Characterisation of mucoid Staphylococcus aureus recovered from the airways of cystic fibrosis patients: prevalence, impact on clinical course and detailed analysis by molecular and imaging techniques

金黄色葡萄球菌是从CF患者呼吸道中发现的首批病原菌之一。在许多患者中，尽管接受了抗生素治疗和宿主防御，金黄色葡萄球菌仍持续了几十年。在呼吸道持续期间，金黄色葡萄球菌需要适应这个充满敌意的利基环境。就在最近，我们将粘液金黄色葡萄球菌分离株描述为一种迄今未见报道的从CF患者呼吸道中恢复的表型。这些粘液分离株在ICA操纵子的基因间隔区iCar-ICAA区域有5bp的缺失，导致生物膜的高表达。到目前为止，无论是粘液型金黄色葡萄球菌的流行还是对CF患者肺部疾病的影响都是未知的。此外，对粘液型金黄色葡萄球菌在呼吸道内的表现(例如厌氧条件、与铜绿假单胞菌粘液/非粘液的相互作用、作为体外标本的痰中的空间分析)的详细分析还不清楚。此外，最近描述的没有5bp缺失的粘液金黄色葡萄球菌分离株的潜在机制尚不清楚，将进一步鉴定。因此，在我们的建议中，我们计划执行(1)。该项目的一个临床部分是在一项横断面研究中确定粘液型金黄色葡萄球菌的流行率，并通过观察粘液型金黄色葡萄球菌患者12个月与一组年龄、性别和铜绿假单胞菌阳性/阴性的金黄色葡萄球菌阳性而没有粘液分离物的匹配组进行比较，确定已识别的粘液型金黄色葡萄球菌对肺部疾病临床病程的影响。在我们项目的第二部分，我们计划描述(2)。粘液金黄色葡萄球菌分离株在体外不同环境条件下(需氧和厌氧)的表现，在生物被膜形成、与宿主细胞的相互作用和宿主防御方面模拟了在CF肺栖息地的体内情况。此外，(3.)我们计划研究粘液分离株的潜在机制，对于粘液分离株，我们不能使用下一代测序和RNA-SEQ分析在iCar-ICAA间隔区检测到5bp的缺失。最后，(4.)我们计划使用一种名为MiPACT的新可视化技术，该技术可以表征粘液型/非粘液型金黄色葡萄球菌与宿主细胞和其他重要的CF病原体(如CF患者痰中的铜绿假单胞菌)作为体外标本的相互作用。

S. aureus is one of the first isolated pathogens recovered from the airways of CF patients. In many patients, S. aureus persists for decades in spite of antibiotic treatment and host defence. During persistence in the airways, S. aureus needs to adapt to this hostile niche. Just recently we described mucoid S. aureus isolates as a so far not reported phenotype recovered from the airways of CF patients. These mucoid isolates carried a 5bp-deletion in the intergenic icaR-icaA region of the ica-operon leading to hyper-expression of biofilm. So far, neither the prevalence of mucoid S. aureus nor the impact on lung disease in CF patients is known. Furthermore, a detailed analysis of the performance of mucoid S. aureus within the airways (e.g. anaerobic conditions, the interaction with P. aeruginosa mucoid/non-mucoid, spatial analysis in sputum as an ex vivo specimen) is not clear. Moreover, the underlying mechanism of mucoid S. aureus isolates without a 5bp-deletion as recently described, which were also isolated from our CF patients, is not clear and will be further characterised.Therefore, in our proposal we plan to perform (1.) a clinical part of the project to determine the prevalence of mucoid S. aureus isolates in a cross-sectional study and the impact of identified mucoid S. aureus on the clinical course of the lung disease by observing patients with mucoid S. aureus for 12 months in comparison with a group of age-, gender- and P. aeruginosa-positive/negative matched group of S. aureus-positive patients without mucoid isolates. In the second part of our project we plan to characterise (2.) the performance of mucoid S. aureus isolates within the airways under different environmental conditions in vitro (aerobic versus anaerobic), which mimic the in vivo situation in the CF lung habitat in terms of biofilm formation, interaction with host cells and host defence. Furthermore, (3.) we plan to investigate the underlying mechanisms of mucoid isolates, for which we could not detect a 5bp-deletion in the icaR-icaA intergenic region using next generation sequencing and RNA-seq analysis. Lastly, (4.) we plan to use a new visualisation technique named MiPACT, which allows to characterise the interaction of mucoid/non-mucoid S. aureus with host cells and other important CF pathogens such as P. aeruginosa in sputa from CF patients as ex vivo specimens.