Respiratory microbiota resistance and resilience to pulmonary exacerbation and subsequent antimicrobial intervention

Abstract

Pulmonary symptoms in cystic fibrosis (CF) begin in early life with chronic lung infections and concomitant airway inflammation leading to progressive loss of lung function. Gradual pulmonary function decline is interspersed with periods of acute worsening of respiratory symptoms known as CF pulmonary exacerbations (CFPEs). Cumulatively, CFPEs are associated with more rapid disease progression. In this study multiple sputum samples were collected from adult CF patients over the course of CFPEs to better understand how changes in microbiota are associated with CFPE onset and management. Data were divided into five clinical periods: pre-CFPE baseline, CFPE, antibiotic treatment, recovery, and post-CFPE baseline. Samples were treated with propidium monoazide prior to DNA extraction, to remove the impact of bacterial cell death artefacts following antibiotic treatment, and then characterised by 16S rRNA gene-targeted high-throughput sequencing. Partitioning CF microbiota into core and rare groups revealed compositional resistance to CFPE and resilience to antibiotics interventions. Mixed effects modelling of core microbiota members revealed no significant negative impact on the relative abundance of Pseudomonas aeruginosa across the exacerbation cycle. Our findings have implications for current CFPE management strategies, supporting reassessment of existing antimicrobial treatment regimens, as antimicrobial resistance by pathogens and other members of the microbiota may be significant contributing factors.

Figure 1

The persistence and abundance (total number of sequence reads) of bacteria taxa present in all longitudinal samples and within each of the five partitioned periods. (All) All samples collected from the 12 CF patients, irrespective of disease state, within the study (n=237, r²=0.7, F_1,182=425.7, P<0.001); (B₀) baseline pre-CFPE (n=56, r²=0.8, F_1,106=429.3, P<0.001); (E) CFPE, 30 days prior to treatment (n=41, r²=0.8, F_1,121=415.1, P<0.001); (T) CFPE treatment period (n=67, r²=0.7, F_1,140=363.3, P<0.001); (R) recovery, 30 days post-CFPE treatment (n=32, r²=0.8, F_1,86=316.7, P<0.001); and (B₁) baseline post-CFPE (n=41, r²=0.7, F_1,92=221.1, P<0.001). Core OTUs were defined as those that fell within the upper quartile (dashed lines), and rare OTUs defined as those that did not. Taxa defined as core across all samples are labelled in each panel: Prevotella melaninogenica (Pm), Pseudomonas aeruginosa (Pa), Streptococcus pneumoniae group (Sp), Streptococcus sanguinis group (Ss), and Veillonella parvula (Vp).

Figure 2

Changes in the rate of OTU turnover for each patient over time. Solid vertical lines indicate the start and end of treatment for CFPE, and dashed lines indicate the start and end of the 30-day period either side of the treatment period. Disease states are denoted by (B₀) baseline pre-CFPE; (E) CFPE, 30 days prior to treatment; (T) treatment for clinical exacerbation; (R) recovery, 30 days post-CFPE treatment; and (B₁) stable post-CFPE. Black lines represent turnover in the whole microbiota, while green and blue lines represent turnover within the core and rare OTUs groups, respectively.

Figure 3

Changes in metacommunity diversity across the disease periods. Given are the whole microbiota, core and rare OTUs groups. Disease periods are denoted by (B₀) baseline pre-CFPE; (E) CFPE, 30 days prior to treatment; (T) treatment for clinical exacerbation; (R) recovery, 30 days post-CFPE treatment; and (B₁) baseline post-CFPE. Richness (S*) was calculated with a uniform re-sample size following 1000 iterations in each instance; re-sample sizes by group were n=55 656 (whole), n=47 092 (core), and n=8712 (rare) equating to the lowest number of sequences by disease period within each group. Error bars represent the standard deviation of the mean. Asterisks denote significant differences between B₀ and subsequent disease periods at the P<0.05 level and determined by two sample t-tests.

Figure 4

Trends in the relative abundance of bacterial species with change in disease period. Parameters are extracted from mixed-effects models based on 237 samples from 12 patients. (B₀) baseline pre-CFPE, (E) CFPE, 30 days prior to antibiotic treatment, (T) period of time patients were receiving treatment for pulmonary exacerbation, (R) recovery, 30 days post-treatment, (B₁) baseline post-CFPE. **P<0.001, *P<0.05. ^‡ indicates rare group membership.