Poster Bruxelles

1
DESIGNING DNA PROBES SPECIFIC TO Pseudomonas
aeruginosas ISOLATES FROM CYSTIC FIBROSIS
PATIENTS A.L. PIMENTA1,2, P. VENUS2, C. HULEN1
M.A. BLIGHT2 1 Université de Cergy-Pontoise,
Dept.Biologie, Lab. ERRMECe - 95302
Cergy-Pontoise Cedex 2 Université de Paris XI,
IGM -, Laboratoire de Pathogenèse Comparée -
91405 Orsay Cedex
Colonization of airways by the opportunistic
pathogen Pseudomonas aeruginosa is responsible
for increased morbidity, deterioration of lung
function and reduced life expectancy amongst
cystic fibrosis (CF) patients. The means of
acquisition and transmission of this pathogen
amongst the population is not yet well
understood. Although transmission of P.
aeruginosa between siblings with CF is well
established, transmission amongst unrelated CF
individuals and the effect of such spread on
cross-infection control is controversial. In
order to estimate the persistence of pulmonary
colonization, patient-to-patient transmission and
the extent of nosocomial spread of P. aeruginosa
we undertook a subtractive hybridization approach
to obtain DNA probes specific for different CF
isolates of P. aeruginosa.
1. Suppression Subtractive Hybridisation (SSH)
3. Confirmation of Tester-Specific Sequences
Isolates showing distinct RAPD profiles were
obtained from CF patients followed by the
Department of Pediatrics, University of British
Columbia, Vancouver, Canada. Genomic DNA was
subtracted between clinical isolates c1138
(Tester) and c0175 (Driver), as described by
Diatchenko et al. and summarized bellow.
Out of the 18 clones identified on the first
dot-blot as containing potential tester-specific
sequences, two were chosen at random (T10 and
T13) for further characterization. PCR products
specific to these two sequences were labeled and
their specificity tested by probing (A) Southern
blots of genomic DNAs of either Tester or the
Driver strains (B) Dot blots of genomic DNAs of
12 other clinic isolates of the same geographic
origin (British Columbia) or (C) Dot blots of
genomic DNA of 38 geographically distinct clinic
strains, isolated either from CF or non-CF
patients (Cochin Hospital, Paris, France)
A) Southern Blot against Tester and Drivers
genomes
Southern blots results confirm the specificity of
T10 and T13 sequences to the strain c1138.
Following sub-cloning of subtracted fragments,
recombinant clones were obtained and the presence
of inserts was verified by PCR, using the Nested
oligonucleotides as primers.
B) Dot blots against geographic related isolates
2. Dot blot analysis of clones carrying
subtracted DNA fragments
46 of these recombinant clones, chosen at random,
had their inserts analyzed by dot-blot
hybridization, using both tester and driver total
genomic DNAs as probes.
In both membranes, spots numbered 1 to 13 contain
DNA from strains isolated in Canada by the
Pedriatic Department of Britsh Columbia
University 12 tester DNA (strain c1138) 13
driver DNA (strain c0175) 14 and 15 no DNA
controls.
T10 and T13 sequences allow specific
discrimination of strain c1138 amongst
geographic-related isolates.
C) Dot blots against geographic distinct isolates
Inserts from individual recombinants were PCR
amplified, and equal amounts of the denatured
amplification products were spotted and fixed
onto two nylon membranes. The two identical dot
blot membranes were hybridised against either the
testers (Panel A) or the drivers (Panel B)
genomic, a32P-dCTP-labelled DNA. Spots marked
with a circle represent specific tester clones.
Squares represent control spots containing either
vector DNA or no DNA.
In both membranes, spots numbered A1 to D2
contain DNA from strains isolated in the
Bacteriology Department of Cochin Hospital,
Paris D4 and D6 tester DNA (strain c1138) D8
and d10 driver DNA (strain c0175) D12 no DNA
control.
T10 and T13 probes allow the identification of
strain c1138 amongst geographically distinct CF
and non-CF isolates.
18 clones (35) were identified as specific to
the tester strain (c1138), representing potential
specific strain-discriminating probes.
? Using the Suppression Subtractive Hybridization
technique we have isolated probes specific to an
P. aeruginosa CF strain (c1138), isolated at
Vancouver, Canada. Characterization of two of
these probes (T10 and T13) clearly demonstrates
that the these sequences allow the
identification of the original strain not only
amongst geographically -related isolates, but
equally amongst unrelated ones. ? Database
searches using sequences obtained from clones T10
and T13 revealed no homologies to any DNA
sequence available in the databanks, indicating
the specificity of these probes to the original
strain c1138. ? These results demonstrate the
suitability of SSH technique for the isolation of
specific probes, useful in epidemiological
studies of P. aeruginosa cross-contamination
amongst CF and non-CF patients.