In Ovo Exposure to Phenanthrene Promotes Antimicrobial Resistance Genes and Hemolysin Virulence Factor in Guts of Chicken Embryos

Antimicrobial resistance genes (ARGs) are increasingly recognized as emerging environmental contaminants that threaten ecological integrity and animal health. Polycyclic aromatic hydrocarbons (PAHs) are widespread pollutants that accumulate in the environment and have been detected in wild bird eggs. Although PAHs have been shown to accelerate ARG dissemination in environmental matrices, their influence on ARG dynamics in birds is unclear. Therefore, it is hypothesized that exposure to elevated levels of PAHs promotes the propagation and enrichment of ARGs in the avian gut. In this study, chicken embryos at Hamburger–Hamilton Stage 1 (HHS 1) were exposed in ovo to phenanthrene (PHE; 1, 100, and 400 nmol/g egg), and the toxic effects were evaluated. High PHE exposure significantly reduced embryonic tarsus length by 14.81% and increased the liver somatic index (LSI) by 26.97%. Functional annotation based on the Virulence Factor Database (VFDB) showed that the relative abundance of hemolysin, a microbial virulence factor associated with gastrointestinal microorganisms, was significantly elevated in the high-PHE group. In a Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, genes involved in nucleotide excision repair (NER) and basal transcription factor (BTF) pathways showed significantly lower relative abundance following high-dose exposure. Additionally, ARGs related to polymyxin and pleuromutilin resistance were significantly enriched in the high-exposure group. Partial least squares path modeling (PLS-PM) indicated that ARG enrichment was jointly shaped by multiple factors. PHE may be directly associated with ARG proliferation or may act indirectly through mobile genetic elements (MGEs), whereas bacterial community abundance might be linked to the potential dissemination of ARGs given its significant positive association with MGEs. Collectively, these findings provide correlative evidence that PHE exposure is associated with increased ARG abundance in avian organisms and highlight the need for an integrated evaluation of PAH-associated hazards to avian health.