Fungal Microbiota in Cystic Fibrosis. FABB_CF

Background: The cystic fibrosis (CF) lung microbiota has been characterized in several studies and the observation between loss of bacterial diversity with increasing age and disease progression has been well-described. However, the dynamic relationship between the change in bacterial microbiota over time and increasing fungal colonization in CF with age remains understudied. Little is known about the role of asymptomatic fungal airway colonisation in CF and its impact on lung disease progression and the airway microbiota. Objectives: To longitudinally study the dynamic changes in sputum fungal composition, airway microbiome rarefication and disease progression in CF. Methods: A prospective, single-centre study including adult and paediatric CF patients. Sputum and/or BAL samples were processed for fungal culture (FC) and high-throughput sequencing for bacteria and fungi. Results: 235 respiratory samples were collected from 70 CF patients. Sixty-one percent (n=43) of cohort included paediatric patients aged 0-18 years. The median age at recruitment was 17 years (range 6 months–59 years) with 32 male participants and a median percent predicted FEV1 of 88% (range 26–135%). Sixty percent of CF participants has a least one positive result for Aspergillus fumigatus during the study period, of which 3% were persistently colonised. The majority of patients who were FC naïve (68%) were aged less than 15 years. Fungal culture positivity, advanced disease stage, use of antibiotics and inhaled steroid was significantly associated with a reduced bacterial diversity compared to fungal diversity. Streptococcus was the predominant bacterial taxa in both the FC positive and negative group (24% vs 22% respectively), followed by Pseudomonas (17% vs 19%). Greater abundance of Haemophilus was noted in the FC positive group in comparison to the FC negative group where higher levels of Neisseria were reported. There was a higher abundance of bacterial and fungal rare taxa in the FC negative groups. Both FC groups showed a predominance of Candida and Aspergillus species. Longitudinal analyses showed a steeper decline in bacterial and fungal diversity in CF participants with evidence of persistent fungal colonisation. The most dominant genus across all FC states over time was Streptococcus. Other bacteria that dominated in the FC naïve and transient groups included Veillonella and Nesisseria species in contrast to the FC colonized group which showed greater predominance of Stenotrophomonas and less Veillonella and Neisseria species. A variable pattern of fungal species was noted in the sub-groups naïve or transient for fungi. Aspergillus species was strikingly dominant in CF patients with persistent fungal colonisation. Conclusion: Asymptomatic fungal culture positivity is associated with changes in the bacterial and fungal microbiota. Persistent fungal airway colonisation is associated with a steeper decline in bacterial and fungal diversity over time. Longitudinal differences in bacterial and fungal microbiota were also observed between differing fungal culture states.

背景：多项研究已对囊性纤维化（cystic fibrosis, CF）患者的肺部微生物群进行了表征，且已有充分研究证实随着年龄增长与疾病进展伴随的细菌多样性丧失现象。然而，囊性纤维化患者中随年龄增长的细菌微生物群动态变化与真菌定植增加之间的关联，目前仍未得到充分研究。目前对于囊性纤维化患者无症状气道真菌定植的作用，及其对肺部疾病进展与气道微生物群的影响，尚不清楚。
研究目的：本研究旨在纵向探究囊性纤维化患者痰液真菌组成、气道微生物群稀疏化特征的动态变化，以及疾病进展情况。
研究方法：本研究为一项单中心前瞻性研究，纳入成人与儿童囊性纤维化患者。对获取的痰液及/或支气管肺泡灌洗液（bronchoalveolar lavage, BAL）样本进行真菌培养（fungal culture, FC）检测，并针对细菌与真菌开展高通量测序。
研究结果：本研究共纳入70例囊性纤维化患者，累计收集235份呼吸道样本。其中61%的研究队列（n=43）为0~18岁的儿童患者。入组时患者的中位年龄为17岁（范围：6个月~59岁），男性患者共32例，预测一秒用力呼气容积（predicted forced expiratory volume in one second, predicted FEV1）的中位百分比为88%（范围：26%~135%）。研究期间，60%的囊性纤维化受试者至少1次检测出烟曲霉（Aspergillus fumigatus）阳性，其中3%为持续定植患者。未检测到真菌定植的患者（FC阴性组）中，68%的患者年龄小于15岁。与真菌多样性相比，真菌培养阳性、疾病晚期、使用抗生素与吸入性糖皮质激素，均与细菌多样性降低显著相关。在真菌培养阳性组与阴性组中，链球菌属（Streptococcus）均为优势细菌类群（占比分别为24%与22%），其次为假单胞菌属（Pseudomonas，占比分别为17%与19%）。真菌培养阳性组的嗜血杆菌属（Haemophilus）丰度更高，而真菌培养阴性组的奈瑟菌属（Neisseria）水平更高。真菌培养阴性组的细菌与真菌稀有类群丰度均更高。两个真菌培养组均以念珠菌属（Candida）与曲霉属（Aspergillus）物种为主。纵向分析显示，存在持续真菌定植证据的囊性纤维化患者，其细菌与真菌多样性下降更为显著。随时间推移，在所有真菌培养状态分组中，链球菌属均为最优势菌属。在真菌培养阴性与一过性定植组中，占主导地位的其他细菌包括韦荣球菌属（Veillonella）与奈瑟菌属物种；而真菌定植组则以寡养单胞菌属（Stenotrophomonas）占比更高，韦荣球菌属与奈瑟菌属占比更低。在真菌培养阴性或一过性定植的亚组中，真菌物种呈现出多样化的分布模式。在存在持续真菌定植的囊性纤维化患者中，曲霉属物种占据绝对优势。
结论：无症状真菌培养阳性与细菌及真菌微生物群的改变相关。持续性气道真菌定植随时间推移，与细菌及真菌多样性的显著下降相关。不同真菌培养状态的患者之间，细菌与真菌微生物群的纵向变化亦存在差异。