Antibiofilm Activity of Actinobacillus pleuropneumoniae Serotype 5 Capsular Polysaccharide

Cell-free extracts isolated from colony biofilms of Actinobacillus pleuropneumoniae serotype 5 were found to inhibit biofilm formation by Staphylococcus aureus, S. epidermidis and Aggregatibacter actinomycetemcomitans, but not by A. pleuropneumoniae serotype 5 itself, in a 96-well microtiter plate assay. Physical and chemical analyses indicated that the antibiofilm activity in the extract was due to high-molecular-weight polysaccharide. Extracts isolated from a mutant strain deficient in the production of serotype 5 capsular polysaccharide did not exhibit antibiofilm activity. A plasmid harboring the serotype 5 capsule genes restored the antibiofilm activity in the mutant extract. Purified serotype 5 capsular polysaccharide also exhibited antibiofilm activity against S. aureus. A. pleuropneumoniae wild-type extracts did not inhibit S. aureus growth, but did inhibit S. aureus intercellular adhesion and binding of S. aureus cells to stainless steel surfaces. Furthermore, polystyrene surfaces coated with A. pleuropneumoniae wild-type extracts, but not with capsule-mutant extracts, resisted S. aureus biofilm formation. Our findings suggest that the A. pleuropneumoniae serotype 5 capsule inhibits cell-to-cell and cell-to-surface interactions of other bacteria. A. pleuropneumoniae serotype 5 capsular polysaccharide is one of a growing number of bacterial polysaccharides that exhibit broad-spectrum, nonbiocidal antibiofilm activity. Future studies on these antibiofilm polysaccharides may uncover novel functions for bacterial polysaccharides in nature, and may lead to the development of new classes of antibiofilm agents for industrial and clinical applications.


Introduction
Biofilm is the predominant mode of growth for bacteria in most natural, industrial and clinical environments [1]. Biofilms typically contain millions of tightly-packed cells encased in a polymeric matrix attached to a tissue or surface. The biofilm mode of growth protects bacteria from cell stressors such as desiccation, predators and antibiotics. Biofilms cause corrosion and biofouling of industrial equipment and chronic infections in clinical settings [2]. New methods for treating and preventing biofilm formation are being sought.
Many biofilm bacteria secrete molecules such as quorumsensing signals [3,4], surfactants [5], enzymes [6,7], polysaccharides [8], and D-amino acids [9] that function to regulate biofilm architecture or mediate the release of cells from biofilms during the dispersal stage of the biofilm life-cycle. These compounds often exhibit broad spectrum biofilm-inhibiting or biofilm-dispersing activity when tested against biofilms cultured in vitro. Such compounds may represent a novel source of antibiofilm compounds for technological development [10].
Previous studies showed that colony biofilms may be a useful source of novel antibiofilm compounds [11]. Colony biofilms are lawns of bacterial cells cultured directly on an agar surface or on a semipermeable membrane that sits on an agar plate [12]. Colony biofilms have been shown to exhibit many properties characteristic of broth-cultured biofilms, including high cell density, extracellular matrix production, spatially dependent microbial growth, chemical gradients, and reduced susceptibility to antibiotics [13][14][15][16][17][18]. Cell-free extracts isolated from colony biofilms [11], or from biofilms cultured in a continuous-flow fermentor that produces a similarly high amount of biofilm biomass [19], have been shown to be enriched for soluble molecules produced within the biofilm matrix.
In the present study we screened a panel of 12 colony biofilm extracts isolated from various bacteria for their ability to inhibit Staphylococcus aureus biofilm formation in a 96-well microtiter plate assay. We found that colony biofilm extracts isolated from Actinobacillus pleuropneumoniae serotype 5 inhibited S. aureus biofilm formation without inhibiting S. aureus growth. Here we present results suggesting that the nonbiocidal antibiofilm activity in the A. pleuropneumoniae extract is due to high-molecular-weight serotype 5 capsular polysaccharide.

Bacterial Strains, Media and Growth Conditions
The bacterial strains used in this study are listed in Table 1. For solid media, Tryptic Soy agar was used for A. pleuropneumoniae, A. actinomycetemcomitans and H. influenzae, sheep blood agar (Catalog No. 221239; Becton, Dickinson and Co.) was used for staphylococci, and LB agar was used for all other bacteria. For broth cultures, Tryptic Soy broth supplemented with 6 g/L yeast extract and 8 g/L glucose was used for A. pleuropneumoniae, A. actinomycetemcomitans, H. influenzae and staphylococci, and LB broth was used for all others. A. pleuropneumoniae cultures were supplemented with 10 mg/L NAD. H. influenzae cultures were supplemented with 10 mg/L NAD and 10 mg/L hemin. Plasmid-harboring A. pleuropneumoniae strains were cultured in 80 mg/L spectinomycin. A. pleuropneumoniae and A. actinomycetemcomitans cultures were incubated in 10% CO 2 , whereas all other cultures were incubated in air. P. carotovorum cultures were incubated at 28uC, while all others were incubated at 37uC.

Preparation of Colony Biofilm Extracts
A 100-ml aliquot of an overnight broth culture (.10 8 CFU) was spread onto the surface of a 100-mm-diam agar plate using a sterile glass spreader. The plate was incubated for at least 48 h until a robust lawn of microbial growth (colony biofilm) developed. Thereafter, the cell paste was scraped from the surface of the plate using a plastic inoculating loop or plastic cell scraper, and the cells were transferred to a microcentrifuge tube containing 750 ml of saline (0.9% NaCl). The tubes were mixed by vortex agitation for 10 min, and the cells were pelleted by centrifugation. The supernatant was sterilized by passage through a 0.22-mm poresize filter, and the resulting colony biofilm extract was stored at 4uC until use.
Screening Colony Biofilm Extracts for Antibiofilm Activity S. aureus inocula were prepared from 18-h-old agar colonies as previously described [20]. A volume of 180 ml of inoculum (ca. 10 4 -10 5 CFU/ml) was transferred to the well of a tissue-culturetreated polystyrene microtiter plate (Falcon no. 353047). A total of 20 ml of colony biofilm extract, or 20 ml of saline as a control, was mixed with the inoculum and the plate was incubated statically at 37uC. After 18 h, biofilms were rinsed with water and stained for 1 min with 200 ml of Gram's crystal violet. Wells were then rinsed with water and dried. The amount of crystal violet binding was quantitated by destaining the wells for 10 min with 200 ml of 33% acetic acid, and then measuring the absorbance of the crystal violet solution in a microplate spectrophotometer set at 595 nm.

Physical and Chemical Analyses of A. pleuropneumoniae Colony Biofilm Extract
Size-exclusion filtration was carried out using a Microcon centrifugal concentrator (Millipore) with a 100-kDa molecular weight cut-off filter. For enzymatic treatments, extracts were incubated for 1 h at 37uC with 100 mg/L DNase I, RNase A, porcine pancreatic lipase or proteinase K (Sigma-Aldrich) or 10 mg/L dispersin B (Kane Biotech). Controls consisted of mocktreated extracts, or enzymes alone with no extract. For sodium metaperiodate treatment, 0.1 vol of 100 mM sodium metaperiodate was added to the extract, and the extract was incubated at 37uC for 1 h. Controls consisted of mock-treated extract and sodium metaperiodate alone with no extract. Following all treatments, extracts and controls were incubated at 100uC for 10 min prior to testing in the S. aureus biofilm assay described above. Purification of A. pleuropneumoniae Serotype 5 Capsular Polysaccharide Capsular polysaccharide was purified from broth cultures of A. pleuropneumoniae strain J45 by Cetavlon precipitation of culture supernatant, extraction of the precipitate with NaCl and aqueous phenol, and Sepharose CL-4B gel filtration chromatography as previously described [21].

Cell Binding Assay
A single-cell suspension of S. aureus (ca. 10 6 -10 7 CFU/ml) was prepared in fresh broth using a filtration protocol as previously described [22]. The cell suspension was supplemented with 10% A. pleuropneumoniae colony biofilm extract, or 10% saline as a control, and then aliquots of cells (0.5-ml each) were transferred to 1.5-ml polypropylene microcentrifuge tubes. Stainless steel rods (0.6-mm diam613-mm length) were placed in the tubes, and the tubes were incubated at 37uC. After 30 or 60 min, rods were removed from the tubes, rinsed three times with saline, and transferred to 15-ml conical centrifuge tubes containing 1 ml of saline. The rods were sonicated on ice (2630 sec) using an IKA Labortechnik sonicator set to 50% power and 50% duty cycle. CFUs in the sonicate were quantitated by dilution plating.

Intercellular Adhesion Assay
S. aureus was cultured in 17-mm 6100-mm glass tubes in 2 ml of broth. The broth was supplemented with 7% (by vol) A. pleuropneumoniae colony biofilm extract isolated from strain J45 or J45-100, or with 7% saline as a control. Bacteria were incubated with shaking (200 rpm). After 7 h, tubes were incubated statically for 10 min and then photographed.

Surface Coating Assay
A volume of 25 ml of A. pleuropneumoniae colony biofilm extract, or 25 ml of saline as a control, was transferred to the center of a well of a 24-well tissue-culture-treated polystyrene microtiter plate (Falcon no. 353047). The plate was incubated at 37uC for 30 min to allow complete evaporation of the liquid. The wells were then filled with 1 ml of broth containing 10 4 to 10 5 CFU/ml of S. aureus. After 18 h, wells were rinsed with water and stained with 1 ml of Gram's crystal violet. Stained biofilms were rinsed with water and dried, and the wells were photographed.

Microscopy
A. pleuropneumoniae inocula were prepared from 24-h-old agar colonies as previously described [20]. Bacterial inocula were diluted in fresh broth to 10 4 -10 5 CFU/ml. Aliquots of diluted cells (250 mL each) were pipetted onto the surface of sterile glass slides, and the slides were placed inside a Petri dish. After incubation for 24 h, the slides were rinsed with water and stained with SYTO9 (Molecular Probes) for 20 min in the dark. Slides were then rinsed with water to remove excess stain. Biofilm bacteria were visualized at 106 magnification using a Nikon Eclipse 80i fluorescent microscope.

Statistics and Reproducibility of Results
All microtiter plate assays were performed in duplicate wells, which exhibited an average variation of ,10%. All assays were performed 2-3 times with similarly significant differences in absorbance values. The significance of differences between means was measured using the Student's t-test. A P value of #0.05 was considered significant.

A. pleuropneumoniae Colony Biofilm Extract Exhibits Antibiofilm Activity
We isolated colony biofilm extracts from 12 different bacteria and tested the extracts for their ability to inhibit biofilm formation by S. aureus in a 96-well microtiter plate assay (Fig. 1). The bacteria that were tested comprised a convenience sample of 11 Proteobacteria and Lactococcus lactis (Table 1). Extracts were tested at a concentration of 10% by vol. Under these conditions, five extracts significantly inhibited S. aureus biofilm formation, while seven extracts had no significant effect on biofilm formation. Inhibition of S. aureus biofilm formation by P. aeruginosa extract was partially due to growth inhibition (data not shown). We selected A. pleuropneumoniae IA5 colony biofilm extract for further analysis because it exhibited a high level of biofilm inhibition, but it did not inhibit S. aureus growth or contain proteases or DNases that are known to inhibit S. aureus biofilm formation (data not shown). A. pleuropneumoniae IA5 colony biofilm extract inhibited biofilm formation by S. aureus, S. epidermidis, and the Gram-negative Aggregatibacter actinomycetemcomitans (Fig. 2). A. pleuropneumoniae colony biofilm extract did not inhibit the growth of S. aureus, S. epidermidis or A. actinomycetemcomitans (data not shown).

The Antibiofilm Activity in A. pleuropneumoniae Colony Biofilm Extract is due to Capsular Polysaccharide
Physical analysis of the A. pleuropneumoniae IA5 colony biofilm extract indicated that the antibiofilm activity in the extract was .100 kDa in mass (Fig. 3A) and heat stable (Fig. 3B). Treatment of the extract with proteinase K, lipase, DNase or RNase had no effect on its antibiofilm activity (Fig. 3C). In contrast, treatment of the extract with the carbohydrate-active agent sodium metaperiodate significantly reduced its antibiofilm activity (Fig. 3C). These data suggest that the antibiofilm activity in the A. pleuropneumoniae colony biofilm extract was due to high-molecular-weight polysaccharide.
To determine whether the A. pleuropneumoniae serotype 5 capsule contributes to the antibiofilm activity of the colony biofilm extract, we isolated extracts from wild-type serotype 5 strain J45, and from the isogenic serotype 5 capsule-mutant strain J45-100. Like extracts isolated from strain IA5, extracts isolated from strain J45 inhibited S. aureus biofilm formation (Fig. 4A). In contrast, extracts isolated from capsule-mutant strain J45-100 did not inhibit S. aureus biofilm formation (Fig. 4A). Extracts isolated from capsule-mutant strain J45-100 transformed with plasmid pJMLCPS5, which restores capsule production, exhibited significantly greater antibiofilm activity than extracts isolated from uncomplemented J45-100 (Fig. 4B). This biofilm inhibition was not due to growth inhibition caused by antibiotic carryover from the genetically-complemented A. pleuropneumoniae culture (data not shown). In addition, purified serotype 5 capsular polysaccharide inhibited S. aureus biofilm formation in a dose-dependent manner (Fig. 4C). These findings confirm that the A. pleuropneumoniae serotype 5 capsule exhibits antibiofilm activity against S. aureus.
A. pleuropneumoniae Colony Biofilm Extract Inhibits S. aureus Cell-to-cell and Cell-to-Surface Interactions S. aureus cells cultured in broth with shaking aggregated and settled to the bottom of the tube, resulting in a visible clearing of the broth (Fig. 5A, left panel). When A. pleuropneumoniae J45 extract was present in the culture, the S. aureus cells exhibited less settling (Fig. 5A, middle panel). S. aureus cells cultured in the presence of extract isolated from serotype 5 capsule-mutant strain J45-100 exhibited the same amount of settling as control cultures (Fig. 5A,  right panel). These data suggest that A. pleuropneumoniae serotype 5 capsule inhibits S. aureus intercellular adhesion.
We also measured binding of S. aureus planktonic cells to stainless steel rods in the presence and absence of A. pleuropneumoniae J45 colony biofilm extract. As shown in Fig. 5B, the A. pleuropneumoniae extract significantly inhibited S. aureus cell binding after both 30 and 60 min.
We also tested whether A. pleuropneumoniae J45 colony biofilm extract could modify the surface properties of an abiotic substrate. To do this, we used evaporation coating to deposit the extract onto the surface of polystyrene wells, and then tested the ability of the coated surfaces to resist biofilm formation by S. aureus. When A. pleuropneumoniae extract was applied to the polystyrene surfaces, the coated surfaces efficiently repelled S. aureus biofilm formation in the area where the extract was deposited (Fig. 5C). Surfaces coated with extract isolated from the serotype 5 capsule-mutant strain did not resist S. aureus biofilm formation (Fig. 5C).

A. pleuropneumoniae Colony Biofilm Extract does not Inhibit A. pleuropneumoniae Biofilm Formation
Extracts isolated from A. pleuropneumoniae wild-type strain J45 and capsule-mutant strain J45-100 were tested for their ability to inhibit biofilm formation by A. pleuropneumoniae J45 and J45-100. There was no significant difference between the amount of biofilm formed by strains J45 and J45-100 when quantitated by spectrophotometry (Fig. 6A). Neither the J45 nor the J45-100    extract significantly inhibited biofilm formation by either strain. Dispersin B significantly inhibited biofilm formation by both strains, indicating that strain J45, like A. pleuropneumoniae serotype 5 strain IA5 [23], produces PNAG-dependent biofilms. Microscopic analysis revealed that biofilms produced by the capsule mutant strain J45-100 appeared much denser than biofilms produced by wild-type strain J45 (Fig. 6B).

Discussion
The Gram-negative bacterium A. pleuropneumoniae is the causative agent of swine pleuropneumonia, a severe and contagious respiratory disease that affects pigs worldwide [24]. A. pleuropneumoniae strains are divided into 15 serotypes based on the structures of their capsular polysaccharide (CPS) [25]. Serotype 5 is a prevalent A. pleuropneumoniae serotype in the U.S., Canada, Brazil, Chile, Korea and Taiwan [26]. A. pleuropneumoniae serotype 5 CPS consists of a linear polymer with the structure R6)-a-D-GlcpNAc-(1R5)-b-D-dOclAp-(2R [27]. A subset of serotype 5 strains (designated serotype 5b) contain an additional b-D-Glcp reside covalently joined to the b-D-dOclAp residue in (1R4) linkage [26]. Mutant strains lacking serotype 5 CPS have been shown to exhibit decreased serum resistance in vitro and decreased virulence in pigs [28,29]. Our findings demonstrate that A. pleuropneumoniae serotype 5 CPS exhibits nonbiocidal antibiofilm activity against other Gramnegative and Gram-positive bacteria. Several other bacteria produce nonbiocidal antibiofilm polysaccharides including Kingella kingae [11], Escherichia coli [8,19], Bacillus licheniformis [30], Lactobacillus acidophilus [31], Streptococcus phocae [32] and Vibrio sp. [33]. The biological functions of these antibiofilm polysaccharides are not known, but they may include water channel formation or biofilm dispersal [34]. The fact that the A. pleuropneumoniae CPS mutant strain exhibited increased biofilm formation compared to the wild-type strain (Fig. 6B) suggests that the serotype 5 capsule may function in water channel formation or biofilm dispersal. Previous studies showed that A. pleuropneumoniae produces dispersin B, which may also function in maintaining biofilm architecture or in biofilm dispersal [23].
We found that A. pleuropneumoniae serotype 5 CPS inhibited biofilm formation by S. aureus, S. epidermidis and A. actinomycetemcomitans, but not by A. pleuropneumoniae serotype 5 itself. Biofilm formation by all four of these species is dependent on the production of poly-N-acetylglucosamine (PNAG) matrix polysaccharide [20,35]. Although PNAG is the major biofilm matrix adhesin in both A. pleuropneumoniae and S. epidermidis biofilms, A. pleuropneumoniae serotype 5 CPS inhibited only S. epidermidis biofilm formation and not A. pleuropneumoniae serotype 5 biofilm formation. In addition, colony biofilm extracts isolated from a PNAGdeficient strain of A. pleuropneumoniae serotype 5 exhibited the same antibiofilm activity as extracts isolated from a wild-type strain (unpublished results). These observations suggest that the anti-biofilm activity of serotype 5 CPS is independent of the intercellular adhesion activity of PNAG.
Our screen of colony biofilm extracts isolated from 12 phylogenetically diverse bacteria identified five extracts that exhibited antibiofilm activity against S. aureus. In a similar screen, Rendueles et al. [19] found that 20% of cell-free biofilm extracts isolated from 122 natural E. coli isolates exhibited antibiofilm activity against a panel of seven biofilm-forming Gram-positive and Gram-negative bacteria. These findings suggest that bacterial biofilms constitute untapped sources of natural bioactive molecules antagonizing adhesion or biofilm formation of other bacteria.