Heat stress-induced enrichment of Klebsiella pneumoniae links mammary microbiota dysbiosis with inflammatory responses.

Heat stress is a major challenge to dairy production and leads to substantial losses in milk yield and quality. Although reduced feed intake is recognized as an important contributor to heat stress-induced production decline, evidence from pair-fed studies suggests that intake reduction alone cannot fully explain impaired mammary performance. Mammary inflammation may represent a potential intake-independent mechanism. However, the biological pathways linking heat stress to mammary inflammation, particularly the role of the mammary microbiota, remain poorly defined. Using a controlled animal model combining heat-stressed and pair-fed Holstein dairy cows, we integrated mammary plasma proteomics, time-resolved milk metagenomics, and mechanistic in vitro validation to investigate heat stress-induced mammary inflammation. Proteomic profiling of mammary vein blood revealed that heat stress induced a global host proteomic shift characterized by suppression of metabolic pathways and enrichment of infection- and inflammation-related signatures, accompanied by elevated SCS (Pgroup < 0.1). Metagenomic analysis of milk demonstrated a sustained reduction in mammary microbiota diversity and modest but structured changes in community composition. Time-series clustering further revealed disruption of coordinated microbial dynamics, identifying heat stress-specific microbial modules. Within these modules, Klebsiella pneumoniae emerged as a key taxon enriched under heat stress, with its abundance positively associated with SCS. Functional analysis revealed enrichment of a virulence-associated type VI secretion system gene in heat-stressed cows. In vitro coculture experiments showed that both live and heat-killed Klebsiella pneumoniae directly induced inflammatory cytokine expression and apoptosis in bovine mammary epithelial cells. Transcriptomic profiling further demonstrated coordinated activation of inflammatory and apoptotic gene programs, implicating cytokine signaling pathways associated with epithelial cell apoptosis. This study provides evidence that heat stress can impair mammary function by inducing dysbiosis of the mammary microbiota, thereby promoting subclinical mammary inflammation. By linking host inflammatory responses, microbial dynamics, and epithelial cell apoptosis, our findings highlight a microbiota-mediated pathway contributing to heat stress-associated milk production loss and offer new insights into mammary health regulation under environmental stress.