INFECTION AND IMMUNITY, Aug. 2011, p. 3087–3095 Vol. 79, No. 80019-9567/11/$12.00 doi:10.1128/IAI.00057-11Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Direct Evaluation of Pseudomonas aeruginosa Biofilm Mediatorsin a Chronic Infection Model�†

Matthew S. Byrd,1 Bing Pang,1 Wenzhou Hong,1‡ Elizabeth A. Waligora,1 Richard A. Juneau,1Chelsie E. Armbruster,1 Kristen E. D. Weimer,1 Kyle Murrah,1 Ethan E. Mann,2,3

Haiping Lu,1 April Sprinkle,1§ Matthew R. Parsek,4 Nancy D. Kock,5,6

Daniel J. Wozniak,2,3,7 and W. Edward Swords1*Department of Microbiology and Immunology,1 Department of Pathology,5 and Department of Pathology/Comparative Medicine,6

Wake Forest University Health Sciences, Medical Center Blvd., Winston-Salem, North Carolina 27157; Department ofMicrobiology, University of Washington, Seattle, Washington 981954; and Center for Microbial Interface Biology,2

Infectious Disease,3 and Microbiology,7 The Ohio State University, Columbus, Ohio 43210

Received 15 January 2011/Returned for modification 26 February 2011/Accepted 26 May 2011

Biofilms contribute to Pseudomonas aeruginosa persistence in a variety of diseases, including cystic fibrosis, burnwounds, and chronic suppurative otitis media. However, few studies have directly addressed P. aeruginosa biofilmsin vivo. We used a chinchilla model of otitis media, which has previously been used to study persistent Streptococcuspneumoniae and Haemophilus influenzae infections, to show that structures formed in vivo are biofilms of bacterialand host origin within a matrix that includes Psl, a P. aeruginosa biofilm polysaccharide. We evaluated three biofilmand/or virulence mediators of P. aeruginosa known to affect biofilm formation in vitro and pathogenesis in vivo—bis-(3�,5�)-cyclic dimeric GMP (c-di-GMP), flagella, and quorum sensing—in a chinchilla model. We show thatc-di-GMP overproduction has a positive impact on bacterial persistence, while quorum sensing increases virulence.We found no difference in persistence attributed to flagella. We conclude from these studies that a chinchilla otitismedia model provides a means to evaluate pathogenic mediators of P. aeruginosa and that in vitro phenotypes shouldbe examined in multiple infection systems to fully understand their role in disease.

The formation of biofilms facilitates chronic bacterial infec-tions and reduces the efficacy of antimicrobial therapy (20, 39).The Gram-negative pathogen Pseudomonas aeruginosa is amodel organism for biofilm studies and causes both acute andchronic infections by exploiting deficiencies in host immunity.P. aeruginosa is thought to exist as a biofilm during infectionsof the cystic fibrosis (CF) airway (5, 48), in acute burn wounds(45), and in chronic suppurative otitis media (13). Biofilmformation in P. aeruginosa is regulated by a complex network ofsignals that includes small molecules, two-component systems,small RNAs, and nutritional cues (27). As a result of thesesignals, a matrix that consists predominately of polysaccharidesand extracellular DNA is formed (1, 17, 25, 33, 35, 52). WhileP. aeruginosa biofilms have been studied extensively in vitro,there have been few studies characterizing biofilm formation invivo (31, 45, 48, 53).

The chinchilla model has been widely used to study Haemo-philus influenzae and Streptococcus pneumoniae otitis mediainfections, establishing that these pathogens form biofilms invivo (3, 15, 19, 24, 26, 42). Addressing biofilms in the context of

an intact immune system and the complex environmental andstructural features present in a mammalian host is necessary todetermine if in vitro phenotypes are relevant to clinical infec-tions. We used a chinchilla otitis media model to characterizeP. aeruginosa biofilm formation and then focused on threesystems involved in pathogenesis and/or biofilm formation—bis-(3�,5�)-cyclic dimeric GMP (c-di-GMP), flagella, and quo-rum sensing—in order to understand their contribution to P.aeruginosa infection.

The intracellular second messenger c-di-GMP positively reg-ulates aggregation and biofilm formation in P. aeruginosa (22,30, 51) and mediates a transition from motile to sessile modesof growth by inducing production of matrix components whilerepressing flagellar motility (6, 22, 30, 47, 51). Biofilms pro-duced by c-di-GMP-overproducing strains are structurallymore heterogeneous, aggregate more strongly, and havegreater resistance to antibiotics than those produced by paren-tal strains (30, 51).

Flagella are critical for swimming and swarming motility,and the highly conserved structural subunit flagellin elicits apotent inflammatory response when it is detected by the innateimmune receptor Toll-like receptor 5 (TLR5) (21). Biofilmformation in vitro relies on flagellum-mediated motility andsurface attachment that precedes elaboration of a matrix (38,44). The presence of flagella results in a more severe infectionwith greater morbidity and mortality in acute models of P.aeruginosa infection but has not been studied in biofilm infec-tions outside airway-associated disease (14, 16, 36).

The P. aeruginosa quorum-sensing systems include proteinsthat, in response to high cell density, synthesize and detect theacyl homoserine lactones (AHLs) N-3-oxo-dodecanoyl-homo-

* Corresponding author. Mailing address: Department of Microbi-ology and Immunology, Wake Forest University Health Sciences,Medical Center Blvd., Winston-Salem, NC 27157. Phone: (336) 713-5049. Fax: (336) 716-9928. E-mail: wswords@wfubmc.edu.

† Supplemental material for this article may be found at http://iai.asm.org/.

‡ Present address: Department of Otolaryngology, Medical Collegeof Wisconsin, Milwaukee, WI 53226.

§ Present address: Department of Neurosurgery, Wake Forest Uni-versity Health Sciences, Medical Center Blvd., Winston-Salem, NC27157.

� Published ahead of print on 6 June 2011.

3087

serine lactone (3OC12-HSL) and N-butanoyl-homoserine lac-tone (C4-HSL), products of the lasRI and rhlRI systems, re-spectively, as well as the Pseudomonas quinolone signal (PQS)(40, 50). The lasRI and rhlRI systems, either singly or redun-dantly, control a large regulon of virulence genes (50). Underquorum-sensing control are exotoxins, proteases, phenazinepigments, and other virulence factors associated with patho-genesis in rat and mouse models (7, 10, 18, 32, 37, 50). Theparticipation of quorum sensing in biofilm formation is stilldebated, but it appears to be conditional on nutritional andenvironmental factors (15, 28, 29, 43, 46).

In this study, we use a chinchilla otitis media model to evaluateP. aeruginosa mutants that have in vitro biofilm phenotypes ordefects in production of virulence factors. Virulence versus per-sistence will be discussed for each of the experiments presented.A more virulent strain of P. aeruginosa would result in a time tomorbidity similar to or shorter than that for the parental PAO1strain, while a strain demonstrating persistence would cause alonger duration of infection without morbidity than PAO1. Inboth virulent and persistent infections, bacteria would be detect-able; otherwise, survival with no detectable bacteria is consideredclearance. We show that P. aeruginosa establishes a persistentinfection in the chinchilla middle ear and that the material pres-ent in the middle ear space, or bulla, contains cells and matricesof both host and bacterial origin. In this model, a c-di-GMPoverproduction mutant has increased persistence, while the pres-ence of flagella does not affect persistence or survival. Finally, weshow that quorum-sensing regulators promote increased viru-lence and decreased survival and thereby present a promisingtarget for reducing host damage in P. aeruginosa infections.

MATERIALS AND METHODS

Bacterial strains and culture conditions. P. aeruginosa strains were routinelygrown in Luria-Bertani (LB) broth (10 g/liter tryptone, 5 g/liter yeast extract)without NaCl and on 1.5% LB agar plates without NaCl (LANS). For chinchillaexperiments, bulla and brain homogenates were plated on 1.5% Pseudomonasisolation agar (Difco). Strains used in this study are shown in Table S1 in thesupplemental material and have been described previously. In each experimentcomparing PAO1 and mutant P. aeruginosa strains, the parental PAO1 strainfrom which the mutant(s) was derived was used as the wild-type strain.

In vitro biofilm assays. Strains were evaluated in vitro by a 30-min polyvinylchloride (PVC) microtiter dish attachment assay, as described previously (9), andby a procedure modified from the MBEC P&G assay (Innovotech). In themodified MBEC assay, 100 �l of mid-log-phase (optical density at 600 nm, 0.5)cultures was added to wells of a microtiter plate; the lid, containing 96 polysty-rene pegs that fit into the wells of the microtiter plate, was placed on the plate;and the plate was incubated at 37°C for 3 h. Pegs were rinsed twice in phosphate-buffered saline (PBS) with agitation to remove loosely attached cells, the lid wasplaced on another 96-well microtiter plate containing 100 �l of 0.1% crystalviolet per well, and the plate was incubated at room temperature for 15 min. Pegswere rinsed three times in PBS with agitation and allowed to air dry, and then thelid was placed on a final microtiter plate containing 200 �l of 95% ethanol perwell for 30 min at room temperature. One hundred microliters of solubilizedcrystal violet from each well was transferred to a flat-bottom microtiter plate, andthe absorbance was read at 540 nm.

Chinchilla infection experiments. A chinchilla otitis media model was used toevaluate persistence of P. aeruginosa in vivo. Healthy adult chinchillas (Chinchillalanigera; body weight, 400 to 500 g) from Rauscher’s Chinchilla Ranch (Larue,OH) were allowed to acclimate to the vivarium for 7 to 10 days prior to infection.Animals were assessed and found to be healthy before any studies were under-taken. Chinchillas were anesthetized by isoflurane inhalation, and both left andright bullae were inoculated via transbullar injection of approximately 100 CFUof P. aeruginosa into the middle ear, in accordance with protocols approved bythe Animal Care and Use Committee of Wake Forest University Health Sciencesand as described previously (3, 24). All inocula were confirmed by plating a single

dose (100 CFU in 0.1 ml sterile PBS) on LANS. For survival experiments, at least4 to 6 animals per strain were used; 3 animals per strain per time point were usedin those experiments with defined harvest times. Animals were euthanized eitherat defined time points (1 day, 2 days, 3 days, or 7 to 8 days) or, for survivalexperiments, whenever the animals began to show signs of distress requiringeuthanasia. In these experiments, distress requiring euthanasia includes severehead tilt, trouble righting, and/or severe lethargy coupled with loss of appetite.Otoscopies were performed regularly to monitor redness, drainage, and tym-panic membrane bulging. Otoscopies showing abundant drainage correlated wellwith distress, typically requiring euthanasia within 12 to 24 h. Left bullae wereaseptically removed and homogenized. In some experiments, right bullae werehomogenized, while in others, the right bullae were fixed intact or had biofilmmaterial removed and stained. In some experiments brain tissue was asepticallyremoved prior to opening of the bullae and homogenized or fixed in 2% para-formaldehyde for histology. Bullae and brain were homogenized in 10 ml sterilePBS for CFU determination by plate count (power setting 4 for bullae, powersetting 3 for brain; Power Gen 1000; Fisher Scientific). Effusion was removedfrom the bullae and combined with a 1-ml PBS wash for determination of thenumbers of CFU of bacteria not associated with biofilm material.

Microscopic examination of bullae. For scanning electron microscopy (SEM),bullae were fixed whole in 2.5% glutaraldehyde-PBS, trimmed of excess bone,and prepared as described previously (42). Samples were visualized with a PhilipsSEM-515 scanning electron microscope. For histopathology, bullae were fixedintact in 4% formalin for at least 24 h and then decalcified for 6 h using aDecalcifier II reagent (Surgipath Medical Industries). The tissues were sagitallysectioned, embedded in paraffin, processed routinely for histology, cut at 6 �m,and stained with hematoxylin-eosin (H&E) according to standard techniques (3).Slides were examined by routine light microscopy using a conventional NikonEclipse 50i microscope in a blinded fashion by a board-certified veterinarypathologist.

Staining and visualization of bulla material. Material removed from bullaewas fixed in 2% paraformaldehyde overnight for histopathology or immunohis-tochemistry staining or was immediately live/dead stained. Viability of unfixedmaterial was evaluated using BacLight LIVE/DEAD staining (MolecularProbes). Material was stained with SYTO 9 and propidium iodide (PI) in PBS for15 min and then washed in PBS (42). Material embedded in OCT (SakuraFinetek) at �20°C was cut into 5-�m sections and stained with rabbit antipseu-domonal or rabbit anti-Psl antiserum (each 1:5,000) (9) with a fluorescein iso-thiocyanate (FITC)-conjugated donkey anti-rabbit IgG secondary antibody (1:50). Some sections were stained with H&E and evaluated by light microscopy asdescribed above. In sections stained with anti-Psl, PI was also used to stain DNA.Fluorescence was visualized using a Zeiss LSM510 confocal microscope.

Video otoscopy. Animals were monitored for redness and/or effusion in theexternal ear using a Digital VetScope system with VetDock (version 2.1) soft-ware (MedRx). Each ear was evaluated over time and scored positively forhaving redness or drainage at any time during the experiment.

Statistical analysis and image processing. Statistical analyses were performedusing GraphPad Prism (version 5) software. Survival curves were compared bythe log-rank (Mantel-Cox) test, and video otoscopy scores were evaluated byFisher’s exact test. Images were processed using Adobe Systems Photoshop CS(version 8.0) and assembled using Photoshop, Prism, and/or ImageJ (version1.42q) (National Institutes of Health) software.

RESULTS

Pseudomonas aeruginosa readily establishes infection in thechinchilla middle ear. Initial experiments evaluated the abilityof the common laboratory strain PAO1 to cause otitis media inchinchillas and established the optimal inoculum for persis-tence. To evaluate gross infection, the thin layer of bone com-prising the superior bullous surface was removed and the bullawas imaged from above. Thick, opaque exudate is visible (ar-rows) in the bullae of chinchillas infected by transbullar inoc-ulation (3, 24) with 20 or 200 CFU PAO1 (Fig. 1A to F) at both3 days postinfection (d.p.i.) (Fig. 1A to D) and 8 d.p.i. (Fig. 1Eand F). This material is similar to what is observed in the bullaeof chinchillas infected with nontypeable Haemophilus influen-zae (NTHi) and Streptococcus pneumoniae, the two most com-mon pathogens causing otitis media in humans (24, 41, 42).

3088 BYRD ET AL. INFECT. IMMUN.

Clear effusions (Fig. 1A and B) are visible in a majority ofinfected animals, and in fewer cases, erythema is visible (Fig.1C and D). In contrast, middle ears infected with 0.1 ml sterilePBS remained clear of obstructing material (Fig. 1G and H).Infection with 20 CFU increased persistence slightly over thatseen with 200 CFU (i.e., the onset of morbidity in animals thatdid not clear the infection was delayed compared to the time toonset of morbidity in animals receiving the higher dose), but at8 d.p.i. with 2/4 animals surviving, 1 animal did not have de-tectable bacteria. Further experiments used an inoculum of100 CFU, as this reliably results in establishment of infectionwhile reducing the morbidity seen with higher inocula. In thisand subsequent experiments, the bacterial load commonly ex-ceeded 109 CFU per bulla at as early as 2 d.p.i., confirming thatP. aeruginosa readily exploits the middle ear environment anddemonstrating the utility of this model in evaluating the estab-lishment of infection.

In this model, less than 20% of PAO1-infected animals rou-tinely survive to 10 days. Survival is defined as the time tomorbidity requiring euthanasia. To investigate the cause ofmorbidity, three chinchillas were infected with PAO1, andplate counts of blood, bullae, and brain were performed at 3 to4 d.p.i. (see Table S2 in the supplemental material). No ani-

mals had detectable bacteria in the blood in this or subsequentexperiments in which blood samples were evaluated. In the twoanimals with detectable bacteria in the bullae, there was ap-proximately 10-fold less bacteria in the left hemisphere of thebrain, which was aseptically removed prior to opening of thebullae. In the one animal that did not have bacteria in the leftbulla, there were likewise no bacteria detected in the brain.This suggested that although none of the animals were mori-bund, P. aeruginosa in the brain could account for the eventualonset of otitis media signs and rapid progression of morbidityoften observed in these experiments.

P. aeruginosa-infected chinchilla middle ears contain bothhost and bacterial components. To determine the compositionof the middle ear material, whole bullae were excised, fixed,and trimmed to expose the inner surface and then prepared forSEM (9). At 4 d.p.i. with PAO1, the middle ear epitheliumclearly shows the presence of both P. aeruginosa and host cells,the latter having morphology consistent with polymorphonu-clear cells (PMNs) (Fig. 2A, top left). At higher magnification,individual P. aeruginosa cells can be seen enmeshed in a matrix(Fig. 2A, top right and bottom), recalling in vitro biofilms, inwhich, at 48 h under static conditions, PAO1 cells are partiallyobscured by a matrix of bacterial origin (see Fig. S1A in thesupplemental material). In PBS-infected animals, no bacterialor immune cells are visible on an intact epithelial surface (seeFig. S1B and C in the supplemental material). Other bullaewere sectioned and stained to observe the material in situ. At3 d.p.i. with PAO1, there was evidence of PMN infiltration intothe middle ear, with a dense network of fibrin surrounding theimmune cells (Fig. 2B). The presence of PMNs associated witha fibrous mesh is similar to that seen in NTHi and S. pneu-moniae otitis media (3, 42). The fibrin forms layers within thebulla, reflecting waves of infiltration likely attempting to isolatethe infection. Material removed from the bulla showed aggre-gates of bacteria, some associated with a weblike structure thatstained positively for nucleic acid (Fig. 2C). Immediately fol-lowing removal from the bulla, some material was stainedusing the live/dead reagents. Confocal images showed livingand dead cells, identified both as host and as bacterial cells bysize and morphology (Fig. 2D). Staining of cryosections withantipseudomonal antiserum revealed both single cells anddense clusters of bacteria distributed throughout an immuno-reactive matrix (Fig. 2E) (9). A Psl-specific antiserum, whichstains both cell- and matrix-associated biofilm polysaccharide(9), showed Psl colocalization with cells stained with the non-viable indicator PI, indicating that Psl remains associated withdead cells or cells with compromised membranes (Fig. 2F andG). Diffuse Psl and DNA staining between cells agrees with thefindings of in vitro analyses of P. aeruginosa biofilms, althoughthe DNA could be of either bacterial or host origin (Fig. 2F)(1, 33, 52). At high magnification, Psl was detected surround-ing individual cells, supporting a model of surface-associatedPsl mediating cell-cell and cell-surface interactions during bio-film formation (Fig. 2G) (33). From this evidence, we con-cluded that the bulla material contains living and dead hostand bacterial cells in a matrix of host-derived fibrin, P. aerugi-nosa-specific material, including Psl, and extracellular DNA ofundetermined origin.

MJK8, a c-di-GMP overproduction mutant, has increasedpersistence in vivo. The second messenger c-di-GMP positively

FIG. 1. Images of chinchilla bullae infected with P. aeruginosa PAO1(D. J. Wozniak). Images were taken from above following removal of acircle of bone from the superior bullous surface. Pairs of bullae (right andleft from the same animal) at 3 d.p.i. (A to D) and 8 d.p.i. (E and F) with200 CFU PAO1 (A and B) or 20 CFU PAO1 (C to F). Arrows denotematerial obstructing bullae. (G and H) PBS control at 3 d.p.i.

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regulates biofilm formation in P. aeruginosa (6, 22, 27, 30). Therugose small colony variant (RSCV) MJK8 is derived fromPAO1 but contains a mutation in the wspA gene resulting inhigh levels of intracellular c-di-GMP (51). Due to overproduc-

tion of c-di-GMP, MJK8 forms biofilms with significantlygreater biomass than PAO1 biofilms (Table 1). We thus hy-pothesized that MJK8 would persist longer than its parentalPAO1 strain in the chinchilla due to its ability to form biofilms

FIG. 2. Characterization of P. aeruginosa-induced chinchilla otitis media. (A) SEM images of bulla epithelial surface at 4 d.p.i. Host andbacterial cells are visible at low magnification (top left; magnification, �2,500; bars, 10 �m). Bacteria enmeshed in a matrix at mediummagnification (top right; magnification, �5,000; bar, 5 �m) and high magnification (bottom; magnification, �10,000; bar, 1 �m). (B) H&E-stainedbulla section at 3 d.p.i. showing PMNs and fibrin infiltration (left, magnification � �4 and bar � 100 �m; right, magnification � �20 and bar �50 �m). (C) H&E-stained cryosection of bulla material at 2 d.p.i. showing single cells, aggregates of P. aeruginosa, and a weblike structure of nucleicacid (left and right, magnification, �100; bar, 10 �m). (D) Live/dead staining of material removed from bulla with both live (green, left) and dead(red, middle) host and bacterial cells (right, merge; bar, 2 �m). (E) Immunofluorescent staining of bulla material at 3 d.p.i. with antipseudomonalantiserum at low magnification (left, FITC; middle, FITC-differential interference contrast merge; bar, 10 �m) and high magnification (right,FITC; bar, 5 �m) shows individual cells in an immunoreactive matrix. Immunofluorescent staining of bulla material at 3 d.p.i. with anti-Pslantiserum (left) and DNA staining with PI (middle) at low (F) and high (G) magnifications. Cell-associated and extracellular Psl and DNA arevisible in panel F (right, merge; bar, 5 �m), while in panel G, Psl is seen surrounding PI-stained regions (right, merge; bar, 1 �m).

TABLE 1. Biofilm phenotypes of strains in this study

Strain In vitro biofilm phenotype in literature (reference)

Mean � SEM absorbance at 540 nma

30 min attachment toPVC wells

3 h attachment topolystyrene pegs

PAO1 Forms robust biofilms on glass and PVC (9, 25, 33–35, 44) 0.180 � 0.011 0.116 � 0.007MJK8 Forms biofilms on PVC with 4-fold greater biomass than PAO1 (30, 51) 0.266 � 0.009 0.228 � 0.016MJK8-psl Forms biofilms on PVC with half the biomass of MJK8 (30, 51) 0.162 � 0.006 0.116 � 0.001MJK8-pel Forms biofilms on PVC with two-thirds the biomass of MJK8 (51) ND 0.179 � 0.015PAO-R1 Yet to be tested ND 0.122 � 0.001PDO111 Yet to be tested ND 0.104 � 0.005PAO-JP3 Yet to be tested ND 0.093 � 0.005WFPA850 Defective attachment on A549 airway epithelial cells (8) 0.013 � 0.003 0.084 � 0.003

a Mean � standard error of the mean absorbance values read at 540 nm following washing, staining, and solubilization of crystal violet with 95% ethanol. ND, not done.

3090 BYRD ET AL. INFECT. IMMUN.

that would potentially limit dissemination and immune systeminvolvement. Chinchillas were inoculated with PAO1 or MJK8and monitored for signs of otitis media for 9 days. Animalswere euthanized when signs became severe, and bullae wereremoved, homogenized, and plated to determine bacterialload. Chinchillas infected with MJK8 survived significantly lon-ger than those infected with PAO1 (Fig. 3; P � 0.013). Six outof 11 animals infected with MJK8 survived until the end of theexperiment without becoming moribund, and of these, 2 haddetectable P. aeruginosa, while 4 did not harbor detectablebacteria. In contrast, one-half of the chinchillas infected withPAO1 had to be euthanized by 4 d.p.i., and only 2 out of 10,with neither having detectable bacteria, survived through 9days. All MJK8-infected animals displaying signs of otitis me-dia at 7 to 9 d.p.i. (5/11) had detectable P. aeruginosa. Thepresence of MJK8 at later time points suggests that this strainis not simply cleared early in infection but, rather, persists withdelayed morbidity compared to PAO1. The bacterial load atthe time of killing was not different between the groups (ap-proximately 5 � 108 average CFU per bulla for both groups)and cannot account for the difference in survival (data notshown). Bulla material from MJK8-infected animals at 8 d.p.i.does not appear to be altered or reduced in size compared tomaterial from PAO1-infected animals (see Fig. S2A and B inthe supplemental material). SEM analysis of an MJK8-infectedbulla at 8 d.p.i. shows both bacterial and host components atthe epithelial surface, similar to those seen in PAO1 in Fig. 2A(see Fig. S2C and D in the supplemental material). These datasupport the argument that differences in MJK8 as a result ofc-di-GMP overproduction are sufficient to explain the survivaldata, the mechanism of which is currently under investigation.

Effects of P. aeruginosa polysaccharides Psl and Pel on per-sistence of MJK8. Expression of P. aeruginosa biofilm polysac-charides Psl and Pel (17, 25, 33, 35) is elevated in MJK8 due toincreased intracellular c-di-GMP (51). Biofilms produced invitro by MJK8 psl and pel mutants contain significantly lessbiomass than MJK8 (Table 1). To test whether loss of individ-ual polysaccharides and, thus, loss of biofilm-forming abilityaffects persistence of MJK8, chinchillas were infected withMJK8, MJK8 �psl, MJK8 �pel, or PAO1. While the mediansurvival of animals infected with MJK8 �psl was 4 d.p.i., com-pared to 6 d.p.i. for MJK8-infected animals, the survival curveswere not significantly different (P � 0.234; data not shown).The progression of infection with MJK8 �pel was identical to

that of MJK8, suggesting that Pel does not contribute to thepersistence of MJK8 in this model. All animals infected withPAO1, however, had severe otitis media signs and had to beeuthanized by 5 d.p.i., while half of the MJK8-infected animalssurvived to the end of the experiment (data not shown). SincePsl and Pel are associated with biofilm formation, we sought todetermine if the bulla material was influenced by loss of eitherpolysaccharide. Macroscopically visible material was present inat least half of the bullae in each group: PAO1, 2/3; MJK8, 2/4;MJK8 �psl, 4/4; MJK8 �pel, 3/4. The composition of the ma-terial was similar to that seen with PAO1 (Fig. 2B) and was notdifferent among the groups by H&E staining (Fig. 4A to F);however, these samples were not evaluated with Psl-specificantiserum. These data suggest that biofilm phenotypes dis-played in vitro are not necessarily recapitulated in vivo but maybe overshadowed by an immune response that includes hostcells, fibrin, and other host-derived factors. As in the previousexperiment, the bacterial loads at the time of killing were notsignificantly different among the groups (data not shown).

Since Psl has previously been shown to be important inPAO1 biofilm architecture (9, 17, 25, 30, 33–35), we sought todetermine if the loss of Psl results in differences in bacterialgrowth rate or spatial distribution within bullae early in infec-tion. To address this, chinchillas were infected with MJK8 orMJK8 �psl, and the bacterial load was determined in the sur-face-attached, or biofilm, material, in middle ear effusion, andin the brain at 2, 3, and 7 d.p.i. (Fig. 4G). A distinction betweeneffusion and the numbers of biofilm CFU was made because ofthe possible role of Psl in maintaining the association of bac-teria with biofilm components present within bullae. There wasno significant difference in bacterial load at any time pointtested in both biofilm and effusion samples, suggesting that Psldoes not affect the growth rate, nor does it influence the distri-bution of P. aeruginosa within the middle ear space (Fig. 4G).Bacterial load in the brain was variable at each time point and wasnot affected by expression of Psl (data not shown). The reducedsize of the MJK8 �psl group at 7 d.p.i. was due to two animalsbeing euthanized at 5 d.p.i., with 4/4 bullae containing copiousexudate and effusion. Signaling by c-di-GMP regulates an array ofbiofilm-associated responses beyond polysaccharide expression(6, 22, 27, 30); therefore, it is likely that future studies addressingthese c-di-GMP-regulated components will elucidate the mecha-nism of persistence of MJK8 and similar clinical isolates (30, 51).

Flagella do not alter the course of chinchilla otitis mediacaused by infection with P. aeruginosa. Flagella are requiredfor swimming and swarming motility and reversible surfaceattachment in vitro (38, 44), as well as proinflammatory signal-ing via interaction with the innate immune receptor TLR5(21). WFPA850, a fliC deletion mutant, adheres to humanA549 airway epithelial cells at less than 35% of the rate ofPAO1 (8) and displays a severe defect in biofilm initiation onPVC surfaces (Table 1). Interestingly, in the 3-h peg assay ona polystyrene surface, the fliC mutant displayed a less severebiofilm defect than on PVC, suggesting that the biofilm phe-notype may be confined to biofilm initiation or to certainsurfaces (Table 1). Flagellar mutants of P. aeruginosa haveattenuated virulence in acute mouse burn and pneumoniamodels (4, 14, 16, 36). We hypothesized that deletion of fliCwould result in increased survival of infected chinchillas with adecreased bacterial burden compared to that of PAO1, due to

FIG. 3. Effect of c-di-GMP on P. aeruginosa infection in chinchillaotitis media. Increased survival of chinchillas infected with c-di-GMP-overproducing MJK8 over PAO1 (M. R. Parsek) (MJK8, n � 11;PAO1, n � 10; P � 0.013, log-rank test).

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the reduced motility and induction of inflammation character-istic of this strain outweighing the potential loss of flagellum-dependent biofilm formation. Six chinchillas per group wereinfected with PAO1 or with the isogenic fliC mutant WFPA850and monitored for signs of otitis media over 10 days. Surpris-ingly, there was no difference in survival between chinchillasinfected with PAO1 and those that received WFPA850 (Fig.5A; P � 0.218). At 10 d.p.i., 2/6 PAO1-infected animals hadsurvived and did not have detectable bacteria in the bullae orbrain, whereas 0/6 WFPA850-infected animals survived, andall had detectable bacteria at the time of killing both in the

bullae and in the brain; however, these counts were not signif-icantly different from those of PAO1 (Fig. 5A and B). Bacteriain the brain are likely not contamination because, although notall brains were examined histologically, encephalitis was diag-nosed in one WFPA850-infected animal, making it reasonableto propose that clinical signs occurred as a result of infectionspreading from the middle ear to the brain. The presence ofbacteria in the brain, especially in animals infected with themotility-impaired WFPA850, indicates that P. aeruginosa canescape the site of infection without flagella, but the mechanismfor this is not understood. Video otoscopy of the auditory canalrevealed both redness (PAO1, 4/12 ears; WFPA850, 3/12) anddrainage (PAO1, 4/12; WFPA850, 6/12) during the course ofinfection for both groups. These results suggest that, unlike inmouse models of acute infection, in the chinchilla otitis mediamodel, deletion of fliC is not sufficient to attenuate PAO1. Theabsence of flagella may affect immune recognition by PMNsand other cell types expressing TLR5. It is also possible thatloss of flagellum-mediated attachment to host surfaces contrib-uted to dispersal to the brain, in effect compensating for themotility defect that we had predicted would limit dispersal.

Regulators of quorum sensing contribute to virulence of P.aeruginosa in chinchilla otitis media. The LasRI/RhlRI quo-rum-sensing systems in P. aeruginosa regulate expression ofnumerous virulence factors, including elastase, rhamnolipid,pyocyanin, exotoxin A, and other proteases (7, 50). Productionof biofilm matrix components, such as extracellular DNA, Pel,and Psl, is influenced by quorum sensing, yet the requirementfor quorum sensing in biofilm formation in vitro is still debated(1, 18, 43, 46). Nutritional factors mitigate the impact of quo-rum-sensing-regulated swarming motility, which directly affects

FIG. 4. Effects of Psl and Pel on P. aeruginosa infection in chin-chilla otitis media. (A to F) H&E-stained bulla sections from chinchil-las infected with MJK8 (A and B), MJK8 �psl (C and D), or MJK8�pel (E and F) at 4 to 6 d.p.i. Magnifications, �4 (A, C, and E) and�40 (B, D, and F). Black arrows, concentrations of fibrin; white ar-rows, concentrations of neutrophils. (G) Bacterial load (mean numberof CFU � standard error of the mean) in bullae (closed symbols) andeffusion (open symbols; number of CFU per ml of effusion plus PBSwash) of animals infected with MJK8 (circles) or MJK8 �psl (dia-monds) at 2, 3, and 7 d.p.i. Dotted line, lower limit of detection.

FIG. 5. Effect of flagella on P. aeruginosa infection in chinchillaotitis media. (A) Survival is not different for chinchillas infected withPAO1 (D. J. Wozniak) or the fliC mutant WFPA850 (PAO1 andWFPA850; n � 6; P � 0.218, log-rank test). (B) Bacterial load (meannumber of CFU � standard error of the mean) in bullae and brain attime of killing (PAO1 and WFPA850; n � 6 bullae; n � 6 brain).Dotted line, lower limit of detection.

3092 BYRD ET AL. INFECT. IMMUN.

biofilm structure (46). Biofilms formed in vitro by lasR (PDO-R1) or rhlR (PDO111) mutants or a lasR rhlR double mutant(PAO-JP3) contained a similar biomass as PAO1, althoughthere was a trend toward decreased biomass in double-mutantbiofilms relative to that in PAO1 (Table 1). The role of quo-rum sensing in P. aeruginosa pathogenesis has been well estab-lished in both rat and mouse models of acute and chronicinfection but has not been evaluated in the chinchilla otitismedia model (10, 32, 37, 50). We hypothesized that the loss ofquorum-sensing-controlled virulence factors would outweighany biofilm defects and lead to a more attenuated infectionwith increased survival. Chinchillas were infected with PAO1,PDO-R1 (�lasR), or PDO111 (�rhlR) and monitored for signsof otitis media for 10 days. There was no significant differencein survival or bacterial load in bullae and brain for eithermutant-infected group compared to the PAO1-infected group(Fig. 6A). One of two brains examined histologically from boththe PAO1 and rhlR groups was diagnosed as having meningitis,

providing additional evidence for bacteria migrating from theear to the brain, causing some of the clinical signs observed inthis experiment. Video otoscopy revealed that the majority ofears in all three groups had drainage, correlating with the rapidonset of morbidity (Fig. 6C).

We next determined if deletion of both lasR and rhlR wouldaffect survival, since the loss of virulence factors would begreater than that for lasR or rhlR single mutants. Chinchillaswere infected with PAO1 or PAO-JP3, an isogenic lasR rhlRdouble mutant that cannot respond to either the 3OC12-HSLor C4-HSL autoinducer, and monitored for 10 days. Survivalwas significantly greater for animals infected with PAO-JP3than PAO1-infected animals (P � 0.001), PAO-R1-infectedanimals (P � 0.003), and PDO111-infected animals (P � 0.009;Fig. 6A). Animals infected with PAO1 displayed otitis signsearlier than the PAO-JP3 group and had a median survival of2.5 days, compared to a median survival of 5 days for PAO-JP3-infected animals. Video otoscopy correlated with the sur-vival data in both of these experiments, with what appeared tobe higher proportions of PAO1-infected ears having drainage(15/22) and redness (4/22) compared to PAO-JP3-infected ears(4/12 and 5/12, respectively); however, only the incidence ofdrainage approached significance (P � 0.075). Animals in-fected with PAO-R1 had a significantly greater incidence ofdrainage (11/11) than those infected with PAO-JP3 (4/12, P �0.001), with a concomitantly lesser incidence of redness (P �0.037), correlating well with the greater survival of the PAO-JP3 group (Fig. 6C). There were no significant differences inthe incidences of redness or drainage between the PDO111-and PAO-JP3-infected groups. As in other experiments, dele-tion of lasR and rhlR did not affect the bacterial load in thebullae at the time of killing (Fig. 6B). The bacterial load in thebrain was likewise not different between the two groups, indi-cating that factors not requiring LasR or RhlR may be respon-sible for the spread. The one surviving PAO-JP3-infected an-imal did not have recoverable bacteria in the left bulla or brainbut did have a small amount of material present in the rightbulla at 10 d.p.i. The presence of material coupled with rednessvisible by otoscopy at 4 d.p.i. suggests that an infection wasestablished in the right bulla but either had not been estab-lished in the left bulla or had been cleared. We concluded fromthese experiments that the ability to respond to one or bothAHLs, which leads to production of regulator-specific andredundantly regulated virulence factors, results in infectionsimilar to that with PAO1 in this model but that loss of bothLasR and RhlR results in increased persistence.

DISCUSSION

The studies presented here suggest a model in which eithera loss of virulence factors, as in the lasR rhlR double mutant, orthe additional production of biofilm components, as in theMJK8 RSCV, increases the persistence of P. aeruginosa inchinchilla otitis media. In the same model of otitis media,expression of flagella does not have a discernible effect onpersistence and may in fact aid containment of infection. Innone of these cases is the bacterial load at the time of killingdifferent, which suggests that there may be a limit, due toeither space or nutritional factors, on the number of viablebacterial cells that can be contained within a bulla at approx-

FIG. 6. Contribution of quorum sensing to P. aeruginosa virulencein chinchilla otitis media. (A) Greater survival of chinchillas infectedwith PAO-JP3 (�lasR �rhlR) than those infected with PAO-R1(�lasR), PDO111 (�rhlR), or PAO1 (U. Ochsner) (P � 0.001, log-ranktest; all groups, n � 6). (B) Bacterial load (mean number of CFU �standard error of the mean) in bullae and brains of chinchillas infectedwith strains shown in panel A. Dotted line, lower limit of detection.(C) Percentage of ears in chinchillas infected with strains from panel Ahaving redness or drainage visible by video otoscopy at any pointduring the experiment (PAO1, n � 22; PAO-R1, n � 11; PDO111, n �12; PAO-JP3, n � 12).

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imately 109 CFU. We cannot exclude the possibility that thereis a difference in bacterial proliferation early in infection ormigration to the brain, but none of these strains have knowngrowth defects in vitro, and the data shown in Fig. 4G, com-bined with the results of previous experiments focused on earlytime points (data not shown), suggest similar growth kinetics invivo for those strains evaluated. The variation in survival foranimals infected with PAO1 in Fig. 3 to 6 is best explained bythe use of the isogenic parental PAO1 strain, from which eachset of mutants (MJK8 series, WFPA850, or quorum-sensingseries) was derived. The PAO1 strain has been widely used bydifferent laboratories, so it is expected that some variationshould emerge. The specific differences between the virulenceof each PAO1 strain are currently being investigated.

An important consideration when studying biofilms in thecontext of infection is the contribution of host cells and com-ponents that can inhibit or possibly facilitate biofilm formation.Bacteria may take advantage of host components as scaffoldingand form aggregates that hinder clearance by phagocytes. Thehyperadherent phenotype of MJK8 may aid persistence byforming bacterial aggregates and by increasing adherence tomucosal epithelium, fibrin, and other host components. Al-though experiments with a greater number of animals will berequired to determine whether survival is significantly differentin the absence of Psl, we hypothesize that Psl plays an impor-tant role in binding to host components, thus limiting its po-tential for dispersal and decreasing the incidence of interac-tions with immune cells. Future work will address thecontribution of Psl to binding to host components and possibleeffects on the function of host immune cells. It is also possiblethat factors critical for in vitro biofilms have different functionsin vivo or lack a structural role altogether (26).

As a consequence of aggregation, both laboratory and clin-ical RSCVs show increased transcription of quorum-sensing-associated genes, but it is not known if this leads to greaterproduction of quorum-sensing virulence factors (30, 51). It ispossible that negative regulators of quorum sensing are alsoinduced by c-di-GMP or that posttranscriptional regulation ofquorum-sensing virulence factors prevents expression of po-tentially damaging effectors. A role for c-di-GMP in persis-tence in CF has recently been proposed (51). Both MJK8 andthe clinical CF RSCV isolate CF39s induced less interleukin-8and NF-�B from airway epithelial cells than their respectiveparental strains, and less interleukin-8 and NF-�B may reducePMN recruitment and inflammatory signaling, respectively(51). Additionally, strong c-di-GMP-mediated repression offlagellar protein expression may further reduce the inflamma-tory response elicited by RSCVs, favoring persistence in thehost (51). We predicted that deletion of the flagellum struc-tural subunit gene fliC in PAO1 would similarly reduce inflam-mation and increase persistence; however, the level of intra-cellular c-di-GMP in PAO1 is substantially lower than that inMJK8, greatly affecting expression of c-di-GMP-regulatedgenes other than those involved in flagellum production. It islikely a combination of highly expressed polysaccharides andproteins with repressed flagella and other inflammatory factorsthat allows MJK8 to persist in the chinchilla middle ear as wehave reported here. Future work investigating induction ofimmune signaling by MJK8 in vivo could reveal a mechanismfor the persistence observed in this study.

If the quorum-sensing regulators LasR and/or RhlR arerequired for biofilm formation, then we would have expected adecrease in persistence of the double mutant compared to thatof PAO1. Instead, we found no difference in persistence eitherfor the single mutant or for the double mutant but did observegreater persistence of the double mutant than PAO1. Therestill may be a role for the LasRI/RhlRI systems in biofilmformation, but perhaps the conditions used by us in vitro andthe environment in the chinchilla middle ear are not conducivefor quorum-sensing involvement in biofilms, or, as we pre-dicted, the loss of virulence factors could have outweighed anyloss in biofilm-forming ability, preventing us from seeing abiofilm effect. As for the loss of virulence factors, quorum-sensing proteases may favor dispersal of infection and a greaterper cell interaction with the host immune system, leading tomore inflammation. In chronic CF isolates, lasR is often mu-tated, likely to facilitate growth in a low-oxygen environment,but it may also indicate that quorum-sensing virulence factorsare detrimental to long-term colonization (23, 49). A previousstudy showed that the addition of a matrix metalloproteaseinhibitor against P. aeruginosa elastase and alkaline phospha-tase, two quorum-sensing-controlled virulence factors, in achinchilla otitis model resulted in less external erythema andotorrhea than in the control (11). More recently, the 3OC12-HSL autoinducer itself has been implicated in inducing pro-duction of inflammatory and chemotactic factors that may ex-acerbate tissue damage and modulate the immune response,potentially in favor of P. aeruginosa (50). We did not addressthe direct effects of 3OC12-HSL in this study, but given that alasR rhlR double mutant could still synthesize AHLs, it ispossible that deletion of lasI in this background would furtherincrease persistence. These results differ from those describedin recent publications of studies examining the lux quorum-sensing system of NTHi in chinchilla otitis media that revealeda positive role for the AI-2 autoinducer synthase LuxS in per-sistence and reducing inflammation (3, 12). This disparityreflects the natural distribution of these two species: the hu-man-adapted NTHi, which likely benefits from quorum-sens-ing-mediated evasion of host immune recognition (3, 24), andP. aeruginosa, in which quorum sensing stimulates productionof virulence and nutrition-acquisition factors that provide anadvantage in polymicrobial or environmental settings (2).

We have presented here a direct in vivo evaluation of threefactors associated with biofilm structure and virulence of P.aeruginosa: c-di-GMP, flagella, and quorum sensing. We haveshown that MJK8, a c-di-GMP-overproducing mutant, hasgreater persistence in the chinchilla model than PAO1, possiblydue to overexpression of Psl. One of these mediators, LasRI/RhlRI, significantly increases virulence, as predicted, while flagel-lin has little effect on virulence, in contrast to the profound effectsseen using other animal models. These data, in addition to theless defined in vivo contributions of biofilm polysaccharides thatare critical to in vitro biofilm formation, demonstrate the necessityof evaluating potential pathogenic mediators within the context ofan intact immune response in multiple infection systems.

ACKNOWLEDGMENTS

We thank Urs Ochsner for quorum-sensing mutants. We thank KenGrant and Mark Willingham for SEM and histopathology assistance,

3094 BYRD ET AL. INFECT. IMMUN.

Will Willner for photography, and Gayle Foster for technical assis-tance. Lauren Bakaletz kindly reviewed our manuscript.

This work was supported by Public Health Service grants AI076561,HL58334, AI061396, and DC007444 (to W.E.S. and D.J.W.) andNRSA fellowship AI07870002 (to M.S.B.).

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