MRSA-dominant colonization and synergistic pathogenicity with Pseudomonas aeruginosa in a polymicrobial bone infection model

Objective: (PA) represents a clinically prevalent subtype of PBI. Using a rat PBI model, this study aimed to elucidate the spatiotemporal dynamics of MRSA-PA co-infection and its impact on host responses, bacterial persistence, and antimicrobial susceptibility. Methods: Titanium screws were pre-contaminated with fluorescently labeled MRSA (mCherry) or PA (green fluorescent protein, GFP) and implanted 5 mm apart into the femoral mid-diaphysis of Sprague-Dawley rats. Animals were assigned to five groups: sterile, MRSA, PA, MRSA + PA, and PA + MRSA. Longitudinal evaluation included hematology, serum biochemistry (CRP, PCT, TNF-α, and IL-6), micro-CT imaging, and hard-tissue sectioning. Colony-forming unit (CFU) counts and histological analyses (H&E, Gram, and DAPI staining) were performed to evaluate bacterial burden, spatiotemporal colonization and biofilm formation. Local inflammation and osteoclast activation were further assessed by TNF-α immunohistochemistry and tartrate-resistant acid phosphatase (TRAP) staining. Antimicrobial susceptibility was evaluated by automated MIC testing and complementary disk diffusion assays. Transcriptomic and whole-genome sequencing analyses were performed to characterize transcriptional adaptations and virulence- and resistance-associated gene profiles. Results: Co-infection induced a synergistic inflammatory response, with a 6.8-fold increase in neutrophil counts relative to baseline, and caused severe osteolysis, with an approximately 80% reduction in bone-implant contact relative to the sterile group. At postoperative day 7, the co-infection groups showed increased local TNF-α-positive and TRAP-positive areas, supporting an association between the amplified inflammatory response and early osteoclast activation. Spatial analyses revealed asymmetric colonization dynamics: MRSA rapidly disseminated to and colonized PA-contaminated sites by postoperative day 7, whereas reciprocal PA dissemination into MRSA-dominant sites was not detected until day 28. Both pathogens concurrently infiltrated the osteocyte lacuno-canalicular network, uncovering a previously unrecognized reservoir for polymicrobial persistence within bone. Multi-omics profiling revealed susceptibility shifts and extensive adaptive reprogramming in PA. Conclusion: MRSA acts as a dominant ecological driver in PBI that amplifies bone destruction and facilitates polymicrobial persistence, emphasizing the need for multi-species therapeutic strategies. The translational potential of this article: This study establishes a reproducible rat model of polymicrobial bone infection and reveals dynamic MRSA-PA interactions, pathogen-specific ex vivo susceptibility shifts, and polymicrobial occupation of the osteocyte lacuno-canalicular network. These findings highlight the limitations of single-pathogen-based treatment strategies and support the development of multispecies-directed antimicrobial, immunomodulatory, and biofilm-targeting approaches. The model provides a clinically relevant platform for mechanistic studies and preclinical evaluation of diagnostics, therapeutics, drug-delivery systems, and orthopaedic implants.