List and origin of Staphylococcus epidermidis used in this study is reported in Additional file 6: Table S3. All strains were isolated from patients hospitalized at the Medical School of the University of Naples Federico II. All strains were molecular identified by means of kat A-RFLP analysis technique described by Blaiotta et al. .
The study does not investigate clinical aspects of the disease, nor it uses human specimen. The study therefore does not require the Ethic Committee approval.
Antibiotic susceptibility of Staphylococcus epidermidis strains
The antibiotic-susceptibility profile of strains was tested using the disk diffusion method on Mueller-Hinton agar, according to the NCCLS guidelines (2002) . The antibiotics used and their concentrations were as follows: amoxicillin (25 μg; AMX25), ampicillin (10 μg; AM10), aztreonam (30 μg; ATM30), bacitracin (10 μg; B2), carbenicillin (100 μg; CB100), ceftazidime (30 μg; CAZ30), cefoxidin (30 μg; FOX30), cephaloridine (30 μg; CD30), cloxacillin (1 μg; CX1), erythromycin (15 μg; E15), fosfomycin (50 μg; FF50), fusidic acid (10 μg; FD10), gentamicin (10 μg; GM10), imipenem (10 μg; IPM10), lincomycin (2 μg; MY2), metronidazole (80 μg; M80), mezlocillin (75 μg; MZ75), netilmycin (30 μg; NET30), nitrofurantoin (300 μg; FM300), novobiocin (30 μg, NB30), oxytetracycline (30 μg, T30), penicillin-G (10 μg; P10), piperacillin (100 μg, PIP100), rifampicin (30 μg; RF30), chlorotetracycline (30 μg; A30), spiramycin (100 μg; SP100), sulfamethoxazole (100 μg; SP100), tetracycline (30 μg; TE30), and vancomycin (30 μg; VA30). All antibiotics were provided by BioMérieux SA, (Marcy l’Etoile, France).
Pulsed-field electrophoresis of Staphylococcus epidermidis strains
The procedure adopted was that described . Briefly, inserts of intact DNA were digested in 200 μl of appropriate buffer supplemented with 40 U of Sma I (Promega, Milan). Pulsed field gel electrophoresis (PFGE) of the restriction digests was performed by using the CHEF system (Bio-Rad Laboratories, Hercules, CA, USA) with 1% (wt/vol) agarose gels and 0.5 x TBE as running buffer, at 10°C. Restriction fragments were resolved in a single run, at constant voltage of 6 V cm2 and an orientation angle of 120° between electric fields, by a single phase procedure for 24 h with a pulse ramping between 1 and 50s.
Antibacterial activity of AMPs
Antibacterial activity of the peptides used in this work was evaluated as described previously . A potential synergism (FIC) between TB-KK and RJI-C (MIX) was evaluated by adding combinations of two peptides in a serial two-fold dilutions (RJI-C 5–100 μg, 40 μl/well; TB-KK 5–100 μg, 40 μl/well;) to wells containing 105 CFU/well in 60 μl . The fractional inhibitory concentration (FIC) index for combinations of two peptides was calculated according to the equation: FIC index = FICA + FICB = A/MICA + B/MICB , where A and B are the MICs of drug A and drug B in the combination, MICA and MICB are the MICs of drug A and drug B alone, and FICA and FICB are the FICs of drug A and drug B. The FIC indices were interpreted as follows: ≤0.5, synergy; 0.51–4.0, no interaction; > 4.0, antagonism .
The growth inhibition percentages of Staphylococcus epidermidis and probiotic strains were assessed under the same conditions.
Inhibition zone assay and test of the haemolytic activity of the antimicrobials
The MIX (RJI-C at 9 μg/ml and TB-KK at 6 μg/ml) was tested for its haemolytic activity using mouse red blood cells and for inhibition zone assay test . The MIX was tested for its haemolytic activity using mouse red blood cells. The blood was collected from the tail of the animals and centrifuged (4x102 g for 3 min). The erythrocytes were washed with saline, suspended at 3x106 erythrocytes/ml, mixed with the peptide combination (RJI-C 9 μg and TB-KK 6 μg in 100 μl saline) and incubated for 1 h at 37°C. The haemolytic activity was measured according to the formula OD peptide - OD negative control/OD positive control - OD negative control X 100 where the negative control (0% haemolysis) was represented by erythrocytes suspended in saline and the positive control (100% haemolysis) was represented by the erythrocytes lysed with 1% triton X100 .
The LC50 values relative to the two peptides and the MIX were calculated as described .
J774 murine macrophages from the American Tissue Culture Collection (ATCC, Rockville, MD,USA) were cultured in Dulbecco's modified Eagle's medium (DMEM, Cambrex Bio Science, Verviers, Belgium). Culture media contained 10% fetal bovine serum (FBS, Sigma, Milan, Italy), 100 IU/ml penicillin, 100 μg/ml streptomycin (all from Gibco, Paisley, Scotland). Cells were seeded on 96-well plates (Falcon, Milan) for the MTT Assay, and on 24-well plates (Falcon, Milan) for NO2− measurements, fluorescence microscopy analysis, and RT-PCR assays. Cell monolayers were grown to adherence before the experiments were started.
Experiments were carried out on female BALB/c mice (aged 8 to 10 weeks) at the animal facility of the University of Naples. Bacteria (107 or 108 CFU/mouse) were inoculated by intravenous routes (i.v.). LPS (250 μg, ~10 mg/Kg) (Sigma-Aldrich Milan), or an equivalent volume of sterile 0,9% saline vehicle (250 μl) was administered intraperitoneally. Blood samples were drawn from the tail vein using 0.5 ml syringes. Spleen and kidney were collected at several time points (4,5,7,8,10, 11and 12 days) after the mice infection with a sub-lethal dose of Staphylococcus epidermidis (107 CFU/mouse). However the same organs were also collected at 3, 6, 9 and 12 hours after infection with a lethal dose of Staphylococcus epidermidis (108 CFU/mouse). Spleens and kidneys were dissected and weighed. One g of each sample was homogenized in 1 ml saline and serially diluted in saline.
Colony forming units (CFU) were evaluated by the plate count assay. Animal experiments were approved by the Animal Care Committee of the University of Naples.
Measurement of cell viability
Analysis of cell viability was performed using the CellTiter 96® AQueous One Solution Cell Proliferation Assay system (MTS assay) (Promega, Madison,WI, USA). J774 cells were seeded at 2500 cells per well in a 96-well plate and incubated at 37°C, in a humidified atmosphere with 5% CO2. TB-KK 15 μg/ml, RJI-C 15 μg/ml, MIX (TB-KK 6 μg/ml + RJI-C 9 μg/ml) or RJII-C (Control 15 μg/ml) were added to the medium immediately after cell adhesion. At each time point 20 μl of CellTiter 96® AQueous One Solution reagent was added to each well, according to the manufacturer's instructions. Absorbance was recorded at 490 nm after 2 h using an EnVision 2102 multilabel reader (PerkinElmer, Waltham, USA).
Nitrite formation in J774 cells stimulated with LPS and treated with RJI-C, TB-KK, and the MIX
Nitrite accumulation (NO2−, μmol/106 cells) in the cell culture medium was determined by the Griess reaction .
Western Blot Analysis COX-2
Cell lysates for Western blotting were prepared by washing cells twice with ice-cold phosphate-buffered saline followed by cell lysis in 500 μl of Fastprep lysis buffer (1X protease inhibitor cocktail tablet (Roche EDTA free) resuspended in 1X PBS) on ice and lysed 20s at 6.5 intensity, 2X intervalling with 5–10 minutes on ice. Cell lysates were centrifuged for 10 min at 7800 g at 4°C, and the supernatants were collected and stored at −80°C until analysis. Lysate protein concentrations were measured using the Bio-Rad protein assay method, as described in the manufacturer’s instructions. Cell lysate volumes corresponding to 20 μg of total protein were diluted 1:1 in Laemmli buffer (Bio-Rad) and boiled for 5 min prior to electrophoresis on a 10% acrylamide gel. The resolved proteins were electroblotted on PVDF membrane (Bio-Rad) by the Bio-Rad semidry transfer method, according to the manufacturer’s instructions. Membranes were stained with PonceauRed to verify uniform protein transfer, and then blocked with blocking buffer (1X TBS, 0.1% Tween-20, 5% w/v non-fat dry milk) for 1 h at RT. Blocked membranes were incubated overnight at 4°C with COX-2 mouse monoclonal antibody (diluted 1/2000), β-actin mouse monoclonal antibody (diluted 1/10,000). Blots were washed three times in TBS-Tween before incubation with the appropriate horseradish peroxidase-conjugated secondary antibody (sheep anti-mouse IgG diluted 1/5000) for 1 h at room temperature.
After three washes with TBS-Tween, the signal was developed using standard procedure. Gel image was acquired in Fujifilm LAS-3000 Chemiluminescence system (Fujifilm Life science).
Real time PCR of pro-inflammatory
Total RNA was isolated from the tissue and the cell line after treatment by using Trizol reagent (Invitrogen, Milan, Italy). RNA was suspended in RNase-DNAse free distilled water, assessed for concentration (by measuring the absorbance at 260 nm) and purity (by ascertaining that the A260/A280 ratio was .1.9). RNA (1 μg) was then treated with 1U RNAse-free DNAse (Promega, Madison, WI). DNA contamination of RNA samples was excluded by PCR with primers specific for the gapdh gene. Reverse transcription was carried out with ImProm-II reverse transcriptase (Promega, Madison, WI) and oligo(dT). Real-time PCR was performed on 50 ng cDNA, using 1x master mix SYBRGreen (Applied Biosystem, Milan) in a StepOne Applied Biosystem instrument (Applied Biosystem, Milan). Reactions were performed in 20 μl in triplicate. The primer list is reported in Additional file 7: Table S4.
ELISA test of pro-inflammatory cytokines
In addition, the ELISA test was used to measure the anti-inflammatory activity of the MIX and its components : RJI-C 9 μg/mL e TB-KK 6 μg/mL.
Briefly, J774 cells (106 cells/well) were stimulated with LPS (10 μg/ml; 1 hour), treated with RJI-C 9 μg/ml or TB-KK 6 μg/ml or MIX (RJI-C 9 μg/ml + TB-KK 6 μg/ml) in presence or absence of LPS (10 μg/ml). The supernatants from these cells (100 μl/well) were transferred into the wells of a plate previously coated with mouse anti-human TNF-α (BD Pharmingen; 50 μl diluted 2 x 10-3/well) or mouse anti-human IFN-γ (Biosciences, 50 μl diluted 2 x 10-3/well) along with a second dose of anti IFN- γ or TNF- α, HRP-labelled rabbit anti mouse IgG diluted 10-3 (100 μl/well) and TMB peroxidase substrate (BIORAD; 100 μL/well), in the order. The optical density of each well was read at 405 nm using a microplate reader (Bio-Rad, Japan). Triplicate positive and negative controls were included in each plate .
CD64 expression in total White Blood Cells was analyzed using a Flow cytometry EPICS Elite (Beckman Coulter, Fullerton, CA). Daily instrument quality control including fluorescence standardization, linearity assessment, and spectral compensation were performed to ensure identical operation from day to day. At least 10.000 events for each sample was analyzed and the data were saved for later analysis on EXPO32 software (Beckman Coulter). Data analysis was performed by using electronic gating on the basis of FSC and SSC excluded cellular debris and nonviable cells. PE-coniugated anti-mouse CD64 expression was measured using a log10 scale. Briefly, 50 ul of whole blood was incubated for 10 minutes at room temperature with saturating amounts of phycoeritrine- conjugated anti-CD64 murine monoclonal antibody (Becton Dickinson) followed by red blood cell lysis with an ammonium chloride–based red cell lysis solution (Beckman Coulter, Fullerton, CA). Samples were then washed once and resuspended with phosphate-buffered saline at pH 7.4, to a volume of 1 mL.
The kidney was fixed in 10% buffered formalin, sectioned (10 μm) and stained with hematoxylin-eosin according to standard protocols. Bacterial counts and cytokine levels were analyzed using Student’s t test.