Abstract
<title>Abstract</title> <p> Background Bacterial endometritis is a major cause of subfertility in mares. Diagnosis relies mainly on endometrial cytology and bacterial culture, interpreted together with clinical findings, but these approaches may not fully explain disease expression in subclinical or complex cases. Sequencing-based and proteomic approaches may complement conventional diagnostics by providing a broader view of the uterine microbial community and local host response. This study aimed to characterize uterine microbial composition, cytological inflammation, and proteomic host-response patterns in mares with bacterial endometritis, with particular emphasis on differences between Gram-positive and Gram-negative bacterial isolates. Results Twenty-nine mares were included: 11 healthy mares and 18 mares with bacterial endometritis, classified according to uterine culture as Gram-positive (n = 12) or Gram-negative (n = 6). Healthy mares harbored a diverse low-biomass uterine microbiota dominated by Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria, despite negative cytology and culture. In contrast, endometritis was associated with shifts in dominant bacterial taxa, including lower relative abundance of Proteobacteria and higher relative abundance of <italic>Streptococcus</italic> and other endometritis-associated taxa. Stratification by Gram classification revealed distinct microbial profiles: Gram-positive endometritis, largely associated with <italic>Streptococcus equi</italic> subsp. <italic>zooepidemicus</italic> , showed low-diversity, <italic>Streptococcus</italic> -dominated communities, whereas Gram-negative endometritis showed greater microbial richness, higher phylogenetic diversity, and more heterogeneous profiles enriched in anaerobic-associated taxa such as <italic>Prevotella</italic> , <italic>Lactobacillus</italic> , and <italic>Mogibacterium</italic> . Cytological severity was also associated with microbial community structure. Moderate inflammation showed the greatest inter-individual variability in microbial richness and composition, whereas severe inflammation was characterized by reduced richness and near-complete <italic>Streptococcus</italic> dominance. Proteomic analysis revealed distinct host-response patterns according to Gram classification. Gram-positive cases showed higher abundance of proteins related to innate immune activation, neutrophil-associated responses, chromatin organization, extracellular components, and cytoskeletal remodeling, whereas Gram-negative cases showed higher relative abundance of a smaller subset of proteins mainly related to redox regulation and metabolic responses. Conclusions These findings indicate that equine bacterial endometritis is a heterogeneous condition in which uterine microbiota, cytological inflammation, and proteomic host response vary according to the bacterial isolates involved. Integrating culture, cytology, 16S rRNA gene sequencing, and proteomics may improve the characterization of uterine disease in mares and provide further insight into endometritis pathophysiology. </p>