Cross-Sectional Analysis of Clinical and Environmental ... · Except for the sequential isolates...

INFECTION AND IMMUNITY, Jan. 2004, p. 133–144 Vol. 72, No. 10019-9567/04/$08.00�0 DOI: 10.1128/IAI.72.1.133–144.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Cross-Sectional Analysis of Clinical and Environmental Isolates ofPseudomonas aeruginosa: Biofilm Formation, Virulence, and

Genome DiversityNathan E. Head and Hongwei Yu*

Department of Microbiology, Immunology and Molecular Genetics, Joan C. Edwards School of Medicine, MarshallUniversity, Huntington, West Virginia 25704-9330

Received 4 April 2003/Returned for modification 14 July 2003/Accepted 23 September 2003

Chronic lung infections with Pseudomonas aeruginosa biofilms are associated with refractory and fatalpneumonia in cystic fibrosis (CF). In this study, a group of genomically diverse P. aeruginosa isolates werecompared with the reference strain PAO1 to assess the roles of motility, twitching, growth rate, and overpro-duction of a capsular polysaccharide (alginate) in biofilm formation. In an in vitro biofilm assay system, P.aeruginosa displayed strain-specific biofilm formation that was not solely dependent on these parameters.Compared with non-CF isolates, CF isolates expressed two opposing growth modes: reduced planktonic growthversus efficient biofilm formation. Planktonic cells of CF isolates showed elevated sensitivity to hydrogenperoxide, a reactive oxygen intermediate, and decreased lung colonization in an aerosol infection mouse model.Despite having identical genomic profiles, CF sequential isolates produced different amounts of biofilm. WhileP. aeruginosa isolates exhibited genomic diversity, the genome size of these isolates was estimated to be 0.4 to19% (27 to 1,184 kb) larger than that of PAO1. To identify these extra genetic materials, random amplificationof polymorphic DNA was coupled with PAO1-subtractive hybridization. Three loci were found within thegenomes of two CF isolates encoding one novel homolog involved in retaining a Shigella virulence plasmid(mvpTA) and two divergent genes that function in removing negative supercoiling (topA) and biosynthesis ofpyoverdine (PA2402). Together, P. aeruginosa biodiversity could provide one cause for the variation of mor-bidity and mortality in CF. P. aeruginosa may possess undefined biofilm adhesins that are important to thedevelopment of an antibiofilm therapeutic target.

Pseudomonas aeruginosa, a gram-negative environmentalbacterium, is responsible for the majority of morbidity andmortality in cystic fibrosis (CF) (27). Lung infections with thisbacterium manifest with varied disease severity. Particularly, inCF chronic lung infections, P. aeruginosa develops a charac-teristic phenotype, surrounded by an overproduced capsularpolysaccharide called alginate (17) and associated into largebacterial conglomerates known as biofilms (9). It is this for-mation that allows the bacteria to survive within the CF lungand elicit unproductive immune responses that ultimately re-sult in the demise of the patient (19).

Biofilms are formed from individual free-floating (plank-tonic) cells and are defined as exopolysaccharide-surroundedbacteria, or microcolonies, growing on biotic or abiotic sur-faces (37). Biofilms are ubiquitous in nature and are also as-sociated with numerous chronic or recurrent bacterial infec-tions and diseases (13). Formation of biofilms by thisbacterium can be viewed as a developmental process (31) thatis roughly divided into four steps: (i) adhesion, (ii) monolayer,(iii) microcolony, and (iv) mature biofilms (10). In recentyears, considerable progress has been made with regard to themolecular components that promote biofilm formation. Sev-eral surface-associated factors, such as flagella and type IV pili,have been shown to be essential for adhesion and microcolony

formation, respectively (32, 49), as well as other undefinedadhesins (12, 35, 52, 53). Flagella act to overcome repulsiveforces between the bacterium and surface to allow the initialcontact (37). Following adhesion of single bacteria into theformation of a monolayer, twitching motility is used to initiatethe grouping of cells into microcolonies (32). Further devel-opment of biofilms leads to the emergence of mature biofilms,which are characterized by alginate-encased bacterial aggre-gates (17).

The genome of the reference strain P. aeruginosa PAO1 isavailable (50). However, the functions of the majority of thegenes within this genome remain elusive. Furthermore, P.aeruginosa isolates are known to possess extensive genomediversity (40), the origin of which is yet to be completely de-fined. There are, however, many isolates that carry additionalgenetic materials that are missing in PAO1 (25, 47). Two suchexamples include the identification of P. aeruginosa genomicislands 1 (26) and 2 (2) (PAGI-1 and -2, respectively). Whilethe functions of PAGI-1 are proposed to play a role in immuneevasion (26), PAGI-2 is involved in glycosylation of the flagel-lin protein, a function that is not present in PAO1 (2). There-fore, genomic diversity may allow this bacterium to expand itspathogenic potentials.

While CF isolates are known to have the ability to attach toa surface and form biofilms (46), it is not clear whether thereis an intrinsic difference in biofilm formation among genomi-cally diverse environmental and clinical isolates of P. aerugi-nosa. In this report, we will examine the roles of four biofilm-related factors in biofilm formation: motility, twitching, growth

* Corresponding author: Mailing address: Department of Microbi-ology, Immunology and Molecular Genetics, Marshall UniversitySchool of Medicine, 1542 Spring Valley Dr., Huntington, WV 25704-9330.Phone: (304) 696-7356. Fax: (304) 696-7207. E-mail: [email protected].

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rate, and overproduction of alginate. Our studies have shownthat while the activities of flagella and type IV pili are requiredfor the initiation of biofilm formation, they play no quantitativerole in the progressive development of the initial biofilm mi-crocolony. Also, we demonstrate increased H2O2 sensitivityand decreased lung colonization in an aerosol infection mousemodel with CF isolates relative to non-CF isolates. Two op-posing modes of growth are indicative of CF isolates: efficientbiofilm formation and reduced planktonic growth rate. Thegenomic diversity found throughout many P. aeruginosa iso-lates appears to be due considerably to the presence of largeportions of DNA not found in PAO1. We also examined thecause of genomic diversity as a function of novel DNA inclu-sion. Rapid amplification of polymorphic DNA (RAPD) cou-pled with subtractive hybridization was used to identify genes

novel to or existing in a highly varied form in PAO1. Ourresults lead us to conclude that CF isolates of P. aeruginosadiffer greatly in many aspects of biofilm formation among eachother and also when compared with non-CF isolates.

MATERIALS AND METHODS

Bacterial strains. The strains used in this study are listed in Table 1. P.aeruginosa and Escherichia coli strains were grown at 37°C in Lennox broth (LB),on LB agar, or Pseudomonas isolation agar (PIA; DIFCO) plates unless other-wise noted. For the flagellar activity assay, P. aeruginosa was grown on motilitytest medium (MTM; Becton Dickinson).

Biofilm assay. The assay for biofilm formation was adapted from the proce-dure previously described (33). Briefly, P. aeruginosa overnight culture was di-luted 1:100 in fresh LB medium, dispensed (125 �l) to wells of a 96-well polyvinylchloride (PVC) microtiter plate and grown for 15 h at 37°C (unless otherwisenoted) with no aeration. Wells were stained with 100 �l of 0.25% crystal violet

TABLE 1. Phenotypic characteristics of P. aeruginosa isolates used in this study

IsolateaIncrease in

biofilmformationb

MotilitycType IV pilus

twitchingactivity (mm)d

Doublingtime (h)e

Mucoidy (�g ofuronic acid/mg

of wet cell massfOrigin Sourceg

CFS1 3.1 0.8 23 1.3 1.2 CF-94 SpeertCFS2 4.7 0.3 11 1.9 0.9 CF-97 SpeertCFS3 1.6 0.3 — 1.0 0.5 CF-98 SpeertCFS4 4.6 0.6 — 1.8 1.5 CF-98 SpeertCF001 4.0 0.2 — 2.3 0.5 CF SpeertCF005 5.5 0.3 — 3.0 1.7 CF SpeertCF017 5.5 0.1 29 2.7 2.4 CF HoibyCF040 4.6 0.8 9 1.3 2.0 CF PhibbsCF049 3.2 0.5 — 1.8 1.0 CF SpeertCF5 2.2 0.3 — 1.4 0.8 CF GovanCF15 3.9 0.4 7 1.3 0.8 CF GovanCF25 2.2 0.4 22 2.6 4.0 CF GovanCF29 1.7 0.1 — 1.8 5.5 (M) CF GovanCF37 3.2 0.3 — 1.0 5.0 CF GovanCF46 1.8 0.8 31 1.1 1.5 CF GovanCF149 3.8 0.7 22 0.8 1.2 CF Pier104035 1.3 0.9 — 1.1 1.8 Biopsy Guymon114199 2.9 0.6 11 1.1 2.1 Sputum Guymon203084 3.5 0.5 15 0.8 1.0 Sputum Guymon203097 6.6 0.6 25 1.2 1.0 Wound Guymon226281 6.0 0.6 25 1.3 0.6 Eye Guymon311058 1.8 0.9 19 1.5 1.9 Sputum Guymon822026 2.9 0.9 30 1.4 3.4 Blood GuymonERC-1 3.8 0.9 22 0.8 0.8 Env.h StoodleyENV42 2.2 0.6 16 0.8 2.3 Env. SpeertOR 2.8 0.3 26 1.5 1.7 Env. SomervillePAO1 1.0 1.0 — 0.8 1.7 Wound PhibbsCF032 0.6 0.6 — 1.7 0.6 CF HoibyCF9 0.1 0.3 16 2.5 0.6 CF GovanCF011 � 0.1 — 1.2 1.9 CF HoibyCF029 � 0.0 — 4.8 0.8 CF HoibyPA14 � 0.8 26 1.0 0.7 Wound Ausubel

a A group of 32 isolates were used. Except for the sequential isolates (CFS1 to CFS4), the genomic profiles of these isolates were different when examined by SpeIdigestion of genomes monitored by PFGE separation.

b Shown is the relative fold increase in biofilm formation in comparison to PAO1. �, the level of biofilm formation was much lower than the level for PAO1.c Motility is expressed as a relative ratio of the swimming zone between the isolate and PAO1.d Type IV pilus-mediated twitching activity was measured as described by Alm and Mattick (1). Shown are the actual twitching zones in millimeters. —, measurement

was below the limit of detection.e Doubling time represents the time required to double the bacterial population in LB medium within the 96-well plate at 37°C, as determined during the log phase

of bacterial growth.f Alginate production was measured based on growth on LB plates at 37°C for 48 h. Mucoidy was expressed as micrograms of uronic acid per milligram of wet cell

mass. The mucoid (M) phenotype was determined after 48 h of growth on LB agar at 37°C.g The P. aeruginosa isolates used in this study were obtained from the following individuals: D. Speert, University of British Columbia, British Columbia, Canada;

N. Hoiby, University of Copenhagen, Copenhagen, Denmark; P. Phibbs, East Carolina University Pseudomonas Genetic Stock Center, Greenville, N.C.; J. Govan,University of Edinburgh, Edinburgh, Scotland; G. Pier, Harvard Medical School, Boston, Mass.; C. Guymon, U.S. Army Institute of Surgical Research, Fort SamHouston, Tex.; P. Stoodley, Montana State University, Bozeman; C. Somerville, Marshall University, Huntington, W.V.; and F. Ausubel, Harvard Medical School,Boston, Mass.

h Env., environmental.

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(CV) for 30 min at room temperature. Stain was discarded, and the plate wasrinsed three to five times in standing water and allowed to dry. Stained biofilmwas solubilized with 200 �l of 95% ethanol for 10 min and read according to theoptical density at 570 nm (OD570). For light microscopy, biofilm samples wereremoved from the stained biofilm assay 96-well microtiter plates with a razorblade and wet-mounted on a specimen slide. Samples were viewed under normallight microscopy.

Determination of motility and twitching activity. Equal concentrations of P.aeruginosa isolates, as adjusted by OD (corresponding to CFU per milliliter)from an overnight culture, were used to inoculate MTM or LB plates. Aninoculation needle was placed in the culture and stabbed vertically into themedium. Plates were incubated overnight at 37°C for the motility assay, and themotility-mediated swimming zone was measured in millimeters. After incuba-tion, the agar was removed from the twitching activity plate, and the plate wasstained with 0.25% (wt/vol) Coomassie blue for 30 min. Stain was removed, andtwitching activity was measured in millimeters.

Determination of planktonic and biofilm growth rate. For static planktonicgrowth rate, OD570 readings of biofilm assay plates were taken every 30 minstarting at t � 0 until the density reached 0.07, at which point, readings weretaken every 15 min for a total time of 15 h. For biofilm growth rate, duplicate96-well plates were tested for biofilm formation at t � 0, 1, 3, 4.5, 6, 8, 10, and15 h. Furthermore, a shaken 200-ml culture was assayed every 30 min for a totaltime of 6.5 h. For determining the viable cell count from biofilm, 96-well plateswere tested for biofilm formation after 15 h. Duplicate plates were washed gentlywith sterile water, and the biofilm growth was removed by sterile swab andplated, and the number of CFU per 16 wells was determined. To calculate thespecific growth rate for all growth modes, the exponential growth phase wasselected on a graph of ln OD versus time (24). The specific growth rate wasdefined as the slope of the regression line (1/h).

H2O2 sensitivity assay. This assay was based on a previously published method(58), except paper disks were impregnated with 10 �l of 12% H2O2.

Catalase assay. P. aeruginosa cell suspensions in phosphate buffer (pH 7.4)were bead beated for 60 s three times while on ice. The supernatant was mea-sured for protein concentration by using the Bio-Rad DC protein assay. Catalaseactivity was determined by catalase assay kit (Cayman Chemical, Ann Arbor,Mich.).

PFGE. Pulsed-field gel electrophoresis (PFGE) analysis of genomic profiles ofP. aeruginosa isolates was adapted from the standard enterobacterial proceduredeveloped by the Centers for Disease Control and Prevention (PulseNet, section5, “Preparation of PFGE plugs from agar cultures” [www.cdc.gov]). Briefly, theP. aeruginosa overnight culture (5 ml) was pelleted and resuspended in 1 ml ofcell suspension buffer (100 mM Tris, 100 mM EDTA [pH 8.0]) to 10% trans-mittance as indicated by the BioMerieux Vitek colorimeter. The cell suspension(200 �l) was combined with 200 �g of proteinase K and inverted six times. Amixture of 1.6% InCert agarose (FMC BioProducts, Rockland, Maine) and 1%sodium dodecyl sulfate (SDS) (0.8 g of InCert agarose, 2.5 ml of 20% SDS, 46.7ml of Tris-EDTA buffer [TE]) (200 �l) was mixed with the cell suspension-proteinase K mixture and dispensed in an agarose plug mold (Bio-Rad). Aftersolidifying, the cells were lysed within the plug using 1.5 ml of cell lysis buffer (50mM Tris, 50 mM EDTA [pH 8.0], 1% N-lauroylsarcosine) and 800 �g of pro-teinase K for 1.5 h in a 54°C water bath with agitation. Plugs were then washedtwice with 10 ml of preheated, sterile, reagent-grade H2O for 15 min and twicewith 10 ml of preheated TE buffer for 15 min, with all washes performed in a48°C water bath with agitation. Portions of the plugs (1.5 mm) were digested withthe restriction enzyme SpeI (20 U), DpnI (14 U), or XbaI (50 U) for 1 h at 37°C.Portions of the plugs (6 mm) were equilibrated with reaction buffer for 3 h at 4°Cand digested with I-CeuI (0.8 U) for 2 h at 37°C. Digested portions were loadedinto an agarose gel of a specific concentration and subjected to certain separationconditions (both noted in the relevant figure). The running buffer was 0.5�Tris-borate EDTA (TBE). After PFGE, the gel was stained with ethidium bro-mide (1 �g/ml) for 30 min and photographed under UV light.

Alginate assay. The alginate assay was based on a previously published method(23) with the following modifications. Isolates were inoculated onto LB platesand incubated for 48 h at 37°C. Bacteria were scraped off the plates and sus-pended in 10 ml of phosphate-buffered saline (PBS; pH 7.4) and centrifuged at1,500 � g for 20 min. The supernatant was used for the alginate assay.

RAPD. RAPD reactions were carried out with purified genomic DNA of P.aeruginosa isolates by using standard primers as previously described (28). Theconditions that were selected as the optimal conditions for obtaining accurateamplified band profiles with the eight primers are as follows: assays were per-formed in 50 �l of EasyStart PCR tubes (Molecular BioProducts) with 2.5 U ofMasterAmp Taq DNA polymerase (Epicentre) and 0.25 �M each primer. Thefollowing temperature cycling was used with a Bio-Rad iCycler system: 94°C for

4 min; 4 cycles consisting of 94°C for 2 min, 45°C for 5 min, and 72°C for 5 min,followed by 30 cycles consisting of 94°C for 1 min, 45°C for 1 min, and 72°C for2 min; and a final extension step consisting of 72°C for 10 min.

Southern hybridization. Agarose gels were soaked in 0.25 N HCl for 30 min,rinsed in H2O, and soaked in 1.5 M NaCl–0.5 M NaOH for 30 min and 1.5 MNaCl–0.5 M Tris-Cl (pH 8.0) for 30 min. A blotting apparatus was constructedwith a filter paper wick and a Hybond-N� membrane (Amersham PharmaciaBiotech) and transferred with 20� SSC (1� SSC is 0.15 M NaCl plus 0.015 Msodium citrate) transfer buffer for 24 h. After transfer, the membrane was rinsedin transfer buffer and UV cross-linked. Hybridization and digoxigenin probelabeling were carried out as described by the manufacturer (Roche MolecularBiochemicals). PAO1 DNA was fully digested with Sau3A, separated by elec-trophoresis on a 1% agarose gel, and stained with ethidium bromide. A partic-ular section of the digested DNA gel corresponding to the size of 0.5 to 2 kb wasremoved, extracted by QIAquick gel extraction (Qiagen) according to the spec-ifications of the manufacturer, and used for labeling.

Transmission electron microscopy. P. aeruginosa overnight culture (2 ml) waspelleted, washed with 500 �l of PBS, pelleted, and resuspended in 100 �l of PBS.The cell suspension (10 �l) and 2% phosphotungstic acid (PTA; 10 �l) werecombined on a glass slide. A Formvar-coated grid was placed on top of themixture for 30 s. The grid was allowed to dry overnight at room temperature.Grids were viewed by transmission electron microscopy (Hitachi).

Examination of genomic islands by PCR. Amplification reactions of PAGI-1and PAGI-2 were carried out with purified genomic DNA by using the followingprimers: for PAGI-1, orf18 forward (5�TAAGGGGTTCTAGCGGC) and reverse(5�AATCGGTGCAAGGGAGTA), orf22 forward (5�GACTTGCATGGGGCTT)and reverse (5�TGCCGAACACGATCAA), and PAO1-specific (PA2221) forward(5�TATCAGTGTCGGGCAAGA) and reverse (5�AGCTCCGGCAACCACTA);and for PAGI-2, orfA forward (5�TATGTTCCGCAAGGTCT) and reverse 5�AATGGTACATGGGGAAGT) and PAO1-specific (PA1089) forward (5�TGTGCGCACTGCCTAC) and reverse (5�GCAAGGTATTGGTTCGG). All primers weremade at the Marshall DNA Core Facility. Assays were performed in 50-�l EasyStartPCR tubes (Molecular BioProducts) with 2.5 U of MasterAmp Taq DNA polymer-ase (Epicentre) and 0.25 �M each primer set. The following temperature cycling wasdone with the Bio-Rad iCycler system: 94°C for 1 min; 34 cycles consisting of 94°Cfor 1 min, 54°C for 2 min, and 72°C for 2 min; and a final extension step consistingof 72°C for 8 min.

P. aeruginosa aerosol infection mouse model. The P. aeruginosa overnightculture (1 ml) was used to inoculate 100 ml of LB and was grown for 14 to 16 hwith aeration at 37°C. The cells were pelleted and resuspended in 10 ml of P-PBS(1% Proteose Peptone–PBS). The cell suspension (5 ml) was dispensed into thenebulizer-Venturi unit of the inhalation exposure system (Glas-Col, TerreHaute, Ind.) and aerosolized for 30 min (compressed air control � 20, vacuumcontrol � 50), cloud decayed for 25 min, and UV decontaminated for 5 min. Atotal of 10 mice were exposed for each strain, with 5 mice terminated at t � 0 andt � 6 h. Mice were sacrificed by carbon dioxide. The right lung was removed,homogenized in 1 ml of P-PBS, and plated via serial dilution on LB agar.

DNA sequencing and bioinformatic analysis of novel genomic sequences.Purified plasmids were sequenced by using universal M13 forward and reverseprimers. Sequencing was performed with 11 �l (approximately 2 �g) of purifiedplasmid, 1 �l (3.2 pmol) of unlabeled sequencing primer, and 8 �l of Big Dyeterminator reaction mix (PE Applied Biosystems). DNA sequencing was per-formed on a LI-COR 4200 DNA sequencer (Lincoln, Neb.) at the Core Facilityat Marshall University School of Medicine. Once the DNA sequences wereavailable, they were compared by BLAST to GenBank and to the annotation ofthe PAO1 genome (www.pseudomonas.com).

RESULTS

P. aeruginosa isolates demonstrate different abilities of invitro biofilm formation. While P. aeruginosa strains possesscomplex, diversified genomes (22) and readily form biofilms(9), it is unclear whether there are any variations in biofilmformation among clonally diverse clinical and environmentalisolates of P. aeruginosa. A total of 151 isolates (101 CF and 50non-CF) collected from geographically diversified areas werefirst examined for their clonal relationship by macrorestrictiondigestions of chromosomal DNA with SpeI, XbaI, and/or DpnI,followed by separation by PFGE. The PFGE pattern of eachisolate was compared with that of the reference strain PAO1

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(50) and the patterns were also compared between these iso-lates. All PFGE patterns were unique except for 16 sequentialCF isolates, which is consistent with previous reports that CFpulmonary infections are mainly associated with a predomi-nant strain (6, 28, 39). Next, all isolates were tested for biofilm-forming ability by using an adaptation of a PVC 96-well mi-crotiter dish method (33). Since some CF isolates areauxotrophic and often require the presence of several aminoacids for growth in minimal media (3, 4), we chose to growthese isolates in complex LB medium. The strain PAO1 wasincluded as a standard in every microtiter dish used to deter-mine biofilm formation. A significant variation in biofilm for-mation was found among all isolates tested (Fig. 1A). To con-firm that the crystal violet-stained samples at the air-liquidinterface on the dish were true biofilm, a sample was preparedand visualized microscopically, which was consistent with char-acteristics of initial biofilms (Fig. 1B). Furthermore, the ma-jority of these isolates showed increased amounts of biofilmcompared with PAO1 (Fig. 1A). A group of 32 phenotypicallydiverse isolates were selected for further analysis. As shown inTable 1, biofilm formation of these samples varied greatly inrelation to PAO1. Only five of the samples (CF032, CF9,CF011, CF029, and PA14) formed less biofilm than PAO1.Sequential isolates (CFS1 to CFS4) (57) from the same patient

over a period of 4 years expressed different levels of biofilmformation. Furthermore, these isolates were also tested forbiofilm formation in a 12-well polystyrene plate, which wasconsistent with the PVC plate results (data not shown). To testwhether the observed variations could be due to the relativehydrophobicity of these isolates, the biofilm formation was alsotested on a glass surface. The dynamics and trend of biofilmformation on this surface were identical to those seen withPVC (data not shown), suggesting that the relative hydropho-bicity of these isolates is not involved in the differences inbiofilm formation. Since some isolates are from the environ-ment, biofilm formation was compared between 30 and 37°C.No difference was seen among these isolates (data not shown).

However, this variation, as observed with CV staining (Fig.1A), could be due to the different amounts of CV-absorbingextracellular materials. To test this, viable counts of biofilmswere measured and compared with the OD of CV-stainedmaterials. There was a correlation between biofilm CFU andOD (Fig. 1C), suggesting that the optical intensity is represen-tative of the cell density in biofilms. Furthermore, three iso-genic strains, PAO1, PAO6862 (PAO1 algD::Gmr) (55), andPAO578 (PAO1 mucA22), a hypermucoid strain known tomaintain mucoid status on LB plates (11), were assayed for thecontribution of mucoidy on biofilm formation. When stained

FIG. 1. Variations in (A) and light microscopy (B) of biofilm formation produced by genomically diversified clinical and environmental isolatesof P. aeruginosa in an in vitro biofilm assay system. For each isolate within each 96-well plate in panel A, the assay was repeated for a total of eighttimes (vertically). Biofilm formation was analyzed along with PAO1 (far-right vertical lane of each plate). (C) Relationship between CV-stainedmaterials (black bars) and biofilm viable counts (grey bars). (D) Relationship between alginate production in PAO1 and its derivatives and biofilmformation.



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with CV, PAO1 showed the largest amount of biofilm followedby PAO6862 and the producer of the least biofilm, PAO578(Fig. 1D). These results are consistent with the recent obser-vation that alginate is probably not required for the initialphase of biofilm formation (18, 54) and also conclude that CVstaining is not indicative of the extracellular matrix but theviable cells inside biofilms.

Flagellum- and type IV pilus-mediated activities are notproportional to biofilm formation. The formation of biofilms isa multistep process that requires participation of structuralappendages, such as flagella and type IV pili (32). Variations inbiofilm formation, as seen in Fig. 1, could be due to alteredactivities of these structural appendages. To test this, we mea-sured the motility and twitching abilities of the isolates inTable 1. The flagellum activity of PAO1 was the greatestamong all strains tested (Table 1 and Fig. 2A). However,PAO1 formed the smallest amount of biofilm compared to theother six CF isolates shown in Fig. 2A. Furthermore, otherisolates with increased motility displayed elevated biofilm for-mation (Table 1, CF040 and ERC-1). Thus, no quantitativecorrelation was seen between the ability of the bacteria toassume a biofilm mode and motility. In addition, when sampleswere negatively stained with 2% PTA and examined by elec-tron microscopy, all isolates showed the presence of flagella(data not shown). We also assayed the twitching activity of the32 isolates. While twitching was not visible in all samples, onethat displayed such activity was strain PA14, which formed thesmallest amount of biofilm compared with other isolates (Fig.2B). There were, however, isolates forming more biofilm cou-pled with increased twitching activity (CF017 and 203097, Ta-ble 1). Therefore, no quantitative correlation was seen be-tween twitching and biofilm formation.

CF isolates express slow planktonic growth in contrast toefficient biofilm formation. Biofilm formation could be affectedby inherent differences in the planktonic growth rate of each

isolate (34). To measure this growth property, we developed amethod to simultaneously measure the specific growth rate ofmultiple samples utilizing the same 96-well microtiter platesused for biofilm formation. P. aeruginosa isolates grown in thissystem displayed a typical bacterial growth curve. While thegeneration time of CF029 was the lowest and equivalent to4.8 h (0.15 h�1), PAO1 had the highest growth rate of about0.8 h (0.9 h�1) under this condition (Table 1). The CF isolatesas a whole were found to grow significantly slower than non-CFisolates (mean, 0.45 versus 0.7 h�1, respectively; P � 0.008, ttest) (Fig. 3). When grouped, the CF isolates were found toform an amount of biofilm similar to that formed by non-CFisolates (P � 0.615, t test; Table 1). Comparatively, the biofilm-specific growth of these isolates at different time points alsomimics this trend (data not shown). Furthermore, in compar-ison with PAO1, the majority of CF isolates displayed slowerplanktonic growth but efficient biofilm formation. For example,the planktonic growth rates of CF005 and CF001 were 3 and2.3 h�1, respectively, but they generated three- and sixfoldmore biofilm than PAO1, respectively (Table 1). To eliminatethe possibility of cell partitioning that could be derived fromthe static culture method, which may affect the measurementof the planktonic growth rate, a shaken culture method wasalso performed. Again, the majority of CF isolates showed thesame trend of reduced planktonic growth rate compared withthe environmental isolates, non-CF isolates, and referencestrains, such as PAO1 and PA14. Furthermore, CF isolatessuch as CF149, CF37, and CF46 showed a pattern of fastplanktonic growth, while CF005, CF029, and CF25 had slowplanktonic growth, consistent with the results obtained withthe static culture method (Table 1). Thus, there was no corre-lation between planktonic growth rate and biofilm formation.

No correlation between mucoidy and initial biofilm forma-tion. Mucoidy (overproduction of alginate) is an importantvirulence factor in chronic P. aeruginosa lung infections in CF

FIG. 2. Motility and twitching activity of P. aeruginosa and the relationship with biofilm formation and planktonic growth rate. Shown aremotility (A) and twitching (B) zones between six isolates and PAO1. The first number under the strain designation indicates the relative foldincrease in biofilm formation versus PAO1; the number in parentheses indicates the doubling time in hours.



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(17). Recently, it has been shown that alginate may not play akey role in initial biofilm formation (18, 54). A correlationbetween uronic acid (an alginate precusor) production andinitial biofilm formation was tested among different isolates.As seen in Table 1, the highest alginate producers, CF25,CF29, and CF37, did not lead to the largest amounts of biofilmformed in this study. Conversely, some strains with low alginateproduction generated large (CFS2, CF005, and 226281) andsmall amounts of biofilms (CF9 and CF029). Therefore, nocorrelation was seen between alginate production and initialbiofilm formation.

Planktonic cells of CF isolates are more sensitive to H2O2.P. aeruginosa biofilms are more resistant to reactive oxygenintermediates (ROI) than their planktonic counterparts (21).However, it is not clear whether individual planktonic cells ofgenomically diverse isolates have altered sensitivity to H2O2,an abundant ROI species within neutrophils. When compared,CF isolates were found to be more sensitive to this particularROI species than those of non-CF origin (Fig. 3B, P � 0.008,t test).

Nonmucoid CF isolates are associated with reduced lungcolonization in an acute aerosol infection mouse model. Totest whether there is a correlation between increased in vitrobiofilm formation and in vivo lung colonization, we utilized anaerogenic infection mouse model (57). A group of age- andsex-matched C57BL/6 mice were exposed to aerosols of sevenCF and three non-CF isolates (Fig. 4). Only nonmucoid iso-lates were aerosolized, because previous work had indicatedthat aerosolized mucoid isolates retained more effectively inthe lungs than isogenic nonmucoid strains (56). Bacterial sur-vival in the lungs was measured for each isolate 6 h afteraerosol exposure in comparison with the initial depositiondose. CF isolates were cleared more effectively (up to 10%retention) from the murine lungs than non-CF isolates (25 to

35% retention, Fig. 4). PAO1 was markedly less capable ofbeing cleared than the CF isolates tested.

CF isolates have increased genome sizes with genomic in-sertions. Horizontal gene transfer is a cause of genomic diver-sity in P. aeruginosa (2, 25, 26, 47). When the genomes of theisolates in Table 1 were examined for genomic diversity by SpeImacrorestriction digestion coupled with PFGE separation(Fig. 5A), the isolates possessed at least one band larger thanthat of the largest PAO1 fragment (SpeA, Fig. 5A). Also, the

FIG. 3. Comparison of planktonic growth rate (A) and sensitivity to hydrogen peroxide (B) between CF and non-CF isolates. Specific growthrates and sensitivity to H2O2 of each isolate in comparison with PAO1 (stars) were determined as described in Materials and Methods. Thehorizontal bar in each group represents the median values of growth rates (P � 0.008, t test) and growth inhibition zones in millimeters (P � 0.008,t test).

FIG. 4. Nonmucoid CF isolates of P. aeruginosa are cleared moreefficiently from the mouse lungs than non-CF isolates in an acuteaerosol infection mouse model. A group of C57BL/6 mice were ex-posed to aerosols of different P. aeruginosa isolates as labeled on the xaxis. For each exposure, five mice each were included for determina-tion of bacterial deposition to the lungs at t � 0 and 6 h.



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PFGE banding pattern was highly diverse among these isolates(Fig. 5A). This suggests that genome size of these isolatescould be hypervariable in relation to PAO1. To determinewhether intrinsic diversity was related to genome size, the chro-mosomes of these isolates were digested by the homing endonu-clease I-CeuI, which specifically cuts at the rrn operons. A selectedgroup of isolates were analyzed for genome size (Table 2). Asshown in Fig. 5B and C, all isolates examined had an equalnumber of four rrn operons, just like PAO1. The genome sizes ofthese isolates ranged from 0.4 to 18.9% larger than that of PAO1(Table 2). Since many clinical isolates carry specific genomic se-quences called PAGI-1 and -2 (2, 26), we examined the presenceor absence of PAGI-1 and -2 by PCR. As shown in Table 2,PAGI-1 and -2 were present within the genomes of CF149 and311058, corresponding with the largest genomes identified.

Identification of novel DNA sequences in CF isolates. Toidentify novel DNA sequences in these isolates, we developeda simple method based on RAPD coupled with subtractivehybridization using PAO1 DNA as a probe. While the resolv-ing power of RAPD mainly allows for strain differentiation(28), it is not clear what genes are amplified by this PCR-based

method. We hypothesized that some of the randomly amplifiedPCR products from the larger genomes could be missing fromthe PAO1 genome and may represent unknown genomic is-lands. A Southern hybridization of a RAPD membrane withrandomly labeled PAO1 genomic DNA yielded two fragments(1.4 and 2.0 kb) that failed to be hybridized (Fig. 6B). Usingthis method, we identified three novel genomic sequences fromtwo CF isolates, CF005 and CF023 (Table 3). The sequenceCF023-228 was matched to a probable nonribosomal peptidesynthetase (PA2402) that had homology with the recently re-leased highly divergent pyoverdine biosynthetic locus from twoCF isolates (47). The CF005-275 and -272 sequences were notpresent in the GenBank database, with the first one encodinga variant of topoisomerase I from P. aeruginosa and the secondencoding a homolog of mvpTA from Shigella flexneri (42) thathas not been identified in P. aeruginosa.

DISCUSSION

Four primary conclusions can be drawn from this cross-sectional analysis of clinical and environmental isolates of P.

FIG. 5. Relationship between genome diversity and genome size among clinical and nonclinical isolates of P. aeruginosa. (A) PFGE separation ofSpeI-digested chromosomal DNA from a selected group of P. aeruginosa isolates. An arrowhead indicates the largest SpeI fragment of PAO1 (SpeA) (57).(B and C) PFGE separation of genomic DNA digested with I-CeuI. Arrowheads indicate the largest PAO1 fragments, 4,064 and 950 kb, respectively.The running conditions were 18 h, 6 V/cm, 120° angle, 1% agarose, and a switch time of 15 to 40 s with a gradient of a � 0.35741 in panel A; 48 h, 2V/cm, 106° angle, 0.8% agarose, and a switch time of 20 s to 30 s with a linear gradient in panel B; and 22 h, 6 V/cm, 120° angle, 0.8% agarose, and aswitch time of 90 s in panel C. The molecular size standards were Schizosaccharomyces pombe (B) and Hansenula wingei (C) (Bio-Rad).



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aeruginosa. First, various P. aeruginosa strains have differentcapacities of in vitro biofilm formation. Second, the majority ofCF isolates in comparison with non-CF isolates demonstratetwo opposing growth modes: reduced planktonic growth versusefficient biofilm formation. Third, nonmucoid CF isolates havea reduced ability of lung colonization compared with PAO1,PA14, and environmental isolates. Fourth, the presence ofnovel DNA is common in CF isolates and represents a majorcause of genomic diversity in P. aeruginosa.

Biofilm formation is an important phenotype associated withchronic P. aeruginosa pulmonary infections in CF (10). In thisstudy, we evaluated the effect of motility, twitching, growthrate, and mucoidy on biofilm formation using a group of 32 P.aeruginosa isolates. A standard method based on bacterialgrowth within 96-well microtiter plates (33) was used to assessthe biofilm-forming ability of these strains. This simple methodallows for a high throughput and reliable analysis of biofilmformation on an abiotic surface. This surface is by no meansindicative of CF lungs; however, it allows for analysis of strain-dependent biofilm-forming ability (Fig. 1A). Furthermore, thebiofilms formed in this system possess the characteristics ofmono- and multilayer community structures (Fig. 1B). Labo-ratory strains such as PAO1 and PA14, although originallyisolated from clinical settings (20, 38), have reduced biofilmformation compared with fresh isolates from clinical and en-vironmental sources. While PAO1 and PA14 grow optimally asplanktonic cells (0.8 and 1.0 h, respectively), it appears that thecapacity of biofilm formation in these two strains is at a basallevel when compared with other isolates (Table 1). This isperhaps related to laboratory attenuation of these two strains.Again, CF isolates form much more biofilm than PAO1 (Table1). This suggests that PAO1 may be equipped with endogenousbiofilm suppression mechanisms, or, conversely, CF isolatespossess mechanisms to promote biofilm formation. Also, mostapproaches in studying biofilm formation focus on screening

mutants defective in biofilm formation (32). However, a com-plementary approach that has not been actively pursued is touse mutants with increased biofilm formation to identify bio-film repressors in these two strains. Furthermore, CF isolatesare proficient biofilm formers, since they exhibit efficient bio-film formation and despite the fact that they are deficient inplanktonic growth (Fig. 3). It is known that P. aeruginosa iscapable of making the transition from an environmental stateto a chronic colonizing state in CF (41). However, such anadjustment comes at a price. For example, most CF isolates areauxotrophic mutants and are incapable of synthesizing methi-onine, leucine, arginine, and/or ornithine (3, 4). Intriguingly,this auxotrophic defect is linked with conversion to mucoidy,known to confer increased resistance to antibiotics and hostdefenses (17). The production of alginate in this chronic stateis a major consumer of cellular energy in return for securingthe survival of the bacteria. This adjustment and rerouting ofcellular resources to adequately suit the new environmentcompromise the planktonic growth of the bacterium (51).

The activities of flagella and type IV pili have been shown toplay a role in biofilm formation (37). Since all isolates exam-

FIG. 6. Identification of novel DNA sequences from CF isolates ofP. aeruginosa. (A) Randomly amplified PCR products using CF005 asa template with primers as indicated above the gel were separated ina 1% agarose gel. After blotting, filters were hybridized with randomprimer-labeled total PAO1 genomic DNA. (B) Southern blot of the gelshown in panel A with arrows indicating two major bands amplified byprimers 272 and 275, respectively, but which failed to be bound byPAO1 DNA.

TABLE 2. Genomic characteristics of a selected group of P.aeruginosa isolates used in this study

Strain Genomesize (kb)

Differencefrom

estimatedsize (kb)a

%Difference

Presence ofb:

PAGI-1 PAGI-2

PAO1 6,264 0 0.0 � �PA14 6,618 354 5.7 � �CF37 6,862 598 9.6 � �CF149 6,990 726 11.6 � �CF017 6,291 27 0.4 � �CF032 6,659 395 6.3 � �CF041 6,386 122 2.0 � �114199 6,875 611 9.8 � �311058 7,448 1,184 18.9 � �

a Genome sizes were estimated as shown in Fig. 5B and C.b The presence or absence of PAGI-1 and -2 was determined by PCR ampli-

fication of the PAGI-specific genes. For PAGI-1 (GenBank accession no.AF241171), three sets of primers were used for PCR. The first set amplified thegene coding for phytoene dehydrogenase (orf22; 1.5 kb), and the second setamplified the gene coding for 1,3-propanediol dehydrogenase (orf22; 1.4 kb).Both genes are present only on PAGI-1. The third set of primers produced aPAO1-specific transposase gene (PA2221; 1.5 kb). �, orf18/orf22 present andPA2221 absent; �, PA2221 present, but orf18/orf22 missing. For PAGI-2 (Gen-Bank accession no. AF332547), two sets of primers were used. The first setamplified orfA (1.2 kb), and the second set produced a partial PAO1-specificgene (PA1089; 0.8 kb).



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ined in this study had flagella, our results about the relation-ship between motility and biofilm formation suggest that theflagellar activity of CF isolates, as manifested by the observedswimming zones, is not the predominant factor involving theprogressive development of biofilms in vitro. When groupingall samples in Table 1, a slight correlation was seen betweenplanktonic generation time and motility (R2 � 0.30). Thistrend is expected in that motility, as measured in this study,may be a reflection of how fast the bacteria are dividing plank-tonically. This is particularly evident with PAO1, which has thefastest growth rate and is the most motile (Fig. 2A). Further-more, twitching, as mediated by type IV pili, was not observedin all of the samples tested (Table 1); however, many of thosewithout this activity formed more biofilm than PAO1. This isassuming the twitching assay is a reflection of the role type IVpili play in microcolony formation. Since both assays wereinoculated with the same number of bacteria, the observedvariations in these activities could not have been caused by adifference in the initial inoculum size. Since some CF isolatesgrow primarily for surface attachment (Table 1) independentof these two known biofilm-related factors, these results sug-gest the presence of a novel biofilm adhesin. To explain whythe activities of flagella and type IV pili do not correlate withthe amount of biofilm formed, these structural appendagesmay only act as biofilm adhesins qualitatively. To support this,a report showed that increased initial biofilm formation waslinked with isogenic variants deficient in flagellum and type IVpilus activities (7, 12). Also, an additional surface structurecalled curli has been implicated with increased biofilm forma-tion in an E. coli K12 mutant (53). However, mucoidy status ofthese isolates did not affect initial biofilm formation in vitro(Table 1), which is in agreement with previous reports thatmucoid exopolysaccharide is needed mainly for maintainingthe altered architecture of mature biofilms (18, 29). Further-more, although the specific adhesins that promote biofilm for-mation on a PVC surface are unknown, our studies showedthat the same type of adhesins are also involved in binding toanother abiotic surface, polystyrene. While four sequential iso-lates have identical genomic profiles, as revealed by PFGEanalysis, there exists a significant variation in biofilm forma-tion, motility, type IV pilus activity, growth rate, and alginateproduction among them (Table 1). These isolates may con-stantly adapt themselves to changes in the CF lung environ-ment over the course of chronic colonization. In support of thisconjecture, sequential isolates can express different pheno-types, such as untypeable O-antigen (47), and can be eitherprototrophic or auxotrophic (3).

When the specific planktonic growth rates of CF andnon-CF isolates were compared, the growth rates of CF iso-lates were significantly lower (Fig. 3). These results suggestthat particular responses taken by CF isolates to evade envi-ronmental stresses begin to tax the cellular resources, at leastto a greater extent than those of the non-CF clinical isolates,thereby expressing a reduced planktonic growth rate. The CFlung may be a more extreme environment to adapt to thanother clinical environments, such as burn wounds, thereforeresulting in a reduced planktonic growth rate. The presentwork presents a model that while the majority of CF isolatesretain their biofilm-forming ability during their transition froman in vivo to in vitro environment, the shift from planktonic tobiofilm growth may be beneficial to bacterial survival (9). As-sociation into a biofilm offers a selective advantage allowingthe collective unit to operate as one, protected from the ex-ternal environment, associated with increased resistance toantibiotics and host defenses (14), cooperating metabolicallyand evolving as a community, potentially by horizontal genetransfer (16).

By inducing a bacterial aerosol-induced lung infection(BAILI) using an inhalation exposure system (57), a cleardistinction in colonization is displayed between the CF andnon-CF isolates (Fig. 4). An environmental sample (ERC-1)(48) and burn patient isolates PA14 (38) and PAO1 (20) allshowed increased retention in the mouse lung compared to theCF isolates. While the biofilm assay determines initial attach-ment to a PVC surface, BAILI verifies not only attachment tolung tissue, but also host defense intervention. The explanationof bacterial clearance from the lung can be approached in twostages. First, the inherent adhesive ability of a bacterial straindetermines whether and how efficiently an initial colonizationwill take place. This may be due to the presence of differentbacterial surface structures. For example, CF isolates can ex-press a novel lipid A portion of lipopolysaccharide (15). Also,fresh clinical isolates fail to bind to an asialo-GM1, generallyconsidered a lung epithelial receptor for a laboratory strain(44), indicating CF isolates may have novel surface proteinsthat mediate attachment to lung tissue. Once this colonizationexists, the host defenses are the next consideration as theability of the isolate to evade clearance is determined. Theinnate pulmonary defenses include antimicrobial defensins andneutrophils that carry an abundance of reactive oxygen species,like hydrogen peroxide. Our results suggest that CF isolates,when in the planktonic phase, are significantly more sensitiveto H2O2 than are non-CF isolates (Fig. 3B), which showedincreased mouse lung retention (Fig. 4). However, a direct

TABLE 3. Novel DNA sequences identified from CF isolates of P. aeruginosa

Strain

Primera

(insertsize in

kb)

GenBankaccession

no.Gene Homolog product (accession no.)

E value(BLASTsearch)

CF005 272 (1.5) AY258908 mvpTA Plasmid maintenance protein (S. flexneri; NP_490590.1) 9E-48275 (2.1) AY265810 topA Topoisomerase I (X. fastidiosa; NP_061659.1) 4E-50

CF023 228 (1.6) AY261781 PA2402 Probable nonribosomal peptide synthetase (AE004667_1) 2E-59

a Primers were used as previously described (28). The number in parentheses represents the fragment amplified by this primer, which was cloned into pCR4-TOPO(Invitrogen) and sequenced with universal M13 primers.



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catalase enzymatic activity assay on the total cellular extracts ofall these isolates indicated no difference between the CF andnon-CF isolates. While this is inconclusive, the first assay ap-pears to be quantitative and explains the clearance data. Inci-dentally, the aerosol mouse model simulates an acute infectioncondition caused by planktonic cells, not biofilms. Using thismodel, bacteria deposited to the alveoli were in a single-cellform (data not shown). Also, neutrophil migration from thebloodstream to the lung occurs within the first 4 h followingaerosol exposure (56). This may in part explain why in vivomouse lung clearance does not correlate with the in vitromicrotiter dish biofilm assay. On the other hand, CFTR knock-out mice have recently been shown to develop lung infectionswhen pretreated with oral antibiotics (8). It would be interest-ing to test the response of this orally treated CFTR mousemodel to aerosolized bacteria to determine whether the mod-ified CFTR-deficient lung environment affects colonization.

The inherent genomic diversity in P. aeruginosa may becaused by four means of novel DNA introduction into thebacterial genome: nucleotide substitution, insertions of trans-posons and bacterial phages, horizontal gene transfer, andpreexisting novel genomic sequences. First, CF isolates of P.aeruginosa often display a high frequency of mutation afterlong-term colonization in CF (30), possibly due to single-nu-cleotide polymorphisms (SNPs). One example of CF-relatedSNPs involves mutations in mucA, giving rise to overproduc-tion of alginate (5). It is known that PAO1 carries transposonsand a bacteriophage (50). Two genomic islands, PAGI-1 and-2, have been identified in isolates of P. aeruginosa (2, 26).Another consequence of horizontal gene transfer is the in-crease in genome size. In this study, we used a low-throughputPFGE technique to estimate the genome size of various iso-lates of P. aeruginosa coupled with highly specific digestionusing I-CeuI. This method allowed accurate determination ofthe genome size of P. aeruginosa clone C, which was 6.52 Mb,about 256 kb larger (4%) than the PAO1 genome (43). Usingthis approach, we determined that a selected group of isolatesin Table 1 had much larger genomes than that of PAO1 (Fig.5). These results agree with data showing the presence ofPAGI-1 and -2 in the largest genomes analyzed (Table 2,CF149 and 311058). It has been shown recently that althoughextensive novel sequences are present in the genomes of CFisolates, the backbone of the PAO1 genome is preserved inseveral P. aeruginosa isolates (25, 47). In combination with ourlung clearance data, this suggests that PAO1 remains an ex-cellent model for studying virulence, even though its genomesize is not representative of most CF isolates (Table 2). Sincebacteria in biofilms have an increased incidence of horizontalgene transfer (16), we also note that while genome size wasincreased, the lung colonization ability was reduced in P.aeruginosa CF isolates (Fig. 4). This is in agreement with thetrend that a CF isolate had an overall reduction in virulencewhen examined in a highly sensitive plant model (45). How-ever, in enterohemorrhagic E. coli O157:H7, the increase ingenome size (19.2%) is associated with increased virulencewhen compared with standard laboratory strain K12 (36). Vari-ations in biofilm formation in different isolates lead us tospeculate whether there are some biofilm-specific genomic is-lands in CF isolates. However, the PAO1 genome can beviewed as a mutant. It could be possible that the ancestor of

PAO1 had slowly deleted genes that were not required forspecific environments, thus maintaining only a minimal num-ber of genes (backbone) for P. aeruginosa survival (47). Thisview contradicts the concept of novel gene acquisition by hor-izontal gene transfer. However, this view can explain why manystrains of P. aeruginosa, including those studied here, havegenes not present in PAO1 (25, 47). The predecessor of PAO1could have previously possessed many of the genes foundthroughout P. aeruginosa isolates, as opposed to the introduc-tion of many novel genes into PAO1 giving rise to these iso-lates. Together, what may actually exist could be a combinationof both mechanisms.

Genomic comparison between clinical and environmental iso-lates can yield useful information on the inherent virulence prop-erties. While shotgun sequencing of multiple bacterial genomes isan ideal choice, the costs and time associated are often high. Analternative is the use of microarray gene expression profiles inconjunction with subtractive genomic hybridization. However,this high-throughput method relies on the correct annotation ofthe existing genomes, which may carry misannotated genes. Tocomplement these methods, we developed a simple method thatallows for a quick comparison between two genomes using RAPDin combination with PAO1 DNA subtractive hybridization (Fig.6). This subtractive method has two key features: (i) RAPD-basedamplification of an unknown genome minus (ii) the existingPAO1 genome. Three novel genetic loci were identified in twoclinical isolates (Table 3). The mvpAT locus is located on a largeShigella virulence plasmid and is involved in plasmid retention(42). However, the order is reversed (mvpTA) in a CF isolate(CF005). In addition, we isolated a topA homolog that encodes atopoisomerase I variant. Two topA genes were found in CF005:one with 100% homology to PA3011 in PAO1 and the other topAvariant that failed to be hybridized by PAO1 genomic DNA.Furthermore, this approach led us to identify a variant of PA2402that has been recently hypothesized to be involved in synthesis ofpyoverdine (47). One limitation of this method is the inability topinpoint the exact location of these novel genes within the PAO1backbone; however, this can be resolved by performing inversePCR. Altogether, RAPD coupled with subtractive hybridizationis an effective way to examine the genomic difference betweenisolates. This method also allows identification of smaller DNAsegments absent in the PAO1 genome. Variations of this ap-proach with random degenerate primers coupled with variousamplification conditions are expected to generate more novelDNA sequences.

In addition to the improved understanding of the phenotypicdiversity with relation to biofilm formation in P. aeruginosa,this report suggests that the extensive genomic diversity in thisspecies may become an unexplored cause for the variation inmorbidity and mortality in CF. Furthermore, it demonstratesthe presence of multiple mechanisms of biofilm formation,which is aided by novel biofilm adhesins that could becomeantibiofilm therapeutic targets.

ACKNOWLEDGMENTS

This work was supported by a student traineeship from the CysticFibrosis Foundation (HEAD01H0) and by Public Health Service grantDK58128 from the National Institutes of Health.



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REFERENCES

1. Alm, R. A., and J. S. Mattick. 1995. Identification of a gene, pilV, requiredfor type 4 fimbrial biogenesis in Pseudomonas aeruginosa, whose productpossesses a pre-pilin-like leader sequence. Mol. Microbiol. 16:485–496.

2. Arora, S. K., M. Bangera, S. Lory, and R. Ramphal. 2001. A genomic islandin Pseudomonas aeruginosa carries the determinants of flagellin glycosyla-tion. Proc. Natl. Acad. Sci. USA 98:9342–9347.

3. Barth, A. L., and T. L. Pitt. 1995. Auxotrophic variants of Pseudomonasaeruginosa are selected from prototrophic wild-type strains in respiratoryinfections in patients with cystic fibrosis. J. Clin. Microbiol. 33:37–40.

4. Barth, A. L., and T. L. Pitt. 1996. The high amino-acid content of sputumfrom cystic fibrosis patients promotes growth of auxotrophic Pseudomonasaeruginosa. J. Med. Microbiol. 45:110–119.

5. Boucher, J. C., H. Yu, M. H. Mudd, and V. Deretic. 1997. Mucoid Pseudo-monas aeruginosa in cystic fibrosis: characterization of muc mutations inclinical isolates and analysis of clearance in a mouse model of respiratoryinfection. Infect. Immun. 65:3838–3846.

6. Burns, J. L., R. L. Gibson, S. McNamara, D. Yim, J. Emerson, M. Rosenfeld,P. Hiatt, K. McCoy, R. Castile, A. L. Smith, and B. W. Ramsey. 2001.Longitudinal assessment of Pseudomonas aeruginosa in young children withcystic fibrosis. J. Infect. Dis. 183:444–452.

7. Chiang, P., and L. L. Burrows. 2003. Biofilm formation by hyperpiliatedmutants of Pseudomonas aeruginosa. J. Bacteriol. 185:2374–2378.

8. Coleman, F. T., S. Mueschenborn, G. Meluleni, C. Ray, V. J. Carey, S. O.Vargas, C. L. Cannon, F. M. Ausubel, and G. B. Pier. 2003. Hypersusceptibilityof cystic fibrosis mice to chronic Pseudomonas aeruginosa oropharyngeal colo-nization and lung infection. Proc. Natl. Acad. Sci. USA 100:1949–1954.

9. Costerton, J. W. 2001. Cystic fibrosis pathogenesis and the role of biofilms inpersistent infection. Trends Microbiol. 9:50–52.

10. Costerton, J. W., P. S. Stewart, and E. P. Greenberg. 1999. Bacterial biofilms:a common cause of persistent infections. Science 284:1318–1322.

11. Deretic, V., J. R. Govan, W. M. Konyecsni, and D. W. Martin. 1990. MucoidPseudomonas aeruginosa in cystic fibrosis: mutations in the muc loci affecttranscription of the algR and algD genes in response to environmentalstimuli. Mol. Microbiol. 4:189–196.

12. Deziel, E., Y. Comeau, and R. Villemur. 2001. Initiation of biofilm formationby Pseudomonas aeruginosa 57RP correlates with emergence of hyperpiliatedand highly adherent phenotypic variants deficient in swimming, swarming,and twitching motilities. J. Bacteriol. 183:1195–1204.

13. Donlan, R. M., and J. W. Costerton. 2002. Biofilms: survival mechanisms ofclinically relevant microorganisms. Clin. Microbiol. Rev. 15:167–193.

14. Drenkard, E., and F. M. Ausubel. 2002. Pseudomonas biofilm formation andantibiotic resistance are linked to phenotypic variation. Nature 416:740–743.

15. Ernst, R. K., E. C. Yi, L. Guo, K. B. Lim, J. L. Burns, M. Hackett, and S. I.Miller. 1999. Specific lipopolysaccharide found in cystic fibrosis airwayPseudomonas aeruginosa. Science 286:1561–1565.

16. Ghigo, J. M. 2001. Natural conjugative plasmids induce bacterial biofilmdevelopment. Nature 412:442–445.

17. Govan, J. R., and V. Deretic. 1996. Microbial pathogenesis in cystic fibrosis:mucoid Pseudomonas aeruginosa and Burkholderia cepacia. Microbiol. Rev.60:539–574.

18. Hentzer, M., G. M. Teitzel, G. J. Balzer, A. Heydorn, S. Molin, M. Givskov,and M. R. Parsek. 2001. Alginate overproduction affects Pseudomonasaeruginosa biofilm structure and function. J. Bacteriol. 183:5395–5401.

19. Hoiby, N., A. Fomsgaard, E. T. Jensen, H. K. Johansen, G. Kronborg, S. S.Pedersen, T. Pressler, and A. Kharazmi. 1995. The immune response to bacte-rial biofilms, p. 233–250. In H. M. Lappin-Scott and J. W. Costerton (ed.),Microbial biofilms. Cambridge University Press, Cambridge, United Kingdom.

20. Holloway, B. W. 1955. Genetic recombination in P. aeruginosa. J. Gen.Microbiol. 13:572–581.

21. Jensen, E. T., A. Kharazmi, N. Hoiby, and J. W. Costerton. 1992. Somebacterial parameters influencing the neutrophil oxidative burst response toPseudomonas aeruginosa biofilms. APMIS 100:727–733.

22. Kiewitz, C., and B. Tummler. 2000. Sequence diversity of Pseudomonasaeruginosa: impact on population structure and genome evolution. J. Bacte-riol. 182:3125–3135.

23. Knutson, C. A., and A. Jeanes. 1968. A new modification of the carbazolereaction: application to heteropolysaccharides. Anal. Biochem. 24:470–481.

24. Koch, A. L. 1994. Growth measurement, p. 248–277. In P. Gerhardt (ed.),General and molecular bacteriology. ASM Press, Washington, D.C.

25. Larbig, K. D., A. Christmann, A. Johann, J. Klockgether, T. Hartsch, R.Merkl, L. Wiehlmann, H. J. Fritz, and B. Tummler. 2002. Gene islandsintegrated into tRNAGly genes confer genome diversity on a Pseudomonasaeruginosa clone. J. Bacteriol. 184:6665–6680.

26. Liang, X., X. Q. Pham, M. V. Olson, and S. Lory. 2001. Identification of agenomic island present in the majority of pathogenic isolates of Pseudomo-nas aeruginosa. J. Bacteriol. 183:843–853.

27. Lyczak, J. B., C. L. Cannon, and G. B. Pier. 2002. Lung infections associatedwith cystic fibrosis. Clin. Microbiol. Rev. 15:194–222.

28. Mahenthiralingam, E., M. E. Campbell, J. Foster, J. S. Lam, and D. P.Speert. 1996. Random amplified polymorphic DNA typing of Pseudomonas

aeruginosa isolates recovered from patients with cystic fibrosis. J. Clin. Mi-crobiol. 34:1129–1135.

29. Nivens, D. E., D. E. Ohman, J. Williams, and M. J. Franklin. 2001. Role ofalginate and its O acetylation in formation of Pseudomonas aeruginosa mi-crocolonies and biofilms. J. Bacteriol. 183:1047–1057.

30. Oliver, A., R. Canton, P. Campo, F. Baquero, and J. Blazquez. 2000. Highfrequency of hypermutable Pseudomonas aeruginosa in cystic fibrosis lunginfection. Science 288:1251–1254.

31. O’Toole, G., H. B. Kaplan, and R. Kolter. 2000. Biofilm formation as mi-crobial development. Annu. Rev. Microbiol. 54:49–79.

32. O’Toole, G. A., and R. Kolter. 1998. Flagellar and twitching motility arenecessary for Pseudomonas aeruginosa biofilm development. Mol. Microbiol.30:295–304.

33. O’Toole, G. A., and R. Kolter. 1998. Initiation of biofilm formation inPseudomonas fluorescens WCS365 proceeds via multiple, convergent signal-ling pathways: a genetic analysis. Mol. Microbiol. 28:449–461.

34. O’Toole, G. A., L. A. Pratt, P. I. Watnick, D. K. Newman, V. B. Weaver, andR. Kolter. 1999. Genetic approaches to study of biofilms. Methods Enzymol.310:91–109.

35. Parkins, M. D., H. Ceri, and D. G. Storey. 2001. Pseudomonas aeruginosaGacA, a factor in multihost virulence, is also essential for biofilm formation.Mol. Microbiol. 40:1215–1226.

36. Perna, N. T., G. Plunkett III, V. Burland, B. Mau, J. D. Glasner, D. J. Rose,G. F. Mayhew, P. S. Evans, J. Gregor, H. A. Kirkpatrick, G. Posfai, J.Hackett, S. Klink, A. Boutin, Y. Shao, L. Miller, E. J. Grotbeck, N. W. Davis,A. Lim, E. T. Dimalanta, K. D. Potamousis, J. Apodaca, T. S. Ananthara-man, J. Lin, G. Yen, D. C. Schwartz, R. A. Welch, and F. R. Blattner. 2001.Genome sequence of enterohaemorrhagic Escherichia coli O157:H7. Nature409:529–533.

37. Pratt, L. A., and R. Kolter. 1999. Genetic analyses of bacterial biofilmformation. Curr. Opin. Microbiol. 2:598–603.

38. Rahme, L. G., E. J. Stevens, S. F. Wolfort, J. Shao, R. G. Tompkins, andF. M. Ausubel. 1995. Common virulence factors for bacterial pathogenicity inplants and animals. Science 268:1899–1902.

39. Romling, U., B. Fiedler, J. Bosshammer, D. Grothues, J. Greipel, H. von derHardt, and B. Tummler. 1994. Epidemiology of chronic Pseudomonas aerugi-nosa infections in cystic fibrosis. J. Infect. Dis. 170:1616–1621.

40. Romling, U., J. Greipel, and B. Tummler. 1995. Gradient of genomic diversityin the Pseudomonas aeruginosa chromosome. Mol. Microbiol. 17:323–332.

41. Romling, U., J. Wingender, H. Muller, and B. Tummler. 1994. A majorPseudomonas aeruginosa clone common to patients and aquatic habitats.Appl. Environ. Microbiol. 60:1734–1738.

42. Sayeed, S., L. Reaves, L. Radnedge, and S. Austin. 2000. The stability regionof the large virulence plasmid of Shigella flexneri encodes an efficient post-segregational killing system. J. Bacteriol. 182:2416–2421.

43. Schmidt, K. D., B. Tummler, and U. Romling. 1996. Comparative genomemapping of Pseudomonas aeruginosa PAO with P. aeruginosa C, which be-longs to a major clone in cystic fibrosis patients and aquatic habitats. J.Bacteriol. 178:85–93.

44. Schroeder, T. H., T. Zaidi, and G. B. Pier. 2001. Lack of adherence of clinicalisolates of Pseudomonas aeruginosa to asialo-GM1 on epithelial cells. Infect.Immun. 69:719–729.

45. Silo-Suh, L., S. J. Suh, P. A. Sokol, and D. E. Ohman. 2002. A simple alfalfaseedling infection model for Pseudomonas aeruginosa strains associated withcystic fibrosis shows AlgT (sigma-22) and RhlR contribute to pathogenesis.Proc. Natl. Acad. Sci. USA 99:15699–15704.

46. Singh, P. K., A. L. Schaefer, M. R. Parsek, T. O. Moninger, M. J. Welsh, andE. P. Greenberg. 2000. Quorum-sensing signals indicate that cystic fibrosislungs are infected with bacterial biofilms. Nature 407:762–764.

47. Spencer, D. H., A. Kas, E. E. Smith, C. K. Raymond, E. H. Sims, M.Hastings, J. L. Burns, R. Kaul, and M. V. Olson. 2003. Whole-genomesequence variation among multiple isolates of Pseudomonas aeruginosa. J.Bacteriol. 185:1316–1325.

48. Stewart, P. S., A. K. Camper, S. D. Handran, C. Huang, and M. Warnecke.1997. Spatial distribution and coexistence of Klebsiella pneumoniae andPseudomonas aeruginosa in biofilms. Microb. Ecol. 33:2–10.

49. Stickler, D. 1999. Biofilms. Curr. Opin. Microbiol. 2:270–275.50. Stover, C. K., X. Q. Pham, A. L. Erwin, S. D. Mizoguchi, P. Warrener, M. J.

Hickey, F. S. Brinkman, W. O. Hufnagle, D. J. Kowalik, M. Lagrou, R. L.Garber, L. Goltry, E. Tolentino, S. Westbrock-Wadman, Y. Yuan, L. L.Brody, S. N. Coulter, K. R. Folger, A. Kas, K. Larbig, R. Lim, K. Smith, D.Spencer, G. K. Wong, Z. Wu, and I. T. Paulsen. 2000. Complete genomesequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Na-ture 406:959–964.

51. Terry, J. M., S. E. Pina, and S. J. Mattingly. 1991. Environmental conditionswhich influence mucoid conversion Pseudomonas aeruginosa PAO1. Infect.Immun. 59:471–477.

52. Vallet, I., J. W. Olson, S. Lory, A. Lazdunski, and A. Filloux. 2001. Thechaperone/usher pathways of Pseudomonas aeruginosa: identification of fim-brial gene clusters (cup) and their involvement in biofilm formation. Proc.Natl. Acad. Sci. USA 98:6911–6916.

53. Vidal, O., R. Longin, C. Prigent-Combaret, C. Dorel, M. Hooreman, and P.



http://iai.asm.org/

Dow

nloaded from

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Lejeune. 1998. Isolation of an Escherichia coli K-12 mutant strain able toform biofilms on inert surfaces: involvement of a new ompR allele thatincreases curli expression. J. Bacteriol. 180:2442–2449.

54. Wozniak, D. J., T. J. Wyckoff, M. Starkey, R. Keyser, P. Azadi, G. A. O’Toole,and M. R. Parsek. 2003. Alginate is not a significant component of theextracellular polysaccharide matrix of PA14 and PAO1 Pseudomonas aerugi-nosa biofilms. Proc. Natl. Acad. Sci. USA 100:7907–7912.

55. Yu, H., J. C. Boucher, N. S. Hibler, and V. Deretic. 1996. Virulence prop-erties of Pseudomonas aeruginosa lacking the extreme-stress sigma factorAlgU (�E). Infect. Immun. 64:2774–2781.

56. Yu, H., M. Hanes, C. E. Chrisp, J. C. Boucher, and V. Deretic. 1998.Microbial pathogenesis in cystic fibrosis: pulmonary clearance of mucoidPseudomonas aeruginosa and inflammation in a mouse model of repeatedrespiratory challenge. Infect. Immun. 66:280–288.

57. Yu, H., and N. E. Head. 2002. Persistent infections and immunity in cysticfibrosis. Front. Biosci. 7:D442–D457.

58. Yu, H., M. J. Schurr, and V. Deretic. 1995. Functional equivalence of Esch-erichia coli �E and Pseudomonas aeruginosa AlgU: E. coli rpoE restoresmucoidy and reduces sensitivity to reactive oxygen intermediates in algUmutants of P. aeruginosa. J. Bacteriol. 177:3259–3268.

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