Bacterial strain and cultures
A viscous material producing clinical isolate of P. intermedia, which was isolated from a periodontitis lesion and designated as strain 17 , was used in this study. A total of 10 frozen culture stocks of isolated strain 17 were used in this study. Stock cultures of strain 17 in each vial were grown on trypticase soy blood agar plates (BAP) supplemented with 0.5% yeast extract (Difco Laboratories, Detroit, MI), hemin (5 mg/l), L-cystine (400 mg/l) and vitamin K1 (10 mg/l) or grown in the enriched-TSB: trypticase soy broth (TSB; BBL Microbiology Systems, Cockeysville, ND) supplemented with 0.5% yeast extract, hemin (5 mg/l), L-cystine (400 mg/l) and vitamin K1 (10 mg/l). Bacterial cultures were grown anaerobically in an anaerobic chamber (ANX-3, Hirasawa, Tokyo, Japan) at 37°C in a 5% CO2, 10% H2, 85% N2 atmosphere.
Biofilm phenotype on strain 17 stock cultures
The ability to produce viscous materials in culture media and form meshwork-like structures on cell surfaces were used as criteria for distinguishing between "biofilm-forming" and "biofilm-non-forming" as described previously . We first examined whether strain 17 met the criteria for being a biofilm-forming bacterium, since more than a decade has passed when we first described the unique phenotypic characteristic of strain 17 for its ability to produce viscous material . Ten culture stocks were plated on BAP respectively and grown for 48 h anaerobically. Single colony from each culture stock was transferred to enriched-TSB and grown for 24 h as the seed culture. One hundred and fifty μl of this seed culture was transferred to enriched-TSB (15 ml) and grown for 48 h. The spent culture medium (550 μl) was put into a rotor, and the viscosity was measured as shearing stress between a rotor and a rotor shaft at 50 rpm, 20°C using a rotary viscometer (Toki-sangyo, Tokyo, Japan).
To examine cell surface structures, scanning electron microscopy (SEM) was performed. Bacteria grown on BAP for 48 h were collected on a piece of filter paper (Glass fiber GA55, Toyo Roshi, Tochigi, Japan), fixed with 2% glutaraldehyde in 0.1 M phosphate buffer for 2 h and 1% OsO4 in 0.1 M phosphate buffer for 1 h at 4°C, and dehydrated through an ethanol series and 2-methyl-2-propanol followed by platinum ion coating (E-1030, Hitachi, Tokyo, Japan). Specimens were examined with a scanning electron microscope (S-4800, Hitachi) at an accelerating voltage of 3 kV.
During the evaluation for the ability of our stock strain 17 cultures to form biofilms, one of the 10 stocks that we tested was a naturally-occurring variant that lacked the ability to form biofilms. A stock strain, designated as strain 17-2, produced neither viscous materials in culture medium nor cell surface-associated meshwork-like structures was obtained and considered as a biofilm-negative variant. These phenotypes were stably maintained in this variant strain despite repetitive passages in vitro or in animals (data not shown). The enzymatic activities of strains 17 and 17-2 were examined using the API ZYM system (bioMerieux, Marcy l'Etoile, France) and there was no significant difference regarding the production of enzymes (data not shown).
Biofilm formation assay
The ability to form biofilm was investigated for strains 17 and 17-2 using crystal violet microtiter plate assay. Briefly, the seed cultures of both strains were prepared as described above and diluted to an OD of 0.1 at 620 nm in the same medium. Next, 150 μl diluted culture was transferred to each of eight sterile polystyrene microtiter plate wells (IWAKI, Tokyo, Japan) per strain. Sterile enriched-TSB was used as a control. The plates were prepared in duplicate and incubated at 37°C for 24 and 48 h, respectively. Biofilm formation was quantified according to Mohamed et al. . This assay was repeated three times. A statistical analysis was performed using Student's t-test.
Sugar composition of viscous materials from strain 17 cultures
The exopolysaccharide was prepared from culture supernatants by the method of Campbell et al. . Briefly, P. intermedia strain 17 was grown at 37°C in enriched-TSB for 24 h. Supernatants were separated by centrifuging the liquid culture at 12,000 × g for 30 min, and sodium acetate was added to a final concentration of 5%. The mixture was stirred for 30 min at 22°C and the exopolysaccharide was isolated by ethanol precipitation from the reaction mixture. The ethanol-precipitated material was collected by centrifugation (18,200 × g for 15 min at 22°C), resolved in 5% sodium acetate, and treated with chloroform: 1-butanol (1: 5 by volume). Water-soluble and chloroform-butanol layer were separated by centrifugation, an equal amount of ethanol was added to the water-soluble layer (this procedure was repeated twice), and the ethanol-precipitated material was freeze-dried and stored at -80°C until use. Contaminated lipopolysaccharides (LPS) were removed from preparations according to the method of Adam et al. . The freeze-dried material was dissolved in distilled water (0.5 mg/ml), and Triton X-114 (MP Biomedicals, Eschwege, Germany) stock solution (lower detergent rich phase) was added to a final concentration of 1% (v/v). After cooling on ice for 30 min, the solution was stirred at 4°C for 30 min and incubated at 37°C until the separation into two layers was complete. The upper aqueous phase was recovered by centrifugation for 30 min (1,000 × g, 30°C). This Triton X-114 treatment was performed twice. The upper aqueous phase was extracted three times with 3 vol CHCl3/CH3OH (2: 1 by volume) to remove detergent. The aqueous phase was concentrated under reduced pressure and freeze-dried. The contaminated-LPS level was measured by Limulus Amebocyte Lysate test according to the manufacturer's protocol (Seikagaku-kogyo, Tokyo, Japan).
The sugar composition of the purified viscous material were determined by means of HPLC for neutral and amino sugars and colorimetry for uronic acid. Briefly, neutral monosaccharides were released from purified exopolysaccharide (5 mg) by hydrolysis in a sealed tube with 2 N trifluoroacetic acid (200 μl) at 100°C for 6 h. The hydrolysate was concentrated in vacuo and dissolved in 500 ml of distilled water. The sugars were identified by HPLC (LC-9A, Shimadzu, Kyoto, Japan) with a TSK-gel sugar AXG column (15 cm × 4.6 mm) (Tosoh, Tokyo, Japan) using 0.5 M potassium tetraborate buffer (pH 8.7) as a carrier at a flow rate of 0.4 ml/min and a column temperature of 70°C. Amino sugars were released from purified exopolysaccharide (5 mg) by hydrolysis in a sealed tube with 4 N HCl (200 μl) at 100°C for 6 h. The hydrolysates were analyzed by HPLC (LC-9A, Shimadzu).
Transmission electron microscopy of purified viscous materials
For negative staining, the ethanol precipitated viscous material was dissolved in distilled water (1 mg/ml). Fifteen microliters of the sample was deposited onto a formvar-coated and carbon-stabilized copper grid. After 1 min, excess fluid was removed with filter paper strips, stained with 2% uranyl acetate for 1 min, and examined in a transmission electron microscope (TEM) (H7100, Hitachi, Tokyo, Japan) at 100 kV.
To create a whole-genome microarray for P. intermedia strain 17, 30 perfect-matched and 30 miss-matched 24-mer probes were designed for all putative open reading frames (ORFs) (2,816 ORFs/array) from a whole genome sequence of P. intermedia strain 17, which is available from the Institute for Genomic Research data base (TIGR) using a Maskless Array Synthesizer (NimbleGen Systems Inc., Madison, WI, USA).
To determine an appropriate time point for total RNA isolation from the cultures of strains 17 and 17-2, morphological changes of cell surface structures associating with growth were examined by SEM. Single colony of Strains 17 and 17-2 grown on BAP for 24 h were inoculated into enriched-TSB and grown for 24 h as the seed culture. Five ml of this seed culture was used to inoculate 500 ml of enriched-TSB. The growth of the culture was monitored by measuring the absorbance at the wavelength of 600 nm. The morphology of cultured cells at a different stage of growth was examined by SEM as described above. RNA isolation was performed at a time point (12 h) when the surface-associated meshwork-like structure had begun to form. Total RNA samples were extracted from 12 h cultures of strains 17 and 17-2 using RNeasy Midi Kit (QIAGEN, Tokyo, Japan) according to the manufacturer's protocol. Samples were quantified and checked for purity using an Agilent 2100 bioanalyzer (Agilent, Hachioji, Japan). Total RNA (12 μg) was primed with random primer (Invitrogen, Tokyo, Japan), and cDNA was synthesized with reverse transcriptase (Superscript II, Invitrogen). The resulting cDNA was fragmented with DNase I (Promega, Madison, WI, USA) and labeled with biotin using terminal deoxynucleotidyl transferase (Promega). Biotin-labeled samples were hybridized onto the strain 17 microarray at 45°C for 16-20 h using NimbleGen's Hybriwheel Hybridization chambers (NimbleGen Systems Inc.).
To compare gene expression profiles of strain 17 in solid and liquid culture conditions, seed cultures of strain 17 were newly prepared as described above. Five ml of this seed culture was transferred to enriched-TSB (500 ml) and 200 μl of the seed cultures was transferred to each of 50 BAPs. Both cultures were incubated for 12 h anaerobically. Total RNA was isolated from the liquid cultures as described above. Two hundred μl of PBS was added to BAPs to harvest growing cells using cell scrapers (IWAKI). Cell suspensions were washed twice with PBS and total RNA was isolated as described above.
Microarray image acquisitions and data analyses
Hybridized-microarray slides containing technical duplicates were imaged with a high resolution array scanner (GenePix 4000B Microarray Scanner, Molecular Devices Corp., Sunnyvale, CA, USA) and the fluorescent signal intensities from each spot were quantified using NimbleScan Software (NimbleGen Systems Inc.). Normalization was performed among four microarray hybridization data sets by means of Robust Multi-chip analysis algorithm  and statistical analyses were performed using t-test and Bonferroni adjustment in the Roche-NimbleGen Microarray soft wears (Roche Diagnostics, Tokyo, Japan). When the individual probes met the criteria that the average signals from the culture of biofilm-positive strain versus the average signals from biofilm-negative strain were different by at least twofold with statistic significance, probes selected were used to find up-regulated regions.
Pertinent information on raw data containing experimental designs and hybridization results for specific oligonucleotide sets is available in CIBEX database .
Quantitative real-time RT-PCR
To confirm the up-regulation of several genes in strain 17 recorded by the microarray, a real-time RT-PCR strategy was employed. Twelve hours cultures of strains 17 and 17-2 were prepared again and total RNA was isolated as described above. Real-time RT-PCR was performed according to the one-step RT-PCR protocol of iScript™ One-Step RT-PCR Kit with SYBR® Green (BIO-RAD Laboratories, Tokyo, Japan). Briefly, 50 ng of total RNA, 200 nM of forward and reverse primers for a target gene, and 25 μl of SYBR® Green RT-PCR Reaction Mix (BIO-RAD Laboratories) were added into a PCR tube containing one μl of iScript Reverse Transcriptase for One-Step RT-PCR. The PCR preparation was brought to a final volume of 50 μl with nuclease-free water (BIO-RAD Laboratories). As an internal control, RT-PCR for 16S rRNA was performed at 50°C for 10 min, 95°C for 5 min, followed by 35 cycles at 95°C for 10 sec and 64°C for 30 sec followed by melt curve analysis. Primers for 16S rRNA and several genes showing up-regulation in microarray assays were as follows: 16S rRNA: forward 5'-AGAGTTTGATCCTGGCTCAG-3', reverse 5'-AAAGGAGGTGATCCAGCC-3'; PIN 0281: forward 5'-TGAACGTAAGCCGCAGCTAC-3', reverse 5'-TTGTTCTTGGCGCAAAGCAG-3'; PINA1058: forward 5'-TGTGAACCCCGACGAAGTGG-3', reverse 5'-GTGCCTGCTGACCAGCATCT-3'; PINA1756: forward 5'-AATACAGCCTTCGAGGGTTT-3', reverse 5'-TTCGGTCAAGACAGTAGGGA-3'; PINA1797: forward 5'-TGAAGATTTGCGCTGTCAAG-3', reverse 5'-TAGCAGGAGTTTCTTCAGGT-3'; PINA1798: forward 5'-AGCGGAGCAGAAAGTAGGTG-3', reverse 5'-CAACAGCAAGAACGTCGCTT-3'; PINA2006: forward 5'-CTTGGAACAACGGGTACAGG-3', reverse 5-AAATCTCGCTTTGCGTCAGT-3'. Annealing temperatures were optimized for each primer pair by the use of melting curve analysis in which the melting curve starts at 55°C and ends at 90°C with temperature increment of 0.2°C and a hold time of 2 sec. The optimized annealing temperature for each target gene was 64.5°C for PIN 0281, 62.0°C for PINA1058, 64.5°C for PINA1756, 65.0°C for PINA1797, 58.7°C for PINA1798 and 57.6°C for PINA2006, respectively. The threshold cycle (CT) values were obtained for the reactions reflecting the quantity of the template in the sample. ΔCT for each gene was calculated by subtracting the calibrator gene 16S rRNA CT value from each of the target values represented the relative quantity of the target mRNA normalized to the level of the internal standard 16S rRNA mRNA level. The target mRNA levels in strains 17 and 17-2 were defined and compared.
To observe how the expression levels of these genes fluctuate through the culture period, single colony of strains 17 and 17-2 grown on BAP for 24 h were inoculated into enriched-TSB and grown for 24 h as the seed culture. One hundred and fifty μl of this seed culture was used to inoculate 15 ml of enriched-TSB. Total RNA samples were extracted from 6, 12, 18, 24 and 30 h cultures of strains 17 and 17-2 using RNeasy Midi Kit (QIAGEN) and applied to the real-time RT-PCR as described above. Changes of the target mRNA levels through the culture period were recorded by the strain.
The virulence of biofilm-forming strain 17 was compared with that of biofilm-non-forming variant strain 17-2 regarding abscess formation in mice. Bacterial strains were cultured in enriched-TSB for 24 h for strain 17-2 and 36 h for strain 17, respectively (early stationary phase; see Fig. 5). Five hundred μl of bacterial suspensions (106 to 1010 CFU/ml) was injected subcutaneously into the inguen of each BALB/c mouse (male, 4 weeks; 3 mice per strain). Changes of abscess lesions were recorded photographically using a camera (Nikon FIII, Nikon, Japan) set at a fixed magnification for five consecutive days.
To compare anti-phagocytic activity of strain 17 with that of strain 17-2, bacterial cells were co-cultured with polymorphonuclear leukocytes (PMNL) obtained from healthy human volunteers (n = 3; age 20–23 years) in accordance with institutional approved procedures. PMNLs were purified by a PolymorphprepTM (Axis-Shield PoC AS, Oslo, Norway) gradient and cultured in RPMI 1640 medium (Gibco BRL, Grand Island, NY) supplemented with 10% heat-inactivated fetal calf serum (FCS) (MultiSer, Trace Scientific Ltd, Melbourne, Australia) and 2 mM L-glutamine at 37°C in 5% CO2. Bacterial cell suspensions (1.5 × 106 CFU/ml) were prepared from strains 17 and 17-2 cultures as described in the animal studies. Three hundred μl of PMNLs (106 cells/ml) was dispensed into the wells of 24-well tissue culture plates (Becton Dickinson, Franklin Lakes, NJ). To these wells, 100 μl of bacterial suspension of different tested strains was added. After incubation for 60–90 min at 37°C, PMNLs co-cultured with bacterial cells were centrifuged at 8,000 × g at 4°C for 5 min and processed for transmission electron microscopy to determine the internalization of tested strains by PMNLs. Cell pellets were fixed with 2% glutaraldehyde in 0.1 M phosphate buffer for 2 h at 4°C, post-fixed with 1% OsO4 in 0.1 M phosphate buffer for 1 h at 4°C, and dehydrated through an ethanol series. Samples were embedded into Epon resin and ultrathin sections were prepared by a ultramicrotome (Ultracut, Leica, Tokyo, Japan). Ultrathin sections were placed on a copper grid, stained with uranyl acetate and lead citrate, and observed in a TEM (H7100, Hitachi).