D-Glucopyranose on Biofilm Formation by Staphylococcus aureus

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 2011, p. 1021–1027 0066-4804/11/$12.00 doi:10.1128/AAC.00843-10 Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Vol. 55, No. 3

Inhibitory Effects of 1,2,3,4,6-Penta-O-Galloyl-␤-D-Glucopyranose on Biofilm Formation by Staphylococcus aureus䌤† Mei-Hui Lin,1 Fang-Rong Chang,2 Mu-Yi Hua,3 Yang-Chang Wu,2* and Shih-Tung Liu1* Research Center for Bacterial Pathogenesis and Department of Medical Biotechnology and Laboratory Science, Chang-Gung University, Kwei-Shan, Taoyuan 333, Taiwan1; Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan2; and Department of Chemical and Materials Engineering, Chang-Gung University, Kwei-Shan, Taoyuan 333, Taiwan3 Received 20 June 2010/Returned for modification 9 August 2010/Accepted 13 December 2010

1,2,3,4,6-Penta-O-galloyl-␤-D-glucopyranose (PGG) is an active ingredient in plants that are commonly used in Chinese medicine to treat inflammation. We demonstrate here that PGG, at 6.25 ␮M, does not inhibit the growth of Staphylococcus aureus, and yet it prevents biofilm formation on polystyrene and polycarbonate surfaces. At the same concentration, PGG is not toxic to human epithelial and fibroblast cells. PGG has an IB50 value, i.e., the PGG concentration that inhibits 50% biofilm formation, of 3.6 ␮M. The value is substantially lower than that of N-acetylcysteine, iodoacetamide, and N-phenyl maleimide, which are known to inhibit biofilm formation by S. aureus. Biochemical and scanning electron microscopy results also reveal that PGG inhibits initial attachment of the bacteria to solid surface and the synthesis of polysaccharide intercellular adhesin, explaining how PGG inhibits biofilm formation. The results of this study demonstrate that coating PGG on polystyrene and silicon rubber surfaces with polyaniline prevents biofilm formation, indicating that PGG is highly promising for clinical use in preventing biofilm formation by S. aureus. lysostaphin, i.e., a peptidoglycan-degrading enzyme, prevents staphylococcal biofilm formation (39); in addition, N-acetylcysteine (NAC) reduces EPS production, subsequently inhibiting biofilm formation (25, 26). Meanwhile, dispersin B, which hydrolyzes ␤-1,6-N-acetylglucosamine, disperses mature biofilm and is an agent potentially useful in antibiofilm therapy (29). Inhibitors of N-acetyl-D-glucosamine-1-phosphate acetyltransferase that catalyzes the biosynthesis of UDP-N-acetylglucosamine, i.e., a precursor of PIA, also inhibit biofilm formation (3). 1,2,3,4,6-Penta-O-galloyl-␤-D-glucopyranose (PGG) (Fig. 1) is an active antioxidative ingredient in geranium (27). In addition to inducing p53 expression and inhibiting STAT3 in prostate cancer cells, PGG suppresses the growth of prostate xenograft tumor in a nude mouse model (18). Meanwhile, PGG resembles insulin in that it activates insulin receptor to stimulate glucose transport in adipocytes and functions as an effective antidiabetic drug (21, 30). This compound is also a vasorelaxant that dilates vascular smooth muscle and suppresses NO/ cGMP signaling (19). Plant materials that contain PGG have been used frequently in Chinese medicine to treat inflammation (16). The present study demonstrates that PGG, either in a solution or coated on solid surfaces, inhibits biofilm formation by S. aureus by inhibiting bacterial attachment and PIA formation.

Staphylococcus aureus forms biofilms on medical devices and causes pneumonia, meningitis, endocarditis, osteomyelitis, and septicemia (13, 14). Formation of biofilm by S. aureus is closely associated with the synthesis of an extracellular polysaccharide substance (EPS), polysaccharide intercellular adhesin (PIA), which is a ␤-1,6-linked N-acetyl (succinyl) glucosamine polymer synthesized by enzymes encoded by the ica operon (7, 9, 31). In addition, the proteins of S. aureus that contribute to biofilm formation include fibronectin-binding proteins A and B (24), collagen-binding protein (40), clumping factor A and B (4), SasG surface protein (6), and the biofilm-associated protein Bap (28, 37). Meanwhile, the pertinacious form of biofilm formation is inhibited by extracellular proteases produced by the organism (35). These examples illustrate that factors on the bacterial surface facilitate attachment of bacteria and establishment of multilayered cell clusters on a solid surface, which are crucial to biofilm formation (13). Owing to the high resistance to antibiotics of biofilm-embedded staphylococci (2, 20), biofilm-associated infections are extremely difficult to treat, necessitating the development of drugs that prevent and destroy biofilm. Earlier investigations identified several substances that are useful for preventing the formation or removal of staphylococcal biofilms. For example, * Corresponding author. Mailing address for S.-T. Liu: Molecular Genetics Laboratory, Department of Microbiology and Immunology, Chang-Gung University, Kwei-Shan, Taoyuan 333, Taiwan. Phone and fax: 886-3-2118292. E-mail: cgliu@mail.cgu.edu.tw. Mailing address for Y.-C. Wu: Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan. Phone: 886-7-3121101, ext. 6986, 2197. Fax: 886-7-3223170. E-mail: yachwu@kmu.edu.tw. † Supplemental material for this article may be found at http://aac .asm.org/. 䌤 Published ahead of print on 20 December 2010.

MATERIALS AND METHODS Bacterial strains and media. S. aureus SA113 (ATCC 35556) is a strain producing biofilm. A mutant derived from this strain, S. aureus SA113⌬ica (9), contains a deletion in the ica operon and does not produce PIA. Clinical strains SA13, SA33, SA41, SA285, SA288, and SA289 are sensitive to methicillin (MSSA); strains SA44, SA130, SA435, SA486, SA703, and SAChu are resistant to methicillin (MRSA). These strains were isolated from Chang Gung Memorial Hospital. The ability of S. epidermidis ATCC 35547 and S. epidermidis RP62A

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FIG. 1. Structure of 1,2,3,4,6-penta-O-galloyl-␤-D-glucopyranose (PGG).

(ATCC 35984) (5) to form biofilm was also tested by the present study. S. carnosus TM300 (12, 32), which does not form biofilm, was used as the negative control. The organisms were cultured in tryptic soy broth (Oxoid) containing 0.5% glucose (TSBg). Chemicals. PGG was purified from 680 g of Eustigma oblongifolium (Hamamelidecae), which was collected from Kaohsiung County, Taiwan. A voucher specimen (Eustigma-01) was deposited at the Graduate Institute of Natural Products, Kaohsiung Medical University. Dry stems of E. oblongifolium were extracted with methanol and ethyl acetate. The extract was purified by silica gel and Sephadex LH-20 chromatography. The structure and purity of PGG were verified by mass and nuclear magnetic resonance spectrometry (36). Iodoacetamide (IDA), N-phenyl maleimide (NPM), and NAC were purchased from Sigma-Aldrich (St. Louis, MO). PGG and NPM were dissolved in dimethyl sulfoxide (DMSO). IDA and NAC were dissolved in sterile distilled water. These chemicals were prepared as a 20 mM stock and stored at ⫺20°C before use. Culturing conditions and counting bacterial cells. An overnight culture of S. aureus was diluted 200-fold with TSBg, 200 ␮l of which was seeded in wells in a 96-well polystyrene microtiter plate, followed by incubation at 37°C for 6 h. The cell density was determined at A578 with a microtiter plate reader (SpectraMax 340; Molecular Devices). To enumerate the numbers of viable cells, cells attached to the well surface were washed with phosphate-buffered saline (PBS) and suspended in PBS by pipetting. The cell suspension was sonicated and subsequently plated on TSBg agar according to a method described earlier (34). Biofilm was also formed on the surface of 13-mm-diameter polycarbonate discs (Thermanox; Nalgene Nunc International) that were placed in a 24-well microtiter plate, which was seeded with 1 ml of bacterial culture in each well. Cells were washed off the polycarbonate surface and enumerated according to method described above. Screening compounds and biofilm assay. S. aureus SA113 cells were seeded in 96-well microtiter plates. Compounds purified from medicinal plants were added to each well at a final concentration of 100 ␮M. At 6 h after seeding, the amount of biofilms formed in the wells was determined by using a crystal violet staining method (8). Cells treated with either distilled water or DMSO were used as a control. The amount of biofilm formation by the control group was set at 100%. Each experiment was repeated at least three times, with the samples in each experiment prepared in six wells. Moreover, the concentration that inhibited formation of 50% biofilms (IB50) was calculated based on logistic regression analysis results. Adherence assay. PGG was added at 0, 0.5, 1, 1.5, and 2 h after seeding to S. aureus culture in a 96-well polystyrene microtiter plate. The amount of biofilm formation in the wells was determined at 6 h after seeding using a crystal violet staining method. Meanwhile, cells adhering to the polystyrene and polycarbonate surfaces at 60 min after seeding were washed with PBS and stained with Syto 9 (Invitrogen), a green florescence dye that stains nucleic acids. Cells were then observed under a fluorescence microscope. Cell viability assay. HepG2 and 293T cells were cultured in Dulbecco modified Eagle medium containing 10% (vol/vol) fetal calf serum. MRC-5 and HEp-2 cells were cultured in Eagle minimum essential medium that contained 10% fetal calf serum. Cells (105 in 500 ␮l of medium) were seeded in the wells in 24-well polystyrene tissue culture plates. After incubation at 37°C for 24 h, PGG was

ANTIMICROB. AGENTS CHEMOTHER. added to each well. The toxicity of PGG to cells was tested by using the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method (27) at 24 h after treatment with PGG. In addition, toxicity of PGG to 293T cells were also determined in medium containing 0% and 2% fetal calf serum. Cells treated with DMSO were used as a negative control. Detection of PIA. PIA was extracted from cells cultured in a petri dish according to a method described elsewhere (10). The extract was blotted onto polyvinylidene difluoride membrane (Millipore) by using a 96-well dot blot apparatus. After blotting, the membrane was dried and soaked in a solution containing 3% bovine serum albumin and 0.05% Tween 20 in PBS. The membrane was then incubated at room temperature for 1 h in a solution containing 0.8 ␮g of wheat germ agglutinin conjugated to biotin (WGA-biotin; Sigma-Aldrich)/ml. After four washes with PBS, the amount of PIA was determined by using horseradish peroxidase (HRP)-conjugated streptavidin (Pierce), followed by chemiluminescence detection according to a method described elsewhere (23, 34). To detect PIA in culture medium, the medium was concentrated by using Amicon-Ultra4 centrifuge filters (Millipore, Billerica, MA) prior to extraction. The relative PIA amount was determined by quantifying the intensity of each spot on the blot using a densitometer (LAS-3000; Fujifilm). The amount of PIA from cells untreated with PGG was set at 100%. SEM. Biofilms were grown on polycarbonate discs. After 6 h of incubation, the discs were washed three times with PBS to remove planktonic cells and prepared for scanning electron microscopy (SEM) examination as described elsewhere (38). Samples were analyzed with a Hitachi S-5000 scanning electron microscope. RNA isolation and real-time RT-PCR. Bacterial cells were suspended in 0.5 mg of lysostaphin (Sigma-Aldrich)/ml and incubated at 37°C for 15 min. RNA was then isolated and purified by using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. The icaA transcript was quantified by using real-time reverse transcription-PCR (RT-PCR; LightCycler; Roche, Mannheim, Germany). The icaA transcript was reverse transcribed and amplified using the primers A1 (5⬘-GTGCAGTTGTCGACGTTGGCTACT-3⬘) and B1 (5⬘-TTGA GCCCATCTCACGCGTTGC-3⬘). The gyrB mRNA, which was used as an internal control to normalize the amount of icaA mRNA, was reverse transcribed and amplified using the primers F1 (5⬘-ACGGATAACGGACGTGGTATC CCA) and R1 (5⬘-GCCACCGCCGAATTTACCACCA-3⬘). In addition, amplified products were detected by using a Light Cycler-RNA Amplification Kit Cyber Green 1 (Roche). Reactions were performed as follows: denaturation for 1 cycle at 95°C for 5 min and for 45 cycles at 95°C for 30 s, 62°C for 30 s, and 72°C for 20 s. To monitor the specificity of RT-PCR, the PCR products were analyzed by melting-curve analysis. Coating of PGG and inhibition of S. aureus biofilm formation on polystyrene and silicon rubber. PGG was coated on the surface of the wells in 96-well polystyrene microtiter plates with 100 ␮l of solution, which contained 60% of PGG in various concentrations and 40% of polyaniline. The polyaniline concentration was kept constant in all samples. The plate was then dried in a vacuum chamber. Overnight culture of S. aureus SA113 was diluted 200-fold in 200 ␮l of TSBg, added to the PGG-coated wells of the microtiter plate, and cultured at 37°C for 24 h. After incubation, the wells were washed twice with PBS, and biofilm formed on the coated wells was determined by the safranin staining method (17). PGG was also coated on 6-mm-diameter silicon rubber discs, which were cut from a sheet of 2-mm-thick silicon rubber with a paper punch. Next, the silicon rubber surface was coated with PGG according to the above-mentioned method. After coating, the plates and discs were sterilized under UV for 30 min. Silicon rubber discs were placed in a 48-well microtiter plate that was seeded with 500 ␮l of bacterial solution. The amount of biofilms formed on the surface of silicon rubber at 24 h after incubation was determined by using the safranin staining method.

RESULTS Screening compounds that inhibit biofilm formation by S. aureus. A total of 48 compounds isolated from plants commonly used in Chinese medicine were screened for their activity to prevent biofilm formation by S. aureus SA113 at 6 h after seeding the bacteria in a 96-well polystyrene microtiter plate. According to screening results, at 100 ␮M, most of the compounds did not inhibit either bacterial growth or biofilm formation. However, several compounds, e.g., AN-3, AN-4, and AN-9, not only exhibited strong antibiofilm activity, reducing biofilm formation by ⬎94%, but also inhibited bacterial growth

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TABLE 1. Screening of compounds that inhibit biofilm formation by S. aureus SA113 in broth culturea 100 ␮M

Compound PGG AN-3 AN-4 AN-9 CSBM4-2 CSBM5-4 a

TABLE 2. Inhibition of biofilm production by S. aureus clinical strains and S. epidermidis by PGGa

10 ␮M

Strain

Growth

Biofilm

Growth

Biofilm

69 ⫾ 5 15 ⫾ 10 40 ⫾ 3 9⫾1 79 ⫾ 19 75 ⫾ 7

2⫾0 0⫾2 6⫾3 2⫾2 8⫾5 27 ⫾ 32

109 ⫾ 8 28 ⫾ 17 37 ⫾ 23 112 ⫾ 11 107 ⫾ 1 101 ⫾ 10

5⫾1 6⫾4 5⫾9 82 ⫾ 18 87 ⫾ 7 101 ⫾ 7

Values are mean percentages ⫾SD.

by ⬎60% (Table 1). CSBM5-4 inhibited bacterial growth by 25% and prevented biofilm formation by 73% (Table 1). PGG (Fig. 1) and CSBM4-2 reduced biofilm formation by 98 and 92% and inhibited bacterial growth by 31 and 21%, respectively (Table 1). At 10 ␮M, PGG was the only compound that did not kill S. aureus SA113 but strongly inhibited biofilm formation; after 6 h of incubation, 10 ␮M PGG inhibited biofilm formation by 95% (Table 1). Inhibition of biofilm formation on polystyrene and polycarbonate surfaces by PGG. Exactly how PGG affects the growth and biofilm formation by S. aureus SA113 was more closely examined by adding PGG to the culture during seeding to polystyrene microtiter plates. After incubation for 6 h, PGG at concentrations below 50 ␮M did not affect the viability of S. aureus SA113 (Fig. 2A). However, PGG at 6.25 and 12.5 ␮M inhibited biofilm formation 93 and 96%, respectively (Fig. 2B); at 25 ␮M, the inhibition increased to 97% (Fig. 2B). Next, the incubation period was extended to 24 h. According to those results, the inhibition persisted and the amount of biofilms detected at 24 h was approximately equal to those observed at 6 h (Fig. 2B). Our results further demonstrated that PGG inhibited biofilm formation on a polycarbonate surface. PGG at 6.25 and 12.5 ␮M inhibited biofilm formation by 75 and 96%, respectively, at 6 h after seeding (Fig. 2C). At 25 ␮M, the inhibition increased to 99% (Fig. 2C). Similar levels of inhibition by PGG on polycarbonate surface were also observed after incubation was extended to 24 h (Fig. 2C). The present study also examined whether PGG influenced biofilm formation by 12 S. aureus clinical strains. At 12.5 ␮M,

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Growth (A578)

Biofilm (A595)

0 ␮M

12.5 ␮M

0 ␮M

12.5 ␮M

0.68 ⫾ 0.02 0.69 ⫾ 0.02

0.65 ⫾ 0.03 0.70 ⫾ 0.01

6.31 ⫾ 0.34 0.12 ⫾ 0.01

0.26 ⫾ 0.28 0.10 ⫾ 0.01

0.50 ⫾ 0.01

0.42 ⫾ 0.01

1.42 ⫾ 0.09

0.15 ⫾ 0.11

0.57 ⫾ 0.09

0.56 ⫾ 0.09

6.43 ⫾ 0.04

4.18 ⫾ 0.54

MSSA SA13 SA33 SA41 SA285 SA288 SA289

0.69 ⫾ 0.09 0.79 ⫾ 0.01 0.65 ⫾ 0.02 0.56 ⫾ 0.09 0.55 ⫾ 0.01 0.77 ⫾ 0.02

0.67 ⫾ 0.06 0.79 ⫾ 0.04 0.61 ⫾ 0.05 0.46 ⫾ 0.03 0.50 ⫾ 0.01 0.72 ⫾ 0.07

0.98 ⫾ 0.13 0.80 ⫾ 0.07 1.67 ⫾ 0.27 0.86 ⫾ 0.09 0.83 ⫾ 0.02 1.16 ⫾ 0.01

0.12 ⫾ 0.04 0.13 ⫾ 0.03 0.36 ⫾ 0.05 0.16 ⫾ 0.02 0.10 ⫾ 0.01 0.35 ⫾ 0.01

MRSA SA44 SA130 SA435 SA486 SA703 SAChu

0.66 ⫾ 0.08 0.61 ⫾ 0.12 0.64 ⫾ 0.10 0.76 ⫾ 0.08 0.74 ⫾ 0.10 0.56 ⫾ 0.02

0.64 ⫾ 0.04 0.62 ⫾ 0.17 0.67 ⫾ 0.09 0.65 ⫾ 0.02 0.70 ⫾ 0.07 0.52 ⫾ 0.04

1.33 ⫾ 0.01 3.05 ⫾ 0.10 0.97 ⫾ 0.09 1.94 ⫾ 0.19 2.33 ⫾ 0.02 8.06 ⫾ 0.10

0.46 ⫾ 0.01 0.32 ⫾ 0.04 0.46 ⫾ 0.06 0.26 ⫾ 0.07 0.88 ⫾ 0.07 0.77 ⫾ 0.11

Staphylococcus spp. S. aureus SA113b S. carnosus TM300c S. epidermidis ATCC 35547 S. epidermidis RP62A

Values are the mean inhibition at the indicated PGG concentration ⫾ SD. S. aureus SA113 is a biofilm-producing strain used as a positive control. c S. carnosus TM300 cannot form biofilm and was thus used as a negative control. a b

PGG, while not influencing the growth of these strains after 6 h of culturing, inhibited their capacity to produce biofilms (Table 2). Notably, 12.5 ␮M PGG did not reduce biofilm formation by strains SA289, SA44, SA435, and SA703 as much as the other strains did; 50 ␮M PGG reduced biofilm formation by 83 to 97% in these four strains (data not shown). Moreover, PGG at 12.5 ␮M reduced 90 and 35% of biofilm formation by S. epidermidis ATCC 35547 and S. epidermidis RP62A, respectively (Table 2). Toxicity of PGG to human cells. The toxicity of PGG to 293T, HepG2, HEp-2, and MRC-5 cells was tested by using an MTT-based colorimetric method (27). According to these results, PGG did not affect the viability of 293T, HepG2, HEp-2, and MRC-5 cells at concentrations below 50 ␮M (see Fig. S1A

FIG. 2. Effects of PGG on bacterial growth and biofilm formation. (A) S. aureus SA113 was cultured in TSBg broth that contains PGG in 96-well microtiter plates at 37°C for 6 h (f) and 24 h (䡺). The cell density was determined at A578. (B) The amount of biofilm formation in the well at 6 h (f) and 24 h (䡺) after seeding was determined at A595 after crystal violet staining. (C) Biofilm formed on polycarbonate discs after 6 h (f) and 24 h (䡺) of incubation. (D and E) The inhibition of S. aureus SA113 (f) and S. aureus SA113⌬ica (u) biofilm formation by PGG in polystyrene wells (D) and polycarbonate discs (E) was also examined. The amount of biofilm formed by S. aureus SA113 untreated with PGG was set at 100%. Experiments were performed three times, and each sample in the experiment was prepared in six wells. The error bar represents the standard error.

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TABLE 3. IB50 values of different compounds on biofilm formation by S. aureus SA113 Compound

IB50 (␮M)a

Cell growth (%)b

PGG NPM IDA NAC

3.6 41.9 120.4 6381.8

100 66 50 88

a IB50 is calculated from the results of the logistic regression equation, which was defined as the concentration of a compound that inhibited 50% biofilm formation. b That is, the percentage of growth in relation to the untreated control at the concentration of IB50.

in the supplemental material). Furthermore, serum concentrations in the culture medium did not appear to influence the toxicity of PGG toward 293T cells (Fig. S1B and C in the supplemental material). Comparison of the efficacies of antibiofilm compounds. S. aureus SA113 was treated with IDA, NPM, NAC, and PGG for 6 h to compare their effectiveness in preventing biofilm formation. According to these results, the IB50 values of NPM, IDA, and NAC were substantially higher than that of PGG (Table 3). The IB50 value of PGG, NPM, IDA, and NAC was 3.6, 41.9, 120.4, and 6381.8 ␮M, respectively (Table 3). Prevention of bacterial adherence to solid surface by PGG. Instead of inhibiting bacterial adherence to a solid surface during the initial stage of biofilm formation, PIA facilitates the accumulation and aggregation of bacterial cells in the biofilm. Consequently, a mutant that is defective in PIA synthesis, S. aureus SA113⌬ica, forms a thin biofilm in microtiter plates (9). As anticipated, the mutant strain formed 50% less biofilm than S. aureus SA113 did (Fig. 2D). Although 1.56 ␮M PGG did not affect the biofilm formation by the mutant, 3.13 ␮M PGG decreased the amount of biofilm by 72% (Fig. 2D). Similar levels of inhibition were also observed on polycarbonate surface; S. aureus SA113⌬ica produced about half of the amount of biofilm produced by S. aureus SA113 (Fig. 2E). At 3.13 and 12.5 ␮M, PGG decreased the amount of biofilm formed by S. aureus SA113⌬ica by 67 and 96%, respectively (Fig. 2E). These

results showed that PGG likely inhibits the adherence of cells to a solid surface during the initial stage of biofilm formation. Furthermore, 6.25 and 12.5 ␮M PGG were added to S. aureus SA113 culture in a 96-well polystyrene microtiter plate at 0, 0.5, 1.0, 1.5, and 2 h after seeding. The formation of biofilm on the surface was evaluated at 6 h after seeding. Notably, biofilm formation was reduced by 62 and 19% when 6.25 ␮M PGG was added at 0.5 and 1 h after seeding, respectively (Fig. 3A). The addition of 12.5 ␮M PGG at 0.5 and 1 h after seeding inhibited biofilm formation by ⬎90% (Fig. 3A). Moreover, formation of biofilm was no longer inhibited if 6.25 or 12.5 ␮M PGG was added at 1.5 and 2 h after seeding (Fig. 3A). The results indicated that the timing of adding PGG is critical to the inhibition of biofilm formation, suggesting that PGG interferes with the initial attachment of S. aureus SA113 to polystyrene. The present study further determined the number of cells attached to the polystyrene microtiter plates and polycarbonate discs. After incubation of the cells for 20, 40, and 60 min, the number of cells adhered to the well in the DMSO-treated control was 4.1 ⫻ 105, 6.4 ⫻ 105, and 7.8 ⫻ 105 CFU, respectively (Fig. 3B). However, PGG at 6.25 ␮M reduced the number of cells attached to the plate to 2.1 ⫻ 105, 2.05 ⫻ 105, and 1.8 ⫻ 105 CFU (Fig. 3B); 12.5 ␮M PGG decreased the number of cells to 3.7 ⫻ 104, 2.3 ⫻ 104, and 1.7 ⫻ 104 CFU, respectively. Similar inhibition was also observed on polycarbonate discs (Fig. 3C). PGG at 6.25 and 12.5 ␮M substantially reduced the number of cells adhered to the surface at 20, 40, and 60 min after seeding (Fig. 3C). The number of cells adhered to polycarbonate in the DMSO-treated control was 7 ⫻ 105, 1.1 ⫻ 106, and 2 ⫻ 106 CFU at 20, 40, and 60 min after seeding, respectively (Fig. 3C). PGG at 6.25 ␮M reduced the number of cells attached to the surface to 2.9 ⫻ 105, 2.3 ⫻ 105, and 3.5 ⫻ 105 CFU; 12.5 ␮M PGG decreased the number of cells to 1.0 ⫻ 105, 8 ⫻ 104, and 5 ⫻ 104 CFU, respectively (Fig. 3C). The cells adhering to the polystyrene wells and polycarbonate discs were also observed using fluorescence staining after treating them with 0, 6.25, and 12.5 ␮M PGG for 60 min. The treatment significantly reduced the number of cells attached to polystyrene and polycarbonate surfaces (Fig. 3D).

FIG. 3. Kinetics of the antiadherence activity of PGG. (A) S. aureus SA113 was seeded in a 96-well microtiter plate. PGG at 6.25 ␮M (f) and 12.5 ␮M (䡺) was added to the culturing medium at 0, 0.5, 1.0, 1.5, and 2 h after inoculation. The amount of biofilm formed in the wells was determined at 6 h after inoculation using a crystal violet staining method. The amount of biofilm that was formed by the cells treated with DMSO was set at 100%. Experiments were performed three times, and each sample in the experiment was prepared in six wells. The error bar represents the standard error. Bacterial cells were treated with 0 (f), 6.25 (䡺), and 12.5 ␮M (u) PGG for 20, 40, and 60 min. Cells adhered to the polystyrene wells (B) and polycarbonate discs (C) were washed and suspended in PBS. The number of cells was counted by plating on TSBg agar. (D) After treatment with 0 ␮M (a and d), 6.25 ␮M (b and e), and 12.5 ␮M (c and f) PGG for 60 min, the cells that adhered on the polystyrene wells (a to c) and polycarbonate discs (d to f) were visualized after staining with Syto 9 and examined under a fluorescence microscope.

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FIG. 5. Effect of PGG coating on biofilm formation. PGG was coated on the surface of the wells in a polystyrene microtiter plate (A) and silicon rubber discs (B) using polyaniline as a carrier. S. aureus was seeded in PGG-coated wells in polystyrene microtiter plate and the wells that contained a PGG-coated silicon rubber disc. At 24 h after incubation, the amount of biofilm formation in the wells and the surface of silicon rubber was determined by safranin staining. The biofilm that formed on the surface of polyaniline-coated wells and silicon rubber discs, which do not contain PGG, were used as 100%. Experiments were conducted three times, and each sample in the experiment was prepared in four wells and discs. Error bars denote the standard errors.

FIG. 4. Inhibition of PIA expression by PGG. SEM images of S. aureus SA113 cultured on polycarbonate discs in the absence (A) or presence (B) of 3.13 ␮M PGG for 6 h. The images shown were taken at a magnification of ⫻30,000. Bar, 1.0 ␮m. (C) S. aureus SA113 was cultured in TSBg that contained PGG. PIA was extracted from the cells (a) and culture medium (b) and detected by using WGA-biotin. After incubation with HRP-streptavidin, the spot was visualized by chemiluminescence detection. PIA produced by S. carnosus TM300, i.e., a strain that does not produce biofilm, was used as a control (NC). PIA produced by clinical isolates of S. aureus after PGG treatment for 6 h was also analyzed (F). (D) The relative intensity of each spot was quantified by using a densitometer. The amount of PIA from cells untreated with PGG was set at 100%. (E) S. aureus SA113 was treated with 0, 3.13, 12.5 and 50 ␮M PGG for 5 h. RNA was then isolated by using TRIzol. Transcription of icaA was determined by LightCycler quantitative RT-PCR. The amount of ica transcript was normalized to the amount of gyrB mRNA. The amount of ica mRNA in the cells untreated with PGG was set at 1.

Inhibition of EPS expression by PGG. As is well known, synthesis of EPS is essential to biofilm formation. According to our SEM study, cells untreated with PGG produced filaments that form a web structure (Fig. 4A), which likely consists of PIA (13, 15), on polycarbonate surfaces. However, treating

cells with 3.13 ␮M PGG caused the web structure to disappear (Fig. 4B). Based on this finding, we further analyzed PIA on bacterial surface after PGG treatment. Extracting PIA from cells cultured in TSBg that contained PGG revealed that the PIA expression was inhibited after PGG treatment (Fig. 4Ca). Evaluating the intensity of dots on the blot (Fig. 4Ca) using a densitometer revealed that the amount of PIA expressed on the cell surface decreased 7, 58, and 87% after treatment with 3.13, 12.5, and 50 ␮M PGG, respectively (Fig. 4Ca and D). PIA released into the culture medium also decreased after PGG treatment in a dose-dependent manner (Fig. 4Cb). Moreover, PGG at 12.5 and 25 ␮M inhibited PIA expression by clinical isolates of S. aureus (Fig. 4F). To determine whether the inhibition manifested at the transcriptional level, RNA was isolated from S. aureus SA113, which had been treated with 0, 3.13, 12.5, and 50 ␮M PGG for 5 h. Furthermore, the transcript of icaA was quantified by using LightCycler RT-PCR. We found that PGG at concentrations below 50 ␮M did not influence the transcription of icaA (Fig. 4E), indicating that PGG did not inhibit transcription of the ica operon. Inhibition of biofilm formation by PGG coated on surfaces of polystyrene and silicon rubber. Wells in 96-well polystyrene microtiter plates were coated with 0.5 to 5.7 ␮M PGG. The inhibition of biofilm formation by PGG was examined by staining with safranin. Experimental results indicated that the wells coated with PGG at concentration of 1.9 ␮M or lower only slightly affected biofilm formation (Fig. 5A). However, the amount of biofilm decreased by ⬎90% in wells that were coated with ⬎3.8 ␮M PGG (Fig. 5A). In addition, PGG was coated on the surface of silicon rubber, a material commonly found in catheters. The results showed that coating with 1.9 ␮M PGG inhibited biofilm formation by 62% (Fig. 5B). When the concentration reached 5.7 ␮M, PGG inhibited biofilm formation by 85% (Fig. 5B).

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DISCUSSION As the major cause of chronic infections, S. aureus forms biofilm on medical devices and implants (1, 23, 33). Because biofilm is extremely difficult to eliminate following its formation on the surface, developing drugs that inhibit or eliminate S. aureus biofilms is vital to solving clinical problems caused by biofilm. We screened here 48 compounds purified from medicinal plants to evaluate their efficacy in inhibiting biofilm formation by S. aureus. Among the compounds screened, only PGG does not kill S. aureus SA113 at concentrations below 50 ␮M (Fig. 2A), and yet it reduces biofilm formation by 93% at 6.25 ␮M (Fig. 2B). PGG also inhibits the biofilm formation by MSSA and MRSA clinical isolates and S. epidermidis strains (Table 2). The inhibitory effect of PGG on biofilm formation by these strains apparently does not correlate with susceptibility to clinical relevant antibiotics, such as methicillin (Table. 2). We also examined how PGG inhibits biofilm formation on polystyrene and polycarbonate surfaces. These two materials are both hydrophobic, possibly explaining our finding that PGG is equally effective in preventing biofilm formation on these two different materials. In addition, PGG inhibits biofilm formation on silicon rubber, i.e., a material commonly used in catheters, demonstrating that PGG is potentially useful for coating medical devices to prevent biofilm formation. Our study also finds that PGG inhibits the attachment of S aureus SA113 on glass coverslips (data not shown), suggesting that PGG also prevents biofilm formation on hydrophilic surfaces. This study demonstrates that PGG likely inhibits the formation of S. aureus SA113 biofilm during the initial attachment stage because PGG is effective only when it is added to a medium within 1 h after seeding (Fig. 3). Because biofilm formation starts from cell attachment, the observations described above indicate that PGG inhibits the initial attachment of the cells to a solid surface. Additional evidence, which supports the notion that PGG inhibits initial cell attachment to a solid surface, comes from the results made on a mutant strain, S. aureus SA113⌬ica. Although not producing PIA, which mediates cell-to-cell adhesion and cell aggregation during biofilm formation, this mutant strain can adhere to a solid surface but form biofilms at a reduced level (9). Our results further demonstrate that PGG inhibits biofilm formation by S. aureus SA113⌬ica in a dose-dependent manner (Fig. 2), implying that PGG inhibits primary attachment ability of the cell. Although PGG inhibits the attachment of S. aureus to a solid surface at the onset of biofilm formation, our results also indicate that PGG reduces the amount of PIA produced by S. aureus strains in a dose-dependent manner (Fig. 4). SEM and biochemical assay verify that PIA production by the biofilm cells is markedly reduced after PGG treatment (Fig. 4). Such a decrease cannot be attributed to the possible promotion of PIA release from the cell surface to the medium by PGG because PIA does not increase in the culture medium after PGG treatment (Fig. 4). The reduced PIA synthesis is also not attributed to the repression of ica operon at the transcriptional level since quantitative RT-PCR reveals that PGG treatment influences little of the amount of ica mRNA expressed by the cells (Fig. 4). Thus far, exactly how PGG affects PIA synthesis remains unclear. Contamination of medical implants by microorganism is a

major risk of bloodstream infection and urinary tract infection (22). Therefore, strategies have been developed that involve coating clinical materials with antimicrobial substances, e.g., triclosan and dispersin B (11), to prevent microbial colonization. Despite the anti-infection and antibiofilm effects of coating, developing resistance to these substances by bacteria may cause adverse consequences. The present study demonstrates the usefulness of polyaniline in coating PGG on polystyrene and silicon rubber, indicating the effectiveness of the coating in preventing biofilm formation. As a natural product purified from a medicinal plant, PGG is commonly used in Chinese medicine. Although this compound is a strong antioxidant (Fig. 1), its antioxidation activity may not be the only factor influencing biofilm formation. Our study indicates that treating S. aureus with other strong antioxidants, such as (⫺)-epigallocatechin gallate and ascorbic acid, does not influence biofilm formation (data not shown). Our results further demonstrate that PGG is far more potent than IDA, NAC, and NPM (3), which have been shown to prevent biofilm formation by S. aureus (Table 3). Furthermore, PGG lacks toxicity to human epithelial and fibroblast cells at concentrations below 50 ␮M, indicating that PGG is highly promising for clinical use in preventing biofilm formation. ACKNOWLEDGMENTS We thank Friedrich Go ¨tz for his critiques, Chih-Jung Chen for providing the S. aureus clinical strains, and Wan-Chun Lai and HongWei Yang for their help in the preparation of PGG and polyaniline. This study was supported by grants from the Chang Gung Memorial Hospital (CMRPD170261) and the National Science Council of the Republic of China, Taiwan (NSC-99-2320-B-182-013-MY3). REFERENCES 1. Brady, R. A., J. G. Leid, J. H. Calhoun, J. W. Costerton, and M. E. Shirtliff. 2008. Osteomyelitis and the role of biofilms in chronic infection. FEMS Immunol. Med. Microbiol. 52:13–22. 2. Brown, M. R., D. G. Allison, and P. Gilbert. 1988. Resistance of bacterial biofilms to antibiotics: a growth-rate related effect? J. Antimicrob. Chemother. 22:777–780. 3. Burton, E., et al. 2006. Antibiofilm activity of GlmU enzyme inhibitors against catheter-associated uropathogens. Antimicrob. Agents Chemother. 50:1835–1840. 4. Cheung, A. L., and V. A. Fischetti. 1991. The role of fibrinogen in mediating staphylococcal adherence to fibers. J. Surg. Res. 50:150–155. 5. Christensen, G. D., et al. 1982. Nosocomial septicemia due to multiply antibiotic-resistant Staphylococcus epidermidis. Ann. Intern. Med. 96:1–10. 6. Corrigan, R. M., D. Rigby, P. Handley, and T. J. Foster. 2007. The role of Staphylococcus aureus surface protein SasG in adherence and biofilm formation. Microbiology 153:2435–2446. 7. Cramton, S., and F. Go ¨tz. 2004. Biofilm development in Staphylococcus, p. 64–84. In M. A. Ghannoum and G. A. O. Toole (ed.), Microbial biofilms. ASM Press, Washington, DC. 8. Cramton, S. E., C. Gerke, and F. Go ¨tz. 2001. In vitro methods to study staphylococcal biofilm formation. Methods Enzymol. 336:239–255. 9. Cramton, S. E., C. Gerke, N. F. Schnell, W. W. Nichols, and F. Go¨tz. 1999. The intercellular adhesion (ica) locus is present in Staphylococcus aureus and is required for biofilm formation. Infect. Immun. 67:5427–5433. 10. Cramton, S. E., M. Ulrich, F. Go ¨tz, and G. Do ¨ring. 2001. Anaerobic conditions induce expression of polysaccharide intercellular adhesin in Staphylococcus aureus and Staphylococcus epidermidis. Infect. Immun. 69:4079–4085. 11. Darouiche, R. O., M. D. Mansouri, P. V. Gawande, and S. Madhyastha. 2009. Antimicrobial and antibiofilm efficacy of triclosan and DispersinB combination. J. Antimicrob. Chemother. 64:88–93. 12. Gotz, F. 1990. Staphylococcus carnosus: a new host organism for gene cloning and protein production. Soc. Appl. Bacteriol. Symp. Ser. 19:49S–53S. 13. Go ¨tz, F. 2002. Staphylococcus and biofilms. Mol. Microbiol. 43:1367–1378. 14. Go ¨tz, F., and G. Peters. 2000. Colonization of medical devices by coagulasenegative staphylococci, p. 55–88. In F. A. Waldvogel and A. L. Bisno (ed.), Infections associated with indwelling medical devices, 3rd ed. ASM Press, Washington, DC. 15. Hall-Stoodley, L., et al. 2008. Characterization of biofilm matrix, degradation

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