Systematic evaluation of phage cocktail-ciprofloxacin combination therapy against multidrug-resistant <i>Salmonella</i> Typhimurium induced gut dysbiosis

Phage therapy, despite its promise as a novel therapeutic alternative for multidrug-resistant (MDR) bacterial infections, is hampered by clinical limitations such as narrow host range and therapeutic inconsistency. This research has engineered a phage-antibiotic synergistic therapy enabling precision eradication of drug-resistant <i>Salmonella</i> Typhimurium (<i>S</i>. Typhimurium). From slaughterhouse wastewater and fecal samples, three <i>Felixounavirus</i> phages (TSP_TW2, TSP_SW1, and TSP_SJ5) were isolated. Compared to the clinical phage repository, these phages demonstrated more superior broad-spectrum activity, successfully lysing over 85% of the tested wild-type isolates. The <i>in vitro</i> study, validated by laser confocal fluorescence imaging demonstrates that compared to solitary phage, phage cocktail delays the emergence of resistance to 24 h while also achieving effective biofilm eradication. In the murine intestinal infection model, the experimental group treated with phage cocktail in combination with ciprofloxacin (CIP) demonstrated significant therapeutic effects, reaching 90% survival rate, restoring the CD4⁺/CD8⁺ T cell balance, and decreasing the levels of pro-inflammatory factors, which were significantly superior to those of phage or CIP alone. In addition, the susceptibility of <i>S</i>. Typhimurium to β-lactams, quinolones, and observed four- to eightfold reduction in tetracycline minimal inhibitory concentration (MIC) demonstrated effective restoration of antibacterial activity after therapeutic intervention. 16S rRNA high-throughput sequencing showed that the index of intestinal flora diversity of mice in the treatment group was not significantly difference to healthy control group, while specifically promoting the growth of beneficial microbial populations such as <i>Lactobacillus</i> spp. The innovative phage-antibiotic combination therapy established in this study demonstrated dual therapeutic advantages: effective clearance of antimicrobial-resistant strains and maintenance of enteric microbiome homeostasis, thereby offering a clinically promising strategy for controlling infections caused by antimicrobial-resistant <i>S</i>. Typhimurium.IMPORTANCEFoodborne <i>Salmonella</i> infections threaten global public health, as conventional antibiotics accelerate resistance and disrupt microbial balance. We pioneer a synergistic phage-ciprofloxacin cocktail strategy that overcomes multidrug-resistant <i>Salmonella</i> infection through three key advances: First, it delays resistance evolution while eradicating biofilm matrices; second, the therapy synergistically enhances antibiotic sensitivity to restore efficacy of critical drugs; and third, the combined approach maintains optimal gut microbiota balance during pathogen clearance. By using environmentally derived phages with minimized antibiotic dosing, this strategy achieves targeted removal of resistant pathogens-including invasive biofilms-without collateral damage to commensal flora. Crucially, it prevents systemic inflammation and preserves intestinal barrier function. This ecologically sustainable paradigm provides a dual-defense mechanism against infections and microbiome dysbiosis, positioning phage-antibiotic synergy as a transformative tool for containing foodborne disease threats.