Bacterial Microbiota in Cystic Fibrosis

Background: Conventional culture methodology detects a limited palette of dominant bacterial CF pathogens, whose role in lung disease progression is well-established. However, the role of asymptomatic filamentous fungal colonization in CF remains uncertain. The decline in bacterial diversity with age appears to coincide with increasing fungal airway colonization in CF. This complex polymicrobial relationship can be better studied using molecular sequencing and may be the first step in understanding the clinical consequences of asymptomatic fungal colonization and its impact on the microbiome. Objectives: To study the bacterial and fungal microbiota over time in relation to fungal culture positivity. Methods: A single-centre study involving adult and paediatric CF patients over a 5-year study period. 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 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 were 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 analysis also showed differences in bacterial and fungal microbiota between differing fungal culture states.背景：常规培养方法仅能检测到有限种类的主要细菌性囊性纤维化（Cystic Fibrosis, CF）致病菌，其在肺部疾病进展中的作用已得到明确证实。然而，无症状丝状真菌定植在囊性纤维化患者中的作用仍不明确。随着年龄增长，细菌性多样性下降似乎与囊性纤维化患者气道真菌定植增加相一致。利用分子测序技术可更好地研究这种复杂的多微生物相互作用，这或是阐明无症状真菌定植的临床结局及其对微生物组（microbiome）影响的第一步。 研究目的：探究随时间推移的细菌性与真菌性微生物组特征，及其与真菌培养阳性结果的关联。 研究方法：本研究为单中心研究，纳入了5年研究周期内的成年与儿科囊性纤维化患者。对痰液和/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，并对细菌与真菌进行高通量测序分析。 研究结果：本研究共从70例囊性纤维化患者中收集了235份呼吸道样本。其中61%（n=43）的研究队列纳入了0~18岁的儿科患者。受试者入组时的中位年龄为17岁（范围：6个月~59岁），其中男性32例，预测第1秒用力呼气容积（Forced Expiratory Volume in 1 second, FEV1）的中位值为88%（范围：26%~135%）。60%的囊性纤维化受试者在研究期间至少出现1次烟曲霉（Aspergillus fumigatus）培养阳性结果，其中3%为持续性定植。多数真菌培养阴性（FC-naïve）的患者（68%）年龄小于15岁。真菌培养阳性、疾病晚期阶段、抗生素使用与吸入性糖皮质激素使用，均与细菌多样性相较于真菌多样性的显著降低相关。链球菌属（Streptococcus）是真菌培养阳性组与阴性组中占比最高的细菌分类群（分别为24%与22%），其次为假单胞菌属（Pseudomonas，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分类群丰度更高。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，存在持续性真菌定植证据的囊性纤维化受试者，其细菌与真菌多样性下降幅度更为显著。随时间推移，在所有真菌培养状态下占比最高的菌属均为链球菌属。真菌培养阴性与一过性定植组的优势菌属还包括韦荣球菌属（Veillonella）与奈瑟菌属，而真菌培养持续定植组则以窄食单胞菌属（Stenotrophomonas）占比更高，韦荣球菌属与奈瑟菌属占比更低。真菌阴性或一过性定植亚组的真菌物种分布模式存在差异，而持续性真菌定植的囊性纤维化患者中，曲霉属物种占据绝对优势。 研究结论：无症状真菌培养阳性与细菌性及真菌性微生物组的改变相关。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。纵向分析还显示，不同真菌培养状态下的细菌与真菌微生物组存在差异。