Case study: Metagenomic analysis of microbial restructuring and nitrogen metabolism under probiotic and Chinese herb applications during post-antibiotic-ban shrimp farming.

China's 2020 aquaculture antibiotic ban has driven widespread use of probiotics and Chinese herbs in shrimp farming, yet their ecological effects on microbial communities remain unclear. This case study investigated three commercial Litopenaeus vannamei ponds in eastern China that exhibited contrasting nitrite accumulation and production outcomes under a post-antibiotic ban regime using probiotics and Chinese herbs. All ponds received daily Bacillus licheniformis probiotics and weekly supplements of Effective Microorganisms and a multi-herb blend, including Coptis, Elsholtzia, Sophora, Ligusticum, and Artemisia argyi. Our analysis revealed that Firmicutes-dominated communities replaced typical Proteobacteria-dominated microbiomes. Pond A, characterized by stable production, maintained low nitrite levels (a peak of 0.5 mg/L) and was dominated by Planococcus. In contrast, Ponds B and C, which exhibited elevated nitrite accumulation (peaks of 1.3 mg/L for Pond B and 1.5 mg/L for Pond C) and reduced production, were dominated by Paenisporosarcina. Metagenomic reconstruction indicated that this difference may result from aberrant nitrogen-transforming pathways. Paenisporosarcina correlated positively with nitrite accumulation, whereas Planococcus exhibited negative correlations. Virulence factor gene analysis revealed low abundance of pathogenic Vibrio spp.-associated genes. Importantly, even high-nitrite ponds exhibited minimal antibiotic resistance genes, including the absence of common aquaculture-associated ones such as those conferring resistance to sulfonamides (sul1, sul2), quinolones (qnr), and tetracyclines (tet), confirming the effectiveness of the antibiotic ban. Our case findings indicate that Paenisporosarcina dominance is linked to nitrite accumulation, highlighting a potential target for microbiome management in antibiotic-free shrimp farming.