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Microbiota and metabolite profiling reveal specific alterations in bacterial community structure and environment in the cystic fibrosis airway during exacerbation.

ABSTRACT: Chronic polymicrobial infections of the lung are the foremost cause of morbidity and mortality in cystic fibrosis (CF) patients. The composition of the microbial flora of the airway alters considerably during infection, particularly during patient exacerbation. An understanding of which organisms are growing, their environment and their behaviour in the airway is of importance for designing antibiotic treatment regimes and for patient prognosis. To this end, we have analysed sputum samples taken from separate cohorts of CF and non-CF subjects for metabolites and in parallel, and we have examined both isolated DNA and RNA for the presence of 16S rRNA genes and transcripts by high-throughput sequencing of amplicon or cDNA libraries. This analysis revealed that although the population size of all dominant orders of bacteria as measured by DNA- and RNA- based methods are similar, greater discrepancies are seen with less prevalent organisms, some of which we associated with CF for the first time. Additionally, we identified a strong relationship between the abundance of specific anaerobes and fluctuations in several metabolites including lactate and putrescine during patient exacerbation. This study has hence identified organisms whose occurrence within the CF microbiome has been hitherto unreported and has revealed potential metabolic biomarkers for exacerbation.

SUBMITTER: Twomey KB

PROVIDER: S-EPMC3866110 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Microbiota and metabolite profiling reveal specific alterations in bacterial community structure and environment in the cystic fibrosis airway during exacerbation.

Twomey Kate B KB Alston Mark M An Shi-Qi SQ O'Connell Oisin J OJ McCarthy Yvonne Y Swarbreck David D Febrer Melanie M Dow J Maxwell JM Plant Barry J BJ Ryan Robert P RP

PloS one 20131217 12

Chronic polymicrobial infections of the lung are the foremost cause of morbidity and mortality in cystic fibrosis (CF) patients. The composition of the microbial flora of the airway alters considerably during infection, particularly during patient exacerbation. An understanding of which organisms are growing, their environment and their behaviour in the airway is of importance for designing antibiotic treatment regimes and for patient prognosis. To this end, we have analysed sputum samples taken ...[more]

PMID: 24358183

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Project description:16S-based sequencing provides broader information on the respiratory microbial community than conventional culturing. However, it (often) lacks species- and strain-level information. To overcome this issue, we used 16S rRNA-based sequencing results from 246 nasopharyngeal samples obtained from 20 infants with cystic fibrosis (CF) and 43 healthy infants, which were all 0 to 6 months old, and compared them to both standard (blind) diagnostic culturing and a 16S-sequencing-informed "targeted" reculturing approach. Using routine culturing, we almost uniquely detected Moraxella catarrhalis, Staphylococcus aureus, and Haemophilus influenzae (42%, 38%, and 33% of samples, respectively). Using the targeted reculturing approach, we were able to reculture 47% of the top-5 operational taxonomical units (OTUs) in the sequencing profiles. In total, we identified 60 species from 30 genera with a median of 3 species per sample (range, 1 to 8). We also identified up to 10 species per identified genus. The success of reculturing the top-5 genera present from the sequencing profile depended on the genus. In the case of Corynebacterium being in the top 5, we recultured them in 79% of samples, whereas for Staphylococcus, this value was only 25%. The success of reculturing was also correlated with the relative abundance of those genera in the corresponding sequencing profile. In conclusion, revisiting samples using 16S-based sequencing profiles to guide a targeted culturing approach led to the detection of more potential pathogens per sample than conventional culturing and may therefore be useful in the identification and, consequently, treatment of bacteria considered relevant for the deterioration or exacerbation of disease in patients like those with CF. IMPORTANCE Early and effective treatment of pulmonary infections in cystic fibrosis is vital to prevent chronic lung damage. Although microbial diagnostics and treatment decisions are still based on conventional culture methods, research is gradually focusing more on microbiome and metagenomic-based approaches. This study compared the results of both methods and proposed a way to combine the best of both worlds. Many species can relatively easily be recultured based on the 16S-based sequencing profile, and it provides more in-depth information about the microbial composition of a sample than that obtained through routine (blind) diagnostic culturing. Still, well-known pathogens can be missed by both routine diagnostic culture methods as well as by targeted reculture methods, sometimes even when they are highly abundant, which may be a consequence of either sample storage conditions or antibiotic treatment at the time of sampling.

Project description:Rationale: Differences in cystic fibrosis (CF) airway microbiota between periods of clinical stability and exacerbation of respiratory symptoms have been investigated in efforts to better understand microbial triggers of CF exacerbations. Prior studies have often relied on a single sample or a limited number of samples to represent airway microbiota. However, the variability in airway microbiota during periods of clinical stability is not well known.Objectives: To determine the temporal variability of measures of airway microbiota during periods of clinical stability, and to identify factors associated with this variability.Methods: Sputum samples (N = 527), obtained daily from six adults with CF during 10 periods of clinical stability, underwent sequencing of the V4 region of the bacterial 16S ribosomal RNA gene. The variability in airway microbiota among samples within each period of clinical stability was calculated as the average of the Bray-Curtis similarity measures of each sample to every other sample within the same period. Outlier samples were defined as samples outside 1.5 times the interquartile range within a baseline period with respect to the average Bray-Curtis similarity. Total bacterial load was measured with droplet digital polymerase chain reaction.Results: The variation in Bray-Curtis similarity and total bacterial load among samples within the same baseline period was greater than the variation observed in technical replicate control samples. Overall, 6% of samples were identified as outliers. Within baseline periods, changes in bacterial community structure occurred coincident with changes in maintenance antibiotics (P < 0.05, analysis of molecular variance). Within subjects, bacterial community structure changed between baseline periods (P < 0.01, analysis of molecular variance). Sample-to-sample similarity within baseline periods was greater with fewer interval days between sampling.Conclusions: During periods of clinical stability, airway bacterial community structure and bacterial load vary among daily sputum samples from adults with CF. This day-to-day variation has bearing on study design and interpretation of results, particularly in analyses that rely on single samples to represent periods of interest (e.g., clinical stability vs. pulmonary exacerbation). These data also emphasize the importance of accounting for maintenance antibiotic use and granularity of sample collection in studies designed to assess the dynamics of CF airway microbiota relative to changes in clinical state.

Dataset Information

Microbiota and metabolite profiling reveal specific alterations in bacterial community structure and environment in the cystic fibrosis airway during exacerbation.

Publications

Microbiota and metabolite profiling reveal specific alterations in bacterial community structure and environment in the cystic fibrosis airway during exacerbation.

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