A Canine Commensal Escherichia coli Exhibits Anti-Proliferative Effects on Mammary Tumor Cells While Displaying a Multidrug-Resistant Phenotype: Implications for One Health

Introduction: Antimicrobial resistance (AMR) is a major One Health challenge, and companion animals may act as reservoirs of resistant bacteria. Concurrently, growing evidence suggests that gut microbiota can influence cancer biology. However, the relationship between AMR phenotypes and anti-tumor activity in canine commensal bacteria remains poorly understood. This study investigated whether a canine commensal Escherichia coli isolate exhibits both anti-proliferative activity against canine mammary tumor (CMT) cells and clinically relevant AMR traits.

Methods: An E. coli isolate obtained from a clinically healthy dog (no reported antibiotic treatment within the previous four weeks) was co-cultured with primary CMT cells at 0, 1×10⁵, and 1×10⁶ CFU/mL for 72 hours. Cell proliferation was assessed using the MTT assay, while reactive oxygen species (ROS), caspase-3/7 activity, and expression of BAX, *BCL-2*, TLR4, and TNF-α were evaluated. Antimicrobial susceptibility was determined using Kirby–Bauer disc diffusion according to CLSI guidelines against eight commonly used veterinary antimicrobials.

Results: The E. coli isolate reduced CMT cell proliferation in a dose-dependent manner, with a 17 percent decrease observed at 1×10⁶ CFU/mL (p = 0.032; R² = 0.84). Exposure to the isolate was associated with increased ROS production (1.68-fold, p = 0.006), elevated caspase-3/7 activity (1.56-fold, p = 0.011), and upregulation of BAX (2.1-fold), TLR4 (1.7-fold), and TNF-α (1.5-fold), suggesting activation of apoptosis-related pathways. Antimicrobial susceptibility testing demonstrated resistance to enrofloxacin, amoxicillin clavulanate, tetracycline, and cefalexin, intermediate susceptibility to gentamicin, and susceptibility to trimethoprim sulfamethoxazole, marbofloxacin, and chloramphenicol, consistent with a multidrug-resistant phenotype.

Conclusions: This preliminary study demonstrates that a canine commensal E. coli isolate can exhibit anti-proliferative effects on CMT cells while simultaneously carrying multidrug-resistant traits of One Health concern. The coexistence of potentially beneficial biological activity and antimicrobial resistance highlights important safety considerations for future microbiome-based applications and reinforces the role of companion animals in AMR surveillance