Ciofu, O.

Høiby, N.

Johansen, H.K.

Jørgensen, K.M.

Molin, S.

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156 Adaptive metabolic changes in Pseudomonas aeruginosa during cystic fibrosis lung infection 

5. Microbiology S39
153 Pseudomonas aeruginosa isolates from cystic ﬁbrosis patients
with and without autoantibodies to bactericidal/permeability
increasing protein (BPI-ANCA) exhibit different gene expression
proﬁle
S. Shukla1, A˚. Petterson1, M. Carlsson1, T. Hellmark1, K. Riesbeck2,
M. Segelmark1. 1Lund University, Department of Clinical Sciences, Lund,
Sweden; 2Lund University, Department of Laboratory Medicine, Malmo¨, Sweden
Prognosis of cystic ﬁbrosis (CF) patients chronically colonized with Pseudomonas
aeruginosa (Pa) is highly variable. We have previously shown that adverse prognosis
is associated with the presence of anti-neutrophil cytoplasm antibodies (ANCA) spe-
ciﬁc for Bactericidal/permeability increasing protein (BPI). Our present hypothesis
is that ANCA development is dependent on bacterial characteristics.
Methods: Two ANCA+, three ANCA− strains and a reference strain (POA1) were
used as sources for a RNA microarray using Affymetrix chips. Real time PCR is
being performed to conﬁrm the results from the array in a larger set of isolates.
Results: Analysis of the chips shows substantial and consistent differences between
ANCA+ and ANCA− strains. The genes from the array were analyzed and
grouped on the basis of their known/putative functions. The microarray analysis
has revealed marked up-regulation in strains from ANCA+ patients in some key
genes involved in polyamine biosynthesis, antibiotic resistance, LPS biosynthesis,
antibiotic resistance, quorum sensing, two component system. Down-regulation was
seen for fatty acid and amino acid metabolism pathways as well as for the ﬂagellar
assembly pathway. Real-time PCR for genes representing the different metabolic
pathways are currently being performed and preliminary data conﬁrm the ﬁndings
of the microarray.
Conclusion: Preliminary results show signiﬁcant differential expression of genes
in the polyamine biosynthesis, suggesting this pathway to be related to the ANCA
formation in CF.
154* Assessing the role of lysogenic bacteriophages in a cystic
ﬁbrosis epidemic strain of Pseudomonas aeruginosa
J.L. Fothergill1,2, A.-A. Lemieux3, C.E. James1, I. Kukavica-Ibrulj3, G. Filion3,
S.J. Charette3, R.C. Levesque3, C. Winstanley1,2. 1University of Liverpool,
Institute of Infection and Global Health, Liverpool, United Kingdom; 2University
of Liverpool, NIHR Biomedical Research Centre, Liverpool, United Kingdom;
3Universite Laval, Institut de biologie inte´grative et des syste`mes (IBIS), Quebec,
Canada
The Liverpool Epidemic Strain (LES) of P. aeruginosa has been identiﬁed in CF
patient sputum across the UK and North America. Genomic studies revealed the
presence of numerous mobile genetic elements in the LES chromosome including
six prophages. Using signature-tagged mutagenesis (STM) we have demonstrated a
role for three of the prophages (LES prophages 2, 3 and 5) in the competitiveness of
isolate LESB58 in a rat chronic lung infection and a Dictyostelium host model. The
aim of this study was to determine the infection dynamics of the LES bacteriophages
and investigate the effect they have on the host bacterium.
We used PCR detection and enumeration to show that ﬁve of the prophages
can produce active phage progeny. The sensitivity of the lytic/lysogenic switch
varies between the prophages. Furthermore, different immunity proﬁles and plaque
morphology were identiﬁed.
Following screening of 10,000 insertion mutants, we analysed mutants with dis-
rupted prophage genes and reduced competitiveness in the rat model when compared
to the wild type. The least successful mutants were disrupted in genes involved in
the phage lytic cycle and had altered levels of phage production and in some
cases, altered growth characteristics. Isogenic knockout mutants for these genes
were constructed to reinforce these data.
Our studies suggest a dynamic interplay between the LES prophages and their
bacterial host. The prophages are important for competitiveness in two infection
models and this may be associated with the switch between lysogenic and lytic
pathways.
We acknowledge funding from the NIHR Liverpool Biomedical Research Centre,
the Wellcome Trust and CIHR in Canada.
155 Fluctuating levels of pyocyanin in cystic ﬁbrosis patient sputum
from stable and acute samples
J.L. Fothergill1,2, G. Grant3, S. Dukie3, S. Macauley3, M.J. Walshaw4,
M.J. Ledson4, C. Winstanley1,2. 1University of Liverpool, Institute of Infection
and Global Health, Liverpool, United Kingdom; 2University of Liverpool, NIHR
Biomedical Research Centre, Liverpool, United Kingdom; 3Grifﬁth University,
School of Pharmacy, Queensland, Australia; 4Liverpool Heart and Chest Hospital,
Liverpool, United Kingdom
Pyocyanin (PCN) is a blue, redox-active phenazine secreted by P. aeruginosa.
Synthesis is controlled by the quorum sensing system and PCN is over-produced
by some isolates. PCN causes many effects of relevance to CF, including effects on
redox processes, the immune system, other microbes and physical processes such
as cilia beating. Studies from our laboratory on populations of the LES in culture,
isolated from patient samples, have suggested a correlation between pulmonary
exacerbation and over-production of PCN. The aim of this study was to determine
whether pyocyanin is directly detectable in the sputum of LES infected patients and
how the levels vary.
Sputum samples obtained from both stable and exacerbation periods were collected
from chronically infected adult CF patients. PCN was extracted using chloroform
and analysed by high performance liquid chromatography. PCN was detected at
280 nm and separated from interfering chemicals on a Phenomenex C18 Gemini
reverse phase column. Sample concentrations were calculated from peak areas using
external standard curves. All experiments were performed in duplicate. The level of
PCN was considerably higher in sputum samples taken during exacerbations than
in samples taken during stable periods. Furthermore, ﬂuctuations between samples
from the same patient were detected.
PCN can have a number of detrimental effects on the host. We present further
evidence that levels may be increased during exacerbation. Therapeutics directed at
preventing PCN production could be beneﬁcial to CF patients, particularly during
periods of exacerbation. We acknowledge funding from the NIHR through the
Liverpool Biomedical Research Centre.
156 Adaptive metabolic changes in Pseudomonas aeruginosa during
cystic ﬁbrosis lung infection
K.M. Jørgensen1, H.K. Johansen2, S. Molin3, N. Høiby1,2, O. Ciofu1. 1University
of Copenhagen, Institute for International Health, Immunology and Microbiology,
Copenhagen N, Denmark; 2Rigshospitalet, University of Copenhagen, Department
of Clinical Microbiology 9301, Copenhagen, Denmark; 3Technical University
of Denmark, Center for Systems Microbiology, Department of Systems Biology,
Lyngby, Denmark
Objectives: Cystic ﬁbrosis (CF) is the most prevalent hereditary fatal disease among
Caucasians. The mortality of this disease is mainly due to the chronic airways
infection with P. aeruginosa, which uses various adaptive mechanisms to persist in
the harsh lung environment.
Methods: By applying the Biolog Phenotype MicroArray we have tested metabolic
changes involving C-, N-, P- and S sources in 11 CF isolates (2−4 isolates/patient)
of P. aeruginosa, which were sequenced in mucA, lasR, algT, rhlR and rpoN.
The isolates were collected 2 to 5 years before and after establishment of chronic
infection from the lungs of 4CF patients harboring 4 different clones.
Results: Mutations in lasR, which have previously been shown to have an impact
on the metabolic shift during chronic lung infection, were found in neither of the
isolates of 3 of 4 patients and a T227C mutation was present in both early and late
isolates of the last patient. No signiﬁcant difference in metabolic activity between
early and late isolates was found in 3 patients. A signiﬁcant loss of metabolic
activity involving 37 of 76 C-sources, 48 of 91 N-sources, 33 of 50 P-sources and
19 of 33 S-sources was found in an isolate which had acquired a rpoN mutation
compared to an earlier isolate. Metabolic changes in 24 of 33 N-sources, 25 of 36
P-sources and 11 of 14 S-sources occurred in the late isolate of another patient
despite no changes in the investigated genes.
Conclusion: Signiﬁcant metabolic changes between pre- and post-chronic infection
isolates were seen in only 1 of 4 CF patients suggesting that the adaptive metabolic
changes occur in the later stages of chronic lung infection.


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156 Adaptive metabolic changes in Pseudomonas aeruginosa during cystic fibrosis lung infection

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