowledge of gut phages in pigs is still limited.

Results: Here, we investigated the gut phageome in 112 pigs from seven pig breeds using PhaBOX strategy based on the metagenomic data. A total of 174,897 non-redundant gut phage genomes were assembled from 112 metagenomes. A total of 33,487 gut phage genomes were classified and these phages mainly belonged to phage families such as Ackermannviridae, Straboviridae, Peduoviridae, Zierdtviridae, Drexlerviridae, and Herelleviridae. The gut phages in seven pig breeds exhibited distinct communities and the gut phage communities changed with the age of pig. These gut phages were predicted to infect a broad range of 212 genera of prokaryotes, such as Candidatus Hamiltonella, Mycoplasma, Colwellia, and Lactobacillus. The data indicated that broad KEGG and CAZy functions were also enriched in gut phages of pigs. The gut phages also carried the antimicrobial resistance genes (ARGs) and the most abundant antimicrobial resistance genotype was diaminopyrimidine resistance.

Conclusions: Our research delineates a landscape for gut phages in seven pig breeds and reveals that gut phages serve as a key reservoir of ARGs in pigs. Video Abstract.

Keywords: Antimicrobial resistance genes; Gut phageome; Metagenomic; PhaBOX; Pig.

Fig. 1

Identification of pig gut phages from metagenomic data and the diversity of gut phages. A Distribution of genome lengths in gut phage genomes. B Classification of gut temperate and virulent phages. C Proportion of taxonomic families identified in gut phage populations. D PCoA of gut phage communities based on the Bray–Curtis distance (DLY, Duroc × [Landrace × Yorkshire]; TM, Tibetan miniature; LW, Laiwu; SZL, Shaziling; CM, Congjiang miniature; HM, Huanjiang miniature; NX, Ningxiang). E Chao index analysis of gut phage communities. F Shannon index analysis of gut phage communities. Data are presented as mean ± SEM (n = 8) and evaluated by Kruskal–Wallis test in E and F. **p < 0.01, *p < 0.05

Fig. 2

Analyses of the taxonomic composition of gut phages in pigs. A Taxonomic composition of gut phage populations at family level. B Heatmap analysis of gut phage populations at family level. C LEfSe analysis of gut phage populations in weaned piglets. D LEfSe analysis of gut phage populations in finishing pigs

Fig. 3

Analyses of core, common, and unique gut phages in pigs. A The proportion of core, common, and unique gut phages in the whole gut phage communities. B Heatmap analysis of core, common, and unique gut phages. The frequencies of phages are marked by different colors and each line is a phage. C–E Proportion of core (C), common (D), and unique (E) gut phages in seven pig breeds, respectively. Data are presented as mean ± SEM (n = 8) and evaluated by the one-way analysis of variance (ANOVA) test in C–E. **p < 0.01, *p < 0.05

Fig. 4

Comparative analysis of the gut phages amongst seven pig breeds and the shifts in the abundances of gut phages with the age of pig. A UpSet plot comparing the gut phage populations amongst seven breeds in weaned piglets. B UpSet plot comparing the gut phage populations amongst seven breeds in finishing pigs. C The number of gut phages whose abundances increased or decreased as the pigs aged. D UpSet plot comparing the gut phages whose abundances increased with the age of pig amongst seven breeds. E UpSet plot comparing the gut phages whose abundances decreased with the age of pig amongst seven breeds. Data are presented as mean ± SEM (n = 8) and evaluated by Wilcoxon rank sum test (C–E) and were shown in detail in Data S1

Fig. 5

Host prediction analysis of gut phages in pigs. The taxonomic compositions after host prediction at phylum (A), genus (B), and species (C) levels, respectively. The number of temperate (D) and virulent (E) phage populations associated with host genera, respectively. The number of temperate (F) and virulent (G) phage populations associated with host species, respectively

Fig. 6

KEGG functions encoded in gut phages. A PCoA of KOs annotated in gut phages. B Heatmap analysis of core, common, and unique KOs. The frequencies of KOs are marked by different colors. C UpSet plot comparing the KOs whose abundances increased with the age of pig amongst seven breeds. D UpSet plot comparing the KOs whose abundances decreased with the age of pig amongst seven breeds. Data are presented as mean ± SEM (n = 8) and evaluated by Wilcoxon rank sum test (C and D) and were shown in detail in Data S2

Fig. 7

CAZy functions encoded in gut phages. A PCoA of CAZys annotated in gut phages. B Heatmap analysis of core, common, and unique CAZys. The frequencies of CAZys are marked by different colors. C UpSet plot comparing the CAZys whose abundances increased with the age of pig amongst seven breeds. D UpSet plot comparing the CAZys whose abundances decreased with the age of pig amongst seven breeds. Data are presented as mean ± SEM (n = 8) and evaluated by the Wilcoxon rank sum test (C and D) and were shown in detail in Data S3

Fig. 8

Identification of ARGs encoded in gut phages. A PCA of ARG profiles. B The mean proportion of ARGs in gut phages of 112 pigs. C Heatmap analysis of antimicrobial resistance types. D The mean proportion of antimicrobial resistance types in gut phages of 112 pigs. E The comparative analysis of diaminopyrimidine resistance type amongst seven breeds. Data are presented as mean ± SEM (n = 8) and evaluated by one-way ANOVA in E. **p < 0.01, *p < 0.05

Fig. 9

Correlation analysis of gut phages and antimicrobial resistance. A Heatmap for the Spearman’s correlation analysis of gut phages and ARGs (top 15 of mean proportion). B Heatmap for the Spearman’s correlation analysis of gut phages and antimicrobial resistance types. **p < 0.01, *p < 0.05