JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 2011, p. 4352–4355
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 49, No. 12
True Microbiota Involved in Chronic Lung Infection of Cystic Fibrosis
Patients Found by Culturing and 16S rRNA Gene Analysis?
Vibeke B. Rudkjøbing,1Trine R. Thomsen,1,2Morten Alhede,3Kasper N. Kragh,3Per H. Nielsen,1
Ulla R. Johansen,4Michael Givskov,3Niels Høiby,3,4and Thomas Bjarnsholt3,4*
Department of Biotechnology, Chemistry, and Environmental Engineering, Faculty of Engineering and Science, Aalborg University,
Aalborg, Denmark1; The Danish Technological Institute, Life Science Division, Aarhus C, Denmark2; Department of
International Health, Immunology, and Microbiology, Faculty of Health Sciences, University of Copenhagen,
Copenhagen, Denmark3; and Department of Clinical Microbiology, Rigshospitalet,
University Hospital of Copenhagen, Copenhagen, Denmark4
Received 13 October 2011/Accepted 13 October 2011
Patients suffering from cystic fibrosis (CF) develop chronic lung infection. In this study, we investigated the
microorganisms present in transplanted CF lungs (n ? 5) by standard culturing and 16S rRNA gene analysis.
A correspondence between culturing and the molecular methods was observed. In conclusion, standard
culturing seems reliable for the identification of the dominating pathogens.
Cystic fibrosis (CF) is the most common lethal autosomal
recessively inherited disorder of Caucasians. Although several
organs are affected, the most severe effect is observed in the
lungs, which is the major cause of deaths of patients (5). Here,
genetic alterations of the chloride channel in epithelial cells
lead to dehydration of the airway mucus, increasing its viscos-
ity. This means that the cilia are unable to transport the mucus
in which inhaled material and, importantly, bacteria are en-
trapped, enabling microorganisms to colonize and establish
infections within the mucus (9). In the early stages of CF,
intermittent colonizations occur, which can be treated with
antibiotics (10). Establishment of chronic infection occurs over
time and is characterized by the formation and establishment
of bacterial aggregates (the so-called biofilms) (1, 5). Forma-
tion of biofilm is problematic since not only does this afford
protection against the different components of the host de-
fense in the lungs but the bacteria also become extremely
tolerant to antibiotics (1, 4, 5). Most pathogenic bacteria are
easily diagnosed by standard culture-based techniques; how-
ever, many less well recognized bacteria can be difficult to
culture due to their growth requirements or being very slow
growing or not growing at all if the patient has been treated
with antibiotics. In these cases, the standard culture techniques
may fail to detect these bacteria and detect only the more
readily culturable bacteria (14). In the CF centers in Denmark,
an intensive antibiotic treatment strategy has been shown to
prolong the life expectancy of the CF patients (10). In recent
studies, the chronically infected lungs of CF patients have been
observed to harbor multiple species (19, 21). However, the
strict antibiotic strategy employed in Denmark has led to only
a small variety of microorganisms being found in the lungs of
CF patients, compared to what is found in other studies (8, 18,
23). In a previous study, we applied fluorescence in situ hybrid-
ization (FISH) using peptide nucleic acid (PNA) probes to
investigate the spatial distribution of Pseudomonas aeruginosa
in the lungs of end-stage Danish CF patients by using both
general and specific probes and found P. aeruginosa to be
present alone (1). The end stage is defined as the time when
the lungs are destroyed and the lung function is reduced to an
extent where lung transplantation is required for the patient to
In the present study, we investigated the true microbiota of
the end-stage CF lung by investigating fresh samples directly
from explanted lungs of Danish CF patients undergoing dou-
ble lung transplantations. This was to avoid possible contami-
nation by the patient’s oral and pharyngeal flora during expec-
toration of sputum, which is the typical type of sample
investigated in CF studies.
We included 34 lung tissue and mucopurulent pus/sputum
samples excised directly and sterilely from the lungs of five
Danish end-stage CF patients undergoing double lung trans-
plantation at Rigshospitalet (Copenhagen, Denmark). The
lungs were collected with the consent of the patients and in
accordance with the biomedical project protocol (KF-
01278432) approved by the Danish Council of Ethics. To in-
vestigate the microorganisms of the true microbiota present
within the lungs of the patients, both standard culturing and
16S rRNA gene analysis were performed. All culture experi-
ments were performed at the Department of Clinical Micro-
biology, Rigshospitalet (Copenhagen University Hospital,
Denmark), according to standard protocols (2). All samples
were incubated both aerobically and anaerobically. Aerobic
culturing was performed on blood agar, chocolate agar, and
eosin-methylene blue (EMB) agar with an incubation time of
up to 1 week. Anaerobic culturing was performed on blood
agar and chocolate agar, using an atmosphere of 7% CO2and
7% H2in N2for up to 2 weeks.
Before extraction of DNA for 16S rRNA gene analysis,
samples were lysed by proteinase K (40 ?l) and ATL buffer
(360 ?l) from the DNeasy blood and tissue kit (Qiagen, Co-
penhagen, Denmark) for each 500 mg of tissue and incubated
overnight at 56°C. The samples were then centrifuged at 13,000
* Corresponding author. Mailing address: Department of Interna-
tional Health, Immunology and Microbiology, University of Copenha-
gen, DK-2100 Copenhagen, Denmark. Phone: 4535457774. Fax:
4535327853. E-mail: firstname.lastname@example.org.
?Published ahead of print on 19 October 2011.
rpm for 1 min, and DNA was extracted using the FastDNA
Spin kit for soil (MP Biomedicals, Illkirch, France) according
to the manufacturer’s protocol (revision 6560-200-07DEC);
starting from step 6, DNA was eluted with 60 ?l diethyl pyro-
carbonate (DEPC)-treated water. Nearly full-length 16S
rRNA genes were amplified as described in the literature (22),
using two different combinations of universal bacterial primers:
1390R (5?-GACGGGCGGTGTCTACAA-3?) or 1492R (5?-T
ACGGYTACCTTGTTACGACTT-3?) (15). The resulting 16S
rRNA gene fragments were pooled and purified using Nucleo-
spin Extract II columns (Macherey-Nagel, Du ¨ren, Germany).
The PCR products were cloned into a pCR4-TOPO vector,
transformed into One Shot Top 10 chemically competent Esch-
erichia coli cells (Invitrogen, Carlsbad, CA), and incubated
overnight at 37°C on LB agar plates containing 50 ?g/ml
kanamycin and 50 ?g/ml X-Gal (5-bromo-4-chloro-3-indolyl-
?-D-galactopyranoside). Either plasmids were purified using
the Illustra TempliPhi DNA amplification kit (GE Healthcare,
Brøndby, Denmark) and sequenced commercially by Macro-
gen (South Korea), or plasmid purification was performed by
Macrogen before sequencing. Sequences were obtained using
FIG. 1. Maximum likelihood tree of the sequences in the clone libraries with their closest relatives. The OTUs from the clone libraries from
the five patients are given with the numbers of sequences in parentheses. The out-group (consisting of 24 sequences of the Chloroflexi phylum) was
set as the root, not shown in the figure. The scale bar represents a 10% deviation of sequence. Asterisks indicate sequences where identification
by BLAST search gave different results. The identities of microorganisms found by culturing are highlighted by a box; these are also the clones
most often identified in the respective clone libraries.
VOL. 49, 2011NOTES4353
the M13F primer (5?-GTAAAACGACGGCCAGT-3?) and
checked for chimeric sequences with the program Bellerophon
(12), using the Huber-Hugenholtz correction and a window
size of 300 nucleotides. The BlastN function in the NCBI
database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used for
initial identification of closest relatives with standard parame-
ter settings, except that the database was set to the nucleotide
Alignment of the sequences was performed using the SILVA
web aligner (17) with default settings and refined manually in
ARB (16). The sequences from the 34 clone libraries were
compiled into overall libraries for each of the 5 patients; within
these, the sequences were grouped into operational taxonomy
units (OTUs) if they had a sequence similarity of more than
97% (13). Representative clones for all OTUs were also se-
quenced using the M13R primer (5?-GCGGATAACAATTT
CACACAGG-3?) in order to obtain consensus sequences cov-
ering the entire length of the fragments. Consensus sequences
representing the different OTUs and their closest relatives in
the nonredundant SSU Ref database from SILVA release 104
were used for calculation of trees by distance matrix, parsi-
mony, and maximum likelihood approaches using default set-
tings in the ARB software but omitting hypervariable regions
of the gene. Twenty-four out-group sequences from the phy-
lum Chloroflexi were added to the tree calculations.
Culture analysis showed the presence of monospecies infec-
tion in the lungs of four patients, and two bacterial species
were found for the last patient (patient 4). No growth of an-
aerobic bacteria was observed. The isolated bacteria were P.
aeruginosa, Stenotrophomonas maltophilia, or Achromobacter
xylosoxidans (Table 1), and the same result was found on all
types of media used. The 5 patients expectorated sputum just
prior to their lung transplantation. The culture analysis of this
sputum revealed the exact same bacteria as those found by the
culture analysis from the explanted lungs (not shown). The
initial identification of clone library sequences (as determined
by BLAST search) showed that the organisms found by culture
analysis were present in high numbers in the clone libraries
(Table 1). The phylogenetic trees (neighbor joining, maximum
parsimony, and maximum likelihood) were constructed to vi-
TABLE 1. Overview of bacteria found in the explanted lung samples by culturing and 16S rRNA gene analysis
16S rRNA gene analysis
Patient 1 Achromobacter xylosoxidans Achromobacter xylosoxidans
Patient 2 Pseudomonas aeruginosa Pseudomonas aeruginosa
Patient 3 Pseudomonas aeruginosa Pseudomonas aeruginosa
Uncultured Bacteroidetes bacterium
Uncultured Saprospiraceae bacterium
Uncultured Bacteroidetes bacterium
Patient 4 Pseudomonas aeruginosa
Patient 5 Achromobacter xylosoxidans Achromobacter xylosoxidans
aThe species found by 16S rRNA gene analysis is given by the closest relatives of the bacterial OTUs in clone libraries for the patients.
bThe number of sequences that make up the OTUs.
cAsterisks indicate clones where the identification by BLAST differed from the identification made by phylogenetic analysis.
4354 NOTESJ. CLIN. MICROBIOL.
sualize the phylogenetic relationship of the microorganisms Download full-text
and showed congruent topology (the maximum likelihood tree
is shown in Fig. 1). The locations of the sequences in the tree
confirmed the result of the BLAST search and in several cases
gave identification of sequences that had been determined to
be uncultured bacteria by BLAST search, as indicated by as-
terisks in Table 1 and Fig. 1. This is due to the fact that, unlike
the BLAST tool at NCBI, only quality-checked sequences were
used in the ARB database used. Another factor is that, in
ARB, the secondary structure of the 16S rRNA gene was taken
into account. Some of the bacteria identified in the clone
libraries have previously been associated with cystic fibrosis,
such as Stenotrophomonas maltophilia (6, 7, 20), Burkholderia
fungorum (6, 19), and Streptococcus sp. (13), but the clinical
relevance of these bacteria and others found in small amounts
in the samples is unknown (2, 11). Compared to the results
obtained by culture analysis, the 16S rRNA gene analysis
showed a greater diversity of bacteria, with sequences distrib-
uted into 4 phyla: Proteobacteria, Bacteroidetes, Actinobacteria,
and Firmicutes. As the bacteria found by culturing were also
represented by the highest numbers of sequences in the clone
libraries, it is very likely that these bacteria were dominant in
the lung. We are currently investigating this thoroughly by
FISH and quantitative PCR.
The results presented here correlate with results that we
have previously published (1) that the end-stage CF lung har-
bors relatively few bacterial species that could be identified by
culturing. However, this might not represent the other levels of
chronic infection in the CF lungs. In fact, many of the non-
end-stage CF patients at the Copenhagen CF Clinic harbor
several species in their lungs, which should also be investigated
Nucleotide sequence accession numbers. The nonredundant,
nearly full-length 16S rRNA gene sequences representing each
OTU obtained in this study were deposited in GenBank under
the accession numbers JN802672 to JN802704.
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