Evaluation of the Activity of a Combination of Three Bacteriophages Alone or in Association with Antibiotics on Staphylococcus aureus Embedded in Biofilm or Internalized in Osteoblasts

Bacterial strains and bacteriophages.The methicillin-susceptible Staphylococcus aureus strain HG001 is a reference strain (31) previously used by Valour et al. (7) to test the activity of antibiotics against intracellular staphylococci and was used in the present study to assess the bactericidal activity of bacteriophages alone or in association with antibiotics. S. aureus SH50-2 is a clinical strain (isolated in France from a bone and joint infection) and was used in the present study for bacteriophage amplification and titrations.

Before each experiment, all strains were grown on Columbia blood agar (COS; bioMérieux, Marcy-l’Etoile, France) for 24 h at 36°C. Three colonies were then used to inoculate brain heart infusion (BHI) medium (bioMérieux) before incubation overnight. The HG001 strain had MICs (determined by microdilution according to EUCAST guidelines) of 1.5 mg/liter for vancomycin and 0.016 mg/liter for rifampin.

Three bacteriophages, PP1493, PP1815, and PP1957, produced and purified by Pherecydes Pharma (Romainville, France), were used and mixed extemporaneously before each experiment in equal proportions and at the indicated concentration. When needed, bacteriophage titration was performed using a spot test assay. For this, 10-fold serial dilutions (from 10⁻¹ to 10⁻⁸) of bacteriophage suspensions in lysogeny broth (LB Lennox; Fisher Scientific, Hampton, NH, USA) were prepared, and 5 μl of these dilutions was spotted on an agar prepared immediately before use by mixing 25 ml of LB 0.75% agar medium and 2 ml of an exponential S. aureus SH50-2 culture. After 24 h of incubation at 36°C, the number of PFU was visually determined.

S. aureus HG001 susceptibility to bacteriophages.S. aureus HG001 susceptibility to the combination of the three bacteriophages was evaluated using two complementary methods. First, the efficiency of plating (EOP) score was calculated. The latter is the ratio between the bacteriophage titer obtained with the HG001 strain divided by the titer obtained with the SH50-2 reference strain used for the production of bacteriophages; the closer this score is to 1, the more efficient the bacteriophages are (strong killers, EOP > 0.1; intermediate killers, 0.005 < EOP < 0.09; weak killers, EOP < 0.005) (32). Second, the growth inhibition of S. aureus by bacteriophages in liquid medium was assessed by culturing bacteria in a 96-well plate at a starting concentration of 10⁷ CFU/ml with or without bacteriophages at two concentrations (multiplicity of infection [MOI] of 1 or 10 bacteriophages per bacteria). The bacterial growth at 36°C was monitored by measuring the optical density at 600 nm (OD₆₀₀).

Assessment of bacteriophages bactericidal activity against biofilm-embedded S. aureus HG001.An overnight culture of S. aureus HG001 in liquid BHI was adjusted to an OD₆₀₀ of 1.0 and was then diluted 100-fold in BHI (Becton, Dickinson GmbH, Heidelberg, Germany). Then, a 96-well polystyrene plate (Greiner Bio-One, Kremsmünster, Austria) was inoculated with 100 μl of bacterial suspension per well and incubated at 36°C for 24 h to allow mature biofilm formation. After supernatants were removed, the biofilm was rinsed three times with saline water and further incubated for 24 h with 200 μl of antibiotic and/or bacteriophages. A range of 3 concentrations of antibiotics (corresponding to the MIC of the strain, 10-fold MIC, and the usual intraosseous concentration reached in humans when using standard therapeutic doses [7]) and 4 concentrations of bacteriophages (10⁵ to 10⁸ PFU/ml of each bacteriophage) were used to assess potential dose effects. Then, the biofilm was again rinsed before being resuspended in 100 μl of phosphate-buffered saline (PBS) by scraping the wells with sterile pipette tips followed by 10 min of sonication (40 Hz, BactoSonic; Bandelin, Berlin, Germany). Then, the residual viable number of bacteria after exposure to bacteriophages and or antibiotics was determined by plating 10-fold serial dilutions of the resulting bacterial suspensions on COS agar.

Intracellular infection of osteoblasts and assessment of bacteriophage activity against internalized S. aureus HG001.Bacteriophage activity against internalized S. aureus HG001 was assessed in an in vitro model of osteoblast infection adapted from a previously published protocol (7). All cell culture reagents were purchased from Gibco (Thermo Fischer Scientific, Waltham, MA, USA). MG63 osteoblastic cells (ATCC CRL-1427TM; LGC Standards, Molsheim, France) were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (culture medium) and 100 U/ml penicillin and 100 mg/liter streptomycin at 36°C in a 5% CO₂ atmosphere. They were passaged once a week and used up to passage 20 after thawing. Prior to infection experiments, cells were seeded at 50,000 cells per well into 24-well tissue culture plates (Falcon, Le Pont de Claix, France) and incubated during 48 h in culture medium with antibiotics.

Intracellular infection of MG63 cells was performed using a bacterial suspension whose concentration was determined as follows. The day before the infection of osteoblastic cells, 2 ml of an overnight bacterial culture in BHI was centrifuged, and the pellet was resuspended in 10 ml of cell culture medium without antibiotics. Serial dilutions of this suspension were plated on Trypticase soy agar (TSA) (bioMérieux) using an easySpiral device (Interscience, Saint-Nom-la-Bretèche, France) for bacterial enumeration. Meanwhile, the bacterial suspension was kept overnight at 4°C. The next day, this suspension was adjusted at an MOI of 10 bacteria/cell and added to the osteoblastic cell culture wells for 2 h at 36°C to allow bacterial internalization. Cells were then washed twice with phosphate-buffered saline (PBS) and were treated with culture medium containing 10 mg/liter lysostaphin (Sigma-Aldrich, Saint-Louis, MO, US) to kill all remaining extracellular bacteria without affecting intracellular bacteria (33). After 1 h, cells were washed twice, and the cells from three wells were lysed in sterile water over 15 min to determine the number of internalized bacteria at this time, defined as time zero. Then, incubation was carried on for 24 h with 1 ml of culture medium containing bacteriophages and/or an antibiotic. Two concentrations of the combination of three bacteriophages were tested as follows: 10⁷ PFU/ml and 10⁹ PFU/ml of each bacteriophage. The antibiotics were used at their usual local intraosseous concentration (6 mg/liter for both antibiotics) (7).

In the first set of experiments, in order to evaluate specifically the intracellular bacteriophage activity, lysostaphin at 10 mg/liter was also added to the culture medium to kill the bacteria released upon host cell lysis throughout the experiment, thus preventing these bacteria from reinfecting new host cells. The absence of effect of lysostaphin against bacteriophage titer stability was verified. After 24 h of incubation in the presence of lysostaphin with or without bacteriophages, the culture supernatants were removed. Then, the cells were washed twice with PBS, treated with citric buffer (40 mM citric acid in PBS, pH 3) for 2 min in order to inactivate any residual phage particles remaining on the surface of the cells, and washed twice again (25). The number of viable intracellular bacteria was determined after lysis of the cells in sterile water during 15 min. In addition, bacteriophage titrations were performed in both cellular culture supernatants and in the cell lysates.

In the second set of experiments, after the 1-h treatment of infected cells with lysostaphin (10 mg/liter), cells were rinsed and incubation was carried out with bacteriophages and/or antibiotics but without lysostaphin to be sure that lysostaphin was not interfering with bacteriophage penetration inside infected osteoblasts. Thus, in the absence of any treatment, bacteria may escape from infected cells and multiply in the extracellular medium before being reinternalized in other cells. This protocol allowed the evaluation of the natural course of intracellular infection, i.e., the effect of bacteriophage and/or antibiotics on intracellular bacteria and extracellular bacteria released following the death of infected cells.

Electron microscopy analysis.After 24 h of incubation of cells with bacteriophages as described above (second set of experiments), cells were washed and fixed in 2% glutaraldehyde. Then, they were washed three times with a buffered solution containing 1 volume of saccharose 0.4 M and 1 volume of cacodylate 0.2 M (pH 7.4) for 1 h at 4°C and postfixed with a solution containing 1 volume of osmium 2% and 1 volume of cacodylate 0.3 M (pH 7.4) for 1h at 4°C. Cells were then dehydrated using an ethanol gradient (5 min in ethanol 30%, 50%, 70%, and 95% and 3 times in absolute ethanol for 10 min). Impregnation was performed with an epon mix. Inclusion was obtained by polymerization at 60°C for 72 h. Ultrathin sections (approximately 70 nm thick) were cut on a UC7 ultramicrotome (Leica Microsystems, Wetzlar, Germany) mounted on 200 mesh copper grids coated with 1:1,000 polylysine, stabilized for 1 day at room temperature, and contrasted with uranyl acetate and lead citrate. Sections were examined using a 1400-JEM transmission electron microscope (Jeol, Tokyo, Japan).

Statistical analysis.Each experiment was performed three times independently in technical triplicates. For cellular experiments, variables were compared using the nonparametric Mann-Whitney U test. A P value of <0.05 was considered significant. For biofilm eradication data analysis, remaining viable inocula after 24 h of treatment, initially determined in CFU/ml, were log transformed. Effects of bacteriophages or antibiotics in monotherapy were compared to the untreated control condition using the Mann-Whitney U test. Significant variations (P < 0.05) of the viable bacteria counts after 24 h of treatment by bacteriophages or antibiotics alone compared to the untreated condition were denoted with asterisks in Fig. 2. Two-way analysis of variance (ANOVA) and Bonferroni post tests were used to compare the effects of combinations of antibiotics and bacteriophages to that of the antibiotic or bacteriophages in monotherapy at the same concentration. A synergy was defined by an effect of the combination significantly greater (P < 0.05) than that of both agents used alone and was denoted with the sign “S” in Fig. 2. All analyses were performed using Prism software (GraphPad, San Diego, CA, USA).