ORIGINAL RESEARCH

A Novel Microbiota Stratication System Predicts Future

Exacerbations in Bronchiectasis
Geraint B. Rogers1, Nur Masirah M. Zain2, Kenneth D. Bruce2*, Lucy D. Burr1, Alice C. Chen1, Damian W. Rivett3,
Michael A. McGuckin1, and David J. Serisier1,4*
1
Immunity, Infection, and Inflammation Program, Mater Research Institute, University of Queensland, and Translational Research
Institute, Woolloongabba, Queensland, Australia; 2Institute of Pharmaceutical Science, Kings College London, and 3Division of Ecology
and Evolution, Department of Life Sciences, Imperial College London, London, United Kingdom; and 4Department of Respiratory
Medicine, Mater Adult Hospital, South Brisbane, Australia

Abstract dominated (n = 36). Patients with P. aeruginosa and H. inuenzae

Rationale: Although airway microbiota composition correlates
with clinical measures in noncystic brosis bronchiectasis, these
data are unlikely to provide useful prognostic information at
the individual patient level. A system enabling microbiota data to
be applied clinically would represent a substantial translational
advance.
Objectives: This study aims to determine whether stratication of
patients according to the predominant microbiota taxon can provide
improved clinical insight compared with standard diagnostics.
Methods: The presence of bacterial respiratory pathogens was
assessed in induced sputum from 107 adult patients by culture,
quantitative PCR, and, in 96 samples, by ribosomal gene
pyrosequencing. Prospective analysis was performed on samples
from 42 of these patients. Microbiological data were correlated
with concurrent clinical measures and subsequent outcomes.
Measurements and Main Results: Microbiota analysis dened
three groups: Pseudomonas aeruginosa dominated (n = 26),
Haemophilus inuenzae dominated (n = 34), and other taxa
dominated communities had signicantly worse lung function,
higher serum levels of C-reactive protein (CRP), and higher sputum
levels of IL-8 and IL-1b. Predominance of P. aeruginosa, followed by
Veillonella species, was the best predictor of future exacerbation
frequency, with H. inuenzaedominated communities having
signicantly fewer episodes. Detection of P. aeruginosa was
associated with poor lung function and exacerbation frequency,
irrespective of analytical strategy. Quantitative PCR revealed
signicant correlations between H. inuenzae levels and sputum
IL-8, IL-1b, and serum CRP. Genus richness was negatively
correlated with 24-hour sputum weight, age, serum CRP, sputum
IL-1b, and IL-8.
Conclusions: Stratication of patients with noncystic brosis
bronchiectasis on the basis of predominant bacterial taxa is more
clinically informative than either conventional culture or quantitative
PCRbased analysis. Further investigation is now required to assess
the mechanistic basis of these associations.
Keywords: prognostic markers; airway inammation;
microbiome

(Received in original form October 1, 2013; accepted in final form December 27, 2013 )
*Joint senior authors.
Supported by the Mater Adult Respiratory Research Trust Fund.
Author Contributions: G.B.R., N.M.M.Z., K.D.B., L.D.B., A.C.C., D.W.R., M.A.M., D.J.S. all contributed to the conception, execution, analysis, and reporting of
the study, and approved the final version. G.B.R. acts as the guarantor of the manuscript.
Correspondence and requests for reprints should be addressed to Geraint B. Rogers, Ph.D., Mater Medical Research Institute, Translational Research Institute,
37 Kent Street, Woolloongabba, QLD 4102, Australia. E-mail: geraint.b.rogers@gmail.com
This article has an online supplement, which is accessible from this issues table of contents online at www.atsjournals.org
Ann Am Thorac Soc Vol 11, No 4, pp 496503, May 2014
Copyright 2014 by the American Thoracic Society
DOI: 10.1513/AnnalsATS.201310-335OC
Internet address: www.atsjournals.org

The pathophysiology of noncystic bacterial infection (1). An association inammation, and a greater risk of
brosis (CF) bronchiectasis is generally between high bacterial load (as exacerbations (2) appears to support
considered to be characterized by an determined by culture-based approaches) this hypothesis. However, although
airway inammatory response to chronic and systemic inammation, airway Pseudomonas aeruginosa infection is

496 AnnalsATS Volume 11 Number 4 | May 2014

 ORIGINAL RESEARCH
associated with more severe disease (35),
the contribution of other species to
airway disease is not known. Further, in
a substantial proportion of patients, no
recognized respiratory pathogen is
isolated through conventional diagnostic
culture, despite elevated inammatory
marker levels (6).
Previously, we have used a meta-
analytical approach based on 16S ribosomal
RNA (rRNA) gene pyrosequencing data
to dene the core bacterial community
associated with bronchoalveolar lavage and
sputum samples from adult patients with
non-CF bronchiectasis (7). This analysis
revealed that, by a considerable margin, the
species that contributed most to bacterial
community similarity in these subjects
was Haemophilus inuenzae. In contrast,
P. aeruginosa was distributed more
unevenly within the patient population,
but was present at high levels in certain
individuals. Members of the genera
Streptococcus and Veillonella were also
notable as present in a majority of
subjects. In keeping with other reports
(8), a large number of other genera not
traditionally associated with airway
infection were also dened as core within
this patient group.
Dening a core microbiota in this way
helps to identify species that are abundant
and frequently detected within a patient
population, and therefore more likely to
be of clinical relevance. However, by
focusing on similarities between patients,
such meta-analysis could give the
impression that airway microbiology is
conserved among patients with non-CF
bronchiectasis, thus supporting a uniform or
nonstratied approach to antibiotic therapy.
Furthermore, while airway microbiota
composition correlates with important
clinical measures in non-CF bronchiectasis
(7), these complex data sets are not readily
converted into clinical or prognostic
information that can be applied in
a treatment setting on an individual patient
level.
We hypothesized that the dominant
bacterial taxon with the airway microbiota
would provide a basis for clinically relevant
stratication of patients, providing
prognostic information superior to that
available using current analytical
approaches. To test this, we compared
recorded clinical measures with
assessments of sputum bacterial
composition, as dened by conventional
Rogers, Zain, Bruce, et al.: Exacerbation Prediction in Bronchiectasis
diagnostic culture, quantitative PCR (Q-
PCR), and 16S rRNA gene pyrosequencing,
in 107 adult patients with bronchiectasis
prospectively enrolled in the Bronchiectasis
and Low-dose Erythromycin Study
(BLESS) (9).
Methods
The BLESS was a 12-month double-blind,
randomized, placebo-controlled study of
low-dose erythromycin in patients with
non-CF bronchiectasis (10). Patients details
have been published previously, and are
summarized in Table 1. Details of patient
recruitment, inclusion criteria, and
assessment protocols are provided in the
online supplement.
Induced sputum samples were collected
at baseline from 107 patients. Clinical
measures were recorded as described
previously (9, 11). Exacerbation frequency
and antibiotic treatment data were collected
for 52 weeks after sampling, and these data
were used as variables in the analysis of
microbiological data from 42 patients from
the placebo arm of the trial.
Sputum induction was performed as
described previously (10), with details
provided in the online supplement. After
collection, sample aliquots were frozen at
2808 C, with separate aliquots used for
molecular microbiology and inammatory
marker analysis.
Nucleic acid extraction and bacterial
tagencoded FLX amplicon pyrosequencing
(bTEFAP) were performed as described
previously (12), with detailed protocols
provided in the online supplement.
Sequence data generated have the accession
number SRP035600.
All statistical analyses were performed
with IBM SPSS Statistics (version 21; IBM,
New York, NY). Boxplots were generated
with the GraphPad Prism program (version
5.00; GraphPad Software, San Diego, CA),
with full details provided in the online
supplement.
Results
Given the clinical importance of
P. aeruginosa in non-CF bronchiectasis,
and the prevalence of H. inuenzae in
airway samples, we initially assessed the
relationships between these species and
disease measures using standard diagnostic
culture, Q-PCRbased detection, and
Q-PCR enumeration.
Bacterial isolation frequencies by
standard diagnostic microbiological
surveillance are shown in Table 1.
Compared with noninfected subjects,
P. aeruginosa detection by culture was
associated with poor lung function (mean
FEV1, 63.8 vs. 74.9%, P = 0.007, Wilcoxon
rank sum test, n = 107) and with the
number of reported exacerbations in the
prior 12 months (P = 0.011). Patients with
H. inuenzae infection had fewer prior
infective exacerbations compared with
other subjects (P = 0.026).
Agreement between culture- and PCR-
based detection of P. aeruginosa was poor
(k = 0.116, P = 0.022), with detection
signicantly higher by PCR (91 of 107 vs.
31 of 107 samples; P , 0.001). The
associations with reduced lung function
(P = 0.021, postbronchodilator FEV1) and
with the number of exacerbations in the
prior 12 months (P = 0.026) that were
observed for culture remained, despite this
group now comprising 85% of patients. The
frequency of H. inuenzae detection in
these samples was also signicantly higher
by PCR than by culture (92 of 107 vs. 42 of
107 samples, P , 0.001; k = 0.094, P =
0.023).
Levels of total bacteria, P. aeruginosa,
and H. inuenzae per unit volume of
sputum were determined for all samples
by Q-PCR (Figure 1). No signicant
relationships between total bacterial load
and neutrophil count or other disease
measures, including lung function, were
observed. Enumeration of P. aeruginosa
and H. inuenzae by this approach was
the most clinically informative of the
three conventional analyses. A negative
correlation was found between P.
aeruginosa load and lung function (FEV1,
r = 0.28, P = 0.004) and the number of
episodes of intravenous antibiotic therapy
in the prior 12 months (r = 0.23, P =
0.016). In addition, H. inuenzae load was
correlated with levels of sputum IL-8 (r =
0.22, P = 0.035), sputum IL-1b (r =
0.33, P = 0.001), and serum C-reactive
protein (CRP) (r = 0.25, P = 0.010).
Bacterial Community Composition
Sputum samples from 96 subjects
were analyzed by 16S rRNA gene
pyrosequencing, with a median of 18 genera
detected per sample (range, 653 genera).
The genera most commonly detected were
497
 ORIGINAL RESEARCH

Table 1. Patient demographic and diagnostic microbiology data On the basis of their predominant
taxon, bacterial communities could be
Demographic Value placed into one of three groups:
P. aeruginosa dominated (n = 26),
Age (yr), mean (SD) 62.5 (9.8) H. inuenzae dominated (n = 34), or
Female sex, no. (%) 59 dominated by a species other than these
Duration of bronchiectasis (yr), mean (SD, range) 40.5 (21.8, 175) two (n = 36). Median relative abundance of
Etiology the dominant taxon, and bacterial genus
Idiopathic, no. (%) 60 (56.0)
Infectious, no. (%) 30 (28.0) richness, for each subgroup, are shown in
Pinks disease, no. (%) 11 (11.3) Table 3.
Ciliary dysfunction, no. (%) 3 (2.8) Bacterial richness was signicantly
FEV1, mean (SD) lower in communities dominated by either
Prebronchodilator 1.82 (0.72)
Prebronchodilator, % predicted 68.0 (18.3)
P. aeruginosa or H. inuenzae, compared
Postbronchodilator 1.92 (0.74) with those dominated by another bacterial
Postbronchodilator, % predicted 71.6 (18.7) species (P , 0.001, one-way analysis of
Ex-smokers, no. (%) 20 (18.7) variance). Hierarchical cluster analysis
Smoking history (pack-years), mean (SD) 12.2 (18.1) using the BrayCurtis similarity measure
Median (IQR) 0.0 (0.0)
24-h sputum weight (g), median (IQR) 18.0 (13.724.7) was used to assess the extent to which
SGRQ total score, mean (SD) 37.9 (14.4) group membership, based on dominant
LCQ score, mean (SD) 14.9 (3.2) taxa, was maintained when clustering was
6-min walk test (m), median (IQR) 510 (475572.5) performed using all detectable genera (see
C-reactive protein (mg/L), median (IQR) 3.55 (1.28.5)
Exacerbations in prior 12 mo
Figure E1 in the online supplement). In the
Total exacerbations, mean (SD) 4.8 (2.8) majority of cases, clustering was closely
>5 Exacerbations, no. (%) 40 (37.4) related to dominant taxon group, with this
Medications, no. effect more pronounced in those patients in
Combination inhalers, ICS, LABA 47 whom P. aeruginosa or H. inuenzae was
Inhaled SABA 47
Inhaled anticholinergics 13 dominant. The median relative abundance
Inhaled corticosteroids alone 13 of the dominant taxa was higher in
Inhaled LABA alone 4 communities dominated by P. aeruginosa
Prednisolone 3 or H. inuenzae.
Nebulized saline 2
Bromhexine 3
Total bacterial load did not differ
Inhaled mannitol 1 signicantly between P. aeruginosa
Nebulized colistin 1 and H. inuenzaedominated samples
Comorbidities, no. (Figure 2). However, bacterial communities
Hypertension 35 dominated by P. aeruginosa had a higher
Ischemic heart disease 10
Cerebrovascular disease 6 total bacterial load compared with all other
Diabetes mellitus 3 samples analyzed (median, 7.5 3 109 vs.
Diagnostic microbiology 5.5 3 109 CFU/ml; P = 0.024). Agreement
No organism reported 47 between detection of P. aeruginosa and
Haemophilus inuenzae 24
Pseudomonas aeruginosa 32
H. inuenzae by culture, Q-PCR, 16S rRNA
Alcaligenes xylosoxidans 1 gene pyrosequencing, and identication of
Moraxella catarrhalis 3 dominant taxa by pyrosequencing, is shown
Pseudomonas stutzeri 1 in Figure E2, with community dominance
by P. aeruginosa and H. inuenzae
Definition of abbreviations: ICS = inhaled corticosteroids; IQR = interquartile range; LABA = long-
acting b-agonists; LCQ = Leicester Cough Questionnaire; SABA = short-acting b-agonists; SGRQ = comparable with the results of culture-
St. Georges Respiratory Questionnaire. based detection (k = 0.72, P , 0.001,
and 0.65, P , 0.001, respectively).
The median relative abundance of the
those associated with the oropharynx and (33.4%), followed by Pseudomonas (23.5%), dominant taxa was higher in communities
reported previously in chronic lower Prevotella (9.5%), Veillonella (8.2%), dominated by P. aeruginosa or
infections (10) (see Table 2). Streptococcus (5.6%), Pasteurella (2.0%), H. inuenzae than in communities
Bacterial communities were typically Neisseria (1.9%), Porphyromonas (1.6%), dominated by other species (93.7 and 37.6%,
dominated by a small number of taxa. On and Moraxella (1.5%). Genus richness was respectively, P , 0.001, Kruskal-Wallis
average, only 5.3 (63.9) genera per sample negatively correlated with 24-hour sputum analysis of variance). Relative abundance
represented more than 1% of total bacterial weight (r = 0.38, P , 0.001), age (r = of the predominant taxon correlated
signal, with the dominant genus typically 0.28, P = 0.005), serum CRP (r = 0.26, signicantly with a number of disease
representing more than 70% (mean, 71.3%; P = 0.010), sputum IL-1b (r = 0.35, P = measures (Table 4). Notably, relative
SD, 27.1). The genus with the highest mean 0.001), and sputum IL-8 (r = 0.51, P , abundance of the predominant taxon
relative abundance was Haemophilus 0.001). (irrespective of identity) was negatively

498 AnnalsATS Volume 11 Number 4 | May 2014

 ORIGINAL RESEARCH
Figure 1. Levels of total bacterial cells, Pseudomonas aeruginosa, and Haemophilus influenzae, as
determined by quantitative PCR (Q-PCR), are shown for 107 sputum samples. Levels are expressed
as colony-forming unit equivalents per milliliter of sputum. The top and bottom boundaries of
each box indicate the 75th and 25th quartile values, respectively, and lines within each box represent
the 50th quartile values. Ends of whiskers show 10th and 90th percentiles. The broken line
indicates an imposed threshold of detection.
correlated with lung function (FEV1) and
sputum macrophage levels, and positively
correlated with duration of bronchiectasis,
serum CRP, sputum IL-8 and IL-1b, and
sputum neutrophil levels.
Bacterial communities in 36 of the
samples (37.5%) were dominated by
a species other than P. aeruginosa or
H. inuenzae. All of the predominant taxa
in these patients have been reported
previously in the context of lower airway
infections (Figure E3). Whereas the
majority of taxa were dominant in one
sample only, members of the genera
Veillonella and Prevotella were dominant in
10 and 9 samples, respectively. Median
bacterial load for this group was 1.4 3 109
CFU/ml (range, 8.9 3 1041.8 3 1010) with
bacterial community proles obtained for
all samples. Despite this, 27 (75%) were
classied as culture negative by standard
diagnostic microbiology.
We next examined the ability of patient
grouping based on predominant taxon to
provide clinically useful stratication.
Patients whose airway bacterial community
was dominated by P. aeruginosa exhibited
disease features more severe than those of
patients dominated by H. inuenzae or
other bacterial species. FEV1 was lower in
these patients (mean, 56.8 6 17.6%) than in
patients whose bacterial community was
dominated either by H. inuenzae (69.9 6
17.9%; P = 0.010) or another species
(73.1 6 16.8%; P = 0.001). Patients with
P. aeruginosadominated bacterial
communities also had a greater number
of exacerbations in the 12 months before
sampling (6.1 6 3.0) compared with both
the patient group as a whole (4.7 6 2.6;
Rogers, Zain, Bruce, et al.: Exacerbation Prediction in Bronchiectasis
P = 0.007) and those whose bacterial
community was dominated by H.
inuenzae (3.5 6 1.4; P = 0.001). Levels of
CRP, sputum IL-8, and sputum IL-1b did
not differ between P. aeruginosa and
H. inuenzaedominated communities;
however, P. aeruginosa and H. inuenzae
dominated communities had lower FEV1
(P = 0.029) and higher levels of CRP
(P = 0.004), sputum IL-8 (P , 0.001),
and sputum IL-1b (P , 0.001) than
communities dominated by another
species.
Although P. aeruginosa was detected
in 22 of the 34 patients in whom H.
inuenzae was dominant, no signicant
differences were observed in clinical
measures between these patients and the
H. inuenzaedominated patients in whom
P. aeruginosa was not detected.
Table 2. Bacterial taxa most frequently detected by 16S ribosomal RNA gene
pyrosequencing, and the frequency of detection of selected recognized respiratory
pathogens
Taxon
Veillonella
Prevotella
Streptococcus
(of which S. pneumoniae)
Neisseria
Haemophilus inuenzae
Pseudomonas aeruginosa
Stenotrophomonas maltophilia
Burkholderia cepacia complex
Moraxella catarrhalis
Staphylococcus aureus
Values in parentheses represent percentages.
Microbiological Measures as
Prognostic Markers
Data were available for 42 of the placebo
subjects regarding the number of
exacerbations and the total days of antibiotic
therapy that occurred in the 12 months
after sample collection (Tables 3 and 5).
Patients whose bacterial community was
dominated by H. inuenzae had fewer
exacerbations (1.1 6 1.1) than those
dominated by P. aeruginosa (3.7 6 2.1; P ,
0.001) or other genera (2.5 6 2.1; P =
0.047). This pattern was reected in the
number of days of antibiotic therapy that
patients received, with those whose
bacterial community was dominated by
H. inuenzae having fewer (10.8 6 13.8 d)
than those dominated by either P.
aeruginosa (52.4 6 35.1; P , 0.001) or
other genera (31.2 6 27.3; P = 0.017). No
signicant relationship was identied
between taxon dominance and 12-month
change in FEV1.
By comparison, analysis based on
culture data revealed signicant
relationships only between P. aeruginosa
and a greater number of days of antibiotic
compared with the group as a whole (P =
0.009), and with patients in whom H.
inuenzae was dominant (P = 0.011). No
signicant relationship was identied
between culture-based microbiology and
exacerbation frequency.
Multiple regression analysis
indicated that P. aeruginosa microbiota
predominance was the single best predictor
of exacerbation frequency (b= 0.501, P ,
0.001) and associated antibiotic burden
(b= 0.595, P , 0.001), followed by
Samples (n = 96)
90 (93.8)
84 (86.5)
82 (85.4)
28 (29.2)
69 (71.9)
92 (95.8)
70 (72.9)
29 (30.2)
27 (28.1)
21 (21.9)
11 (11.5)
499

Table 3. Median relative abundance and bacterial genus richness of samples grouped according to dominant taxon
All samples
Pseudomonas aeruginosa dominated
Haemophilus inuenzae dominated
Dominated by another genus
Veillonella spp. dominated
Prevotella spp. dominated
Definition of abbreviation: IQR = interquartile range.
dominance by a Veillonella species (b=
0.385, P = 0.005 and b= 0.385, P = 0.003,
respectively). The relative risk of a patient
having ve or more exacerbations in the
12 months after analysis was greater by a
factor of 1.60 when P. aeruginosa was
dominant, and by a factor or 1.44 when
a Veillonella species was dominant. By
comparison, the relative risk associated with
P. aeruginosa culture positivity was 1.35.
Discussion
We describe a system of microbiological
stratication of patients based on
predominant bacterial taxa that is more
closely correlated with clinical measures of
disease, inammation, and disease-related
outcomes than the detection of recognized
pathogens either by culture or PCR
assays. This stratication system allows
microbiota composition data to be directly
clinically relevant for individual patients, as
well as providing potential mechanistic
insights in relation to hostbacteria
interaction.
An initial assessment of P. aeruginosa
and H. inuenzae detection through culture
and PCR assays indicated they were
comparable in terms of their correlation
with disease measures. Both approaches
indicate a signicant association between
P. aeruginosa detection and poor lung
function, as reported previously (5). A
signicant association was also observed
between P. aeruginosa detection and
subsequent exacerbation frequency, the rst
time this has been shown to our knowledge.
Culture performed slightly better than
PCR-based detection, with a signicant
relationship between H. inuenzae isolation
and low exacerbation frequency identied
by culture alone. The relatively weak
relationship between PCR-based detection
500
ORIGINAL RESEARCH
Dominant Species Relative Abundance (%) Bacterial Richness (Genus)
n Median IQR Median IQR
96 82.1 52.3 18.0 15.0
26 92.4 14.2 17.0 12.0
34 96.2 9.2 15.5 10.0
36 37.6 26.0 28.5 11.3
10 35.4 12.2 24.0 16.0
9 36.8 19.8 28.0 8.0
and disease measures may result from high substantial bacterial population in the lower
assay sensitivity, resulting in most patients airways is arguably a clinical concern.
being classied positive when bacteria Converting complex bacterial
may be below clinically relevant levels; community data into a readily accessible
a model supported by the nding that measure for patient stratication,
Q-PCRbased enumeration of bacteria particularly at the individual patient level, is
provided greater clinical insight than a major challenge. For the rst time, our
detection alone. Here, in addition to stratication system conveys complex
a signicant negative correlation between microbiota data in a clinically relevant and
P. aeruginosa load and lung function, clinically recognizable output that has the
signicant positive correlations were also potential to be applied at the individual
observed between H. inuenzae load and patient level (by clinicians) and also
serum levels of CRP and sputum IL-8 provides prognostic information that is
and IL-1b levels. superior to existing culture-based methods.
In keeping with previous studies of This stratication system therefore
non-CF bronchiectasis, and in studies of represents a substantial translational
chronic airway disease more widely, advance toward a clinically relevant tool
microbiota proling indicated that from bacterial community proling.
a substantial proportion of the detected Bacterial community diversity as
bacteria represented taxa other than a gross measure of community structure has
recognized respiratory pathogens. Although the potential to be informative in itself,
the clinical signicance of many of these requiring relatively little in-depth analysis. A
species in airway disease is as yet correlation between lower diversity and
undetermined, the presence of any more severe disease has been reported in
Figure 2. Levels of total bacterial cells are shown for each of the three sample groups defined by
predominant taxa: Pseudomonas aeruginosa, Haemophilus influenzae, and other species. The top
and bottom boundaries of each box indicate the 75th and 25th quartile values, respectively, and
lines within each box represent the 50th quartile values. Ends of whiskers show 10th and 90th
percentiles. The bar indicates that the bacterial load in samples dominated by P. aeruginosa was
significantly greater than in patients dominated by a species other than P. aeruginosa or H. influenzae.
AnnalsATS Volume 11 Number 4 | May 2014
 ORIGINAL RESEARCH
Table 4. Signicant correlations between the relative abundance of the dominant
bacterial taxon and clinical measures of disease
Clinical Measure Spearmans r Signicance (P Value) n including poorer lung function, frequent
Pre-BD FEV1 20.225 0.027
Post-BD FEV1 20.223 0.029
Bronchiectasis duration, yr 0.221 0.031
CRP 0.306 0.003
Sputum IL-8 0.585 ,0.001
Sputum IL-1b 0.626 ,0.001
Sputum neutrophils, absolute 0.240 0.031
Sputum neutrophils, % 0.347 0.001
Sputum macrophages, absolute 20.340 0.002
Sputum macrophages, % 20.389 ,0.001
24-h sputum volume, g 0.370 0.002
Definition of abbreviations: BD = bronchodilator; CRP = C-reactive protein.
other chronic respiratory infections (1315), exacerbation history revealed by culture,
and was also observed here. It is not clear additional signicant correlations with
whether this relationship is reective of the serum and sputum inammatory marker
impact of virulent pathogens (such as levels were observed by this strategy.
P. aeruginosa) outcompeting other species, Furthermore, grouping of patients
increased antibiotic exposure in more according to dominant taxon was also more
severe disease, or a direct role of reduced informative in terms of predicting future
diversity as a primary driver of disease. clinical course. For example, whereas
Although the high antibiotic treatment H. inuenzae culture positivity showed no
burden in patients with more severe disease signicant clinical correlations, patients
(16, 17) seems a likely explanation, no whose bacterial community was dominated
relationship was observed here between by H. inuenzae had signicantly fewer
bacterial diversity and the number of exacerbations, and received signicantly
courses of antibiotics a patient received in fewer days of antibiotics, compared with
the year before sampling. patients dominated by either P. aeruginosa
However, richness measures do not or other species. In addition, community
reect the relative abundance of clinically dominance by P. aeruginosa or a Veillonella
important species. Here, dening samples species was associated with a signicantly
according to their predominant taxon greater risk of frequent future
provided a substantial advantage. In exacerbations.
addition to the relationship between The results of this study suggest distinct
P. aeruginosa, poor lung function, and clinical and prognostic associations
Table 5. Median relative abundance and bacterial genus richness of samples used for prognostic analysis, grouped according to
dominant taxon
Dominant Species Relative Abundance (%)
n Median
All samples 42 86.3
Pseudomonas aeruginosa dominated 12 95.2
Haemophilus inuenzae dominated 14 96.2
Dominated by another genus 16 34.5
Veillonella spp. 8 34.6
Prevotella spp. 4 33.8
Burkholderia spp. 1 (58.1)
Flavobacterium spp. 1 (16.7)
Leptotrichia spp. 1 (34.6)
Pasteurella spp. 1 (99.6)
Definition of abbreviation: IQR = interquartile range.
Values in parentheses indicate single measures, rather than median values.
Rogers, Zain, Bruce, et al.: Exacerbation Prediction in Bronchiectasis
according to bacterial taxa might exist.
Pseudomonas predominance is associated
with markers of more severe disease
exacerbations, and greater antibiotic use.
96 In contrast, Haemophilus predominance
96 is associated with fewer pulmonary
96 exacerbations (historically and
96 prospectively). However, Haemophilus
86
86 bacterial load was associated with
81 inammatory activation assessed both
81 locally (in sputum) and systemically (in
81 blood); it is tempting to speculate that
81
96
the host inammatory response to
Haemophilus infection, while increased,
contains infection and prevents
exacerbation, in contrast to infection with
Pseudomonas and other dominant taxa.
Although providing a superior basis for
the stratication of non-CF bronchiectasis
airway infections compared with standard
microbiology, identication of dominant
taxa through deep sequencing represents
a much greater investment of resources.
However, if airway microbiota are
conserved over time within individuals,
infrequent assessment could still provide
important prognostic information. Further,
unlike culture-based analysis, which
indicated that approximately one-quarter of
samples analyzed were culture negative,
sequencing identied a dominant bacterial
taxon in all samples. Despite bacterial loads
being signicantly lower than in samples
from other patients, they were still
substantially above levels that might be
expected in lower airway samples from
healthy individuals (18), suggesting strongly
that they represent chronic infection.
Bacterial Richness (Genus)
IQR Median IQR
60.4 19.5 18.5
6.4 18.5 8.8
15.7 11.0 5.8
13.9 29.5 13.0
10.0 34.5 13.0
8.6 29.5 6.3
(11.0)
(38.0)
(27.0)
(11.0)
501
 ORIGINAL RESEARCH

In many cases, the predominant taxa in
culture-negative samples were recognized
to be associated with lower respiratory
infection, for example, Moraxella,
Stenotrophomonas, or Burkholderia species.
However, the genera Prevotella and
Veillonella were both the dominant taxa in
about one-quarter of the samples that were
not dominated by either P. aeruginosa
or H. inuenzae. This observation is
consistent with culture-based work
described elsewhere (8). Further,
Veillonella predominance was predictive
of a high frequency of subsequent
exacerbation, a nding that highlights the
contribution of bacterial species outside
those traditionally considered important
in a respiratory context. As Veillonella
species would not be isolated under
conditions used for routine diagnostic
testing of respiratory samples, they are
likely to be underreported. The data
presented here suggest their potential to
act as a marker for disease progression, or
indeed to play a direct role in
pathogenesis in this context.
Some genera detected in high relative
abundance here, including Veillonella
species, are commonly associated with the
oral cavity, and the potential for sample
contamination is difcult to exclude. The
absence of a signicant divergence in
microbiota composition between matched
induced sputum and bronchoalveolar
lavage samples has been demonstrated
previously, with a high prevalence
of species including members of the
genera Veillonella and Prevotella in
bronchoalveolar lavage uid from this
patient group (7). Further, there is
evidence that although contamination of
CF sputum by oral species occurs at low
relative levels, the presence of oral
anaerobes in sputum in high relative
abundance is likely to represent lower
airway colonization (19). Despite this
evidence, the isolation of these bacteria
directly from areas of bronchiectasis
within the lung, perhaps by directed
bronchoalveolar lavage, represents an
important next step in determining
the extent of their contribution to disease.
Given their potential clinical
importance, it is worth considering the
nature of Veillonella species. Members of
this genus are anaerobic, gram-negative
cocci and are residents of the oral cavity,
gastrointestinal tract, and vagina (20).
Veillonella species are commonly recovered
from abscesses, aspiration pneumonias,
burns, bites, and sinuses (21). In many
instances, these infections are polymicrobial
(21). Perhaps partly because samples from
chronic respiratory infections are rarely
subjected to anaerobic diagnostic
microbiology, the role played by this genus
in disease progression is poorly understood.
However, Veillonella species are generally
susceptible to most antibiotics used for the
treatment of anaerobic infections, including
b-lactam antibiotics, clindamycin, and
metronidazole (21).
Limitations of this study include the
limited number of patients from whom
samples were obtained, and the basis of
analysis in a single time-point sample. Here,
the variability of airway microbiology both
within and between patients is an important
consideration, and one that points to an
extension of the analysis presented here to
a larger patient population. The limitations
of using 16S rRNA gene pyrosequencing to
characterize the airway microbiota must
also be considered. The relatively short read
lengths mean that identication of bacteria
to a species level is not always possible,
with additional methods needed when such
species-level identication is important
(species-specic PCR assays were used here
to conrm identication of P. aeruginosa
and H. inuenzae).
In conclusion, this stratication system
provides highly clinically relevant output
from complex bacterial community data
that surpasses culture-based techniques. In
subjects with non-CF bronchiectasis this
system enabled future exacerbation risk to
be predicted, including in the substantial
subset of subjects with dominant bacterial
taxa that are not ordinarily identied by
culture and have not previously been
considered to be pathogenic. n
Author disclosures are available with the text
of this article at www.atsjournals.org.

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