Journal of orthopaedic translation · 2026
MRSA-dominant colonization and synergistic pathogenicity with Pseudomonas aeruginosa in a polymicrobial bone infection model.
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Zhou Hang, Liu Wanchang, Peng Xinliang, Li Maohui, He Zhongyuan, Liu Hang, Yang Xingyu, Guo Zhaoyang, Zhang Wentao, Fu Wei, Liang Hanchao, Shi Lei, Deng Rui, Chen Yaohua, Zhang Ruixiang, Dang Yongjun, Xie Chao, Wang Yunying, Deng Zhongliang, Chu Lei, Ren Youliang
Аннотация
OBJECTIVE: Polymicrobial bone infections (PBI) remain difficult to treat due to multi-drug resistance and poorly defined microbial interactions. Co-infection with methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (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.
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.