A polymicrobial perspective of pulmonary infections exposes an enigmatic pathogen in cystic fibrosis patients

Abstract

Lung disease is the leading cause of morbidity and mortality in cystic fibrosis (CF) patients. A modest number of bacterial pathogens have been correlated with pulmonary function decline; however, microbiological and molecular evidence suggests that CF airway infection is polymicrobial. To obtain a more complete assessment of the microbial community composition and dynamics, we undertook a longitudinal study by using culture-independent and microbiological approaches. In the process, we demonstrated that within complex and dynamic communities, the Streptococcus milleri group (SMG) can establish chronic pulmonary infections and at the onset of 39% of acute pulmonary exacerbations, SMG is the numerically dominant pathogen. We report the comprehensive polymicrobial community dynamics of a CF lung infection in a clinically relevant context. If a given organism, such as Pseudomonas aeruginosa, becomes resistant to antibiotic therapy, an alternative treatment avenue may mediate the desired clinical response by effectively managing the composition of the microbial community.

Fig. 1.

T-RFLP analysis of the microbial communities in sputum samples collected at admission to hospital for the first exacerbation and during the following year (days 1 to 368) and 5 retrospective samples (days −1784 to −121). The retrospective samples are shown left of the dashed line. The size in base pairs of each of the T-RFs detected during the 6-year period is shown on the left. * indicates T-RFs whereby bacterial species can be assigned by using in silico analysis. The abundance of each T-RF is plotted by using the legend. The names of the bacterial species cultivated from sputum are shown beside the T-RF for which they correspond. Green and red are the P. aeruginosa and S. constellatus T-RFs, respectively.

Fig. 2.

Cluster analysis of longitudinal T-RFLP samples from Fig. 1. The calculated Shannon's diversity indices (H′) are plotted next to the corresponding T-RFLP profile. Those from sputums collected at the onset of pulmonary exacerbation are shown in red. The pink cluster highlights the relatedness of the microbial communities present upon admission to hospital.

Fig. 3.

Detection of SMG by culture-dependent and culture-independent approaches. (A) Microbial cultivation of admission sputum in patient #1, pulmonary exacerbation 1, revealed that S. constellatus was the numerically dominant organism on BHI agar and TSY agar. CBA failed to grow S. constellatus (10⁻⁵ dilution plates shown) after 5 d at 37°C in 5% CO₂. (B) The relative abundance of the 584-bp T-RF corresponding to S. contellatus is shown throughout patient #1's first pulmonary exacerbation (day 1–14) and period of clinical stability after discharge from hospital (day 15–200). * indicates the failure to detect S. constellatus by BHI culture.

Fig. 4.

Examples of pulmonary exacerbations caused by the SMG. The relative abundance of the T-RF corresponding to the S. constellatus during patient #1's second (A) and third (B) pulmonary exacerbation and the S. intermedius exacerbation described in detail in the text (C) are shown. (D–F) Additional examples of cases where McKay agar culture detected SMG as the numerically dominant pathogen at the time of hospital admission (day 1). The SMG species identified in each exacerbation and the patients' sex and age are shown in the top right of each image. Antibiotic therapy is described in the legend. SMG levels are always shown with a bold solid line. Exacerbations (A–E) were also associated with P. aeruginosa (bold dashed line) and exacerbation (F) was associated with S. aureus.