Estrobolome dysbiosis in canine mammary gland tumors: Eggerthella lenta as a candidate microbial marker for hormone-dependent mammary tumorigenesis.

Canine mammary gland tumors (MGTs) are the most prevalent neoplasms in intact female dogs, yet the role of the gut microbiome in their pathogenesis remains largely unexplored. This pilot study investigated the gut microbiota composition of 40 female dogs classified into MGT (n = 11), Non-MGT (n = 20), and under 2 years (U2Y, n = 9) groups using 16S rRNA gene sequencing. Alpha diversity indices showed no significant differences between the MGT and Non-MGT groups, whereas beta diversity analysis revealed significant overall community-level differences across all three groups (PERMANOVA, p = 0.0001), with the U2Y group compositionally distinct from both adult groups. In contrast, MGT and Non-MGT groups showed substantial overlap in community structure after correction for multiple comparisons. Differential abundance analysis identified Eggerthella as a candidate MGT-associated taxon, and species-specific quantitative PCR (qPCR) demonstrated a significantly higher normalized abundance of Eggerthella lenta in MGT samples than in Non-MGT controls (Mann-Whitney U test, p = 0.0159). Conversely, potentially beneficial short-chain fatty acid (SCFA)-producing bacteria, including Clostridium and Megamonas, showed reduced relative abundance in tumor-bearing dogs (Clostridium, ANCOM-BC; Megamonas, exploratory post hoc Mann-Whitney test). PICRUSt2 analysis suggested a trend toward enrichment of polyamine biosynthesis pathways in the MGT group, although no pathway remained significant after multiple-testing correction in the MGT vs. Non-MGT comparison. To our knowledge, this study provides the first evidence linking the estrobolome-associated bacterium Eggerthella lenta with canine MGTs. Together with the depletion of beneficial SCFA-producing bacteria, these findings suggest a pattern of gut microbial dysbiosis that may influence hormone-dependent mammary tumorigenesis through microbiome-associated alterations in estrogen metabolism. Although E. lenta was detected in both MGT and Non-MGT dogs, its significantly higher normalized abundance in MGT samples supports its potential as an abundance-based candidate microbial marker that warrants validation in large, independent prospective cohorts before clinical application. Collectively, these findings provide new insights into the gut-mammary axis and support future investigations into microbiome-based diagnostic and therapeutic strategies for canine mammary gland tumors.