JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 2011, p. 281–291
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 49, No. 1
Analysis of the Bacterial Communities Present in Lungs of Patients
with Cystic Fibrosis from American and British Centers?
Franziska A. Stressmann,1Geraint B. Rogers,1Erich R. Klem,2Andrew K. Lilley,1
Scott H. Donaldson,2Thomas W. Daniels,3Mary P. Carroll,3Nilesh Patel,4
Benjamin Forbes,4Richard C. Boucher,2Matthew C. Wolfgang,2,5
and Kenneth D. Bruce1*
Molecular Microbiology Research Laboratory, Pharmaceutical Science Research Division, King’s College London, 150 Stamford Street,
Franklin-Wilkins Building, London, SE1 9NH, United Kingdom1; Cystic Fibrosis/Pulmonary Research and Treatment Center,
University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 275992; Cystic Fibrosis Unit,
Southampton University Hospitals NHS Trust, Tremona Road, Southampton, SO16 6YD,
United Kingdom3; Pharmaceutical Science Division, King’s College London,
150 Stamford Street, Franklin-Wilkins Building, London, SE1 9NH,
United Kingdom4; and Department of Microbiology and Immunology,
University of North Carolina at Chapel Hill, Chapel Hill,
North Carolina 275995
Received 16 August 2010/Returned for modification 24 September 2010/Accepted 1 November 2010
The aim of this study was to determine whether geographical differences impact the composition of bacterial
communities present in the airways of cystic fibrosis (CF) patients attending CF centers in the United States
or United Kingdom. Thirty-eight patients were matched on the basis of clinical parameters into 19 pairs
comprised of one U.S. and one United Kingdom patient. Analysis was performed to determine what, if any,
bacterial correlates could be identified. Two culture-independent strategies were used: terminal restriction
fragment length polymorphism (T-RFLP) profiling and 16S rRNA clone sequencing. Overall, 73 different
terminal restriction fragment lengths were detected, ranging from 2 to 10 for U.S. and 2 to 15 for United
Kingdom patients. The statistical analysis of T-RFLP data indicated that patient pairing was successful and
revealed substantial transatlantic similarities in the bacterial communities. A small number of bands was
present in the vast majority of patients in both locations, indicating that these are species common to the CF
lung. Clone sequence analysis also revealed that a number of species not traditionally associated with the CF
lung were present in both sample groups. The species number per sample was similar, but differences in species
presence were observed between sample groups. Cluster analysis revealed geographical differences in bacterial
presence and relative species abundance. Overall, the U.S. samples showed tighter clustering with each other
compared to that of United Kingdom samples, which may reflect the lower diversity detected in the U.S. sample
group. The impact of cross-infection and biogeography is considered, and the implications for treating CF lung
infections also are discussed.
Cystic fibrosis (CF) is one of the most common genetic
diseases in Europe and North America, with an incidence of
around 1 in 2,500 (7). Due to the altered lung physiology, CF
patients suffer chronic bacterial lung infections (25). The host
immune response to these infections leads to an irreversible
loss of lung function, with 85 to 95% of CF patients ultimately
succumbing to respiratory failure (11, 20, 25, 34). Therefore,
gaining a better understanding of the characteristics and dy-
namics of these infections is of the greatest importance.
A limited number of bacterial species have been tradition-
ally considered key pathogens in CF lung disease progression,
including Pseudomonas aeruginosa, Staphylococcus aureus,
Haemophilus influenzae, Burkholderia cepacia complex, and
Stenotrophomonas maltophilia (18, 20, 22, 25). Conventional
diagnostic microbiology focuses on the detection of a limited
group of species, including those listed above (16). However,
more recent studies have indicated that the range of bacterial
species harbored in sputa from adult CF patients is much wider
(28, 29, 31). These findings emerged through the application of
culture-independent analytical strategies that detect bacterial
species signatures in nucleic acids extracted directly from clin-
ical samples. In particular, the combination of 16S ribosomal
RNA (rRNA) gene terminal restriction fragment length poly-
morphism (T-RFLP) profiling and 16S rRNA clone sequence
analysis has revealed the widespread presence of anaerobic
bacterial species within the lower CF airways (15, 29).
Patient characteristics, such as cystic fibrosis transmembrane
conductance regulator (CFTR) genotype, the presence of dis-
ease manifestations (e.g., CF-related diabetes), and patient
demographics, vary according to the country of treatment (5,
11, 17). Although median survival for the United Kingdom and
the United States is similar, with approximately 35 years for
both (2, 9), patient and disease characteristics have been
shown to vary, e.g., 40% of patients over 40 years in a United
Kingdom CF center were pancreatic sufficient, compared to
only 16% of patients in an equivalent U.S. center (17). There
* Corresponding author. Mailing address: King’s College London,
FWB, Pharmacy, 150 Stamford St., London, SE1 9NH, United King-
dom. Phone: 207 848 4670. Fax: 207 848 4500. E-mail: kenneth.bruce
?Published ahead of print on 10 November 2010.
TABLE 1. Patient and sample informationa
Last i.v. antibiotics given
Last oral antibiotics given
INTRON 17 POL?
711 ? 3A7G
3849 ? 10Kb-?T
Tobramycin every other
Colistin due to
282 STRESSMANN ET AL.J. CLIN. MICROBIOL.
are many possible reasons why this may be the case, including
socioeconomic factors, neonatal screening practices, and dif-
ferences in treatment practices (14). Recommendations for the
latter differ to some extent between the United Kingdom and
United States (6, 10, 39). In addition, how these recommen-
dations are implemented depends on individual treatment cen-
ters and can vary considerably.
However, as CF lung disease involves infection by bacteria
of an apparently wide range of origins, it may be important to
consider the environment as a source of these species and thus
as another disease-modifying factor. The concept of early mi-
crobiological studies that “everything is everywhere, but the
environment selects” infers that despite the purging effect of
selection in different habitats, microbial dispersal is wide-
spread, and as such it offers little opportunity for the spatial
differentiation of bacterial species (8, 26).
Evidence is emerging that the composition of bacterial com-
munities in different habitats can vary from region to region (4,
12, 13, 19). The degree to which endemism (localized popula-
tions) is important in terms of its impact on species presence in
the CF lung is not clear. This could, however, be of clinical
importance, given that many of the species regarded as patho-
gens are present in a range of natural environments, e.g., P.
aeruginosa, B. cepacia complex, and S. maltophilia. For exam-
ple, P. aeruginosa has been shown to occur worldwide, but
clones with increased virulence and enhanced antibiotic resis-
tance occasionally arise, leading to localized epidemic spread
in the environment and CF centers, such as the Liverpool
epidemic strain (27, 33).
In this study, bacterial community profiles, generated from
adult CF patients in two distinct geographical regions (the
United States and the United Kingdom), were compared to
investigate geographical differences in the bacterial CF airway
community. Culture-independent approaches of terminal re-
striction fragment length polymorphism (T-RFLP) profiling
and clone sequence analysis were used to analyze the bacterial
content of sputum samples. Patients were grouped in pairs,
with one United Kingdom and one U.S. patient matched on
parameters considered clinically relevant. The analysis of these
data showed that, both in terms of species presence/absence as
well as the relative prevalence of species in the lung, no clear
transatlantic divide could be defined between bacterial com-
munities. More-subtle differences in community composition
and structure were, however, observed.
MATERIALS AND METHODS
Clinical samples. Spontaneously expectorated sputum samples were collected
from 19 adult CF patients attending the Adult Cystic Fibrosis Clinic at
Southampton General Hospital, United Kingdom, and 19 patients attending the
University of North Carolina Cystic Fibrosis/Pulmonary Research and Treat-
ment Center, Chapel Hill, NC, under full ethical approval. After collection,
samples were stored at ?80°C prior to nucleic acid extraction. All nucleic acid
extractions, as well as T-RFLP analysis, were carried out by one person in the
laboratory in the United Kingdom. Clone sequence analysis was performed on
these same nucleic acid extracts by one person in the United States. Patient age,
sex, lung disease severity, CFTR genotype, body mass index (BMI), and antibi-
otic therapy details relating to these samples are shown in Table 1. Patients
recruited to this study represented a random cross-section of CF patients. All
were between 18 and 47 years old, with 12 females and 7 males per group.
Enrolled patients from the United Kingdom and United States were paired
according to age, gender, and lung obstruction category (forced expiratory vol-
ume in 1 second [FEV1%] predicted), as assessed clinically (Table 1). An age
3% HS with
aPatients are numbered in pairs. Lung obstruction categories: normal, ? 80%; mild, 60 to 80%; moderate, 40 to 59%; severe, ?40%. NK, not known; HS, hypertonic saline.
VOL. 49, 2011BACTERIA IN THE LUNGS OF U.K. AND U.S. CF PATIENTS 283
difference of less than 10 years together with a difference of 15% or less in
FEV1% predicted but in the same lung obstruction category (i.e., normal, mild,
moderate, severe) was considered acceptable. Mean age difference was 5.4 years
(standard deviation [SD], 4.1) and mean difference in FEV1% predicted was
10.1% (SD, 7.7%). Exceptions where only one parameter could be matched
acceptably where UK/US1, UK/US3, UK/US4, and UK/US9, with an age differ-
ence and difference in FEV1% predicted of 8 years and 30%, 7 years and 18%,
17 years, and 2% and 9 years and 20%, respectively. Additional criteria, such as
distance to hospital, were recorded. Patients were clinically stable at the time of
sampling (at least 21 days had passed before or after a treatment for pulmonary
exacerbation), with eight exceptions (U.S. patients 4 and 19 and United Kingdom
patient 19, who were receiving intravenous [i.v.] antibiotics for pulmonary exac-
erbation; U.S. patient 2, United Kingdom patient 2, and United Kingdom patient
4, who had finished i.v. antibiotics 12, 14, and 13 days previously, respectively;
and U.S. patients 7 and 17, who were clinically stable but were receiving antibi-
otic therapy for reasons other than pulmonary exacerbation). In all cases, clinical
status was given priority over antibiotic therapy for patient matching.
DNA extraction. Prior to DNA extraction, sputum samples were washed three
times in phosphate-buffered saline (PBS; Fisher Scientific, Loughborough,
United Kingdom) to remove adherent saliva. DNA extraction from clinical
samples was adapted from a previously described procedure (29). Briefly, 200 ?l
of each sputum sample was resuspended in 800 ?l of 200 mM PBS (pH 8.0) and
300 ?l guanidium thiocyanate-EDTA-Sarkosyl. After the addition of 0.2 g of
0.18-mm-diameter glass beads (B. Braun Biotech International GmbH, Melsun-
gen, Germany), samples were homogenized for 60 s at 30 Hz in a Mixer Mill 300
(Qiagen, Crawley, United Kingdom). Samples were heated at 70°C for 20 min
and placed on ice for 20 min, and beads and cell debris were removed by
centrifugation (13,000 ? g for 5 min) at room temperature. Supernatants were
transferred to fresh microcentrifuge tubes, followed by the addition of NaCl (to
a final concentration of 0.5 mM) and polyethylene glycol (to a final concentration
of 15%). Nucleic acids were precipitated at 4°C for 1 h.
DNA was pelleted at room temperature by centrifugation at 13,000 ? g for 10
min and resuspended in 300 ?l nuclease-free water. Supernatants were trans-
ferred to fresh microcentrifuge tubes, and 300 ?l Tris-acetate-EDTA (TAE)-
saturated phenol (pH 8.0) (Sigma-Aldrich, Gillingham, United Kingdom) was
added. After the mixture was vortexed vigorously, the phases were separated by
centrifugation at 13,000 ? g for 5 min. The upper phase was transferred to a fresh
microcentrifuge tube, 300 ?l phenol-chloroform-isoamylalcohol (25:24:1 ratio)
(Sigma-Aldrich) was added, and samples were vortexed vigorously and centri-
fuged at 13,000 ? g for 10 min. Supernatants were transferred to fresh micro-
centrifuge tubes, and DNA was precipitated at ?20°C for 1 h after the addition
of an equal volume of isopropanol (Sigma-Aldrich) and 0.1 volume of 10 M
ammonium acetate. The DNA was pelleted by centrifugation at 13,000 ? g for 10
min and washed three times in 200 ?l 75% ethanol. Pellets were briefly air dried,
resuspended in 50 ?l of nuclease-free water, and stored at ?20°C.
DNA verification and quantification. Extracted genomic DNA and PCR prod-
ucts were verified by TAE-agarose gel electrophoresis stained with GelRed
(Biotium, Hayward, CA) and visualized on a UV transilluminator (Herolab,
Wiesloch, Germany). Images were captured by using a Herolab image analyzer
with E.A.S.Y. Stop win 32 software (Herolab). DNA and PCR products were
quantified by spectrophotometry by applying 1.5 ?l directly to a NanoDrop
ND-1000 spectrophotometer (LabTech International, Ringmer, United King-
PCR and T-RFLP profiling. The universal oligonucleotide primers for the
amplification of a region of the 16S rRNA gene specific for the domain bacteria
926r (5?-CCG TCA ATT CAT TTG AGT TT-3?) and 8f700IR (5?-AGA GTT
TGA TCC TGG CTC AG-3?) were used as described previously (24). Primer
926r was unlabeled, and primer 8f700 was labeled with the dye IRD700 at the 5?
end. Both primers were synthesized by TAGN, Newcastle, United Kingdom. The
constituents of the PCR mixture per reaction were the following: 25 ?l of the
Sigma readymix REDTaq (Sigma-Aldrich), each primer at a final concentration
of 0.2 mM, 50 ng of DNA template, made up to a final volume of 50 ?l with
Cycling conditions comprised an initial denaturation at 94°C for 2 min, fol-
lowed by 32 cycles of denaturation at 94°C for 1 min, annealing at 56°C for 1 min,
and extension at 72°C for 2 min, with a final extension step at 72°C for 10 min.
Amplifications were carried out in a Gene Amp PCR system 9700 (Applied
Biosystems, United Kingdom). PCR products were verified on TAE-agarose gels
as described above and stored at ?20°C for T-RFLP analysis.
Approximately 20 ng of each PCR product was digested to completion with 1
U of the restriction endonuclease CfoI (Sigma-Aldrich) for 5 h at 37°C, in
accordance with the manufacturer’s instructions. The restriction enzyme was
inactivated by heating to 90°C for 20 min. Approximately 10 ng of digested PCR
product was denatured at 95°C for 1 min and separated by length using a 25-cm
SequagelXR denaturing polyacrylamide gel (National Diagnostics, Hessle,
United Kingdom), with the addition of 8.3 M urea and formamide (to a final
concentration of 10%). Electrophoresis was performed at 55°C and 1,200 V on
an IR2 automated DNA sequencer (LI-COR Biosciences, Lincoln, NE).
T-RFLP profile analysis. T-RFLP gel images were analyzed using Phoretix
one-dimensional advanced software (version 5.10; Nonlinear Dynamics, New-
castle upon Tyne, United Kingdom). The lengths of the bands detected on the
gel were determined by comparison to the positions of the size marker
microSTEP 15a (700 nm) (Microzone, Lewes, United Kingdom). Additionally,
Phoretix software was used to determine the band volume (the product of the
area over which the band was detected and the intensity of signal recorded over
that area). The band volume was expressed as a percentage of the total volume
of bands resolved in one particular electrophoretic profile. The resolution of
T-RFLP bands was over the region of 50 to 950 bases. T-RF bands shorter than
50 bases were not included in the analysis, as this region is susceptible to high
levels of background signal. The threshold of band detection used in this study
was 0.1% of total signal in one profile in the specified region.
In silico sequence analysis and band identification. Band identification was
performed as described previously by Rogers and coworkers (28, 29). Pub-
lished bacterial 16S rRNA gene sequences were retrieved from GenBank
MapSort (Wisconsin Package, version 10.3; Accelrys, Cambridge, United King-
dom) was used to predict T-RFLP band lengths (in bases) from the 5? end of
primer 8f700IR to the first site of cleavage for CfoI in each recovered 16S rRNA
Generation of 16S rRNA clone libraries and sequence analysis. 16S rRNA
gene fragments were generated by PCR using primers 8f700 and 926r and the
high-fidelity proofreading enzyme PFU-Ultra (Stratagene, La Jolla, CA) as de-
scribed above. The pool of 16S rRNA gene fragments was cloned into the
pCR-Blunt II-TOPO plasmid vector (Invitrogen, Carlsbad, CA) according to
manufacturer’s instructions. Plasmids then were used to transform competent
Escherichia coli DH5? by heat shock at 42°C and plated on selective LB agar
plates with 50 ?g/ml kanamycin. Individual colonies were used to inoculate 2 ml
of LB broth containing 50 ?g/ml kanamycin in 96-deep-well blocks and grown for
20 h with vigorous shaking at 37°C. One hundred microliters of culture was saved
as a glycerol stock, and the remaining 1.9 ml of culture was centrifuged at 500 ?
g for 10 min. Supernatants were removed and plasmid DNA isolated from the
bacterial pellet with the Wizard SV 96 plasmid purification system (Promega
Corporation, Madison, Wisconsin). The quality of resulting cloned 16S rRNA
gene fragments containing plasmid DNA was assessed by TAE-agarose gel
electrophoresis. Aliquots of purified plasmid were sequenced with M13 vector
primer (MWG Biotech, High Point, NC). A total of 871 clones derived from
samples taken from five patients from each geographical location were se-
Phylogenetic tree construction. For each patient, sequences were vector
trimmed with Sequencher v4.8 (Gene Codes Corporation, Ann Arbor, Michigan)
and aligned at 100% identity and clustered. The longest sequence of each cluster
was conserved and shorter redundant sequences removed. Sequences were
aligned with the Ribosomal Database Project (RDP; Michigan State University,
Michigan) Pyrosequencing Alignment and Clustering Program (http://pyro.cme
.msu.edu/) and clustered into bins of 98% similarity with the “farthest nearest
neighbor” complete linkage clustering algorithm. To reduce bin complexity, bins
containing three or more sequences were aligned using the Greengenes align-
ment tool (http://greengenes.lbl.gov/cgi-bin/nph-NAST_align.cgi) using a batch
size of 100, a minimum length of 100, and a minimum percent identity of 75%.
The bins of three or more aligned sequences were transferred to ClustalX v2.0.12
(Conway Institute, UCD, Dublin, Ireland), and a multiple alignment was carried
out with an output in the form of a phylogenetic tree. For each bin, the longest
most-similar branch was chosen as the representative sequence, labeled to rep-
resent the number of sequences it contained (at 100 and 98% sequence identity),
and the remaining sequences removed. Overall, this reduced sequence complex-
ity from 871 to 136. To construct the phylogenetic tree for all patients, these 136
binned sequences were put into the Greengenes alignment tool and ClustalX as
described above, together with the sequences of type strains that all sequences
were most similar to, including CF pathogens, and the phylogenetic tree was
constructed with the same parameters as those described above. Clusters were
named according to the type strains that the sequences clustered most closely
Statistical analysis. Correlations, t tests, and one-way analysis of variance with
tests of normality and homogeneity of variance, as well as Bonferroni and Tukey
post hoc tests, were performed using SPSS software version 15.0.1 (Chicago, IL).
Raup and Crick as well as Morisita similarity coefficients were used to
284STRESSMANN ET AL.J. CLIN. MICROBIOL.
evaluate similarities in community composition. Similarity coefficients were
calculated and dendrograms plotted using PAST software version 1.74 (http:
//folk.uio.no/ohammer/past/; University of Oslo, Norway). Raup and Crick, a
probability-based similarity index (SRC), was calculated from binary matrices
of OTU presences and absences. The probabilities were calculated using
2,000 Monte Carlo simulations to compare the number of bands shared by
two samples and the number of bands predicted if two samples were randomly
selected from an amalgamation of all the samples studied.
Nucleotide sequence accession numbers. Sequences determined in the course
of this work are available through the EMBL nucleotide sequence database
(http://www.ebi.ac.uk/embl/) under accession numbers FN825913 to FN826783.
Community composition: species absence/presence. In the
38 sputum samples analyzed from both United Kingdom and
U.S. patients by T-RFLP, a total of 173 bands representing 73
different T-RF lengths were detected. The number of individ-
ual bands resolved in each sample ranged from 2 to 10 for the
U.S. patients and 2 to 15 for the United Kingdom patients. The
mean number of T-RF bands per patient of 4.6 (standard
deviation [SD], 3.6) in the United Kingdom sample set was
similar to that of the U.S. sample set at 4.5 (SD, 2.3) and not
significantly different [P ? 0.87; t(df 31)? 0.159]. A modest
significant correlation was observed between the number of
bands detected for the paired U.S. and United Kingdom pa-
tient sets [P ? 0.03; r(df 17)? 0.496].
Of the 173 T-RF bands detected, 32.4% were “singletons,”
defined here as bands that occurred in only one sample.
Singletons were detected in nine United Kingdom and eight
U.S. samples. The mean number of singletons in these patients
was 1.7 (SD, 3.0) and 1.2 (SD, 1.8) for United Kingdom and
U.S. patients, respectively. This difference was not significant
[P ? 0.51; t(df 30)? 0.66].
T-RF band lengths that occurred more than once were an-
alyzed in detail. All band lengths that occurred more than four
times in the overall sample set were present in at least one
United Kingdom and U.S. patient. Certain band lengths were
commonly found in both sample sets. Ten band lengths ac-
counted for 57% of all bands and were found in at least one
United Kingdom and U.S. patient. Species consistent with
these band lengths included Pseudomonas aeruginosa, Staphy-
lococcus aureus, and Stenotrophomonas maltophilia, certain an-
aerobic species of the Prevotella genus, as well as two band
lengths for which no species prediction could be made.
Clone sequence analysis was carried out for five patients
from the United Kingdom (UK1, UK10, UK12, UK14, and
UK15) and the United States (US1, US7, US11, US17, and
US18). Samples for this analysis were chosen on the basis of
T-RFLP results to include two patients from each group with
high diversity (at least 10 different T-RF band lengths), one
patient with low diversity (5 or fewer bands), and two patients
for which the band at 155 bases, consistent with P. aeruginosa,
was not detected. Clone sequence analysis showed that clones
FIG. 1. Phylogenetic tree of clones from United Kingdom and U.S.
patients. Clone sequences are clustered with the bacterial type strains
to which they showed highest sequence similarity (minimum of 98%
similarity for cluster membership). Clones were grouped into unique
sequences; each labeled sequence indicates the patient it was isolated
from and the number of redundant clone sequences it comprises.
Clones isolated from United Kingdom patients are shown in green,
clones isolated from U.S. patients are shown in blue.
VOL. 49, 2011 BACTERIA IN THE LUNGS OF U.K. AND U.S. CF PATIENTS285
clustered with 30 different typed strains. These typed strains
were used to name the clusters of the phylogenetic tree con-
structed (Fig. 1, Table 2) and here will be referred to as
species. A wide range of species was identified from 22 differ-
ent genera. These included one species, Phenylobacterium ko-
reense, which had not previously been associated with the CF
lung, as well as six species (20%) of obligate anaerobes. Over-
all, 9 genera of 22 were detected in both patient groups (Table
2, Fig. 1). Individual species within eight bacterial genera de-
tected, namely, Pseudomonas, Streptococcus, Actinomyces,
Staphylococcus, Parvimonas, and Neisseria, as well as two gen-
era of the obligate anaerobes Prevotella and Veillonella, were
present in at least one individual from both the United King-
dom and United States as analyzed by this approach (Table 2,
Fig. 1). In the United Kingdom patient group, the most abun-
dantly identified clones were P. aeruginosa and S. maltophilia
(31% each), and in the U.S. patient group they were P. aerugi-
nosa (30%) and S. aureus (33%). Overall, 12 (40%) species
were found for the United Kingdom patient group alone, 7
(23%) species were found for the U.S. patient group alone,
and 11 (37%) species occurred in both patient groups (Fig. 1,
Table 2). Type strain clusters that comprised four or more
unique sequences all contained sequences from both United
Kingdom and U.S. patients, with the exceptions of Phenylobac-
terium koreense and S. maltophilia, which both were detected in
the United Kingdom patient group only (Table 2, Fig. 1).
Similarities in community composition. The similarity of the
bacterial content of samples was statistically assessed on the
basis of the T-RFLP data. The patterns in shared species
composition between the two sample sets were assessed
through the generation of a Venn diagram (Fig. 2). From the
data set as a whole, the four most commonly occurring band
lengths, consistent with bands produced by P. aeruginosa (155
bases), Prevotella spp. (103 bases), Achromobacter/Bordetella/
Pseudomonas spp. (565 bases), and an unassigned band at 580
bases, were examined in detail here. The two largest sets (band
lengths of 155 and 565 bases) contained high numbers of both
U.S. and United Kingdom patients. In total, this represented
79% of both patient groups. Overall, all four of the most
commonly detected band lengths were found in at least one
patient from each geographical group. In two of the samples
studied (UK14 and US16), none of the four most commonly
occurring band lengths were detected, so they are presented as
outliers in Fig. 2.
To assess the significance of similarities and differences in
species composition between United Kingdom and U.S. sam-
ples, Raup and Crick similarity indices (SRC) were calculated
for the 38 T-RFLP profiles from United Kingdom and U.S.
TABLE 2. Type strain cluster details from Fig. 1a
Type strain cluster
No. of unique
present in type
No. of United
No. of unique
No. of U.S.
Total no. of
aThe number of unique sequences for each cluster, as well as the number of patients from which unique sequences were identified for United Kingdom and U.S.
samples, are shown. Clusters that contained sequences from both United Kingdom (UK1, UK10, UK12, UK14, and UK15) and U.S. (US1, US7, US11, US17, and
US18) patients are shown in boldface.
286STRESSMANN ET AL.J. CLIN. MICROBIOL.
patients, and a cluster diagram was constructed using un-
weighted pair-group averages (UPGMA) (Fig. 3). Values
above 0.95 indicate pairs of samples that were more similar
than expected by chance, and thus indicated significant clus-
tering. Using this 0.95 threshold, Fig. 3 is comprised of three
clusters; cluster 1 of four U.S. patients (US5, US10, US11, and
US13), cluster 2 of five United Kingdom patients (UK7, UK8,
UK17, UK18, and UK19), and a substantial mixed cluster (3)
of 10 U.S. patients and six United Kingdom patients (US1,
US2, US4, US7, US8, US12, US14, US15, US17, US19, UK1,
UK5, UK9, UK11, UK13, and UK16).
The degree of similarity of the U.S. and United Kingdom
samples was further assessed using the Raup and Crick prob-
ability-based similarity index calculated on species presences
and absences. This statistically resolved whether the samples
were significantly similar (SRC? 0.95), significantly dissimilar
(0.05 ? SRC), or neither (0.05 ? SRC? 0.95). Table 3 shows
the numbers and proportions of significant and nonsignificant
SRCvalues for a series of pairwise comparisons of SRCvalues
for United Kingdom with United Kingdom patients, U.S. with
FIG. 2. Venn diagram grouping patients according to the presence
of one or more of the four most commonly occurring T-RF band
lengths in the sample set. Circles correspond to T-RF band lengths;
patients within one or more circles shared these particular T-RF band
lengths. T-RF band lengths corresponded to the following organisms:
103 bases, Prevotella spp.; 155 bases, P. aeruginosa; 565 bases, Pseudo-
monas/Bordetella/Achromobacter spp.; 580 bases, not assigned.
FIG. 3. Cluster diagram of the Raup and Crick similarity coefficient assessment of United Kingdom and U.S. patient T-RFLP data.
VOL. 49, 2011 BACTERIA IN THE LUNGS OF U.K. AND U.S. CF PATIENTS287
U.S. patients, and the matched United Kingdom and U.S.
patients. Here, 23% of United Kingdom-U.S. pairs showed a
significant level of similarity. United Kingdom-U.S. pairs were
more similar than United Kingdom-United Kingdom pairs
(15%) but less similar than U.S.-U.S. pairs (41%). A significant
level of dissimilarity was detected for 4% of United Kingdom-
U.S. pairs. For United Kingdom patients, 4% were more dis-
similar from each other than expected by chance, and only 1%
of U.S. patients were significantly dissimilar from each other.
Further, this meant that many United Kingdom samples were
more similar to U.S. samples than to each other.
Community structure. In addition to species presence, the
similarity of the abundance of these species in samples was
investigated using T-RFLP band volume data. Morisita simi-
larity coefficients (IM) were calculated in a pairwise manner for
the relative volumes of bands of all 38 T-RFLP profiles, and a
cluster dendrogram was plotted using UPGMA (Fig. 4).
Two distinct clusters of exclusively United Kingdom patients
were formed with four and six patients, respectively: cluster 1
(UK7, UK8, UK17, and UK19) and cluster 2 (UK4, UK5,
UK11, UK13, UK15, and UK16). A third cluster of 11 U.S. and
one United Kingdom patient also was observed (US1, US4,
US7, US8, US11, US12, US13, US14, US15, US17, US19, and
UK9). The average IMvalue for United Kingdom-United
Kingdom patient comparisons was 0.27 (SD, 0.39; n ? 171), for
U.S.-U.S. comparisons was 0.59 (SD, 0.38; n ? 171), and for
United Kingdom-U.S. comparisons was 0.35 (SD, 0.38; n ?
361). These three average Morisita index values all were sig-
nificantly different at [P ? 0.0001, F(df 2, 700)? 33.21, and post
hoc tests]. Again the U.S. samples showed a stronger associa-
tion with each other than the United Kingdom samples showed
TABLE 3. Numbers and percentages of significant and
nonsignificant Raup and Crick similarity indicesa
No. (%) with SRCvalue:
Total no. (%)
aIndices are shown for pairwise comparisons for United Kingdom with United
Kingdom patients (U.K.-U.K.), U.S. with U.S. patients (U.S.-U.S.), and United
Kingdom with U.S. patients (U.K.-U.S.) from T-RFLP profiling. Values equal to
or above 0.95 indicate pairs of samples that are more similar than expected by
chance, and values equal to or less than 0.05 indicate pairs of samples that are
less similar than expected by chance.
FIG. 4. Cluster diagram for the Morisita similarity coefficient assessment of United Kingdom and U.S. patient T-RFLP data.
288 STRESSMANN ET AL.J. CLIN. MICROBIOL.
with each other, and the United Kingdom samples were sig-
nificantly more associated with the U.S. samples than with one
In summary, the comparison of the data presented in Fig. 3
and 4 showed that some degree of overlap was identified in
terms of cluster membership in relation to the two different
strategies of data analysis used. Figure 3 identified three clus-
ters, one cluster of 4 patients (US5, US10, US11, and US13),
one of 5 patients (UK7, UK8, UK17, UK18, and UK19), and
another of 16 patients (US1, US2, US4, US7, US8, US12,
US14, US15, US17, US19, UK1, UK5, UK9, UK11, UK13, and
UK16). Figure 4 identified another three clusters, one of 4
patients (UK7, UK8, UK17, and UK19), one of 6 patients
(UK4, UK5, UK11, UK13, UK15, and UK16), and another of
12 patients (US1, US4, US7, US8, US11, US12, US13, US14,
US15, US17, US19, and UK9).
Comparison of T-RFLP and clone sequence data. For 90%
of species identified by clone sequence analysis, a T-RF band
length for the corresponding cut site was detected. For Bre-
vundimonas diminuta and Parvimonas micra, no corresponding
T-RF band length could be detected, and for Oribacterium
sinus, the first CfoI cut site lay outside the T-RFLP detection
range. For 26% of T-RF bands a species with a corresponding
cut site was detected by clone sequence analysis. The detection
of species by clone sequencing that corresponded to T-RF
band lengths in individual patient’s profiles ranged from 13%
(United Kingdom patient 1) to 67% (United Kingdom patients
10 and 12).
The aim of this study was to compare the bacterial commu-
nities present in sputum collected from adult CF patients at-
tending CF centers in the United States and United Kingdom.
Patients in the two countries were paired prior to microbio-
logical assessment according to clinical parameters. Two cul-
ture-independent, molecular strategies were used to analyze
sample pairs, T-RFLP profiling and 16S rRNA clone sequence
analysis. Both relied on the PCR amplification of the 16S
rRNA gene from DNA extracted directly from bacteria in the
sputum samples collected. These approaches have been used
previously to characterize the communities of bacteria in the
adult CF lung (3, 15, 28–32, 35, 36). To the best of our knowl-
edge, this is the first application of such techniques to assess
the impact of geographical location on bacterial community
composition in CF respiratory samples.
To normalize for variation other than geographical group-
ing, patients were matched into pairs to align major clinical
parameters considered important by the treating clinicians
(age, sex, lung obstruction, BMI, and CFTR genotype). An
attempt to match for other parameters, such as distance to
clinic and treatment, also was made. However, due to differ-
ences in the size of the catchment area and some aspects of the
treatment regimes, lower priority was given to these parame-
ters. While we recognize that no system of matching patients is
ideal, this approach allowed 19 pairings to be defined based on
The T-RFLP profiling of the generated 16S rRNA PCR
products was used to assess the bacterial community. A total of
73 distinct T-RF band lengths were generated for the 38 pa-
tients studied, which each were regarded as an individual bac-
terial species. The paired U.S. and United Kingdom patients
showed similar numbers of bacterial species within their sam-
ples, suggesting that one or more of the clinical parameters
used to assign patients to their pair were relevant. No marked
difference was identified in the mean number of species per
sample for the United Kingdom or U.S. patient group. While
no comparable studies could be identified in the literature, the
mean number of species obtained here was lower than that in
earlier studies of stable adult CF patients (29, 30, 36), with this
difference accounted for by the selection of a higher detection
threshold. Statistical assessment showed a modest significant
correlation in species number in relation to the patient pairs.
As such, the number of species per sample did not provide
evidence for a geographical difference in samples from the
United Kingdom or U.S.
No difference was identified in the number of singletons
from patients in the United Kingdom or U.S. Overall, approx-
imately one third of all species detected occurred as singletons.
Taken together, these findings imply that the lung community
has a strong component that is patient specific regardless of
patient geographical location. Other evidence reinforcing the
highly different nature of bacterial communities between pa-
tients was found in particular for two patients, one from the
United Kingdom and one from the United States, whose sputa
contained no species that were common with those found in
the other 36 patient samples studied here.
An examination of the species most frequently detected in
the sample set (detected in four or more patients and at least
once in each geographical cohort) suggested that certain spe-
cies not traditionally associated with CF respiratory infections
are common inhabitants of the CF lung. This is particularly
noteworthy given the high degree of microbial diversity re-
ported in this and earlier studies (29, 30, 36). The presence of
species not recognized as key CF pathogens in the airways of a
substantial number of patients may have important implica-
tions for treatment, and this is an area that warrants further
Indeed, a wide range of different bacterial species other than
the key CF pathogens has been identified in the past decade
(15, 28, 29, 36). Clone sequence analysis from 10 patients, with
five patients representing each geographical group, revealed 30
different species from 22 different genera. Species traditionally
associated with the CF lung, e.g., Pseudomonas aeruginosa,
Staphylococcus aureus, and Stenotrophomonas maltophilia (1),
as well as a number of species previously detected in CF sam-
ples primarily by culture-independent means, were identified
here (15, 28, 29, 36), including one species that had not pre-
viously been reported in the context of CF, Phenylobacterium
koreense. Eleven (37%) of the species, identified within the
genera Pseudomonas, Streptococcus, Actinomyces, Staphylococ-
cus, Parvimonas, Neisseria, Prevotella, and Veillonella, were
common to both the United Kingdom and U.S. patient group.
Interestingly, species from within the latter two genera require
anaerobic conditions for growth. Anaerobes previously have
been detected in CF sputum (3, 15, 29, 37), nonetheless it was
striking that even in geographically distinct locations, similar
lung physiological conditions were reflected in the species
present. However, the majority of species were uniquely found
VOL. 49, 2011BACTERIA IN THE LUNGS OF U.K. AND U.S. CF PATIENTS 289
in one geographical location, suggesting a strong local domi-
nance in their biogeography.
To determine whether there is an uneven geographical dis-
tribution of the four most commonly detected T-RF bands,
patients were grouped according to whether each band was
present in their sputum sample. These groupings are illus-
trated in the form of a Venn diagram (Fig. 2). Here, two large
clusters formed that contained both U.S. and United Kingdom
patients to approximately 80% of all patients studied. This
suggests that certain species are core to the CF lung by adult-
hood. Here, it was found that species common by this defini-
tion were present within at least one patient from both sample
pools. As such, this provides evidence that these species are
not endemic to either geographical region.
Similarity indices were employed to identify the degree of
similarity between United Kingdom and U.S. samples. The
first of these used here was the Raup and Crick similarity
index. This analysis generated a set of three clusters represent-
ing similar bacterial communities. None of these clusters was
found to relate to patient pairs. In contrast, these three clusters
comprised one containing only U.S. patients, another contain-
ing only United Kingdom patients, and the last containing 10
patients from both the United States and United Kingdom. As
such, this provides evidence both for and against the transat-
lantic distribution patterns of members of the communities
Raup and Crick probability-based similarity indices (SRC)
were analyzed to determine the degree of significance of the
similarities identified between bacterial community member-
ships (38). Here, a large number of sample pairs with nonsig-
nificant values (0.05 ? SRC? 0.95) would indicate stochastic
dispersal, while a large number of significant SRCvalues would
indicate a structuring influence, such as niche selection. The
paired patients from the United States and United Kingdom
showed little tendency to cluster on the basis of community
composition, with evidence of this in only ca. 25% of patient
pairs. In patient groups from either of the two geographic
regions, where no attempt was made to pair on clinical
grounds, U.S. patients had communities that clustered to a
greater degree (ca. 40%) than U.S.-United Kingdom pairs,
whereas United Kingdom patients showed a lower degree of
significant clustering (ca. 15%). This suggests that the pairing
had only a limited impact on the community findings of this
aspect of the study. The reason that the United Kingdom
samples as well as the United Kingdom-U.S. matches differed
so much in terms of this analysis is not clear, although possible
explanations may rest in the differential treatments, exposure
to different microbes, or factors that were not assessed. It also
is possible that the lower diversity in U.S. samples, as con-
firmed within the detection limit of this study, allows for the
tighter grouping of these samples.
Even though samples may contain similar numbers or types
of species, the abundance of those species in the samples could
vary substantially, presenting completely different types of eco-
systems. Morisita similarity coefficients were used to determine
whether relative bacterial species abundance within samples
differed between patient groups in the United Kingdom and
U.S. As for the Raup and Crick analysis based on species
presence or absence, three clusters were identified. Two dis-
tinct clusters of exclusively United Kingdom patients (com-
prised of 4 and 6 patients, respectively) were formed with a
third cluster of 11 U.S. and 1 United Kingdom patient. Fur-
ther, when average Morisita index values for United Kingdom,
U.S., and paired United Kingdom-U.S. patient groups were
examined, U.S. samples again showed a greater level of simi-
larity. Thus, relative species abundance was more affected by
biogeography than was community membership.
Comparative studies of bacterial presence in the lungs of
patients attending different CF centers previously have only
focused on one or more species regarded as pathogens. Johan-
sen et al. (21) found that P. aeruginosa and B. cepacia complex
were more frequently detected in patients attending a CF cen-
ter in Toronto than in those attending an equivalent center in
Copenhagen. This focus on single species made it difficult to
carry out a full comparative analysis with the findings here. In
our study, bacterial species found to be common to both geo-
graphical patient groups included both known CF pathogens,
such as P. aeruginosa, and those species not traditionally asso-
ciated with CF lung infections, such as members of the genus
Prevotella. However, the analysis of larger patient groups will
be required to better characterize the geographic distribution
of these species that were commonly detected here but as yet
are not recognized as CF pathogens.
Certain bacterial species, typically regarded as being of en-
vironmental origin, can act as opportunistic pathogens. A num-
ber of such species were detected in the samples analyzed here;
however, whether these are derived from the wider environ-
ment, the immediate treatment environment, or directly from
other CF patients is not known. While some such species were
detected in both United Kingdom and U.S. patient groups,
others showed an uneven geographic distribution. This distri-
bution may be related to differences in the treatment regimes
between the two centers or environmental and biogeographical
factors. Again, a wider study of this topic is warranted to
determine the likely origin of the differences under the condi-
tion of patients attending different CF centers (23).
In conclusion, this study has considered the bacterial species
present in the lungs of CF patients in two centers in the United
States and United Kingdom. Species numbers per sample were
similar in the two sites, but differences in terms of species
identities were detected, as could be confirmed by the detec-
tion limit here. Despite this, the four most frequently detected
T-RF bands were present in 80% of all patients, suggesting
that certain species are common to the CF lung on both
continents. Overall, the U.S. samples showed a stronger asso-
ciation with each other than the United Kingdom samples,
which may reflect the lower diversity detected in U.S. samples.
Geographical differences were identified in species composi-
tion and more strongly in species prevalence in samples taken
from patients attending the two CF centers. Overall, it is clear
that the bacteriology of the CF lung is complex. Given the
importance of lung function to CF patient health, it is by
extension important to be able to understand this complexity as
the first step in advancing therapy for these patients.
This work was supported by the Anna Trust.
Work conducted at UNC-Chapel Hill was supported by a grant from
the National Institutes of Health (HL092964).
290STRESSMANN ET AL. J. CLIN. MICROBIOL.
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