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Infect Immun. Aug 2007; 75(8): 3715–3721.
Published online Jun 11, 2007. doi:  10.1128/IAI.00586-07
PMCID: PMC1952004

Pseudomonas aeruginosa Forms Biofilms in Acute Infection Independent of Cell-to-Cell Signaling[down-pointing small open triangle]

Abstract

Biofilms are bacterial communities residing within a polysaccharide matrix that are associated with persistence and antibiotic resistance in chronic infections. We show that the opportunistic pathogen Pseudomonas aeruginosa forms biofilms within 8 h of infection in thermally injured mice, demonstrating that biofilms contribute to bacterial colonization in acute infections as well. Using light, electron, and confocal scanning laser microscopy, P. aeruginosa biofilms were visualized within burned tissue surrounding blood vessels and adipose cells. Although quorum sensing (QS), a bacterial signaling mechanism, coordinates differentiation of biofilms in vitro, wild-type and QS-deficient P. aeruginosa strains formed similar biofilms in vivo. Our findings demonstrate that P. aeruginosa forms biofilms on specific host tissues independently of QS.

Bacterial biofilms are communities of microorganisms residing within a polysaccharide matrix that have been imaged in dental plaques, medical prostheses, and contact lenses (7, 31, 33). It is well accepted that biofilms play important roles in bacterial persistence and antibiotic resistance in chronic infections, such as cystic fibrosis and otitis media (3, 8, 9, 30). However, the existence and/or roles of biofilms in acute infections, which are defined by short time courses and high severity, have not been examined. The opportunistic gram-negative pathogen Pseudomonas aeruginosa causes both chronic and acute infections and is one of the leading causes of morbidity and mortality in thermally injured patients (27, 37). In this study we examined the production of P. aeruginosa biofilms in the thermally injured mouse model of acute infections.

The differentiation or maturation of P. aeruginosa biofilms in vitro depends on intercellular signaling systems or quorum sensing (QS) (5, 22). QS systems in many gram-negative bacteria rely on acylated homoserine lactones (AHLs), which are produced at high levels when cell density is high and act as ligands for transcriptional regulators. The P. aeruginosa synthases LasI and RhlI synthesize two AHLs, N-3-oxododecanoyl homoserine lactone (3OC12-HSL) and N-butyryl-homoserine lactone (C4-HSL), which bind and modulate the activity of the transcriptional regulators LasR and RhlR, respectively (28). These transcriptional regulators then regulate the transcription of many genes whose products, including proteases, elastases, toxins, and hemolysins, are thought to be crucial for virulence (28). P. aeruginosa strains lacking functional QS systems are less virulent than wild-type strains (29) and form flat, undifferentiated biofilms on glass surfaces (5). These undifferentiated biofilms are less stable than the differentiated biofilms formed by wild-type P. aeruginosa as they can be easily disrupted by the detergent sodium dodecyl sulfate (5). However, the role of QS in biofilm formation has not previously been examined in vivo. Therefore, in this study we have also examined the role of QS in P. aeruginosa biofilm formation in the acute infection model.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions.

Pseudomonas aeruginosa strains PAO1 (14) and PAO-JP2 (24) containing the green fluorescent protein (GFP) plasmid pMRP9-1 (5) and the rhlI GFP promoter fusion pTdK-rhlI-GFP (6) have been described previously. Bacteria were grown in Luria-Bertani broth overnight with shaking at 37°C, subcultured to logarithmic phase, and diluted to 102 CFU in sterile phosphate-buffered saline (PBS) before inoculation into the mouse.

Thermally injured mouse model.

P. aeruginosa was examined in the thermally injured mouse model as previously described (12, 29). Mice were housed and studied under protocols approved by the Institutional Animal Care and Use Committee in the animal facility of Texas Tech University Health Sciences Center (Lubbock, TX).

Construction of alginate-deficient mutant.

An alginate-deficient mutant of P. aeruginosa was constructed as follows. Briefly, approximately 2,380 nucleotides of a DNA fragment carrying 1,131 nucleotides of the algD open reading frame and 1,050 nucleotides of the flanking sequences was PCR amplified from strain FRD1, a cystic fibrosis isolate of P. aeruginosa. The DNA was amplified with the thermostable DNA polymerase Pfu (Stratagene, La Jolla, CA) to avoid potential errors. The amplified algD fragment was cloned into pUC19 as a BamHI-HindIII fragment, and moriT (36) was subcloned as a HindIII fragment to make the plasmid conjugable. The resulting plasmid, pSS336, was further digested with BclI to delete approximately 510 nucleotides from the algD open reading frame, and a nonpolar tet cassette that codes for tetracycline resistance (36) was inserted into the Bcl site as a BamHI fragment in the same orientation as the algD gene to generate the algD1301::tet deletion allele. To construct an algD deletion mutant, the plasmid carrying the deletion allele (pSS350) was introduced into PAO1 via triparental mating as previously described (35) and the resulting exconjugants that had undergone allelic exchange were selected by tetracycline resistance, screened for carbenicillin sensitivity, and verified by PCR amplification of the mutant allele. To complement the algD deletion, pALG2, which carries the complete alginate operon (20), was introduced into the algD mutant via triparental mating and selected for carbenicillin resistance. Because pALG2 cannot replicate autonomously in P. aeruginosa, the plasmid integrated into the genome via homologous recombination.

Sample preparation, microscopy, and image analysis.

Confocal scanning laser microscopy (CSLM), transmission electron microscopy (TEM), scanning electron microscopy (SEM), and fluorescence microscopy were used to examine tissue sections for biofilm formation. For CSLM tissue sections were removed after euthanasia from the underlining burned tissue using a scalpel, washed in sterile PBS, and placed in imaging chambers (Molecular Probes, Eugene, OR) containing an antifade reagent (Molecular Probes). CSLM and fluorescent images were obtained using an Olympus IX71 upright microscope equipped with the Fluoview 300 CSLM package (Olympus America, Melville, NY). Image analysis and reconstruction were performed using MetaMorph 6.1 (Universal Imaging Corporation, PA) and COMSTAT (13) software. Bright-field images were acquired using a Nikon DXM1200 color digital camera equipped with the ACT-1 software analysis package (Nikon, Melville, NY). Skin tissue was prepared for SEM by being fixed overnight at 4°C in 1.5% glutaraldehyde and 3% paraformaldehyde in 0.1 M Millonig's buffer with dextrose. Tissue was then frozen in liquid nitrogen and fractured with a frozen blade to expose more surface. Tissues were postfixed in 1% osmium tetroxide, taken through a graded series of alcohols, and critically point dried. The sections were mounted on edge on stubs coated with carbon tape and gold coated. Images were acquired with a Hitachi S-500 scanning electron microscope (Hitachi America, Ltd., Brisbane, CA). TEM with ruthenium red staining was performed as previously described (11), and images were viewed with a Hitachi H600 transmission electron microscope. Fluorescence imaging of perivascular cuffing (PVC) was performed on paraformaldehyde-fixed tissue sections using a fluorescence in situ hybridization (FISH) probe specific for P. aeruginosa (5′-Cy3/GCTGGCCTAGCCTTC-3′ [IDT, Coralville, IA]) as previously described (38). Fluorescence immunohistochemistry for alginate was performed on formalin-fixed paraffinized tissues. The tissues were deparaffinized, treated for 15 min with 20-μg/ml proteinase K, and incubated overnight at 4°C in a 1:100 dilution of human antialginate monoclonal antibody (G. B. Pier, Harvard Medical School/Brigham and Women's Hospital). Tissue sections were then rinsed three times for 10 min each in PBST (PBS plus 0.1% Tween 20) and incubated for 1 h at room temperature with a 1:30 dilution of anti-human Alexa Fluor 488 antibody (Molecular Probes). Sections were rinsed three times for 10 min each in PBST and imaged as described above. For TEM visualization of alginate, tissue sections were fixed in 4% paraformaldehyde in Dulbecco's PBS. Samples were cut with a 1-mm punch biopsy instrument, high pressure frozen using 200-μm-deep aluminum planchettes in a BAL-TEC HPM-010 high-pressure freezer, and stored under liquid nitrogen (4, 18). Samples were freeze substituted in 0.2% anhydrous glutaraldehyde and 0.1% uranyl acetate in acetone using a Leica EM AFS and subsequently embedded in LR White using microwave polymerization (17). One-hundred-nanometer thin sections were cut for immunolabeling on 200-mesh, Formvar- and carbon-coated nickel grids and subsequently labeled with primary antialginate antibody (polyclonal rabbit antialginate; G. B. Pier, Harvard Medical School/Brigham and Women's Hospital) at 1:200 dilution in a blocking buffer containing bovine serum albumin and cold water fish gelatin overnight at 4°C, followed by 10-nm-gold secondary antibody (Electron Microscopy Sciences) at 1:20 dilution for 1 h at room temperature. Sections were poststained for 4 min using 2% uranyl acetate in 70% methanol and imaged on a Philips CM200 transmission electron microscope at 200 keV with a Tietz TemCam F214 charge-coupled device.

RESULTS AND DISCUSSION

P. aeruginosa pathogenesis in burn wounds has been extensively examined using the thermally injured mouse model, which closely resembles human burn wound sequelae (29, 34). In this mouse model, a low infecting dose (102 CFU) of P. aeruginosa causes up to 100% mortality within 48 h (29). In order to determine if biofilms form in acute burn wound infections, we used microscopic approaches to visualize bacterial infections in situ in mice administered full-thickness, third-degree scald burns and infected with a GFP-expressing, wild-type strain of P. aeruginosa, PAO1 (14). We previously reported that PAO1 proliferates rapidly within the burn eschar, multiplying from a starting dose of 102 CFU to 109 CFU in less than 24 h (29). Bacteremia is apparent in these mice as early as 24 hours post-burn/infection by the presence of P. aeruginosa in the blood, liver, and spleen, and >90% of mice die within 48 h post-burn/infection (29).

P. aeruginosa forms microcolonies in burned tissue by 8 h post-burn/infection.

Tissues were harvested from the burn eschar at 8, 24, and 46 h post-burn/infection. A third-degree burn completely destroys the ultrastructure of the epidermis and dermis, leaving only hypodermis, which is composed primarily of vascular, connective, muscle, and adipose tissues. Thus, the burned epidermis and dermis were peeled away, homogenized, and used to determine CFU (see Fig. S1 in the supplemental material). Thin layers (approximately 1 mm) of the hypodermis were rinsed in sterile PBS and placed directly on slides for image analysis. Small clusters or microcolonies of GFP-expressing bacilli were visualized by CSLM at 8 h post-burn/infection in all mice examined (n = 6) (Fig. (Fig.1A).1A). Microcolonies ranged in size from 14 to 33 μm. The CFU in these tissues had increased from the starting dose of 102 to 4.4 × 107 ± 1.8 × 107 (see Fig. S1 in the supplemental material). Green fluorescence was not observed in burned but noninfected tissue samples or burned tissue infected with non-GFP-expressing PAO1 (data not shown). The CFU in the burned skin had increased to 1.3 × 109 ± 3.9 × 108 by 24 h post-burn/infection, and large bacterial aggregates or macrocolonies, ranging in size from 38 to 53 μm, were visualized in the tissues in 91% of the mice (10/11). These macrocolonies were primarily located surrounding adipocytes and veins (Fig. (Fig.11 and and2).2). All tissues harvested from PAO1-infected mice at 46 h post-burn/infection exhibited extensive surface coverage (n = 11), and aggregates measured 15 to 25 μm (Fig. (Fig.1).1). Individual bacterial cells that were not associated with structures were also observed at all time points (Fig. (Fig.11).

FIG. 1.
Biofilms are present around adipocytes in PAO1- and PAO1-JP2-infected tissue. Burned skin sections were harvested from mice infected with PAO1 or PAO1-JP2 after 8, 24, or 46 h. The burned epidermis and dermis layers were removed, and the underlying hypodermis ...
FIG. 2.
PAO1 and PAO1-JP2 form biofilms around veins. (A and B) Burned skin sections from mice infected with PAO1 (A) or PAO1-JP2 (B) were harvested after 24 or 46 h, respectively, and longitudinal sections of P. aeruginosa PVC around veins were imaged by CSLM. ...

P. aeruginosa preferentially congregates around blood vessels.

Two distinctive clinical features of P. aeruginosa bacteremia are invasion and necrosis of blood vessels (32). Historically, blood vessel invasion by P. aeruginosa has been associated with the presence of bacilli in a circumferential pattern surrounding the vessel, where the bacterial cells are aligned single file or in stacks between cells of the venous walls (32). The formation of these structures is termed PVC (21). PVC was visualized in PAO1-infected tissues by CSLM, TEM, and fluorescence microscopy using a specific P. aeruginosa FISH probe (Fig. (Fig.2C;2C; see also Fig. S2 in the supplemental material). PVC similar to that seen in P. aeruginosa-infected mouse tissues is commonly observed in human skin lesions termed ecthyma gangrenosum (19). Ecthyma gangrenosum is primarily associated with infections by the Pseudomonas and Aeromonas species, and clinical diagnosis of P. aeruginosa infection is often based entirely on the recognition of these lesions (21). However, the mechanisms controlling the formation of PVC by P. aeruginosa and the role of PVC in pathogenesis are not fully understood. In this study, the detection of PVC in PAO1-infected tissue correlated strongly with the systemic spread of the bacteria to the liver and/or blood (n = 14/15). Therefore, biofilm formation around blood vessels may be an important step leading to invasion of the vasculature and systemic spread of the bacteria.

P. aeruginosa possesses an extracellular, alginate-associated matrix in vivo.

Bacterial biofilms have been defined as groups of bacteria attached to a surface and enclosed in a matrix, typically made of polysaccharides, nucleic acids, and proteins (3). Our CSLM images revealed large aggregates of P. aeruginosa, which were not removed by rinsing the tissue (Fig. (Fig.11 and and2).2). P. aeruginosa aggregates were visualized by SEM and TEM of the burned tissue to determine if they were associated with a biofilm matrix (BFM) (Fig. (Fig.1B,1B, ,2D,2D, and and3).3). SEM images revealed matrix-like structures and/or “bacterial flocs” in association with the P. aeruginosa aggregates (Fig. (Fig.1B).1B). These structures are consistent with the polysaccharide BFMs that have been described in P. aeruginosa biofilms in vitro (38, 39). For visualization by TEM, tissue sections were treated with ruthenium red, a polyanionic stain that stabilizes the structural integrity of the polysaccharide-rich BFM, which can be lost during the dehydration process (10, 11). Ruthenium red-treated tissue, counterstained with uranyl acetate and lead citrate, revealed dark fiber-like structures between P. aeruginosa cells in TEM (Fig. (Fig.3),3), which are consistent with previously demonstrated biofilms (10, 11). These fibrous structures were not visualized in areas devoid of P. aeruginosa.

FIG. 3.
P. aeruginosa PVC in infected mouse tissue was viewed with ruthenium red staining and TEM. A cross section of a vein displaying P. aeruginosa PVC was imaged at the following magnifications: ×5,500 (A), ×16,500 (B), ×68,750 (C), ...

The extracellular polysaccharide alginate is composed of mannuronic and guluronic acids and is a component of the P. aeruginosa BFM that may assist in protecting bacteria from antibiotics and host defenses in an infection (15). Alginate is produced by PAO1 in vivo, and alginate antibodies are detected in patients with extant P. aeruginosa infections (2, 25). We examined whether alginate was associated with P. aeruginosa vascular biofilms in thermally injured mice. Deparaffinized, PAO1-infected tissue sections were incubated with a monoclonal human antialginate antibody (26) and detected by fluorescence microscopy. A strong fluorescent signal was observed around blood vessels and adipocytes in samples from PAO1-infected tissues (Fig. 4A and B) but not in noninfected tissues, tissues incubated with secondary antibody alone, or tissues treated with an irrelevant primary antibody (see Fig. S3 in the supplemental material). To further confirm the specificity of the alginate antibody, we performed immunohistochemical analysis on thermally injured mice infected with either an isogenic alginate mutant derived from PAO1 (PAO1 algD1301::tet) or a mutant strain complemented with a plasmid carrying the alginate synthesis genes (PAO1 algD1301::pALG2). Although equivalent levels of PVC were detected in tissue sections infected with PAO1 algD1301::tet and in those infected with PAO1 algD1301::pALG2, alginate signal was detected only in mice infected with the complemented mutant (compare Fig. 4C and D). To obtain higher-resolution images, we utilized TEM to visualize tissues incubated with alginate primary antibodies and immunogold-labeled secondary antibodies. Gold particles were evident between individual bacterial cells in vivo (Fig. 4E and F), confirming that alginate is a component of the BFM surrounding bacteria in vivo. As with the fluorescent immunohistochemical analysis, signal was not detected in tissues incubated with secondary antibody alone or tissues treated with an irrelevant primary antibody (data not shown). Taken together, these results indicate that P. aeruginosa rapidly forms aggregates that possess extracellular matrices in an in vivo acute infection model.

FIG. 4.
Alginate is present in the BFM surrounding P. aeruginosa in vivo. (A and B) Immunohistochemical images of longitudinal sections of skin tissues from a thermally injured mouse infected with PAO1 after incubation with alginate primary monoclonal antibody ...

QS is not related to P. aeruginosa in vivo biofilm formation in mouse burn wounds.

In order to determine if a functional cell-to-cell signaling system is required for biofilm formation in vivo, we compared biofilm formation in thermally injured mice infected with either PAO1 or an isogenic P. aeruginosa QS mutant strain (PAO1-JP2). PAO1-JP2 carries deletions in the lasI and rhlI genes and does not synthesize 3OC12-HSL or C4-HSL (29). PAO1-JP2 is also defective in twitching motility (1) and is significantly less virulent in the thermally injured mouse model (29). Tissues from PAO1- and PAO1-JP2-infected mice were evaluated for bacterial load, the presence of micro- or macrocolonies, and PVC. Additionally, several features of PAO1 and PAO1-JP2 biofilms were quantitatively analyzed using COMSTAT (13), an image analysis program developed for analyzing structural elements in biofilms (Table (Table1).1). CFU in the burn eschar were similar for the two strains at 8, 24, and 46 h, indicating that both can proliferate rapidly (see Fig. S1 in the supplemental material). Morphological analyses revealed no major differences between the biofilms formed by PAO1 and those formed by PAO1-JP2 (Fig. (Fig.22 and and3;3; Table Table1).1). Specifically, PVC was visualized in six/nine PAO1-infected mice and four/nine PAO-JP2-infected mice at 24 h post-burn/infection. Similarly, five/six PAO1-infected mice and four/six PAO1-JP2-infected mice displayed PVC at 46 h post-burn/infection. In order to discount the possibility that the formation of PVC biofilms by PAO1-JP2 was due to reversion to wild type during passage in the mouse, we examined 3OC12-HSL synthesis in PAO1-JP2 colonies obtained from the liver and skin at 46 h post-burn/infection utilizing the standard autoinducer bioassay (23). None of the PAO1-JP2 colonies examined produced 3OC12-HSL (data not shown). Analysis of COMSTAT data revealed no significant differences between any of the parameters studied, except that PAO1-JP2 displayed significantly less surface area coverage than PAO1 at 46 h post-burn/infection (Table (Table1).1). This supports our previous findings that PAO1-JP2 does not spread through the burn eschar as efficiently as PAO1 (29), and this phenotype is likely due to its defect in type IV fimbria-mediated twitching motility which facilitates bacterial translocation over moist surfaces (16). However, in most regards the in vivo biofilms made by PAO1-JP2 were similar to those made by PAO1. These data indicate that AHL-based cell-to-cell signaling is not required for rapid biofilm formation by P. aeruginosa within a burn wound.

TABLE 1.
COMSTAT analysis of biofilm parameters from tissues infected for 24 h with PAO1 or PAO1-JP2

We have previously determined that PAO1-JP2 causes less bacteremia and lower percent mortality than PAO1 (29), and these results were confirmed in this study (see Fig. S1 in the supplemental material). However, the diminished systemic spread and decreased virulence of PAO1-JP2 were not due to its inability to form a biofilm. It is likely that the differences in virulence between PAO1 and PAO1-JP2 are due to defects in the expression of QS-regulated virulence factors in the mutant strain. It is possible that one or more of these factors are needed for efficient blood vessel invasion subsequent to biofilm formation. Using a PAO1 strain carrying a GFP reporter fused to the rhlI promoter, we detected GFP expression around blood vessels similar to that seen with the constitutive GFP reporter (Fig. (Fig.5).5). This supports the contention that the role of biofilms in acute infections may be to achieve the high local cell density needed for expression of QS-controlled virulence factors crucial for systemic spread.

FIG. 5.
QS is induced in the aggregates surrounding veins. Burned skin sections from three mice infected with PAO1 carrying an rhlI::gfp promoter fusion were harvested after 24 h, and longitudinal sections of P. aeruginosa PVC around veins were imaged by CSLM ...

Supplementary Material

[Supplemental material]

Acknowledgments

We thank G. B. Pier for the generous gift of the alginate antibodies, S. C. Williams and M. R. Parsek for critical review of the manuscript, R. H. Veeh for critical assessment of biofilm images, M. Zemla for assistance in thin sectioning for immunolabeling, R. Wolcott for facilitating the TEM experiments, and B. Iglewski for PAO1-JP2 and pTdK-rhlI-gfp.

This study was supported by the American Lung Association and South Plains Foundation (K.P.R.).

Notes

Editor: V. J. DiRita

Footnotes

[down-pointing small open triangle]Published ahead of print on 11 June 2007.

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

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