Journal of Cystic Fibrosis 4 (2005) $34 $58
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157" Use of suppression subtracSve hybridization to idenSfy novel sequences in epidemic strains of Pseudomonas aeruginosa
159 Investigation of the use of Tandem Repeat Polymorphism for typing of Clinical P. aeruginosa isolates
C.H. Smart 1, F. Scott 2, T. Pitt 2, C.A. Hart 1, M.J. Walshaw3 , C. Winst anley 1
H.C. Ryley, J. Weeks
Department of Medical Microbiology a~u] Genitourinary Medicine, University of Liverpool, UK, 2HeaIth ProtectionAgeney, CoIindale, Lorulorg UK, 3RegionaI AduIt CF Unig CatJiothoracic Centre, Liverpool, UK
D ept of Medical Microbiology, School of Medicine, CaMiff University, Cardiff, UK
I n r m d u e t l o n ha 1996 w e reported the spread of an epidemic strain of Pseudomonas aeruginosa (Liverpool epidemic strain, LES) among CF patients in Liverpool. A recent survey investigating t~aeruginosa genotypes among CF patients in E n g l a n d and Wales found LES to be the most common genotype and identified other transmissible strains, including the Midlands epidemic strain (Mid1). W e h a v e previously used suppression subtractive hybridisation (SSH) to identify a small number of D N A sequences present in the LES and absent from the reference strain PA01. One sequence (PS21) is currently targeted in a diagnostic PCR test for the LES. A i m s (i) To identify novel D N A sequences present in the LES and M i d l epidemic strains of t~aeruginosa and absent from PA01. (ii) To screen a panel of CF t~aeruginosa isolates to determine the distribution of these sequences. Methods SSH was used to identify a number of subtracted sequences for each of the M i d l and LES strains. PCR amplification assays w e r e used to screen a panel of CF t~aeruginosa isolates in order to determine the prevalence of such sequences. Results A substantial database of novel D N A sequences has n o w b e e n obtained for the LES (>1C~)) mad M i d l (>50) genotypes. N o v e l sequences w e r e identified for the development of diagnostic tests for epidemic strains. Conclusions Both the LES and M i d l epidemic strains possess a number of sequences not widely represented a m o n g our CF t~aeruginosa panel. These sequences may play a role in the unusual characteristics displayed by these epidemic genotypes.
W e h a v e found that multilc~us restriction typing (MLRT) is a rapid and convenient method o f / ~ aeruginosa typing but the level of discrimination currently achieved means that similar M L R T isolates need to b e confirmed by another typing method, n a m e l y Random Amplified Polymorpbism D N A (RAPD) PCR. H e r e w e report our investigation of an alternative PCR typing method recently proposed by Onteniente et aI (J Clin Microbio12003: 41; 4991 97) based on tandem repeat polymorpbism of a number of different g e n e loci as a possible alternative to RAPD. Methods: Isolates collected from CF patients over the past 2 years w e r e used. Within this collection, there w e r e 16 M L R T groups containing isolates from 2 or more patients. 15 of 75 (20%) of these isolates had a different R A P D type from other isolates w i t h the same M L R T type. Tandem repeat PCR typing protc~ols w e r e similar to those used by Onteniente et aI. E a c h loci was scored according to the differences in polymotphism found. Results. Preliminary w o r k indicated that, of the 7 loci proposed by Onteniente et al, only three (tr077,tr142, tr172) had sufficient polymotphism and stability to be of use in routine cross infection screening. A combination of the three loci scores (ie. 3.1.4) g a v e a tandem repeat (TR) genotype w h i c h was then estimated ha the 7 5 isolates (see above). 9 of the 15 isolates, identified as different by R A P D had a different TR genotype from their shared M L R T isolates. 15% of the isolates identified as the same by RAPD, had a different TR genotype from their shared M L R T isolates. Conclusions. Tandem repeat polymorphism typing has a poorer discrimination overall compared w i t h M L R T for /~ aeruginosa typing mad, e v e n it" used in combination w i t h MLRT, could not replace the need for confirmatory R A P D typing
This work was funded by the UK CF Trust.
158 Analysis of Liverpool Epidemic Strain Pseudomonas aeruginosa distribution using T-RLFP profiling
160 IdenSfication of Burkholderia cepacia complex species by SNuPE analysis of recA and gyrB genes
D.W.M. Ketruish 1, G.B. Roger s 1, D.J. Serisier~, M.P. Carroll2, C.A. Hart~, K.D. Bruce 1
P. Cc~chi 1, L. Ferri 1, C. Papaleo 1, G. Marmo 9, M. Mentasti9, N. Ravenni s, S. Campana 3, G. Taccetti ~, S. Tabacchioni 4, C. Dalmastri4, L. Chiarini4, A. Bevivino 4, R. Fani 1 1Dept of Genetics, Fire~e; 2G Gaslini lnstitute, Genoa; 3Meyer Hospital, Fire~e;
iDivision of Life Sciences, Kings College London, UK, 2Soutlu~rrq2ton Univemity Hospital, Southampton, UK, 3Mater Adult Hospital, Brisbane, Australia, 4Royal Liverpool UniversityHospital, Liverpool, UK Cross infection by Pseudomonas aeruginosa between cystic fibrosis patients is increasingly prevalent. A t least three different strains h a v e been identified ha different CF treatment centres. T h e Liverpool Epidemic Strain (LES) has a distribution of 48 % in tested centres. LES is associated w i t h poor clinical pr o~nosis. Environmental contamination of LES has been shown to occur ha areas associated w i t h infected patients. LES can remain in the environment ha excess of 48h, although other nontransmissble strains h a v e better environmental survival rates. Airborne dissemination plays an important role in the transmissibility of this strain. The distribution of LES may be the result of the movement of CF patients between treatment centres throughout the U.K. However, the prevalence of LES in those centres outside the Pitt et aI., 2CO4 study is unknown. Here, w e assayed samples from the Southampton Adult CF centre for the presence of LES. D N A was collected from sputum taken from CF patients at Royal Liverpool University hospital and Southampton General hospital. Further, T R F L P analysis was carried out on samples w i t h and without LES strains. Comparison of their bacterial profiles was performed to determine the effect of the LES strain on the other microbial flora colonising the CF lung. A t the t i m e of writing w e h a v e found no evidence for the LES strain ha the 12 Southampton patients tested. A relatively small number of Southampton CF patients h a v e been tested so far. Increasing the number of samples w i l l allow us to determine the prevalence of LES in Southampton and its effects on microbial lung flora.
#ENEA, S. Maria di Galeria, Italy Respiratory infections are the m a i n cause of morbidity and mortality in cystic fibrosis, and bacteria belonging to the Burkholderia cepacia complex (Bee) represent the most important agents of infection due to their h i g h level intrinsic antibiotic resistance and easy patient to patient spread. Bee comprises n i n e species that are characterized by a h i g h degree of D N A homology, mad thus are difficult to identify by routine clinical analysis. Nowadays a combination of molecular techniques is required for a correct identification of Bee strains; however, these methodologies are not applicable in clinical routine, ha this study w e apply the Single Nucleotide Primer Extension (SNuPE) technique, w h i c h allows the investigation of single nucleotide polimorphysms, to discriminate Bee isolates. W e firstly focused our attention to the recA gene. To this purpose a set of 130 recA nucleotide sequences (newly determined or retrieved from databases) was analyzed and allowed us to design some primers that w e r e successfully used in the SnttPE analysis. However, the analysis of the sole recA g e n e did not permit to discriminate all Bee species. Therefore, w e h a v e also investigated another marker: the gytB gene. W e amplified mad sequenced a gytB D N A fragment of about 1900 bp from 64 strains representatives of all of the genomovars. The analysis of the gytB sequences allowed to identify several SNPs located in different modules of the gene. T h e polymorpbic sites w e r e successfully revealed by SNttPE using a d kmc designed oligonucleotides.
This work was supported by Italian Cystic Fibrosis Fowulatio~ Grant n° 5/2003