Journal of Microbiological Methods 76 (2009) 58–69

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Journal of Microbiological Methods

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RISA–HPLC analysis of lung bacterial colonizers of cystic fibrosis children

S. Nazaret a,b,⁎, F. Assade a,b, E. Brothier a,b, A.-M. Freydière a,c, G. Bellon a,d, B. Cournoyer a,b

a Université de Lyon, Franceb Research Group on « Bacterial Opportunistic Pathogens and Environment », CNRS, Ecole Nationale Vétérinaire de Lyon, and Université Lyon 1, UMR 5557 Ecologie Microbienne, Francec Laboratoire de Bactériologie, Hôpital Debrousse, Université Lyon 1, and Hospices Civils de Lyon, Franced Service de Pédiatrie-Pneumologie-Allergologie, Hôpital Debrousse, Université Lyon 1, and Hospices Civils de Lyon, France

⁎ Corresponding author. Mailing address: UMR 5557 ELyon1,Mendel Bldg., 5th Floor, F-69622VilleurbanneCedefax: +33 4 26 23 44 68.

E-mail address: nazaret@biomserv.univ-lyon1.fr (S. N

0167-7012/$ – see front matter © 2008 Elsevier B.V. Aldoi:10.1016/j.mimet.2008.09.019

a b s t r a c t

a r t i c l e i n f o

Article history:

Microbiological analysis of s Received 9 June 2008Received in revised form 5 September 2008Accepted 16 September 2008Available online 30 September 2008

Keywords:16S-23S intergenic spacerDHPLCCystic fibrosisOpportunistic pathogensBacterial communitiesLung

putum samples, from children affected by cystic fibrosis (CF) and showing signsof acute or chronic infections, is routinely performed by culture-dependent approaches involving selectivemedia and biochemical tests. These identification schemes are time-consuming, and may lead to falsenegative results. The aim of this work was to evaluate the efficacy of a Ribosomal Intergenic Spacer Analysis(RISA) coupled to high performance liquid chromatography (HPLC) for the detection and monitoring of CFlung microbial colonizers including Staphylococcus aureus, Haemophilus influenzae, Pseudomonas aeruginosa,the Burkholderia cepacia complex, Stenotrophomonas maltophilia, and Achromobacter xylosoxidans. TheseRISA–HPLC analyses were performed over a 10-months period on infants (below 18 months) and childrenthat were or were not yet known to be colonised by P. aeruginosa. The RISA–HPLC profiles were foundspecific of the patients' microbial communities. A specific P. aeruginosa RISA–HPLC peak corresponding to550 bp PCR products was recorded, and used to investigate P. aeruginosa persistence through time and aftervarious therapeutic treatments. The RISA–HPLC profiles showed the CF children to be colonized by fewbacterial species, and sometimes revealed peaks corresponding to bacterial species that were not detectedby the selective plating approaches. Significant RISA–HPLC infra-specific variations were observed formost bacterial colonizers of CF lungs except P. aeruginosa. These species could yield as much as 5 distinctRISA–HPLC peaks, with some of these profiles being strain-specific. RISA–HPLC shows a great potential forrevealing colonization by novel emerging pathogens, and for evaluating the efficacy of therapeutictreatments on the global bacterial community of CF lungs.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

Cystic fibrosis (CF) is an autosomal recessive hereditary diseasecaused by mutations in the CF transmembrane conductance regula-tory gene. The most common complication of CF is the recurrence ofpulmonary infections caused by bacterial pathogens. These infectionscontribute considerably to morbidity and mortality through anintense host inflammatory response leading to an irreversible loss oflung function. Pseudomonas aeruginosa, Staphylococcus aureus, Hae-mophilus influenzae, and Burkholderia cepacia complex (Bcc) organ-isms are the most common CF infectious agents (Saiman and Siegel,2004), but Stenotrophomonas maltophilia, Achromobacter xylosoxidans(former Alcaligenes xylosoxidans), Ralstonia pickettii, Klebsiellae,Mycobacteria (Steinkamp et al., 2005), Nocardia (Petersen et al.,2007), Inquilinus limosus (Coenye et al., 2002; Chiron et al., 2005) and

cologie Microbienne, Universitéx, France. Tel.: +33 4724313 24;

azaret).

l rights reserved.

Pandoraea sp. (Segonds et al., 2003) can also be recovered from CFpatients suffering of pulmonary infections.

In most routine hospital microbiology labs, the analysis of CFclinical samples involves the isolation and culture of microorganismsusing selective media. Phenotypic (biochemical and physiologicalproperties, serotyping, antibiotic resistance profiles, etc) markers arethen used to confirm the identity of the isolates. Selective media showseveral limitations in the analysis of CF bacterial colonizers. Their useis dependent upon the physiological states of the plated bacteria, withsome cells being non-culturable. They are also often used with theassumption that particular bacterial species are more likely to befound than others, and to be the etiological agent of the observedinfection. This bias can prevent the detection of novel infectiousagents, and lead to inaccurate diagnosis by clinical practicians.Nevertheless, progress in molecular biology has led to the develop-ment of new methods for the detection and identification of bacteriawhich circumvent their isolation and growth on selective media. Forexample, specific and sensitive PCR screenings have been developedfor the detection of P. aeruginosa e.g. Lavenir et al. (2007), and the Bcc(Brown and Govan, 2007).

Table 1Description of the samples used in this study and the bacterial strains recovered fromthese samples

Patienta Sampletype

Samplingdate

Bacterial isolates HPLC peak forP. aeruginosa

InfantC1 Swab 25/11/2004 H. influenzae (2 strains) −C2 Swab 18/05/2005 No pathogen −D1 Swab 15/11/2004 H. influenzae, S. aureus −D2 Swab 30/11/2004 No pathogen −D3 Swab 27/01/2005 No pathogen −D4 Swab 29/03/2005 No pathogen −H1 Sputum 04/11/2004 P. aeruginosa, H. influenzae +H2 Swab 30/11/2004 P. aeruginosa −H3 Swab 20/01/2005 No pathogen −H4 Swab 04/03/2005 No pathogen −H5 Swab 30/03/2005 P. aeruginosa (2 strains) −L1 Swab 09/11/2004 S. aureus −L2 Swab 22/02/2005 S. aureus −L3 Swab 10/05/2005 S. aureus −J1 Swab 10/11/2004 P. aeruginosa, S. aureus −J2 Swab 16/03/2005 No pathogen −N1 Swab 25/11/2004 S. aureus, H. influenzae −N2 Swab 10/02/2005 S. aureus −

Children 10 to 12 years oldA1 Sputum 03/11/2004 P. aeruginosa (2 strains) +A2 Sputum 26/01/2005 P. aeruginosa (2 strains) +A3 Sputum 16/03/2005 P. aeruginosa (3 strains) +A4 Sputum 14/04/2005 P. aeruginosa (2 strains) +A5 Sputum 30/06/2005 P. aeruginosa (1 strains) +A6 Sputum 22/08/2005 P. aeruginosa (1 strain) +B1 Sputum 15/11/2004 A. xylosoxidans, H. influenzae +B2 Sputum 18/11/2004 A. xylosoxidans, H. influenzae +B3 Sputum 17/03/2005 H. influenzae (2 strains),

S. aureus−

E1 Sputum 24/11/2004 S. aureus +E2 Sputum 10/02/2005 S. aureus −E3 Sputum 09/03/2005 H. influenzae, S. aureus −E4 Sputum 14/04/2005 P. aeruginosa (2 strains) +E5 Sputum 19/05/2005 P. aeruginosa (2 strains),

H. influenzae, S. aureus+

F1 Swab 06/12/2004 P. aeruginosa, S. aureus +F2 Swab 07/03/2005 P. aeruginosa, H. influenzae,

S. aureus+

G1 Sputum 10/11/2004 Inquilinus limosus +G2 Sputum 02/12/2004 I. limosus, S. maltophilia +G3 Sputum 07/03/2005 I. limosus, S. maltophilia −G4 Sputum 01/04/2005 No pathogen −G5 Sputum 12/09/2005 No pathogen −I1 Sputum 15/11/2004 S. aureus +I2 Sputum 14/02/2005 S. aureus −I3 Sputum 30/05/2005 S. aureus −

Children 14 to 17 years oldK1 Sputum 02/11/2004 S. aureus +K2 Sputum 22/02/2005 S. aureus −K3 Sputum 17/05/2005 S. aureus −M1 Sputum 10/11/2004 S. aureus −P1 Sputum 01/12/2004 B. cepacia, P. aeruginosa +P2 Sputum 20/01/2005 B. cepacia, P. aeruginosa +P3 Sputum 30/06/2005 B. cepacia, P. aeruginosa +S1 Sputum 29/06/2005 P. aeruginosa, S. aureus,

Pandoraea spp.+

S2 Sputum 13/07/2005 P. aeruginosa, S. aureus +S3 Sputum 07/09/2005 P. aeruginosa +

P. aeruginosa RISA–HPLC detection data are indicated.a The first letter code refers to the patient, with a same letter indicating a sample

coming from the same patient at a different date.

59S. Nazaret et al. / Journal of Microbiological Methods 76 (2009) 58–69

Denaturing high performance liquid chromatography (DHPLC) isa novel technology which was recently adapted for high throughputscreenings of single-nucleotide polymorphisms among human DNA(Jones et al., 1999; Betz et al., 2004; Chen et al., 2008). This attractivetechnology was tested on microbial DNA, and successfully applied tothe identification of antibiotic resistance gene alleles (Eaves et al.,2002; Hannachi-M'Zali et al., 2002; Hurtle et al., 2003), and of bacteria(Hurtle et al., 2002) and fungi (Goldenberg et al., 2005). Its potentialfor the characterization of complex bacterial assemblages amongparticular clinical (Domann et al., 2003; Goldenberg et al., 2005) andenvironmental samples (Barlaan et al., 2005) was recently investi-gated. These studies made use of the denaturing mode of the DHPLCsystem, with the aim of exploiting DNA sequence genetic variations,but this system also offers other possibilities through the use ofits non-denaturing mode. For example, this mode can allow analysisof multiple DNA fragments of doubled-stranded DNA. Evans et al.(2004) used the DHPLC non-denaturing mode for typing strainsof Mycobacterium tuberculosis through an analysis of PCR amplifiedDNA regions varying in their number of tandem repeats.

Here, the benefits of the DHPLC non-denaturing mode weretested on another approach generating DNA fragments of varyingsizes, the Ribosomal Intergenic Spacer Analysis (RISA), and appliedto the analysis of bacterial diversity among CF sputa. RISA impliesa PCR amplification of the DNA between the rrs (16S rRNA) andrrl (23S rRNA) genes (internal transcribed spacer, ITS), from DNAdirectly extracted from the samples, and has been, so far, successfullyapplied on highly complex microbial communities for the estimationof their species richness (Ranjard et al., 2000; Hernandez-Raquet et al.,2006). The efficacy of the combined RISA–HPLC approach at differ-entiating bacterial pathogens involved in CF lung infections (S. aureus,H. influenzae, P. aeruginosa, the Bcc, S. maltophilia, and A. xylosoxidans)was tested. The approach was used for the monitoring of P. aeruginosacolonization of infants and children by direct analysis of sputumbacterial communities. The datasets were compared with the onesobtained by the classical culture-dependent approaches.

2. Materials and methods

2.1. Clinical samples

Sputum samples and oro-pharyngeal swabs were obtained fromCF children attending the Pneumology Unit at Debrousse Hospital(Hospices Civils de Lyon) which recently moved to the HôpitalFemme-Mère-Enfant. Six infants up to 18 months, 6 children from 10to 12 years old, and 4 children from14 to 16 years old were selected forthis study. Sputum was collected immediately after a standardizedsession of physiotherapy. Patients provided between 1 and 6 samplesbetween November 2004 and September 2005 leading to a total of52 samples (Table 1). Sputawere split into two parts in sterile (100ml)plastic disposable containers; one part was kept for the culture-dependent analyses, and one for the RISA–HPLC analysis. Samples formolecular analysis were kept at −20 °C, prior their processing.Samples for culture-dependent analyses were processed within 4 hafter their recovery.

2.2. Bacterial strains

Reference strains of P. aeruginosa,B. cepacia complex (B. cenocepaciaand genomovar 1), S. maltophilia, A. xylosoxidans were obtained fromthe ATCC (American Type Culture Collection, Rockville, Maryland,USA), CFBP (Collection Française de Bactéries Phytopathogènes, INRA,Angers, France), DSMZ (Deutsche von Mikroorganismen und Zellk-ulturen Gmbh, Braunschwieg, Deutschland), LMG/BCCM (BelgianCoordinated Collections of Microorganisms, Gent, Belgium) andNCBB (The Netherland Collection of Bacteria, Ultrecht, Nederland)collections and are listed in Table 2. H. influenzae and S. aureus strains

from non-CF patients were obtained from the “Laboratoire deBactériologie”, Debrousse Hospital (Lyon) or from “Ecole NationnaleVétérinaire de Lyon”. Some bacterial strains of P. aeruginosa (n=9),S. aureus (n=4), S. maltophilia (n=1), B. cenocepacia (n=1),A. xylosoxidans (n=1), I. limosus (n=1), Pandoraea sp. (n=1) wereisolated from CF children selected for this study. The “Observatoire

Table 2Bacterial strains used in this study

Pseudomonas aeruginosa Others species

From soils: Achromobacter xylosoxidans:7NSK2a, ATCC 21776, ATCC 31479, CFBP 5036, CFBP 5037, CIP 104590b, DSMZ 6195, LMD 50.34, LMD 68.7. LMG 1231Tc, 05A04171c, 04A08248c

From waters: Stenotrophomonas maltophilia:LMD 25.27, LMG 9009. ATCC 13637Tc, 05A04582c, 05A04433c, 05A04558c, 05A04603From plants: Burkholderia cenocepacia:ATCC 14425, ATCC 33818, CFBP 5031, CFBP 5032, CFBP 5033, CFBP 5034, LMG 1272, LMG 2107, LMG 5031, LMG5032, LMG 5033, LMG 6855, LMG 10643.

LMG 16656, LMG 16826, LMG 18828, LMG 12614, LMG 18830, LMG18829, 9.29, 403272071, C3B1M, H111, 2.20, 6.46

From patients: Burkholderia cepacia:ATCC 15691, ATCC 27853b, CIP 72.26b, PA5b, PA6b, PA12b, PAK6AR2d, PAO1 R,e, UCBPP-PA14f. ATCC 25416Other origin: Staphylococcus aureusA14b, ATCC 27014, ATCC 33988, CFBP 2466T, CFBP 5035, CIP 58.35b, CIP 58.39b, CIP 58.41b, CIP 58.46b, CIP59.20b, CIP 59.33b, CIP 59.34b, CIP 59.35b, CIP 59.37b, CIP 59.38b, CIP 59.39b, CIP 59.40b, CIP 59.43b, CIP 59.44b,CIP 59.45b, CIP 60.92b, CIP 62.3b, CIP 100720T, LMG 15153.

88271g, 01/41.8g

Haemophilus influenzaeH1h, H2h

ATCC: American Type Culture Collection, Rockville, Maryland, USA. CBP: Collection Française de Bactéries Phytopathogènes, INRA, Angers, France. CIP: Collection de l'Institut Pasteur,Paris, France. DSMZ: Deutsche Sammlung von Mikroorganismen und Zellkulturen Gmbh, Braunschweig, Deutschland. LMG/ BCCM: Laboratorium Microbiology Gent CultureCollection / Belgian Coordinated Collections of Microorganisms, Gent, Belgium. NCBB: The Netherland Culture Collection of Bacteria (in the past LMD and Phabagen), Utrecht,Netherlands. T Type Strain. R Genomic and genetic reference strain.

a Collection of M. Höfte, University of Gent, Gent, Belgium.b Collection of J.M. Meyer, University of Strasbourg 1-Louis Pasteur, Strasbourg, France.c Collection of G. Chabanon, Laboratoire de Bactériologie-Hygiène, Hôpital Rangueil, Toulouse.d Collection of J. Wang, University of Arkansas for medical sciences, Little Rock, USA.e Collection of A. Lazdunski, University of Luminy, Marseille, France.f Collection of L.G. Rahme, Harvard medical school, Boston, USA.g Collection of A. Kodjo, Ecole Naionale Vétérinaire de Lyon, Marcy l'Etoile, France.h Collection of F. Vandenesh, HCL Lyon, France.

60 S. Nazaret et al. / Journal of Microbiological Methods 76 (2009) 58–69

Cepacia” (Laboratoire de Bactériologie-Hygiène, Hôpital Rangueil,Toulouse) kindly provided S. maltophilia (n=3) and A. xylosoxidans(n=2) strains isolated from CF patients of other French hospitals.

2.3. Bacteriological analysis of the sputum samples

Fresh sputum was mixed with an equal mass (1:1) of Sputasol(Oxoid Ltd., Dardilly, France). This processed sputum (10 μl) was thenplated on non-selective and selective media: Columbia blood agar(CBA), chocolate Polyvitex agar, MacConkey Agar, Baird Parker Agar,B. cepacia selective agar, Candida ID 2 agar (BioMérieux, Marcy l'Etoile,France) and cetrimide agar for P. aeruginosa (AES, Combourg, France).Plates were incubated aerobically for 24 h to 5 days at 36 °C. Growingmicroorganisms of each patient were identified on the basis of theircolony morphology, Gram staining, oxidase, catalase and coagulaseactivities, and biochemical profiles on ID32 STAPH, Rapid ID32 STREP,API NH, API 32GN, Rapid 32E strips (BioMérieux), depending on theirpresumed identity.

2.4. DNA extraction

Genomic DNA of bacterial cultures was extracted using DNeasyminispin columns, according to the manufacturer's instructions(QIAGEN, Courtaboeuf, France). DNA from sputum samples wereextracted using the FastDNA SPIN kit (BIO101, Inc., Carlsbad, CA),after having performed three Sputasol washes, in accordance withthe manufacturer's instructions (Oxoid Ltd.). Extracted DNA werevisualized after their agarose gel electrophoresis, staining by ethidiumbromide, and UV light exposure. They were quantified accordingto spectrophotometric analyses performed on a nanodrop reader(NanoDrop®ND-1000 UV–Vis Spectrophotometer, LabTech, Palaiseau,France).

2.5. PCR amplification of specific gene markers for P. aeruginosa

Standard PCR amplification was performed for three DNAtargets (ecfX, gyrB, 16S-23S ITS) in 50 μl reaction volume. The reactionmixture contained2.5UTaqpolymerase (MPBiomedicals, Illkirch, France),200 μM dNTP, 1X PCR reaction buffer, 2 mM MgCl2, and 0.5 μM each ofthe primers. The DNA primers were 5′-TCCAAACAATCGTCGAAAGC-3′

and5′-CCGAAAATTCGCGCTTGAAC-3′ for the16S-23S ITS (Tyleret al.,1995),5′- CCTGACCATCCGTCGCCACAAC-3′ and5′-CGCAGCAGGATGCCGACGCC-3′for gyrB (Qin et al., 2003), and 5′-ATGGATGAGCGCTTCCGTG-3′and 5′-TCATCCTTCGCCTCCCTG-3′ for ecfX (Lavenir et al., 2007). PCRwas performed directly on bacterial cells or on 100 ng of total microbialDNA of the samples. PCR cycles were done on a RapidCycler PTC-100(MJ Research Inc., Waltham, USA) with an initial denaturation for 5 minat 95 °C, 30 cycles (45 s at 94 °C, 45 s at the appropriate annealingtemperature (58 °C for ecfX and 16S-23S ITS; 66 °C for gyrB), 45 s or 1min30 s at 72 °C) and a final elongation for 5min at 72 °C. PCR products wererevealed by electrophoresis using a 2% or 1% agarose gel, stained withethidium bromide, exposed to UV (lambda=254 nm) and photographedwith a camera (Biocapt, Vilber Lourmat,Marne laVallée, France). Each PCRwas triplicated.

2.6. Taq polymerase PCR conditions for RISA analysis

BacterialDNAwasamplifiedusingPCRprimers ITSF (5′-GTCGTAACAA-GGTAGCCGTA-3′) and ITSReub (5′-GCCAAGGCATCCACC-3′) (Cardinaleet al., 2004). The ITSF and ITSReub primers match, respectively, po-sitions 1423 to 1443 of the rrs, and positions 38 to 23 of the rrl genes, of E.coli. PCR reaction mix (50 μl) contained 5 μl of 10X Taq buffer, 0.5 μMof primers, 200 μM dNTPs, 2.0 U of Taq polymerase (Invitrogen,Cergy Pontoise, France) and 50 ng of purified DNA. PCR cycles wereperformed on a RapidCycler PTC-100 (MJ Research Inc.) using the fol-lowing program: 94 °C for 3 min; 25 cycles consisting of 94 °C for 1 min,55 °C for 30 s, and 72 °C for 1 min; and extension of incomplete productsfor 5 min at 72 °C.

2.7. WAVE system conditions

RISA PCR products were purified using theMiniElute PCR purificationkit (QIAGEN), and separated by HPLC using the WAVE apparatus and a4 mm diameter DNA Sep® HT Cartridge (Transgenomic, Glasgow, UK).The cartridge was containing alkylated non porous polystyrene–divinylbenzene copolymer microspheres (2.1+/−0.12 μm diameter)specifically designed for the separation of nucleic acids. Products wereanalysed using the double-stranded multiple fragment DNA sizingprogram. The principle behind fragment sizing by the WAVE apparatusoperating in a non-denaturing mode is the differential retention

61S. Nazaret et al. / Journal of Microbiological Methods 76 (2009) 58–69

of the DNA, by the ion-pairing reagent triethylammoniumacetate (TEAA)to the column, depending on the length of the sequence. A gradientof acetonitrile is applied on the column to progressively release DNA. Theshorter the fragment is, the weaker the absorption to the resin, and thenthe lower the amount of acetonitrile is needed. Five to 10 μl of purifiedconcentrated or diluted PCR products were used for each injection.Separation was achieved by applying a flow rate of 0.35 ml min−1 at anoven temperature of 40 °C, with a gradient formed by the WAVEOptimized® Buffer A, consisting of 0.1 M TEAA and WAVE Optimized®Buffer B, consisting of 0.1 M TEAA in 25% (v/v) acetonitrile. Varyingparameters such as the concentration of Optimized® Buffer B, thenumberof segments, themin/100pbvalue andthe rangeof fragment sizeaffected the retention time. Twomain series of parameters were defined,with bothmaking use of an Optimized® Buffer B concentration of 49.8%,of 7 segments, and2.3bp/min. Series 1wasdefinedwithaminimumbasepair (bp) detection of 450 and a maximum bp detection of 970 whileseries 2 was defined with a minimum bp detection of 300 and amaximum bp detection of 800. Separated PCR products were detectedand visualised as peaks under UV light (wavelength of 260 nm) or with aHSX-3500 Fluorescence Detector using the HS Staining Solution I(Transgenomic) to improve the sensitivity.

Fig. 1. RISA–HPLC profiles of DNA extracted from clinical samples. Profiles of the bacterial comsampling times, lanes K1, K2 and K3, over 6 months (B) of a 18 months old CF infant colon6months later, lane C2 and (C) of a 9months baby colonized byH. influenzae and S. aureus straparenthesis are indicated bacterial species detected by the plating approaches. PCR product

3. Results

3.1. RISA–HPLC profiles of clinical samples

RISA–HPLC profiles were generated fromDNA extracted from sputaand oro-pharyngeal swabs, obtained from infants (5 to 18months old)and children (10 to 17 years old) attending the hospital for their regularroutine controls. Culture-dependent microbiological analyses showedthese samples to be free of infectious agents or to harbour up to threebacterial species (Table 1). The RISA–HPLC profiles showed from1 to 10peaks matching DNA fragments going from about 300 to 900 bp, asestimated by polyacrylamide gel electrophoresis analyses (data notshown). Themost complex profileswere those generated fromsputumDNA extracted from children colonized by S. aureus (Fig. 1A–C) orH. influenzae (Fig. 1B–C). Sputum DNA analysis of a same patientbut from samples collected at different times through a 6 monthperiod showed RISA–HPLC profile changes (patient K, Fig. 1A) despitethe colonization by the same species, S. aureus, according to theplating approaches. These profiles showed the conservation of someRISA–HPLC peaks through time, and the emergence of novel peaksrelated to new bacterial invaders, i.e. new strain of the same species or

munities (A) of a 14.5 years old CF child colonized with S. aureus, and analysed at threeized by two H. influenzae strains at the first sampling time, lane C1, and not colonizedins at the first sampling time, lane N1, and onlywith S. aureus 3months later, lane N2. Ins were detected by UV light, and visualised as peaks on the chromatograms.

Fig. 2. Variations among RISA–HPLC profiles of samples collected at different times from a same CF child. (A) Patient F was a 10 years old child infected with P. aeruginosa, andS. aureus at the first sampling time, lane F1, and infected with P. aeruginosa, S. aureus and H influenzae at the second sampling time (lane F2). (B) Patient A was a 11.5 years old childinfected with one (lanes A5, A6), two (lanes A1, A2, A4) or three (lane A3) strains of P. aeruginosa based on culture detection and antibiotic profiles, and followed over 10 months.This patient received several antibiotic treatments. In parenthesis are indicated bacterial species detected by the plating approaches. PCR products were detected with a UV (A) or afluorescence (B) detector. a the strain code corresponds to the patient and sample code as described in Table 1.

Fig. 3. Specificity of the RISA–HPLC P. aeruginosa 550 bp peak. (A) patient Hwas a 8months old baby known to be colonized by P. aeruginosa through selective plating approaches at theH1, H2 andH5 sampling times but not declared as colonized by this species for theH3 andH4 sampling times; analysiswas performed on swab samples except for H1; (B) patient Ewasa 10 years old child not known to be infected by P. aeruginosa according to selective culture plating, during the first fivemonths (samples E1 to E3) of the study; analysiswas performedon sputum samples. In parenthesis are indicated bacterial species detected by the plating approaches. PCR products were detected with a UV (B) or a fluorescence (A) detector.

62 S. Nazaret et al. / Journal of Microbiological Methods 76 (2009) 58–69

Table 3Detection of Pseudomonas aeruginosa among CF children clinical samples

SM+/PCR+ SM+/PCR− SM−/PCR+ SM−/PCR−

ecfX 20 0 7 25psa 18 2 7 25gyrB 18 2 7 25RISA-DHPLC 17 3 7 25

Sensitivity of P. aeruginosa PCR screenings involving various gene targets (ecfX, 16S-23SITS, gyrB, and RISA-DHPLC), was compared to detection schemes making use ofselective media. Fifty-two clinical samples were analysed.+=detected, −=not detected; all analyses were triplicated.SM=detection using selective media.

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new species (Fig. 1). Among samples found to be pathogen-freeaccording to the plating approaches, some yielded RISA–HPLC profilesfrom their DNA extracts (Fig. 1B). It has to be noted that most samplesalso showed an intense common peak whose size estimated byacrylamide gel was 270 bp. A cloning/sequencing strategy was per-formed on inserts from two samples and derived sequences weresubmitted to Blast search. Results showed the sequences (266 bpand 278 bp) to have high similarities with Streptococcus pneumoniaeCGSP14 sequence (data not shown; EU784158, EU784159) suggestingeither external contamination of the sample with nasal flora or a truecolonization of the CF lungs.

DNA samples from sputa of children infected by P. aeruginosa ledto RISA–HPLC profiles showing, inmost cases, the presence of a 550 bppeak (size estimated by polycrylamide gel), as illustrated by theexamples shown on Figs. 2, 3, and summarized in Table 1. Thispeak size matches the expected size of the P. aeruginosa ITS bet-ween the rrs–rrl genes according to GB accessions e.g. accessionsNC002516, NC009656 and NC008463. This peak could be detectedfrom complex bacterial lung communities (according to the com-plexity of the RISA–HPLC profile; patient F, Fig. 2A) of the CF childreneven after an antibiotic treatment (patient A, Fig. 2B). However,this RISA–HPLC 550 bp peak could not be detected from the H2 andH5 oro-pharyngeal swabs of a CF baby (8 month old) known to becolonized by P. aeruginosa at these two sampling dates (according toselective platings) (Fig. 3A). Similarly swab samples from a baby ledto the detection of the P. aeruginosa peak only in two out of the 5PCR reaction tests (J1 sample). On one hand, this suggested thatthe RISA–HPLC approach was not sensitive enough for the analysis oforo-pharyngeal swabs or that the strain detected in these samples bythe plating approach had a specific RISA–HPLC profile not previouslyrecorded. The first hypothesis is more likely since the H1 samplefrom the same baby generated a RISA–HPLC profile with the 550 bppeak which was recorded from a sputum sample known to containP. aeruginosa cells by the plating approach (Fig. 3A). On the other hand,we observed situations of positive results corresponding to thedetection of a 550 bp P. aeruginosa peak whereas the patient was

Fig. 4. RISA–HPLC profiles of bacterial species isolated from children CF samples. PCR productdescribed in Table 1.

not infected based on culture data suggesting that platings could alsogive variable results when sputum samples were used. The E1 sampleof a 10 year old child was found to generate RISA–HPLC profiles withthe 550 bp peak observed among samples containing P. aeruginosacells but without leading to detectable P. aeruginosa cells on selectivemedia (Fig. 3B; Table 1). Six other samples considered not to containP. aeruginosa cells according to the plating approach (i.e. sputumsamples from four children) yielded RISA–HPLC profiles with a 550 bppeak (Tables 1–3).

3.2. Identification of the RISA–HPLC peaks of clinical samples

The RISA–HPLC profiles obtained from clinical samples showedconserved and variable peaks. These profiles can be analysed globally tomonitor major changes among the bacterial communities of the sam-pled patients according to the various therapeutic treatments performedbut might also be used to directly confirm or infirm a colonization byparticular bacterial pathogens. The next step of this study was thus toattempt a direct detection of bacterial CF pathogens through an analysis ofthe RISA–HPLC profiles. Individual DNA extracts of bacterial pathogensisolated from theCF children selected for this studywere thus analysed byRISA–HPLC (Fig. 4). These chromatograms showed specific profiles madeof one or two peaks representing DNA fragments ranging from 440 to800 bp for each single isolate tested of the following species, P. aeruginosa,B. cenocepacia, S. maltophilia, I. limosus, A. xylosoxidans and Pandoraea sp.Comparison of the RISA–HPLC chromatograms of strains and clinicalsamples showed that we were able to detect the peaks corresponding tothe isolated strain (Fig. 5) except for the Pandoraea one (data not shown).These DNA size estimations were found to match those inferredfrom polyacrylamide gel electrophoresis analyses for the dominantpeaks (data not shown). The lack of detection of the Pandoraea peak inthe clinical samplemight be explainedbya lower abundance of that straincompared to the other detected pathogens, P. aeruginosa and S. aureus.

Analysis of RISA–HPLC profile variations among P. aeruginosa CFchildren isolates was performed, and showed 8 out of the 9 profilesto be highly similar, with a single 550 bp peak (Fig. 6A), matchingthe peak observed among the clinical samples known to containP. aeruginosa cells (section above). The RISA–HPLC profile, found tobe slightly different from the others, yielded two peaks, one matchingthe expected 550 bp peak and one of about 530 bp. This led us toinvestigate RISA–HPLC infra-specific variations among an additionalset of 60 strains of P. aeruginosa originating from clinical and envi-ronmental samples. DNA from all these strains generated a singleRISA–HPLC 550 bp peak (Fig. 6B), confirming that direct detection ofP. aeruginosa from RISA–HPLC profiles could be performed.

RISA–HPLC analysis of six S. aureus strains including 4 strainsisolated from CF children of this study (Fig. 7A) and two strains fromother clinical samples (Fig. 7B) was performed. S. aureus RISA–HPLCprofiles were made of several minor and dominant peaks (from 2 to 4)

s were detected by UV light. In parenthesis are indicated the patient and sample codes as

Fig. 5. RISA–HPLC profiles with the detection of peaks matching the pathogens identified by the selective plating approaches. In parenthesis are indicated bacterial species detectedby the plating approaches. (A) patient P was a 16 years old child known to be infected with P. aeruginosa and B. cepacia through selective plating approaches at the P1 and P2 samplingtime (B) patient B was a 12 years old child known to be infected with A. xylosoxidans and H. influenzae at B1 and B2 sampling time, and infected with H. influenzae and S. aureus at theB3 sampling time; (C) Patient G was a10.5 years old child infected with I. limosus at the G1 sampling time and infected with I. limosus and S. maltophilia at the G2 sampling time. Thefour G1 and the two G2 profiles are replicates of injection of the same PCR products. a the strain code corresponds to the patient and sample code as described in Table 1.

64 S. Nazaret et al. / Journal of Microbiological Methods 76 (2009) 58–69

that varied between strains. The great infra-specific variations ob-served among the S. aureus RISA–HPLC profiles made impossible theidentification of S. aureus species-specific RISA–HPLC peaks, prevent-ing any direct detection of this species from RISA–HPLC profilesof clinical samples without the joined analysis of individual specificisolates by RISA–HPLC. Nevertheless, strain-specific analysis of bac-terial resistance or persistence after various therapeutic treatmentscould be performed once the isolate RISA–HPLC profile would beknown. Similarly, distinct RISA–HPLC profiles were observed forthe four S. maltophilia strains, three A. xylosoxidans strains, andseven strains of B. cenocepacia tested (Fig. 8), making impossible theidentification of species-specific RISA–HPLC peaks. Nevertheless,some peaks were found conserved between some strains of thesespecies. Infra-specific RISA–HPLC variations within I. limosus andPandoraea sp. were not assessed.

3.3. PCR detection of P. aeruginosa

To clarify the situation concerning the conflicting resultsobtained by the RISA–HPLC and selective plating approaches, the16S-23S ITS, ecfX, and gyrB P. aeruginosa specific PCR screenings were

performed on DNA extracted from the clinical samples. The resultsare presented in Table 3. The plating approach showed 20 samples tobe positive for P. aeruginosa while the molecular screenings gave 25positive samples. Considering the ecfX PCR screening, all samplesfrom the patients that were known to be colonized with P. aeruginosa(20 patients), according to the plating approach, gave a positive PCRproduct. The psa and gyrB PCR screenings failed to detect P. aeruginosafrom twooro-pharyngeal swabs foundpositivebyplatings. Seven samplesgave positive P. aeruginosa-specific amplification products while thepatients were not previously shown to be colonized by P. aeruginosa(according to selective platings). The RISA–HPLC profiles of these sampleshad been shown to harbour the P. aeruginosa 550 bp peak.

3.4. Discussion

Characterisation of environmental bacterial communities usinggenetic fingerprinting methods have been developed and used toinvestigate various aspects of their spatio-temporal dynamics. Thesefingerprinting approaches avoid the biases and labour intensivework associated with culture-dependent selective plating approaches.Some of these approaches make use of the internal transcribed spacer

Fig. 6. RISA–HPLC profiles of P. aeruginosa strains isolated from children CF samples (A), and of various P. aeruginosa strains isolated from other kind of clinical sources andenvironmental samples (B). PCR products were detected by UV light. a the strain code corresponds to the patient and sample code as described in Table 1.

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(ITS) between the rrs (16S rRNA) and rrl (23S rRNA) genes, and exploitits variations in length and sequence content among microorganisms.The ITS length variations observed among the eubacteria led to thedevelopment of the RISA fingerprinting approach which allows ana-lysis of such variations from PCR amplified rrs–rrl ITS. Here, this RISAapproach was coupled, for the first time, to the recently developedDHPLC technology, in order to analyse the bacterial species richnessof CF children sputum and oro-pharyngeal samples, and improveour understanding of the microbial communities of CF lungs. Thisapproach was shown efficient for the direct analysis of P. aeruginosacolonization and associated microbial colonizers. RISA–HPLC profilescan be used to investigate the efficacy of therapeutic treatments.

3.5. RISA–HPLC profiles of sputumand oro-pharyngeal swab samples fromCF patients

Using the RISA–HPLC approach, monitoring of CF lung bacterialcommunities was achieved. P. aeruginosa populations could bedirectly tracked by this approach, without prior analysis of theisolates. The RISA–HPLC profiles obtained from clinical sampleswere relatively simple and characterized by a number of peaks whichrarely exceeded ten. Most complex profiles were obtained fromCF children samples containing S. aureus and H. influenzae strains,and the simplest ones were found to be generated from samplescontaining P. aeruginosa. These data were in line with those derivedfrom the RISA–HPLC analysis of bacterial CF children isolates. TheRISA–HPLC approach was found very efficient, reproducible and easyto handle for the analysis of sputum samples but results were more

variable with DNA extracted from oro-pharyngeal swabs. However,detection schemes involving platings also gave variable resultswith these swabs. The limiting step for the analysis of oro-pharyngealswabs by RISA–HPLC was found to be related to the poor DNA ex-traction yields obtained with the procedure used in this study. Someattempts at performing the RISA step directly from a swab suspensionled to PCR products but the results were not reproducible (data notshown). Improvements of the DNA extraction and cell recoveryprocedures from swabs could be performed, and will be one of theforthcoming tasks to be tackled for further development of thisclinical RISA–HPLC application.

When compared to previously reported studies using DNA cloningstrategies or the T-RFLP fingerprinting approach (Rogers et al., 2003,2004), our results showed a different picture regarding the complexityof CF lung bacterial communities. The bacterial communities char-acterized in our study were found much less diverse than thosepreviously analysed. This reduced complexity could be related to theage of the analysed patients. Here, only CF children were analysedwhile the other studies strictly considered CF adults. It is also possiblethat the clinical state of the analysed patients at the time of samplingcould have affected the results of our analyses. In our study, someof the samples were coming from highly infected children, showinga very high number of P. aeruginosa cells, and it was observed that ata level above 106 cells of P. aeruginosa per sample, the RISA ampli-fication could only detect DNA from the dominant population (datanot shown). A modified RISA making use of primers avoiding PCRamplification of P. aeruginosa DNA revealed DHPLC peaks which werenot previously detectable (data not shown). It is noteworthy that

Fig. 7. RISA–HPLC profiles of various S. aureus strains isolated from children CF samples (A), and profiles of strains of S. aureus and H. influenzae from clinical samples (B). PCR productswere detected by UV light. In parenthesis are indicated the patient and sample codes as described in Table 1. a the strain code corresponds to the patient and sample code as describedin Table 1.

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the number of identical rrn copies per bacterial genome could havealso created a preferential RISA amplification. Data from the literature(Tyler et al., 1995; Gurtler and Stanisich, 1996), from the genomesequence of PA01 (Stover et al., 2000) and various databases, indicatethat there are 3 to 4 rrn operons in P. aeruginosa with an ITS sizevarying from 468 to 471 bp. These size estimations are consistentwith our data sets. RISA PCR fragment obtained was of 550 bp butincluded the ITS, a short rrs segment and the primer sequences.

RISA–HPLC peaks were detected from samples not showingbacterial pathogens according to the selective plating approaches.This observation suggested the presence of bacterial cells thatmight have not been culturable by the plating approaches in use inthe hospital microbiology unit. However, it is also possible thatthe recorded peaks could have been generated from DNA eitherextracted from bacterial species not targeted by the plating ap-proaches or released from dead cells. Culturing on non-selectivemedia or the use of molecular strategy strictly targeting active cellssuch as the one developed by Rogers et al. (2006) could clarify theseissues.

3.6. Variability of RISA/DHPLC profiles within bacterial species infectingCF patients

PCR-based analysis of length polymorphisms among rrs–rrl ITShave been previously suggested as a strategy for typing clinicalisolates of various species and to distinguish either strains or species(McLaughlin et al., 1993; Dasen et al., 1994; Welsh and McClelland,1992; Gurtler and Stanisich, 1996). RISA–HPLC profiles generated fromthe bacterial strains isolated from CF children showed P. aeruginosa,B. cepacia, S. maltophilia, A. xylosoxidans, S. aureus, I. limosus peaks tobe identifiable from complex clinical sample profiles, and to allow ananalysis of the fate of particular strains according to the therapeutictreatments in use. However, infra-specific variability among mostof these bacterial species except P. aeruginosa made difficult direct

identification of the RISA–HPLC peaks without a comparative analysiswith bacterial isolate profiles of each sample. P. aeruginosa isolatesled to a specific RISA–HPLC peak of 550 bp, making possible a directinterpretation of the fate of P. aeruginosa cells following varioustreatments.

The most complex and variable RISA–HPLC profiles were ob-tained for the S. aureus strains. This is likely due to the high number ofS. aureus rrn operons and their respective observed ITS size variationsgoing from 303 to 551 bp (Gürtler and Barries, 1995; Stewart andCavanaugh, 2007). In fact, ITS size variations among Staphylococcusspecies were reported to be common, and S. aureus was found to bethe species showing the greatest polymorphism (Jensen et al., 1993;Dolzani et al., 1994; Couto et al., 2001). Regarding H. influenzae,the strains analysed in our study showed similar profiles with twodominant peaks matching sizes inferred (478 bp and 716 to 723 bp)from the 6 rrn operons identified in the genome of H. influenzae Rd(Fleischmann et al., 1995; Stewart and Cavanaugh, 2007). However,Privitera et al. (1998) reported a single size of rrn operon ITS regionsover a set of 17 strains isolated from several patients or individuals(not infected or with a chronic infection).

Distinct RISA–HPLCprofileswere also observed amongB. cenocepacia.ITS size variations among the cepacia complex was previously reported(Kostman et al., 1992; Dasen et al., 1994; Tyler et al., 1995) and usedto characterize strains but the genomovar or species status of thesestrains was not clear at the time of these studies. Here, ITS infra-specificvariations among the B. cenocepacia species of the Bcc were clearlyobserved.

The RISA–HPLC profile of S. maltophilia ATCC 13637 consisted ofa single peak whose size matched the one of the Genbank accession(AY116914) for that strain. The other S. maltophilia strains analysedshowed profiles different from the one of S. maltophilia ATCC 13637,and showed variations between each other. Similarly, variationswere observed between the RISA–HPLC profiles of the A. xylosoxidansreference strain and of those generated from CF sputum isolates.

Fig. 8. Infra-specific variations of RISA–HPLC profiles within S. maltophilia (A), A. xylosoxidans (B), and B. cenocepacia (C) species. PCR products were detected by UV light. a the straincode corresponds to the patient and sample code as described in Table 1.

67S. Nazaret et al. / Journal of Microbiological Methods 76 (2009) 58–69

3.7. Discrepancies between selective platings and RISA–HPLC approaches

The RISA–HPLC profiles derived from the sputum samples some-times evidenced a peakmatching to the size of the ITS of P. aeruginosawhile selective plating approaches had not allowed its isolation.To ensure that the RISA-HPLC approach was reliable and that theobserved peak was not an artefact, P. aeruginosa PCR screeningswere used (Lavenir et al., 2007). As previously demonstratedwith environmental samples, the sensitivity of the ecfX screeningwas better than the ones of the gyrB and the 16S-23S ITS screenings,and allowed to detect P. aeruginosa in all culture positive sam-ples including swabs. This sensitivity was also better than the oneobserved for the RISA–HPLC approach despite a higher copy num-ber of the rrn target. It appears that the universal and specific primerstargeting the ITS region were less sensitive than the ecfX ones.These observations confirm the high suitability of this newlydesigned ecfX PCR screening for the detection of P. aeruginosa. In-terestingly, all three PCR screenings allowed the detection ofP. aeruginosa in samples where the plating approach did not. SuchPCR positive/culture negative results have been previously reported(Xu et al., 2004; Clarke et al., 2003; Van Belkum et al., 2000; Da Silva

Filho et al., 1999; Karpati and Jonasson, 1996; McIntosh et al., 1992)using several genetic loci such as algD, groE, oprL or rrs. Explanationsof these results were that DNA from dead cells could have yieldedthe observed PCR products or that cells were in a physiological statenot allowing their growth on selective media. Other possibilitiescould have also been that the plating approaches were affected bythe overgrowth of other bacterial species (Xu et al., 2004). Thesediscrepancies could also be due to misleading identification schemes(Spilker et al., 2004).

Although the classical microbiological techniques currentlyin use in hospital labs are necessary for drug susceptibility testing,a molecular (PCR) approach independent of the use of selectiveculture media represents a more rapid and sensitive tool whichmight be helpful for investigating the fate of the global micro-bial community of CF lungs, and investigate the efficacy of theprescribed therapeutic treatments. One major observation was thatthe fate of a P. aeruginosa population among a CF lung microbialcommunity could be investigated directly by RISA–HPLC, with-out comparison with the RISA–HPLC profile of the sputum isolates.Nevertheless, exploitation of ITS sequence divergences amongP. aeruginosa is under consideration in order to perform strain-

68 S. Nazaret et al. / Journal of Microbiological Methods 76 (2009) 58–69

specific persistence analyses among a CF patient, and investigatephenomena of multiple strains colonization. Such analysis wouldinvolve the use of DHPLC under its denaturing mode. It is note-worthy that RISA–HPLC can be performed in a high throughputmanner, allowing the handling of tens of samples per day, andthat several hospitals are already equipped with DHPLC systemsbecause of their current use for the identification of human geneticdisorders.

Acknowledgements

The authors thank A. Idirian and S. Jaffrezic from Transgenomicfor helpful technical advices on the WAVE system. This work wassupported by the BQR (Bonus Qualité Recherche) program from theUniversité Lyon 1 and Hospices Civils de Lyon. This work was alsopartly funded by the Agence Nationale de la Recherche (ANR) 05-SESTproject 009-01, the CNRS and the Rhône-Alpes Region “ClusterEnvironnement” (PARMIC).

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