Metagenomics and targeted metabolomics uncover concomitant gut microbiota dysbiosis and bile acid metabolism alteration in norfloxacin-exposed Bufo gargarizans tadpoles.

Norfloxacin (NOR) is a fluoroquinolone antibiotic widely detected in aquatic environments, yet little is known about its toxic effects on amphibians. Bile acids (BAs) are crucial metabolites derived from gut microbiota-host co-metabolism and play vital roles in maintaining host health. BA composition is regulated by the gut microbiota through specific enzymes: bile salt hydrolases (BSHs) deconjugate primary BAs; bile acid-inducible enzymes (BAIs) and hydroxysteroid dehydrogenases (HSDHs) then convert them into secondary BAs. This study investigated the effects of NOR on Bufo gargarizans tadpoles using a combination of intestinal-targeted BA metabolomics, metagenomics, and histopathological analysis. Tadpoles were exposed to 10 and 100 μg/L NOR from Gs26 to Gs36, with 4 independent biological replicates per group. Our results showed that NOR exposure significantly increased the relative abundance of gut microbiota encoding BAIs, HSDHs, and/or BSHs, which was accompanied by a decrease in the ratios of primary/secondary BAs and conjugated/deconjugated BAs. Meanwhile, NOR treatment elevated antibiotic resistance gene abundance and induced intestinal histopathological alterations in tadpoles, characterized by reduced epithelial cell height and hypertrophy of smooth muscle cells (SMCs). In summary, environmentally relevant concentrations (10 and 100 μg/L) of NOR affected the intestinal microbiota, thereby disrupting BAs biotrasformation, ultimately potentially compromising intestinal health in tadpoles. This highlighted the potential ecological risks posed by NOR pollution in aquatic ecosystems.