Background: 

Bacterial biofilms pose a challenge in treating implant-associated infections. Biofilms provide bacteria with protection against antimicrobial agents and the immune response and often are invisible to the naked eye. As a biofilm-disclosing agent, methylene blue (MB) has shown promise, but lacks rigorous in vitro evaluation. The purposes of the present study were to assess MB as a biofilm-disclosing agent in vitro for common biofilm-forming organisms and to determine performance characteristics across implant materials and healthy tissue types.

Methods: 

Staphylococcus aureus (ATCC 6538) and Pseudomonas aeruginosa (ATCC 27853) biofilms were grown on culture for 2 days in CDC biofilm reactors on titanium, cobalt chromium, polyethylene, and polyether ether ketone (PEEK) coupons. Biofilms were stained with MB solutions of either 0.005% or 0.01% and then were washed with normal saline solution. Digital photographs were obtained to compare the visual sensitivity of the blue dye at these dilutions. Scanning electron microscopy (SEM) was performed to confirm the absence or presence of biofilm on MB-stained areas. Uninoculated controls were also assessed. Healthy adult sheep tissues were also stained to determine the staining characteristics of the host tissue. ImageJ was used to determine the relative blue intensity of stained implants and tissues compared with standard curves.

Results: 

S. aureus and P. aeruginosa biofilms stained avidly on titanium, cobalt chromium, polyethylene, and PEEK coupons. There was visible dose-dependent staining based on dye concentration. MB was visible only where biofilms were present as confirmed by SEM. MB did not stain uninoculated controls. Articular cartilage and meniscus demonstrated appreciable staining; bone, tendon, muscle, nerve, and fat did not. Bacterial biofilms demonstrated both dose-dependent and species-specific staining.

Conclusions: 

MB is an effective disclosing agent for S. aureus and P. aeruginosa biofilms in vitro. MB did not stain implant materials, nor did it stain most healthy tissues in vitro. MB may allow surgeons to see biofilms and may allow for enhanced debridement once visualized.

中文翻译：

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亚甲蓝是用于鉴定骨科植入物上细菌生物膜的有效披露剂

背景： 

细菌生物膜在治疗植入物相关感染方面提出了挑战。生物膜可为细菌提供抗微生物剂和免疫反应的保护，并且肉眼通常看不见它们。作为生物膜公开剂，亚甲基蓝（MB）已显示出希望，但缺乏严格的体外评估。本研究的目的是评估MB作为体外常见生物膜形成生物的生物膜公开剂，并确定跨植入物材料和健康组织类型的性能特征。

方法： 

金黄色葡萄球菌（ATCC 6538）和铜绿假单胞菌（ATCC 27853）生物膜在CDC生物膜反应器中的钛，钴铬，聚乙烯和聚醚醚酮（PEEK）试片上培养2天。生物膜用0.005％或0.01％的MB溶液染色，然后用生理盐水溶液洗涤。获得数字照片以比较在这些稀释度下蓝色染料的视觉灵敏度。进行扫描电子显微镜（SEM）以确认MB染色区域上是否存在生物膜。还评估了未接种的对照。健康的成年绵羊组织也被染色以确定宿主组织的染色特性。与标准曲线相比，ImageJ用于确定染色的植入物和组织的相对蓝色强度。

结果： 

金黄色葡萄球菌和铜绿假单胞菌生物膜在钛，钴铬，聚乙烯和PEEK试样上狂热染色。根据染料浓度可见可见的剂量依赖性染色。通过SEM确认，MB仅在存在生物膜的地方可见。MB未染色未接种的对照。关节软骨和半月板表现出明显的染色。骨骼，肌腱，肌肉，神经和脂肪没有。细菌生物膜表现出剂量依赖性和物种特异性染色。

结论： 

MB是一种有效的金黄色葡萄球菌和铜绿假单胞菌生物膜的体外揭示剂。MB不会染色植入物材料，也不会在体外染色大多数健康组织。MB可以使外科医生看到生物膜，并且一旦可见就可以增强清创。