Abstract：
OBJECTIVE: Staphylococcus aureus is the most common and impactful multi-drug resistant pathogen implicated in (periprosthetic) joint infections (PJI) and fracture-related infections (FRI). Therefore, the present proof-of-principle study was aimed at the rapid detection of S. aureus in synovial fluids and biofilms on extracted osteosynthesis materials through bacteria-targeted fluorescence imaging with the \'smart-activatable\' DNA-based AttoPolyT probe. This fluorogenic oligonucleotide probe yields large fluorescence increases upon cleavage by micrococcal nuclease, an enzyme secreted by S. aureus.
METHODS: Synovial fluids from patients with suspected PJI and extracted osteosynthesis materials from trauma patients with suspected FRI were inspected for S. aureus nuclease activity with the AttoPolyT probe. Biofilms on osteosynthesis materials were imaged with the AttoPolyT probe and a vancomycin-IRDye800CW conjugate (vanco-800CW) specific for Gram-positive bacteria.
RESULTS: 38 synovial fluid samples were collected and analyzed. Significantly higher fluorescence levels were measured for S. aureus-positive samples compared to, respectively, other Gram-positive bacterial pathogens (p < 0.0001), Gram-negative bacterial pathogens (p = 0.0038) and non-infected samples (p = 0.0030), allowing a diagnosis of S. aureus-associated PJI within 2 h. Importantly, S. aureus-associated biofilms on extracted osteosynthesis materials from patients with FRI were accurately imaged with the AttoPolyT probe, allowing their correct distinction from biofilms formed by other Gram-positive bacteria detected with vanco-800CW within 15 min.
CONCLUSIONS: The present study highlights the potential clinical value of the AttoPolyT probe for fast and accurate detection of S. aureus infection in synovial fluids and biofilms on extracted osteosynthesis materials.
METHODS: Synovial fluids from patients with suspected PJI and extracted osteosynthesis materials from trauma patients with suspected FRI were inspected for S. aureus nuclease activity with the AttoPolyT probe. Biofilms on osteosynthesis materials were imaged with the AttoPolyT probe and a vancomycin-IRDye800CW conjugate (vanco-800CW) specific for Gram-positive bacteria.
RESULTS: 38 synovial fluid samples were collected and analyzed. Significantly higher fluorescence levels were measured for S. aureus-positive samples compared to, respectively, other Gram-positive bacterial pathogens (p < 0.0001), Gram-negative bacterial pathogens (p = 0.0038) and non-infected samples (p = 0.0030), allowing a diagnosis of S. aureus-associated PJI within 2 h. Importantly, S. aureus-associated biofilms on extracted osteosynthesis materials from patients with FRI were accurately imaged with the AttoPolyT probe, allowing their correct distinction from biofilms formed by other Gram-positive bacteria detected with vanco-800CW within 15 min.
CONCLUSIONS: The present study highlights the potential clinical value of the AttoPolyT probe for fast and accurate detection of S. aureus infection in synovial fluids and biofilms on extracted osteosynthesis materials.
摘要：
目的：金黄色葡萄球菌是与关节假体周围感染（PJI）和骨折相关感染（FRI）相关的最常见和最有影响的多药耐药病原菌。因此,本原理验证研究的目的是通过基于\'智能激活\'DNA的AttoPolyT探针的细菌靶向荧光成像,快速检测滑液和提取的骨接术材料上的生物膜中的金黄色葡萄球菌.这种荧光寡核苷酸探针在被微球菌核酸酶切割时产生大量荧光增加，金黄色葡萄球菌分泌的一种酶。
方法：用AttoPolyT探针检测疑似PJI患者的滑液和疑似FRI创伤患者的骨合成材料的金黄色葡萄球菌核酸酶活性。用AttoPolyT探针和对革兰氏阳性细菌具有特异性的万古霉素-IRDye800CW缀合物(vanco-800CW)成像骨接合材料上的生物膜。
结果：收集并分析了38个滑液样本。金黄色葡萄球菌阳性样品的荧光水平明显高于，分别,其他革兰氏阳性细菌病原体（p<0.0001），革兰氏阴性细菌病原体（p=0.0038）和未感染样品（p=0.0030），允许在2小时内诊断金黄色葡萄球菌相关的PJI。重要的是，用AttoPolyT探针对FRI患者提取的骨合成材料上的金黄色葡萄球菌相关生物膜进行准确成像，允许它们与vanco-800CW在15分钟内检测到的其他革兰氏阳性细菌形成的生物膜正确区分。
结论：本研究强调了AttoPolyT探针在滑液和提取的骨合成材料生物膜中快速准确检测金黄色葡萄球菌感染的潜在临床价值。
方法：用AttoPolyT探针检测疑似PJI患者的滑液和疑似FRI创伤患者的骨合成材料的金黄色葡萄球菌核酸酶活性。用AttoPolyT探针和对革兰氏阳性细菌具有特异性的万古霉素-IRDye800CW缀合物(vanco-800CW)成像骨接合材料上的生物膜。
结果：收集并分析了38个滑液样本。金黄色葡萄球菌阳性样品的荧光水平明显高于，分别,其他革兰氏阳性细菌病原体（p<0.0001），革兰氏阴性细菌病原体（p=0.0038）和未感染样品（p=0.0030），允许在2小时内诊断金黄色葡萄球菌相关的PJI。重要的是，用AttoPolyT探针对FRI患者提取的骨合成材料上的金黄色葡萄球菌相关生物膜进行准确成像，允许它们与vanco-800CW在15分钟内检测到的其他革兰氏阳性细菌形成的生物膜正确区分。
结论：本研究强调了AttoPolyT探针在滑液和提取的骨合成材料生物膜中快速准确检测金黄色葡萄球菌感染的潜在临床价值。
