Tetracycline resistance genes (TRGs) in stylo silage: distribution and mitigation by low intrinsic TRGs inoculants

To address tetracycline resistance genes (TRGs) contamination in silage, a primary ruminant feed source, we conducted systematic analysis of TRGs distribution patterns and their relationships with bacterial communities, followed by strategic inoculation with low-intrinsic-TRG LAB candidates. Metagenomic analysis revealed that TRGs were abundant, with tet(W/32/O) , tetB(58) , tetA(58) , tetB(60) , tetA(60) , tet(O/W) , tet(T) , tetA(46) , and tetB(46) being the dominant TRGs in stylo silage. These TRGs were positively correlated ( p < 0.001) with the main bacterial communities, particularly Lactiplantibacillus and Lacticaseibacillus . To mitigate TRGs in silage, three lactic acid bacteria (LAB) strains, PP1, PP2 ( Pediococcus pentosaceus ) and LC1 ( Loigolactobacillus coryniformis ) without 11 TRGs were screened and inoculated. These strains significantly reduced ( p < 0.05) pH, ammonia-N content and coliform bacteria counts, while increasing ( p < 0.05) lactic acid content of stylo silage. Among 61 identified TRGs, more than 40 were downregulated by PP1 and PP2. Both strains significantly increased the relative abundance of Pediococcus ( p < 0.05), while suppressing Lactiplantibacillus ( p < 0.05). PP1 and PP2 not only improved fermentation quality but also reduced TRGs by reducing Lactiplantibacillus and Lacticaseibacillus , the potential hosts of TRGs in silage. This strategy provides a practical and economical biotechnological solution to reduce ARGs, highlighting the potential of low-intrinsic-TRG LAB as functional additives to promote safe feed production and sustainable livestock health.