Metagenomics of Antimicrobial and Heavy Metal Resistance in the Cecal Microbiome of Fattening Pigs Raised without Antibiotics

This study aimed to assess the cecal microbiome, antimicrobial resistance (AMR), and heavy-metal resistance genes (MRGs) in fattening pigs raised under antibiotic-free (ABF) conditions compared with ordinary industrial pigs (control, C) using whole-genome shotgun sequencing. ABF pigs showed enrichment of Prevotella (33%) and Lactobacillus (13%), whereas Escherichia colt (40%), Fusobacterium, and Bacteroides (each at 4%) were notably observed in the C group. Distinct clusters of cecal microbiota of ABF and C pigs were revealed; however, microbiota of some C pigs (C1) appeared in the same cluster as ABF and were totally separated from the remaining C pigs (C2). For AMR genes, the most abundant genes, tet(Q) (35.7%) and mef(A) (12.7%), were markedly observed in the ABF group, whereas tet(Q) (26.2%) and tet(W) (10.4%) were prominently shown in the C group. tet(Q) was positively correlated with Prevotella in ABF and C1 samples. In the C2 group, the prominent tet(W) was positively correlated with Fusobacterium and Bacteroides. The pigs studied here have never received tetracycline, but pregnant sows received chlortetracycline once every 7 days before parturition. Chromosomal Cu and Zn resistance genes were also shown in both groups regardless of the received Cu and Zn feed additives. A higher abundance of multimetal resistance genes was observed in the C group (44%) than with the ABF group (41%). In conclusion, the microbiome clusters in some C pigs were similar to that in ABF pigs. High-abundance tetracycline resistance genes interrelated to major bacteria were observed in both ABF and C pigs. MRGs were also observed. IMPORTANCE AMR is an increasing problem in farm animals, and raising farm animals without antibiotics is one method that could solve this problem. Our study showed that only some tetracycline and macrolide resistance genes, tet(Q), tet(W), and mef (A), were markedly abundant in ABF and C groups. The tet(Q) and tet(W) genes interrelated to different predominant bacteria in each group, showing the potential role of major bacteria as reservoirs of AMR genes. In addition, chromosomal Cu and Zn resistance genes were also observed in both pig groups, independent of the use of Cu and Zn additives on both farms. The association of MRGs and AMR genotypes and phenotypes, together with the method to resensitize bacteria to antibiotics, should be studied further to unveil the cause of genes conferring high-level resistance and solve these problems.