Bacterial strain and growth conditions
The strain S. pseudintermedius DSM 25713 was isolated from a wound of a haematologic patient recently admitted to “Santo Spirito” Hospital in Pescara, Italy . Strain identification was carried out using biochemical tests (API system; bioMerieux, Marcy l’Etoile, France), and confirmed by 16S RNA sequencing. Bacterial stocks were stored at −80 °C until their use, when they were thawed, inoculated into Trypticase Soy broth (TSB; Oxoid SpA, Garbagnate M.se, Italy), and incubated at 37 °C for 24 h. An aliquot was then plated twice on Mueller-Hinton agar (MHA; Oxoid SpA) to check for the purity of the culture. A standardized suspension of 1.0 × 108 CFU/mL (corresponding to OD of 1.0 at 550 nm) was prepared in TSB and used immediately for all experiments.
Standardization and optimization of S. pseudintermedius biofilm growth on polystyrene
Since the optimal conditions for S. pseudintermedius biofilm formation on polystyrene surfaces are not known, preliminary experiments were carried out to optimize and standardize the in vitro model for biofilm formation. The following basic parameters for biofilm growth were considered for optimization: i) inoculum size (suspensions at 105, 106, and 107 CFU/ml were prepared starting from standardized inoculum); ii) dynamic (cultures were incubated under agitation at 200 rpm) (IKA agitator KS 260; IKA, Milan, Italy) or static conditions; and iii) incubation time (24, 48, and 72 h).
Based on our results, an inoculum size of 107 CFU/ml, and static incubation were used for S. pseudintermedius biofilm formation, while susceptibility to antibiotics was tested by exposing 48 h-biofilms to antibiotic for a further 24 h.
Quantitative measurement of static biofilms
In brief, 200 μl of the standardized inoculum at desired concentration prepared in TSB (Oxoid SpA) was added aseptically to each well of a 96-well polystyrene tissue culture plate (Falcon BD; Becton, Dickinson and Company, Milan, Italy), and incubated at 37 °C under static conditions. Wells that only contained TSB were considered as controls. At the end of the incubation, spent medium was discarded and each well was washed twice with PBS (pH 7.2) (Sigma-Aldrich Srl, Milan, Italy) to remove non-adherent cells. Biofilm formation was then assessed by crystal violet assay or viable cell count. i) Crystal violet microtiter plate assay . Biofilm samples were fixed by incubating plates at 60 °C for 1 h, then stained for 5 min with 200 μl Hucker-modified crystal violet . Excess stain was rinsed off with running tap water, and then the plates were air-dried. Crystal violet was extracted by exposure at room temperature for 15 min to 200 μl glacial acetic acid 33 % (Sigma-Aldrich), and biofilm biomass (including adherent bacteria and EPS) was then assessed by measuring the optical density at 492 nm (OD492) (SpectraMax 190; Molecular Devices, Sunnyvale, CA, USA). ii) Total viable cell count. In each well, the biofilm sample was scraped by using a pipette tip after 5-min exposure to 200 μl trypsin-EDTA 0.25 % (Sigma-Aldrich), then resuspended in sterile PBS by vortexing. Serial 10-fold dilutions of each sample were prepared in sterile PBS and 100 μl of each dilution was plated on MHA and incubated at 37 °C for 24 h. Colonies were counted to estimate biofilm viability.
Continuous flow through biofilm
Biofilm was allowed to form in a polycarbonate flow through chamber (The Technical University of Denmark, Lyngby, Denmark) for microscopic studies . The flow cell is composed of three parallel channels in perspex (poly[methyl methacrylate]), covered with a no. 1 24 × 50 mm glass coverslip which serves as the biofilm substratum. Each channel has a dimension (length × width × height) of 40 × 4 × 4 mm and was cleaned with 96 % (v/v) ethanol prior to use.
In brief, the chamber was inoculated with standardized inoculum diluted in TSB at 5 × 105 CFU/ml, then inverted to allow microorganisms to attach for 3 h, under static conditions, at 37 °C. The flow cell was then placed upright and the pump started with a TSB flow rate of 0.5 ml/min. Biofilm was allowed to form for 24 h at 37 °C, then washed with PBS (2 min at 0.5 ml/min), and finally observed by a confocal laser scanning microscope.
Time course of biofilm formation
Biofilms were allowed to form in each well of a 24-well flat-bottom polystyrene tissue-treated microtiter plate (BD Company), as described above. At selected times (30 min, 1, 2, 4, 8, 24, 48, and 72 h of incubation) biofilm viability was assessed by viable colony count as described above. In a parallel series of experiments, wells were broken and fragments representative of each time point were observed by scanning electron microscopy.
Effect of human serum and pH on biofilm formation
Serum for testing was pooled from multiple samples. Serum samples were collected from 30 blood donors, which were selected based on their health status as non-smokers with no other known current diseases, and because they were not on any medications. The serum samples were then pooled, aliquoted, and stored at −20 °C until use. Since it was observed that albumin and total protein levels were significantly higher in serum than in wound fluid , serum was tested against biofilm formation at different dilutions (1:2, 1:10, and 1:100) prepared in TSB. Serum was tested both as free (soluble) and adhered to polystyrene. In the latter case, serum-coated microplates were prepared immediately before use. In brief, 200 μl of serum was added to each well of a 96-well tissue culture plates (BD Company), incubated for 2 h at 37 °C, then washed by PBS to remove excess serum.
The effect of pH and serum on S. pseudintermedius biofilm formation was simultaneously assessed. To this end, 96-well microtiter plates containing free or adsorbed serum were inoculated with the standardized inoculum prepared in TSB that was previously corrected at different pH values (5.5, 7.1, and 8.7) by using HCl or NaOH 1 M solution, then incubated at 37 °C for 24 h. Biofilm biomass levels were then spectrophotometrically measured as described above.
Susceptibility of S. pseudintermedius strain DSM 25713 to chloramphenicol, gentamicin, cefoxitin, linezolid, rifampicin, vancomycin, tetracycline, and tigecycline (all were purchased, as reference powders, from Sigma-Aldrich) was determined by microdilution technique, in accordance with CLSI M100-S20 guidelines . MIC was calculated as the lowest concentration of the test agent that completely inhibited visible growth. MBC was evaluated as the lowest concentration of the test agent killing of at least 99.99 % of the original inoculum. E. faecalis ATCC29212 and E. coli ATCC25922 were used as reference strains.
Antibiotic activity against biofilm formation
In each well of a 96-well flat-bottom polystyrene tissue-culture microtiter plate (Becton, Dickinson and Company), 5 μl of a standardized inoculum (1–5 × 107 CFU/ml) were added to 100 μl of cation-adjusted Mueller-Hinton broth (CAMHB; Oxoid SpA) containing antibiotic at 1/2×, 1/4×, and 1/8×MIC. After incubation at 37 °C for 24 h, non-adherent bacteria were removed by washing twice with 100 μl sterile PBS, then biofilm levels were spectrophotometrically measured as described above.
Antibiotic activity against preformed biofilms
The activity of antibiotics against 48 h-old biofilms was assessed by viable colony count. Biofilms were allowed to form in each well of a 96-well flat-bottom polystyrene tissue-treated microtiter plate (Becton, Dickinson and Company), as described above. Following 48 h-incubation, biofilms samples were washed twice with PBS, then exposed to 200 μl of drug-containing CAMHB (prepared at 1, 2, 4, 8, 16, 32, 64, and 128 × MIC). After incubation at 37 °C for 24 h, non-adherent bacteria were removed by washing twice with 200 μl sterile PBS, and biofilm samples were scraped as described above. Cell suspension was then vortexed for 1 min to break up bacterial clumps. Bacterial counts were performed by plating serial 10-fold dilutions of this suspension on MHA plates. Control biofilm samples were not exposed to antibiotics. Minimum Biofilm Eradication Concentration (MBEC) was calculated as the minimum concentration of tested antibiotic able to eradicate preformed biofilm.
Kinetics of biofilm formation by S. pseudintermedius strain DSM 25713 and its architecture were assessed by scanning electron microscopy (SEM) and environmental-SEM (ESEM), respectively. The effects of exposure to several gentamicin concentrations as well as the ultrastructure of biofilm formed under dynamic incubation were evaluated by confocal laser scanning microscopy (CLSM). i) SEM and ESEM assays. Biofilm formation kinetics in TSB was monitored - under static conditions, without serum, at 37 °C, and at pH 7.1 - in 35 mm-tissue culture polystyrene dish (Becton, Dickinson and Company) at different time periods (30 min, 1, 2, 4, 8, 24, 48, and 72 h). Samples were then fixed in a mixture of 2 % paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) + 2 % glutaraldehyde (Sigma-Aldrich) [vol/vol] in 0.15 M sodium cacodylate buffer (pH 7.4; Fluka), with 0.1 % alcian blue (Sigma-Aldrich). Samples were post-fixed for 90 min at room temperature in 1 % OsO4 [vol/vol] (Electron Microscopy Sciences) in 0.15 M cacodylate buffer, then dehydrated in an ascending ethanol series (50, 70, 80, 95, and twice 100 %; 10 min/each), dried for 30 min with hexamethyldisilazane (Polysciences Inc., Warrington, PA, USA), and finally air-dried. Specimens were coated with gold-palladium by Polaron E5100 II (Polaron Instruments Inc.), and then observed with a Philips XL30CP scanning electron microscope in the high-vacuum mode at 15 kV. In a parallel experiment, a 72 h-old biofilm sample was fixed and post-fixed as described above, and directly observed using a Zeiss EVO (Carl Zeiss SpA, Arese, Milan, Italy). ii) CLSM assay. Briefly, 48 h-biofilms were allowed to grow on polystyrene as described for SEM analysis, then exposed to gentamicin at different concentrations (from 1x to 128xMIC) for a further 24 h. Untreated biofilms were used as controls. In a parallel series of experiments, biofilm was allowed to grow under dynamic conditions in a flow cell system in the absence of antibiotics, as described above. Both static and flow cell biofilms were stained with Live/Dead BacLight kit (Molecular Probes Inc., Eugene, USA) and Concanavalin A (Alexa Fluor 647 coniugate; Molecular Probes Inc.). Static biofilm samples formed on polystyrene were placed in an Attofluor cell-chamber (Molecular Probes Inc.) before observation. CLSM analysis was performed with an LSM 510 META laser scanning microscope attached to an Axioplan II microscope (Zeiss Italia, Arese, Milan, Italy). Depth measurements were taken at regular intervals across the width of the device. To determine biofilm structure, a Z-series of 25 optical planes at xy resolution of 512×512 pixel (68.4 × 68.4 μm) with a thickness of 1.00 μm was taken throughout the biofilm. Both SEM and CLSM representative images were captured and processed for display using Photoshop (Adobe Systems Inc., San Jose, California) software.
Statistical analysis and biofilm interpretative criteria
All experiments were carried out at least in triplicate and repeated at least on two different occasions. Differences were assessed by unpaired-t test (standardization of in vitro model of biofilm formation), ANOVA + Newman-Keuls multiple comparison post-test (effect of serum and pH both on biofilm formation and bacterial growth), chi-square test (percentage of reduction of both biofilm biomass formation and biofilm viability), or Kruskall-Wallis + Dunn’s multiple comparison post-test (kinetic of biofilm formation). Statistical analysis of results was conducted with GraphPad Prism version 6.00 (GraphPad software Inc.; San Diego, CA, USA), considering as statistically significant a p value of < 0.05.
The low cut-off value for biofilm formation was represented by 3 SDs above the mean OD492 of control wells (containing bacteria-free medium) .
To evaluate the effect of serum and pH on biofilm formation, biofilm levels were normalized for bacterial growth by calculating the specific biofilm formation (SBF) index: SBF = (ODbiofilm - ODNC)/ODgrowth in which ODbiofilm is the OD492 of the stained biofilm, ODNC is the OD492 of the stained negative control wells (to eliminate unspecific or abiotic OD values), and ODgrowth is the OD600 of cells grown in broth.
The percentage of inhibition of biofilm formation by antibiotics tested at sub-inhibitory concentrations was calculated as follows: (1 - ODexp/ODUC) × 100 in which ODexp is the OD492 of the stained antibiotic-exposed biofilm, and ODUC is the OD492 of the stained untreated control biofilm.