Endotracheal Tube Mucus for the Growth and Analysis of Pseudomonas aeruginosa Biofilms

Introduction: Muco-obstructive pulmonary diseases (MOPD) including cystic fibrosis (CF) are characterized by the accumulation of hyperconcentrated mucus within the airways that promotes chronic inflammation and infection.1 With the implementation of highly effective modulator therapies (HEMTs), the availability of CF sputum has decreased drastically, limiting the biophysical and biochemical analyses that can be performed on physiologically relevant mucus specimens. Further Pseudomonas aeruginosa infection continues to persist despite HEMT usage. Endotracheal tube (ETT) mucus has previously been shown to be compositionally and rheologically similar to human bronchial epithelial (HBE) mucus that is often used for CF research.2 Herein, the utilization of ETT mucus for the growth and analysis of Pseudomonas aeruginosa biofilms is described in comparison to HBE mucus to serve as a physiologically relevant growth model for infection research in CF. Methods: Endotracheal tube mucus was collected from the UNC surgical unit post-op as previously described.2 Mucus samples from a minimum of 10 patients were pooled, corrected for tonicity, and diluted to 4% total solids and characterized with SEC-MALS, macrorheology, and microrheology. Mucus from HBE cultures was prepared at 4% solids for comparison. P. aeruginosa biofilms were grown in HBE and ETT mucus as previously described,3 treated with tobramycin, and characterized for changes in viscoelastic moduli and viability. Mucociliary transport rates in coordinated ciliated cultures were quantified via fluorescence video microscopy. Results: Mucin complexes in ETT were an order of magnitude larger than those in HBE mucus. Biofilms grown in ETT and HBE mucus were composed of ~1010 CFU/mL and were similar susceptible to tobramycin with a 5-log reduction in viability after treatment at 1 mg/mL. Biofilms grown in ETT mucus were mechanically robust and exhibited complex viscosity values 4x greater than those grown in HBE mucus. Transport of ETT mucus was faster than HBE mucus at 4% solids. While biofilm transport rates were slower than those of mucus, biofilms grown in ETT mucus likewise transported more rapidly than those grown in HBE mucus. Conclusions: Endotracheal tube mucus represents an attractive and physiologically relevant growth model for biofilm infection in MOPD such as CF. Similar to HBE mucus, the rheological properties can be tuned via adjusting concentration. Uniquely, ETT mucus contains a higher non-mucin protein content than HBE mucus which may contribute to altered rheological behavior of biofilms grown therein. As ETT mucus is directly collected from human subjects, this mucus may serve as a more relevant growth environment model of P. aeruginosa biofilms. Both mucus types support the formation of mechanically robust biofilms with similar bacterial loads and antibiotic susceptibility, but viscoelastic properties and mucociliary transport rates vary between mucus types. This phenomenon may be due in part to the disparity in mucin complex size between ETT and HBE mucus.

引言：以囊性纤维化（cystic fibrosis, CF）为代表的黏液阻塞性肺部疾病（Muco-obstructive pulmonary diseases, MOPD），其特征为气道内积聚高浓度黏液，进而诱发慢性炎症与感染。1 随着高效调节剂疗法（highly effective modulator therapies, HEMTs）的临床应用，CF患者痰液的获取量大幅减少，限制了针对生理相关黏液标本开展的生物物理与生化分析研究。即便采用HEMT治疗，铜绿假单胞菌（Pseudomonas aeruginosa）感染仍会持续存在。既往研究表明，气管插管（endotracheal tube, ETT）黏液的组成与流变学特性，与常应用于CF研究的人类支气管上皮（human bronchial epithelial, HBE）黏液相似。2 本研究将ETT黏液与HBE黏液进行对比，探讨其用于铜绿假单胞菌生物膜培养与分析的可行性，以期作为CF感染研究的生理相关体外培养模型。

方法：按照既往研究方案，从北卡罗来纳大学（UNC）外科病房收集术后患者的ETT黏液。2 将至少10名患者的黏液样本混合，调整渗透压后稀释至总固体含量为4%，并通过尺寸排阻色谱-多角度激光光散射（size-exclusion chromatography-multi-angle light scattering, SEC-MALS）、宏流变学与微流变学对其进行表征。为便于对照，将HBE培养所得黏液同样制备为总固体含量4%的样品。按照既往研究方法，分别在HBE黏液与ETT黏液中培养铜绿假单胞菌生物膜，3 随后用妥布霉素（tobramycin）处理，对其粘弹性模量与活菌存活率的变化进行表征。通过荧光视频显微镜对协同纤毛培养体系的粘液纤毛运输速率进行定量分析。

结果：ETT黏液中的黏蛋白复合物尺寸比HBE黏液大一个数量级。在ETT与HBE黏液中培养的生物膜，其活菌密度均约为10^10菌落形成单位（colony-forming unit, CFU）/mL，且对妥布霉素的敏感性相近：经1 mg/mL妥布霉素处理后，两者的活菌存活率均降低5个对数级。在ETT黏液中培养的生物膜机械稳定性更强，其复数粘度值比HBE黏液中培养的生物膜高4倍。总固体含量为4%时，ETT黏液的运输速率快于HBE黏液。尽管生物膜的运输速率慢于纯黏液，但在ETT黏液中培养的生物膜运输速率仍快于HBE黏液中培养的生物膜。

结论：气管插管黏液可作为CF等MOPD患者生物膜感染研究的理想且生理相关的体外培养模型。与HBE黏液类似，其流变学特性可通过调整浓度进行调控。值得注意的是，ETT黏液的非黏蛋白含量高于HBE黏液，这可能会改变在其中培养的生物膜的流变学行为。由于ETT黏液可直接从人体受试者中获取，其可作为更贴近生理状态的铜绿假单胞菌生物膜培养环境模型。两种黏液均可支持形成机械稳定性良好的生物膜，且细菌载量与抗生素敏感性相近，但二者的粘弹性特性与粘液纤毛运输速率存在差异。这一现象的部分原因可能是ETT与HBE黏液的黏蛋白复合物尺寸存在差异。