Mechanism of Corynebacterium striatum-mediated inhibition of Staphylococcus aureus-induced epithelial barrier injury and inflammation activation in chronic rhinosinusitis

Microbes are an important trigger of chronic rhinosinusitis (CRS). Staphylococcus aureus is a CRS-related pathogen that disrupts the epithelial barrier and activates mucosal inflammation. Given the ineffectiveness of antimicrobial therapy in CRS, we explored a potential treatment based on the interaction of bacteria within the dysbiotic microenvironment of the nasal mucosa. Microbiome analyses of clinical isolates from CRS patients and healthy controls were employed to identify Corynebacterium striatum as a potential inhibitor of pathogenic S. aureus. A co-infection culture model using human nasal epithelial cells (hNECs) was constructed to investigate the inhibition of S. aureus-induced disruption of the host epithelium by C. striatum. Further analysis of the inhibition of S. aureus by C. striatum was carried out using a bacteria co-culture model. Overall design: To explore the competition between C. striatum and S. aureus, a bacterial co-culture model was established. Briefly, the two bacterial strains were recovered and amplified in LB liquid medium. The bacterial cells were then harvested, washed, and quantified to an OD600 of 1.0 in PBS. Next, each bacterial suspension was diluted 100-fold in LB liquid medium and incubated on a shaker at 37? and 220 rpm. Referring to the growth curves of the two strains and aiming to simulate the natural colonization state of C. striatum, it was cultured for 9 hours, while S. aureus was cultured for only 3 hours to simulate the invasion condition. Subsequently, equal volumes of the two bacterial cultures were fully mixed for 12 hours of shake co-cultivation, then collected for experimental analysis. As an experimental control, each bacterial culture was mixed with an equal volume of LB liquid medium (Figure 3B). For colonies counting, the cultures after co-cultivation were diluted and plated onto solid LB medium.

微生物是慢性鼻鼻窦炎（chronic rhinosinusitis, CRS）的重要触发因素。金黄色葡萄球菌（Staphylococcus aureus）是一种与CRS相关的病原菌，可破坏上皮屏障并激活黏膜炎症。鉴于抗菌治疗对CRS疗效有限，本研究基于鼻黏膜菌群失调微环境内的细菌相互作用，探索了一种潜在治疗方案。通过对CRS患者与健康对照的临床分离株进行微生物组分析，本研究鉴定出纹状棒状杆菌（Corynebacterium striatum）可作为致病性金黄色葡萄球菌的潜在抑制剂。本研究构建了基于人类鼻上皮细胞（human nasal epithelial cells, hNECs）的共感染培养模型，以探究纹状棒状杆菌对金黄色葡萄球菌诱导的宿主上皮屏障破坏的抑制作用。此外，通过细菌共培养模型进一步分析了纹状棒状杆菌对金黄色葡萄球菌的抑制效果。实验整体设计：为探究纹状棒状杆菌与金黄色葡萄球菌的竞争关系，本研究建立了细菌共培养模型。具体步骤如下：首先将两种菌株于LB液体培养基中复苏并扩增；随后收集细菌菌体，洗涤后用磷酸盐缓冲液（phosphate-buffered saline, PBS）将其浓度调整至OD600值为1.0；接着将各菌悬液以100倍稀释于LB液体培养基中，置于37℃、220转/分钟的摇床中培养。参考两种菌株的生长曲线，并为模拟纹状棒状杆菌的自然定植状态，将其培养9小时，而金黄色葡萄球菌仅培养3小时以模拟其侵袭状态；随后将两种细菌培养液以等体积充分混合，进行12小时的振荡共培养，收集样品用于后续实验分析。作为实验对照，将每种细菌培养液与等体积的LB液体培养基混合（图3B）。对于菌落计数实验，将共培养后的培养液稀释后涂布于LB固体培养基上。