Directly sampling the lung of a young child with cystic fibrosis reveals diverse microbiota

Abstract

Rationale: The airways of people with cystic fibrosis (CF) are chronically infected with a variety of bacterial species. Although routine culture methods are usually used to diagnose these infections, culture-independent, DNA-based methods have identified many bacterial species in CF respiratory secretions that are not routinely cultured. Many prior culture-independent studies focused either on microbiota in explanted CF lungs, reflecting end-stage disease, or those in oropharyngeal swabs, which likely sample areas in addition to the lower airways. Therefore, it was unknown whether the lower airways of children with CF, well before end-stage but with symptomatic lung disease, truly contained diverse microbiota.

Objectives: To define the microbiota in the diseased lung tissue of a child who underwent lobectomy for severe, localized CF lung disease.

Methods: After pathologic examination verified that this child's lung tissue reflected CF lung disease, we used bacterial ribosomal RNA gene pyrosequencing and computational phylogenetic analysis to identify the microbiota in serial sections of the tissue.

Measurements and main results: This analysis identified diverse, and anatomically heterogeneous, bacterial populations in the lung tissue that contained both culturable and nonculturable species, including abundant Haemophilus, Ralstonia, and Propionibacterium species. Routine clinical cultures identified only Staphylococcus aureus, which represented only a small fraction of the microbiota found by sequencing. Microbiota analysis of an intraoperative oropharyngeal swab identified predominantly Streptococcus species. The oropharyngeal findings therefore represented the lung tissue microbiota poorly, in agreement with findings from earlier studies of oropharyngeal swabs in end-stage disease.

Conclusions: These results support the concept that diverse and spatially heterogeneous microbiota, not necessarily dominated by "traditional CF pathogens," are present in the airways of young, symptomatic children with early CF lung disease.

Keywords: cystic fibrosis; infection; lobectomy; microbiota.

Figure 1.

Radiographs of the patient at 33 months, 2 months before right lower pulmonary lobectomy. (a) Chest X-rays (posteroanterior and lateral), revealing dense right lower lobe and some right middle lobe atelectasis (arrow), with mediastinal shift to the right and hyperexpansion of the left upper lobe. (b) Chest computed tomography, illustrating dense right lower lobe atelectasis with extensive bronchiectasis.

Figure 2.

(a) Anatomy and (b) microbiota of the lung tissue removed from a young child with cystic fibrosis. (a) Schematic of the right lower lobectomy sample, indicating the locations of tissue sections that were analyzed by sequence-based microbiota methods. Section 1 was analyzed by both postoperative culture and sequencing; the remaining sections were analyzed by sequencing only. (b) Bar graph indicating the relative abundances and identities of the species detected by bacterial 16S ribosomal RNA gene sequencing in each lung tissue section and the intraoperative throat swab. Bacterial species shown in the legend comprised at least 1% of bacterial reads detected within an individual sample.

Figure 3.

Sections of the right lower lobe, illustrating bronchiectatic airways (arrows) filled with mucus and with necrotizing airway wall inflammation. Hematoxylin and eosin stain. Original magnification: left panel, ×40; right panel, ×200.