True microbiota involved in chronic lung infection of cystic fibrosis patients found by culturing and 16S rRNA gene analysis.
ABSTRACT 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.
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ABSTRACT: PCR targeting the gene encoding 16S ribosomal RNA (commonly named broad-range PCR or 16S PCR) has been used for 20years as a polyvalent tool to study prokaryotes. Broad-range PCR was first used as a taxonomic tool, then in clinical microbiology. We will describe the use of broad-range PCR in clinical microbiology. The first application was identification of bacterial strains obtained by culture but whose phenotypic or proteomic identification remained difficult or impossible. This changed bacterial taxonomy and allowed discovering many new species. The second application of broad-range PCR in clinical microbiology is the detection of bacterial DNA from clinical samples; we will review the clinical settings in which the technique proved useful (such as endocarditis) and those in which it did not (such as characterization of bacteria in ascites, in cirrhotic patients). This technique allowed identifying the etiological agents for several diseases, such as Whipple disease. This review is a synthesis of data concerning the applications, assets, and drawbacks of broad-range PCR in clinical microbiology.MÃ©decine et Maladies Infectieuses 07/2013; · 0.75 Impact Factor
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ABSTRACT: Achromobacter xylosoxidans is an environmental opportunistic pathogen, which infects an increasing number of immunocompromised patients. In this study we combined genomic analysis of a clinical isolated A. xylosoxidans strain with phenotypic investigations of its important pathogenic features. We present a complete assembly of the genome of A. xylosoxidans NH44784-1996, an isolate from a cystic fibrosis patient obtained in 1996. The genome of A. xylosoxidans NH44784-1996 contains approximately 7 million base pairs with 6390 potential protein-coding sequences. We identified several features that render it an opportunistic human pathogen, We found genes involved in anaerobic growth and the pgaABCD operon encoding the biofilm adhesin poly-β-1,6-N-acetyl-D-glucosamin. Furthermore, the genome contains a range of antibiotic resistance genes coding efflux pump systems and antibiotic modifying enzymes. In vitro studies of A. xylosoxidans NH44784-1996 confirmed the genomic evidence for its ability to form biofilms, anaerobic growth via denitrification, and resistance to a broad range of antibiotics. Our investigation enables further studies of the functionality of important identified genes contributing to the pathogenicity of A. xylosoxidans and thereby improves our understanding and ability to treat this emerging pathogen.PLoS ONE 01/2013; 8(7):e68484. · 3.53 Impact Factor
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ABSTRACT: ABSTRACT Urinary tract infections (UTIs) are one of the most commonly acquired bacterial infections in humans, and uropathogenic Escherichia coli strains are responsible for over 80% of all cases. The standard method for identification of uropathogens in clinical laboratories is cultivation, primarily using solid growth media under aerobic conditions, coupled with morphological and biochemical tests of typically a single isolate colony. However, these methods detect only culturable microorganisms, and characterization is phenotypic in nature. Here, we explored the genotypic identity of communities in acute uncomplicated UTIs from 50 individuals by using culture-independent amplicon pyrosequencing and whole-genome and metagenomic shotgun sequencing. Genus-level characterization of the UTI communities was achieved using the 16S rRNA gene (V8 region). Overall UTI community richness was very low in comparison to other human microbiomes. We strain-typed Escherichia-dominated UTIs using amplicon pyrosequencing of the fimbrial adhesin gene, fimH. There were nine highly abundant fimH types, and each UTI sample was dominated by a single type. Molecular analysis of the corresponding clinical isolates revealed that in the majority of cases the isolate was representative of the dominant taxon in the community at both the genus and the strain level. Shotgun sequencing was performed on a subset of eight E. coli urine UTI and isolate pairs. The majority of UTI microbial metagenomic sequences mapped to isolate genomes, confirming the results obtained using phylogenetic markers. We conclude that for the majority of acute uncomplicated E. coli-mediated UTIs, single cultured isolates are diagnostic of the infection. IMPORTANCE In clinical practice, the diagnosis and treatment of acute uncomplicated urinary tract infection (UTI) are based on analysis of a single bacterial isolate cultured from urine, and it is assumed that this isolate represents the dominant UTI pathogen. However, these methods detect only culturable bacteria, and the existence of multiple pathogens as well as strain diversity within a single infection is not examined. Here, we explored bacteria present in acute uncomplicated UTIs using culture-independent sequence-based methods. Escherichia coli was the most common organism identified, and analysis of E. coli dominant UTI samples and their paired clinical isolates revealed that in the majority of infections the cultured isolate was representative of the dominant taxon at both the genus and the strain level. Our data demonstrate that in most cases single cultured isolates are diagnostic of UTI and are consistent with the notion of bottlenecks that limit strain diversity during UTI pathogenesis.mBio 01/2014; 5(2). · 6.88 Impact Factor
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, 2011NOTES 4353
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 2Pseudomonas aeruginosa Pseudomonas aeruginosa
Patient 3 Pseudomonas aeruginosaPseudomonas aeruginosa
Uncultured Bacteroidetes bacterium
Uncultured Saprospiraceae bacterium
Uncultured Bacteroidetes bacterium
Patient 4Pseudomonas aeruginosa
Patient 5 Achromobacter xylosoxidansAchromobacter 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.
4354NOTES J. CLIN. MICROBIOL.
sualize the phylogenetic relationship of the microorganisms
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.
1. Bjarnsholt, T., et al. 2009. Pseudomonas aeruginosa biofilms in the respira-
tory tract of cystic fibrosis patients. Pediatr. Pulmonol. 44:547–558.
2. Bjarnsholt, T., X. C. Nielsen, U. Johansen, L. Nørgaard, and N. Høiby. 2011.
Methods to classify bacterial pathogens in cystic fibrosis, p. 143–171. In M. D.
Amaral and K. Kunzelmann (ed.), Cystic fibrosis: diagnosis and protocols,
vol. II. Methods and resources to understand cystic fibrosis. Humana Press,
3. Braun, A. T., and C. A. Merlo. 2011. Cystic fibrosis lung transplantation.
Curr. Opin. Pulm. Med. 17:467–472.
4. Conese, M., E. Copreni, S. D. Gioia, P. D. Rinaldis, and R. Fumarulo. 2003.
Neutrophil recruitment and airway epithelial cell involvement in chronic
cystic fibrosis lung disease. J. Cyst. Fibros. 2:129–135.
5. Costerton, J. W., P. S. Stewart, and E. P. Greenberg. 1999. Bacterial biofilms:
a common cause of persistent infections. Science 284:1318–1322.
6. Demko, C. A., R. C. Stern, and C. F. Doershuk. 1998. Stenotrophomonas
maltophilia in cystic fibrosis: incidence and prevalence. Pediatr. Pulmonol.
7. Ferroni, A., et al. 2002. Use of 16S rRNA gene sequencing for identification
of nonfermenting gram-negative bacilli recovered from patients attending a
single cystic fibrosis center. J. Clin. Microbiol. 40:3793–3797.
8. Harrison, F. 2007. Microbial ecology of the cystic fibrosis lung. Microbiology
9. Heijerman, H. 2005. Infection and inflammation in cystic fibrosis: a short
review. J. Cyst. Fibros. 4:3–5.
10. Høiby, N., B. Frederiksen, and T. Pressler. 2005. Eradication of early Pseu-
domonas aeruginosa infection. J. Cyst. Fibros. 4(Suppl. 2):49–54.
11. Høiby, N., and T. Pressler. 2006. Emerging pathogens in cystic fibrosis. ERM
12. Hugenholtz, P., and T. Huber. 2003. Chimeric 16S rDNA sequences of
diverse origin are accumulating in the public databases. Int. J. Med. Micro-
13. Juretschko, S., A. Loy, A. Lehner, and M. Wagner. 2002. The microbial
community composition of a nitrifying-denitrifying activated sludge from an
industrial sewage treatment plant analyzed by the full-cycle rRNA approach.
Syst. Appl. Microbiol. 25:84–99.
14. Kolak, M., F. Karpati, H.-J. Monstein, and J. Jonasson. 2003. Molecular
typing of the bacterial flora in sputum of cystic fibrosis patients. Int. J. Med.
15. Lane, D. J. 1991. 16S/23S rRNA sequencing, p. 115–175. In E. Stackebrandt
and M. Goodfellow (ed.), Nucleic acid techniques in bacterial systematics,
1st ed. John Wiley & Sons, London, United Kingdom.
16. Ludwig, W., et al. 2004. ARB: a software environment for sequence data.
Nucleic Acids Res. 32:1363–1371.
17. Pruesse, E., et al. 2007. SILVA: a comprehensive online resource for quality
checked and aligned ribosomal RNA sequence data compatible with ARB.
Nucleic Acids Res. 35:7188–7196.
18. Rogers, G. B., et al. 2004. Characterization of bacterial community diversity
in cystic fibrosis lung infections by use of 16S ribosomal DNA terminal
restriction fragment length polymorphism profiling. J. Clin. Microbiol. 42:
19. Rogers, G. B., F. A. Stressmann, A. W. Walker, M. P. Carroll, and K. D.
Bruce. 2010. Lung infections in cystic fibrosis: deriving clinical insight from
microbial complexity. Expert Rev. Mol. Diagn. 10:187–196.
20. Saiman, L. 2004. Microbiology of early CF lung disease. Paediatr. Respir.
21. Stressmann, F. A., et al. 2011. Analysis of the bacterial communities present
in lungs of patients with cystic fibrosis from American and British centers.
J. Clin. Microbiol. 49:281–291.
22. Thomsen, T. R., K. Finster, and N. B. Ramsing. 2001. Biogeochemical and
molecular signatures of anaerobic methane oxidation in a marine sediment.
Appl. Environ. Microbiol. 67:1646–1656.
23. Worlitzsch, D., et al. 2009. Antibiotic resistant obligate anaerobes during
exacerbations of cystic fibrosis patients. Clin. Microbiol. Infect. 15:454–460.
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