Multi-approach integrating water quality variables, remote sensing, eDNA and histopathology for assessing the impact of microbial pollution on Nile Tilapia from Kitchener Drain, Northern Egypt.

This study integrated environmental DNA (eDNA) metabarcoding, conventional bacteriology, histopathology, water quality variables, and remote sensing indices to investigate how environmental gradients are associated with pathogen-associated taxa communities and influence Nile tilapia (Oreochromis niloticus) health in Kitchener Drain, Northern Egypt. Physicochemical analyses revealed elevated nutrient concentrations, chlorophyll-a (59.21-226.35 µg L⁻1), with localized hypoxia (dissolved oxygen as low as 2.14 mg L⁻1 in site 2). High-throughput 16S rRNA metabarcoding revealed microbial communities dominated by Actinobacteriota (27.35%) and Proteobacteria (26.73%), with beta-diversity patterns driven primarily by species turnover, indicating strong environmental filtering along the pollution gradient. eDNA metabarcoding detected pathogen-associated taxa, including Mycobacterium, Legionella, Burkholderiaceae, Erysipelothrix, Leptospira, and Flavobacterium, many of which were not recovered by culture-based methods. Cross-database integration of SILVA (v138) and GTDB (r214) references identified 256 ASVs assigned to pathogen-associated taxa across 32 genera, with the dual-database strategy detecting 20-52% additional pathogen-associated ASVs relative to either database alone. Spearman correlation was examined for the 27 taxa with physicochemical parameters; phosphate, chlorophyll-a, dissolved oxygen, and BOD came up most often. Spearman correlation analysis revealed two perfect taxon-environment associations: a positive correlation between G_Pseudomonas and nitrate (ρ = + 1.00) and a negative correlation between G_Acinetobacter and chlorophyll-a (ρ = - 1.00). Both associations were nominally significant before multiple-testing correction (uncorrected p = 0.017); however, neither remained statistically significant after correction for multiple comparisons. These findings were therefore interpreted as exploratory associations rather than evidence of environmental drivers or causal relationships. Integration of remote sensing indices (NDWI, NDTI, NDCI, CDOM, NDVI) and water variables aids in identification of the surrounding environment associated with the presence of species. Culture-dependent analyses identified Aeromonas spp. as the dominant intestinal isolate and the only genus consistently observed by both approaches, suggesting selective host filtering between environmental and gut-associated communities. Histopathological examination revealed epithelial necrosis, villous atrophy, and goblet cell hyperplasia, with the most severe lesions at sites of highest organic loading. This approach can aid as a database for further future work, and for establishing more advanced models of pathogen-associated eDNA distributions and targeted fish-health surveillance, and for maintaining biodiversity.