Bromhexine hydrochloride enhances the therapeutic efficacy of tiamulin against experimental Staphylococcus aureus infection in dogs: targeting bacterial virulence, boosting antioxidant defense, and improving histopathology

Abstract

Introduction: Staphylococcus aureus is a prominent pathogen capable of causing systemic infections and multi-organ damage, primarily driven by its high virulence and induction of oxidative stress. This study evaluated the therapeutic efficacy of tiamulin alone and in combination with bromhexine in a canine model of systemic S. aureus infection, focusing on oxidative stress biomarkers, bacterial burden, tissue histopathology, and the expression of cardiac and bacterial virulence-related genes. Methods: Experimental infection was induced in dogs, except for a healthy control group. Animals were assigned to five groups: uninfected control, infected untreated, tiamulin-treated, bromhexine-treated, and tiamulin plus bromhexine-treated. Oxidative stress was assessed through measurements of malondialdehyde (MDA) and total antioxidant capacity (TAC) in cardiac, hepatic, and renal tissues. Bacterial load was quantified, and minimum inhibitory concentrations (MICs) of the treatments were determined. Quantitative PCR was performed to evaluate the expression of S. aureus virulence genes including hla (alpha-hemolysin), ebpS (extracellular matrix-binding protein S), and icaA (intercellular adhesion A). Histopathological analyses of heart, liver, and kidney tissues were conducted, and hematological and biochemical parameters (total protein, albumin, and globulin) were measured. Cardiac injury markers, cytochrome P450 1B1 (CYP1B1) and interleukin-1 beta (IL-1β), were also assessed. Results: Infected untreated animals exhibited significantly elevated MDA, decreased TAC, high bacterial loads, severe histopathological alterations, and upregulated expression of IL-1β and CYP1B1. Tiamulin monotherapy produced moderate reductions in oxidative stress and bacterial burden. The combination of tiamulin and bromhexine resulted in a significant reduction in MDA, restoration of TAC, lower MIC values, suppressed expression of virulence genes (p < 0.05), and near-normal tissue architecture. Cardiac gene expression analysis showed substantial downregulation of IL-1β and CYP1B1 in the combination-treated group, indicating alleviation of inflammation and cardiac injury. Discussion: The combination therapy of tiamulin and bromhexine exhibited superior protective effects against S. aureus infection compared to monotherapy or untreated infection. These benefits appear to be mediated through synergistic antimicrobial, anti-virulence, and antioxidant mechanisms. The findings support the potential of this therapeutic approach for managing drug-resistant S. aureus infections and justify further clinical investigation.