Isolation of bacterial strains
The bacterial strains used in this study were initially obtained from an orange-colored sponge, Spongia officinalis, collected from Mazara del Vallo (Sicilia, Italy), from a depth of 10 m. The sponge sample was transferred soon after collection to a sterile falcon tube and transported under frozen condition to the laboratory for the isolation of associated microbes. The sponge was then mixed with sterile saline water and vortexed. A small fraction of the liquid was serially diluted up to 10-3 dilutions and then spread on plates of Tryptone Yeast agar (TY). The plates were incubated at 37°C for 2 days till growth of colonies was observed. Single bacterial colonies were isolated on the basis of distinct colony morphologies from the TY plates. Isolates were maintained on TY agar plates at 4°C until use.
The isolated bacteria were sub-cultured on M63 (minimal medium) agar plates and incubated at 37°C for two days. A loopful of the bacterial culture from each plate was inoculated into M63 broth (in duplicate), incubated at 37°C for 24 h and then centrifuged at 7000× g for 20 minutes to separate the cell pellets from the fermentation medium. The supernatants were filtered through 0.2 μm-pore-size Minisart filters (Sartorius, Hannover, Germany). To ensure that no cells were present in the filtrates, 100 μl were spread onto TY agar plates, and 200 μl were inoculated in separate wells in the microtiter plate.
Screening for bioactive metabolites for biofilm inhibition
Filtered supernatants from the marine sponge-associated isolates were used to perform the assay for biofilm formation. The method used was a modified version of that described by Djordjevic et al. . Overnight cultures of E. coli PHL628 strain grown at 37°C in M63K10 broth (M63 broth with kanamycine, 10 μg ml-1), were refreshed in M63K10 broth and incubated again at 37°C for 5 to 6 h. 200 μl of inocula were introduced in the 96 well polystyrene microtiter plate with an initial turbidity at 600 nm of 0.05 in presence of the filtered supernatants from the different marine sponge associated isolates. The microtiter plate was then left at 30°C for 36 h in static condition.
To correlate biofilm formation with planktonic growth in each well, the planktonic cell fraction was transferred to a new microtiter plate and the OD570 was measured using a microtiter plate reader (Multiscan Spectrum, Thermo Electron Corporation). To assay the biofilm formation, the remaining medium in the incubated microtiter plate was removed and the wells were washed five times with sterile distilled water to remove loosely associated bacteria. Plates were air-dried for 45 min and each well was stained with 200 μl of 1% crystal violet solution for 45 min. After staining, plates were washed with sterile distilled water five times. The quantitative analysis of biofilm production was performed by adding 200 μl of ethanol-acetone solution (4:1) to de-stain the wells. The level (OD) of the crystal violet present in the de-staining solution was measured at 570 nm. Normalized biofilm was calculated by dividing the OD values of total biofilm by that of planktonic growth. Six replicate wells were made for each experimental parameter and each data point was averaged from these six.
Identification and purification of anti-biofilm compound
144 ml of cell free bacterial broth cultures were extensively dialyzed against water for two days, using a membrane tube of 12000-14000 cut-off; this procedure allowed us to remove the large amount of glycerol in the bacterial broth as confirmed by 1H- 13C-NMR experiments recorded on lyophilized broth before and after dialysis; the inner dialysate (25 mg) was fractionated by gel filtration on Sepharose CL6B, eluting with water. Column fractions were analyzed and pooled according to the presence of saccharidic compounds, proteins and nucleic acids. Fractions were tested for carbohydrate qualitatively by spot test on TLC sprayed with α -naphthol and quantitatively by the Dubois method . Protein content was estimated grossly by spot test on TLC sprayed with ninhydrin and by reading the column fractions absorbance at 280 nm. The active fractions were tested by the Bio-Rad Protein System, with the bovine serum albumin as standard . Finally, the presence of nucleic acids was checked by analysis of fractions absorbance at 260 nm. Furthermore, the grouped fractions were investigated by 1H-NMR spectroscopy. 1H and 13C NMR spectra, were recorded at 600.13 MHz on a BrukerDRX-600 spectrometer, equipped with a TCI CryoProbeTM, fitted with a gradient along the Z-axis, whereas for 31P-NMR spectra a Bruker DRX-400 spectrometer was used.
The gel filtration fractions were tested for anti-biofilm activity and the active fraction resulted positive to carbohydrate tests; this latter was a homogenous polysaccharide (6.6 mg) material. Preliminary spectroscopic investigations indicated the presence of a compound with a simple primary structure; the molecular mass of polysaccharidic molecule was estimated by gel filtration on a Sepharose CL6B which had previously been calibrated by dextrans (with a Mw from 10 to 2000 kDa). It's worthy to notice that some resonances in 13C NMR spectrum (78.32, 70.76, 65.63, 67.15 ppm) were split; this suggested the presence of 31P (JC-P from 4 to 9 Hz, see table 1) and its position into the polysaccharide repeating unit.
The phosphate substitution was confirmed by recording a 31P-NMR spectrum; it showed a single resonance at 1.269 ppm .
The GC-MS analysis of the high-molecular-weight polymer was carried out on an ion-trap MS instrument in EI mode (70eV) (Thermo, Polaris Q) connected with a GC system (Thermo, GCQ) by a 5% diphenyl (30 m × 0.25 mm × 0.25 um) column using helium as gas carrier. Nuclear Overhauser enhancement spectroscopy experiments (NOESY) were acquired using a mixing time of 100 and 150 ms. Total correlation spectroscopy experiments (TOCSY) were performed with a spinlock time of 68 ms.
Heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond correlation (HMBC) experiments were measured in the 1H-detected mode via single quantum coherence with proton decoupling in the 13C domain. Experiments were carried out in the phase-sensitive mode and 50 and 83 ms delays were used for the evolution of long-range connectivities in the HMBC experiment. The 2D 1H-31P HSQC experiment was recorded setting the coupling constants at 10 and 20 Hz.
Growth curve analysis
The effect of the bioactive compound on the planktonic culture was checked by growth curve analysis on both E. coli PHL628 and Pseudomonas fluorescens. The supernatant of the isolate was added to a conical flask containing 50 ml of M63 broth, to which a 1% inoculum from the overnight culture was added. The flask was incubated at 37°C. Growth medium with the addition of bacterial inoculum and without the addition of the supernatant was used as a control. OD values were recorded for up to 24 h at 1-h intervals.
Antibacterial activity by disk diffusion assay
Antimicrobial activity of the supernatant was assayed by the disc diffusion susceptibility test (Clinical and Laboratory Standards Institute, 2006). The disc diffusion test was performed in Muller-Hinton agar (MHA). Overnight cultures of E. coli PHL628 and P. fluorescens were subcultured in TY broth until a turbidity of 0.5 McFarland (1 × 108 CFU ml-1) was reached. Using a sterile cotton swab, the culture was uniformly spread over the surface of the agar plate. Absorption of excess moisture was allowed to occur for 10 minutes. Then sterile discs with a diameter of 10 mm were placed over the swabbed plates and 50 μl of the extracts were loaded on to the disc. MHA plates were then incubated at 37°C and the zone of inhibition was measured after 24 h.
For visualization of the effect of the sponge-associated bacterial supernatant against the biofilm forming E. coli PHL628 and Pseudomonas fluorescens, the biofilms were allowed to grow on glass pieces (1 × 1 cm) placed in 6-well cell culture plate (Greiner Bio-one, Frickenhausen, Germany). The supernatant at concentrations ranging from 1 to 10 times was added in M63K10 (for E. coli PHL628) and M63 broth (for P. fluorescens) containing the bacterial suspension of 0.05 O.D. at 600 nm. The wells without supernatant were used as control.
The plate was incubated for 36 h at 30°C in static condition. After incubation, each well was treated with 0.4% crystal violet for 45 minutes. Stained glass pieces were placed on slides with the bio-film pointing up and were inspected by light microscopy at magnifications of ×40. Visible bio-films were documented with an attached digital camera (Nikon Eclipse Ti 100).
Anti-biofilm effect on various strains and growth conditions
Some laboratory strains such as Acinetobacter, Staphylococcus aureus, Staphylococcus epidermidis, Salmonella typhimurium, Shigella sonneii, Listeria monocytogenes, Bacillus cereus, Bacillus amyloliquefaciens, Bacillus pumilus and Bacillus subtilis were selected. All strains were grown in Tryptone Soya Broth (TSB) (Sigma) supplemented with 0.25% glucose and the same medium was used during the biofilm assay in the presence of SP1 supernatant.
Competitiveness between quorum sensing factors and bioactive compounds
For this experiment the E. coli PHL628 supernatant was prepared by using the same conditions as for that of the sponge-isolated strain. Equal volumes of the two supernatants were added either in combination or alone in the microtiter plate containing a culture of E. coli PHL628 at an initial turbidity of 0.05 at 600 nm and biofilm formation was measured as described above. Each result was an average of at least 6 replicate wells.
Pre-coating of microtiter plate
Wells were treated with 200 μl of the B. licheniformis supernatant for 24 h and then the un-adsorbed supernatant was withdrawn from the wells. Such pre-coated wells were inoculated with E. coli PHL628 cultures having an OD of 0.05 at 600 nm. In another set of wells that were not coated with the supernatant, the fresh culture of E. coli PHL628 having the same density mentioned above were added together with the supernatant (5% v/v). The microtiter plate was then incubated for 36 h in static conditions and biofilm formation was estimated. The control experiments were carried out in wells that were not pre-coated or initially added with the supernatant. Each result was an average of at least 6 replicate wells and three independent experiments.
In a parallel microtiter plate, the supernatant was added to the 36-h biofilm culture in the microtiter plate and was then left at 30°C in static conditions for another 24 h. The experiment was repeated six times to validate the results statistically.
Microbial cell surface hydrophobicity (CSH) assay
Hydrophobicity of the culture of E. coli PHL628 and P. fluorescens were determined by using MATH (microbial adhesion to hydrocarbons) assay as a measure of their adherence to the hydrophobic hydrocarbon (toluene) following the procedure described by Courtney et al. 2009 . Briefly, 1 ml of bacterial culture (OD530 nm = 1.0) was placed into glass tubes and 100 μl of toluene along with the supernatant (5% v/v) was added. The mixtures were vigorously vortexed for 2 min, incubated 10-min at room temperature to allow phase separation, then the OD530 nm of the lower, aqueous phase was recorded. Controls consisted of cells alone incubated with toluene. The percentage of hydrophobicity was calculated according to the formula: % hydrophobicity = [1-(OD530 nm after vortexing/OD530 nm before vortexing)]×100.