Data_Sheet_1_A Relevant Wound-Like in vitro Media to Study Bacterial Cooperation and Biofilm in Chronic Wounds.docx

Biofilm on the skin surface of chronic wounds is an important factor in the pathology, inhibiting wound healing. The polymicrobial nature of these infected wounds and bacterial interactions inside this pathogenic biofilm are the keys for understanding chronic infection. The aim of our work was to develop an innovative in vitro medium that closely mimics the chronic wound emphasizing the microbiological, cellular, and inflammatory environment of chronic wounds but also focusing on the pH found at the wound level. This new medium, called chronic wound medium (CWM), will thus facilitate the study of pathogenic biofilm organization. Clinical Staphylococcus aureus and Pseudomonas aeruginosa strains coisolated from diabetic foot infection were collected and cultivated in this new medium for 24 h in monoculture and coculture. Bacterial growth (growth curves), presence of small colony variant (SCV), biofilm formation (BioFilm Ring Test® assay, biofilm biomass quantification), and virulence (survival curve in a Caenorhabditis elegans model) were evaluated. After 24 h in the in vitro conditions, we observed that P. aeruginosa growth was not affected, compared with a control bacterial medium, whereas for S. aureus, the stationary phase was reduced by two logs. Interestingly, S. aureus growth increased when cocultured with P. aeruginosa in CWM. In coculture with P. aeruginosa, SCV forms of S. aureus were detected. Biofilm studies showed that bacteria, alone and in combination, formed biofilm faster (as soon as 3 h) than the bacteria exposed in a control medium (as soon as 5 h). The virulence of all strains decreased in the nematode model when cultivated in our new in vitro medium. Taken together, our data confirmed the impact of the chronic wound environment on biofilm formation and bacteria virulence. They indicated that P. aeruginosa and S. aureus cooperated in coinfected wounds. Therefore, this in vitro model provides a new tool for bacterial cooperation investigation and polymicrobial biofilm formation.

慢性创面皮肤表面的生物膜（biofilm）是影响其病理进程、阻碍创面愈合的重要致病因素。这类感染性创面的多微生物特性，以及致病性生物膜内部的细菌相互作用，是解析慢性感染机制的核心要点。本研究旨在开发一种创新性体外培养基，其不仅能够精准模拟慢性创面的微生物、细胞与炎症微环境，同时还可匹配创面局部的pH值特征。这种被命名为慢性创面培养基（chronic wound medium, CWM）的新型培养基，将为致病性生物膜的结构研究提供便利。研究人员收集了从糖尿病足感染患者体内共分离得到的临床金黄色葡萄球菌（Staphylococcus aureus）与铜绿假单胞菌（Pseudomonas aeruginosa）菌株，并将其分别以单培养与共培养的方式在该新型培养基中孵育24小时。随后对以下指标进行了检测：细菌生长情况（生长曲线）、小菌落变异株（small colony variant, SCV）的存在情况、生物膜形成能力（采用生物膜环试验（BioFilm Ring Test®）分析法及生物膜生物量定量检测），以及菌株毒力（通过秀丽隐杆线虫（Caenorhabditis elegans）模型测定存活曲线）。在体外培养条件下孵育24小时后，与对照细菌培养基相比，铜绿假单胞菌的生长未受显著影响，而金黄色葡萄球菌的稳定期菌量下降了两个数量级。值得注意的是，当在CWM中与铜绿假单胞菌共培养时，金黄色葡萄球菌的生长得以增强。在与铜绿假单胞菌共培养的体系中，可检测到金黄色葡萄球菌的小菌落变异株。生物膜相关实验结果显示，无论单培养还是共培养的细菌，其生物膜形成速度均快于对照培养基中培养的细菌：前者仅需3小时即可形成生物膜，而后者则需至少5小时。在新型体外培养基中培养的所有菌株，其在秀丽隐杆线虫模型中的毒力均有所下降。综合上述结果，本研究证实了慢性创面微环境对生物膜形成及细菌毒力的影响，同时表明铜绿假单胞菌与金黄色葡萄球菌在合并感染的创面中存在协同作用。因此，该体外模型为细菌协同作用研究及多微生物生物膜形成研究提供了全新的实验工具。