Staphylococcus aureus MFP03 was isolated from the skin of a healthy volunteer and was fully characterized (Hillion et al., 2013). The strain was stored at -140°C in a cryofreezer. For the bacterial culture, an aliquot of bacteria was mixed in 25 ml of Luria Bertani medium (LB) and incubated for 24 h at 37°C under agitation.

Cutibacterium (former Propionibacterium, Scholz and Kilian, 2016) acnes strains ribotype 4 (RT4) HL045PA1 and ribotype 5 (RT5) HL043PA2 (acneic strains), initially isolated by Fitz-Gibbon et al. (2013), were obtained from BEI Resources American Type Culture Collection (VA, United States). These strains are associated with severe forms of acne and differ from non-acneic strains (ribotype 6 and in a lower extend ribotypes 1, 2, and 3) by a large plasmide (Locus 3), which should confer their virulence properties (Fitz-Gibbon et al., 2013). Bacteria stored at -140°C were initially plated on Reinforced Clostridial Medium (RCM) agar. As these strains are strictly anaerobic, the plates were incubated for 72 h in a BD GasPack^TM under anoxic conditions at 37°C. Colonies were then transferred into sterile conical 50 mL tubes (Falcon) filled to maximal capacity with RCM and grown for 72 h at 37°C.

Human ANP (Alfa Aesar, United States) has a molecular weight of 3080.47 g/mol and CNP (PolyPeptide, Strasbourg, France) has a molecular weight of 2196.1 g/mol. Peptides were reconstituted in milli-Q water and stored dissolved at -20°C with molar concentration 1.623 × 10^-4 M and 4.554 × 10^-4 M for ANP and CNP, respectively. According to data in the literature on the physiological NUP concentrations in humans (Edwards et al., 1988; Stingo et al., 1992) and data of NUP concentrations used in experiments with eukaryotic cells (Klinger et al., 2013), we studied the effect of the peptides at concentrations ranging from 10^-6 to 10^-8 M, as well as with a mix of the two peptides at a concentration of 10^-6 M. Peptides were added to experimental medium immediately before the experiment’s start.

The effects of ANP and CNP on monospecies culture growth were studied in a Bioscreen C system (Finland) using 100-well flat-bottom Honeycomb microtiter plates (Growth Curves, United States). On the bottom of the well, an aliquot of NUP was distributed. Then 200 μl of growth medium were injected in each well. S. aureus MFP03 was cultivated in tryptic soy broth (TSB, Sigma) with addition of 0.25% glucose (Sigma). C. acnes strains were cultivated in RCM medium. Before plating, bacterial cultures were washed twice with physiological saline (PS) at pH 7.0. Then OD₅₈₀ of cell suspension in PS was adjusted to 1.0. Finally, 16 μl of culture were injected into each well containing medium and peptides. Some wells were maintained without a culture medium (negative control) and some others without peptides (positive control). S. aureus MFP03 was cultivated aerobically at 37 or 33°C (closer to the skin’s physiological temperature). C. acnes was also cultivated at 33 and 37°C as acneic strains naturally develop in anoxic deep skin regions. In the case of C. acnes, peripheral wells were filled with a CO₂-producing solution and prepared in anoxic conditions using a GasPack^TM system and sealed with parafilm before incubation. The optical density of the cultures was determined automatically every 15 min. Growth curves were determined over a minimum of three independent experiments.

Monospecies biofilms of S. aureus MFP03 and C. acnes RT5 were obtained in 24-well plates with flat glass bottoms (Greiner Bio-One, Germany). Biofilms were grown according to Gannesen et al. (2018a,b). Briefly, prepared cultures were washed twice with sterile PS and OD₅₈₀ was adjusted to 1.0. In each well 300 μl of cell suspension were added, and plates were incubated 2 h at room temperature (time for cell adhesion). C. acnes strains were incubated in a GasPack^® (BD) system in an anaerobic atmosphere. In each well the suspension was removed, and wells were washed twice with sterile PS to remove non-attached cells. One well was dried and fixed in order to verify adhesion control. One milliliter of medium containing ANP or CNP, or no peptides (positive control), was injected into the wells. To find out the probable effect of cultivation conditions on biofilm growth, different conditions for each bacterium were tested. They are summarized in Table 1.

Biofilms were grown for 24 h for S. aureus MFP03 and for 72 h for C. acnes. In each case, where anaerobic atmosphere was necessary, a GasPack^® system (BD) was used. After incubation time, biofilms were washed twice with sterile PS and stained with SYTO 9^® Green fluorescent dye for 20 min. The staining dye was then removed and the biofilms were rinsed and fixed with ProLong^® Diamond Antifade Mountant (Molecular Probes^TM). Observations were realized using an LSM 710 inverted confocal laser-scanning microscope (Zeiss, Germany). Three-dimensional (3D) images and orthocuts were obtained using Zen^® 2009 software. Biofilm thickness was quantified with the same software. Images are representative of the biofilm structure observed in a mean of 20 different fields over a minimum of four independent studies. Biofilm density and thickness were calculated over the same number of observations using Zeiss Zen Image Analysis software for light microscopy (ImageJ software package). Statistical differences were determined using the Mann–Whitney non-parametric test. The differences between the experimental and control variants were considered reliable at a confidence coefficient >95% (P < 0.05).

Binary biofilms were obtained on Petri dishes with RCM agar. Whatman GF/F glass fiber filters 21 mm in diameter were used as carriers. Cultures were prepared as described above. Sterile filters were placed onto the RCM surface, after which 20 μl of each culture were dropped onto filter center. Biofilms were incubated for 72 h in an anaerobic atmosphere at 37 or 33°C. At 33°C both C. acnes RT5 and S. aureus MFP03 were used, and different types of biofilms were constructed and are described in Table 2. Before plating the secondary colonizers, all filters with biofilms were replaced on Petri dishes with fresh RCM agar. Secondary colonizers were plated onto the biofilm as a drop of a culture prepared as described above.

In parallel, as control conditions monospecies biofilms were grown and manipulated in the same way as binary biofilms. All samples were divided into two parts: one was dedicated to metabolic activity analysis of the cells, while the second one was used for S. aureus MFP03 counting. Experiments were conducted independently at least three times. Statistical differences were determined using the Mann–Whitney non-parametric test. The differences between the experimental and control variants were considered reliable at a confidence coefficient >95% (P < 0.05).

The metabolic activity of cells in biofilms was determined as described by Gannesen et al. (2018a,b), using the method of Plakunov et al. (2016). Briefly, filters with biofilms were stained with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT). MTT is an acceptor of electrons from electron-transport pathways; when reduced, it becomes formazan which is non-soluble in water. The amount of formazan generated is proportional to the metabolic activity of the bacteria. Filters with biofilms were stained with a 0.2% solution of MTT in sterile LB for 30 min. Later, filters were washed with distilled water in order to remove MTT, then dried, and formazan was extracted with dymethylsulfoxide (DMSO, Sigma). Subsequently, the optical density of extracts was measured at OD590.

After plating the biofilms, the amount of CFU in 20 μl of the initial culture was determined by plating on Petri dishes for control. After the maturation of biofilms, i.e., after 24 or 72 h of culture, filters were homogenized in 5 ml of sterile PS: first by stirring with a glass rod, followed by vortexing for 30 s. Glass fibers played the role of an additional abrasive material for the disintegration of cell aggregates, allowing for maximal recovery of the bacteria (tests for S. aureus biofilm dispersal and the role of glass fibers as abrasive agent were conducted. Data not shown). After biofilm dispersal, cell suspensions were diluted and aliquots were plated on tryptic soy agar (TSA, Sigma) Petri dishes supplemented with 0.25% glucose. Petri plates were incubated at 37°C for 48 h, and CFU numbers were counted. Experiments were repeated independently at least three times.

The general principle of the determination of the effects of ANP and CNP were performed as described in Gannesen et al. (2018a,b). Briefly, biofilms were grown on glass fiber filters Whatman GF/F in RCM in 12-well plates (Thermo Fisher Scientific, United States). One filter was placed on the bottom of the wells, where 3 ml of RCM containing NUP were previously added. Only concentrations of NUPs with the strongest effect on monospecies biofilms found in CLSM experiments were studied. Samples without NUPs constituted positive controls. Subsequently, 50 μl of cell cultures, prepared as described above, were added. In parallel, monospecies cultures of biofilms for each bacterium were cultivated in the same conditions as the controls. Biofilms were incubated at 33°C for 72 h under anaerobic atmosphere (GasPack^®). All samples in each experiment were duplicated on two filters; after incubation one filter of each sample type was stained with MTT and one was processed to count CFU. Experiments were conducted at least in triplicate.

Biofilms were grown in 24-well plates with a flat glass bottom (Greiner Bio-One, Germany). Three types of binary biofilms were constructed and studied (Table 3).

In case of simultaneously grown biofilms and primary colonizers in pre-formed biofilms, biofilms were obtained as described above. For simultaneously grown biofilms, 300 μl of each culture per well were injected. Simultaneously, grown biofilms were cultivated for 72 h. All biofilms were incubated in an anaerobic atmosphere at 33°C in RCM without agitation. In case of pre-formed biofilms, when the incubation time of the primary colonizer was over, biofilms were washed twice with sterile PS. Then, 1 ml of fresh medium with ANP or CNP was added in each well, and 40 μl of culture of the secondary colonizer were added. As in previously described experiments, we studied the most effective concentrations of NUPs found by CLSM in monospecies biofilms. Then binary biofilms were incubated for the necessary time. In parallel, controls for hybridization were made by preparing monospecies biofilms of each bacteria by the same pathway as binary ones. The positive control was without the addition of NUPs. All experiments were made independently in triplicates.

FISH-labeling was performed as described by Nistico et al. (2014). Briefly, when the incubation time was over, biofilms in wells were washed twice with sterile PS and fixed with 100% methanol for 30 min at room temperature. Subsequently, methanol was removed, and biofilms were dried overnight at room temperature. Fixed biofilms were treated with a lysozyme (Sigma) solution (0.1 mg/ml in buffer 0.1 M Tris–HCl pH 8.0 + 0.05 M EDTA) at 200 μL per well. A piece of paper towel soaked with water was placed in a hermetically sealed bag from the GasPack^® system to maintain high humidity and closed plates with biofilms were introduced inside. Lysozyme treatment was conducted at 37°C for 3 h. Subsequently, the lysozyme solution was removed gently by pipetting, and 200 μl of the lysostaphin solution [Sigma; 10 mg/ml in Tris–HCl pH 8.0 + 0.01% of sodium dodecylsulfate (SDS) + 20% formamide] was added in each well. Plates were incubated in the same system for 10 min at 37°C. Then, the lysostaphin solution was removed, and wells were washed twice with a phosphate buffer (pH 7.4). Cell membranes were permeabilized by exposing them for 3 min to increasing concentrations of ethanol (50, 80, and 100%) in the pH 7.4 phosphate buffer at room temperature. After treatment, the wells with biofilms were properly dried at room temperature.

For C. acnes RT5 hybridization, a 5′-GCCCCAAGATTACACTTCCG-3′ (Eurogentec) probe was used, as described by Poppert et al. (2010). On 5′ the probe was marked with the fluorescent dye Alexa Fluor 546. The probe was dissolved in 50 μl of sterile milli-Q water and stored at -20°C before use. For experiments, a hybridization buffer was prepared in a 2-ml Eppendorf tube by mixing 360 μl of 5 M NaCl in 40 μl of 1 M Tris–HCl (pH 8.0) and 600 μl of formamide (30% of volume). The solution volume was adjusted to 2 ml with milli-Q water, and 2 μl of 10% SDS were added. Tubes with buffer were enveloped in aluminum foil to avoid light flashing. Directly before experiments, 1.5 μl of probe solution was added into the buffer so that the final concentration of the probe reached 50 ηg/μl. In each well with biofilms, 200 μl of hybridization mix were added. Plates were quickly closed and placed into GasPack^® hermetic bags with wet paper towels inside; bags were closed and immediately enveloped in aluminum foil to avoid any light. Hybridization was conducted for 1 h at 46°C. While this occurred, a washing buffer was prepared. In conical 50 ml centrifuge tubes (Corning), 1 ml of 1 M Tris–HCl (pH 8.0), 1.02 μl of 5 M NaCl and 500 μl of 0.5 M sodium ethylene diamine tetraacetate (EDTA) were mixed. The volume was then adjusted to 50 ml with sterile milli-Q water, and 50 μl of 10% SDS were added. After hybridization, the liquid was removed from the wells, 1 ml of washing buffer was added in each well, and plates were incubated in darkness at 48°C for 15 min. Then, the liquid was removed and each well was rinsed with milli-Q water, then dried at room temperature in darkness. Afterward, 100 μl of DAPI-containing ProLong Diamond Antifade Mountant (Thermo Fisher Scientific) were added in each well to fix the biomass and stain it with DAPI. Plates were covered with aluminum foil and incubated at 4°C overnight.

CLSM analysis was conducted in the red and blue modes of fluorescence. For Alexa Fluor 546, the laser line 488 nm was used. DAPI was used as total biomass of S. aureus MFP03 and C. acnes RT5 indicator. For every sample, analysis was realized over at least 20 observations and by scanning at least 5 of the most representative areas. LMSM 710 was upgraded with a 405 nm laser diode, allowing the visualization of the DAPI staining, and scanning was performed in the red, blue, and simultaneously red-blue modes. The biomass density of C. acnes RT5 and S. aureus MFP03 biofilms was studied and calculated using the Comstat 2 plug-in of ImageJ to determine the variations of S. aureus and C. acnes biomass after exposure to ANP or CNP.

According to Rosay et al. (2015), the amidase operon regulator AmiC of P. aeruginosa has been identified as the receptor/sensor for CNP. We searched for proteins in Propionibacteriaceae and Staphylococcaceae taxon representatives, which could be similar to AmiC of P. aeruginosa. After this, the search for C. acnes amidase homologies in taxons Pseudomonas, P. aeruginosa, and Staphylococcaceae was conducted. A protein-BLAST search was made on the NCBI website of the National Institutes of Health in the United States¹ .

All experiments were conducted in at least three independent repeats. The statistical significance of results was evaluated using the non-parametric Mann–Whitney test. A significant difference was marked with ^∗ for p-value <0.05.

At 37°C (Figure 1A), the growth of S. aureus MFP03 (0.6 h^-1), generation time (61 min) and death constant after stationary phase 0.009 h^-1 were measured. The maximal OD₅₈₀ in control conditions reached 0.8 and the mean lag-phase length was about 1 h. Adding ANP or CNP 10^-8 to 10^-6 M did not significantly affect any parameter of S. aureus MFP03 growth. The same observations were made using ANP and CNP (10^-6 M each) in association. At 33°C, the growth curves of S. aureus MFP03 were slightly different in comparison with ones at 37°C (Figure 1B). The growth constant was higher (0.63 h^-1), and the generation time was slightly increased (63 min), but cells did not enter into a death phase characterized by reduction of OD, maybe because they grew slower (lag-phase 1.5 h) and with higher maximal OD₅₈₀ (about 0.97). As observed at 37°C, addition of NUPs did not significantly modify S. aureus growth parameters.

In contrast, we observed that the growth of C. acnes RT4 and RT5 at 37°C was impacted by NUP exposure (Figure 2). More precisely, in control conditions the strain RT5 showed a growth constant of 0.12 h^-1, a generation time of 7 h and a death rate constant at 0.0003 h^-1. Adding ANP and CNP markedly increased the generation time of this bacterium. The maximum reached +112.4 and +60.5% for ANP and CNP, respectively. However, the association of the two NUPs only increased the generation time of +15.7%, and no additive or synergistic effect of combining ANP and CNP was observed. In the presence of both ANP and CNP the growth constant varied from 0.05 to 0.08 h^-1.

Concerning death rate constants, no variation was observed for any NUP concentration that was tested. For the strain RT4 in the control condition, we measured a generation time of 7.8 h, a growth constant of 0.09 h^-1 and a death rate constant at 0.015 h^-1. The addition of both peptides increased the generation time to 10.28 h (ANP 10^-6 M) or 12.25 h (CNP 10^-8 M) (Figure 2A). When the bacteria were exposed to a combination of the two NUPs, no additional or synergistic effect was observed. The growth constant in the presence of NUPs was slightly decreased, varying from 0.06 to 0.08 h^-1. This was probably because of the reduction in growth rate, however, the death rate was constant and varied near zero. The RT4 strain’s maximum OD increased in the presence of CNP and slightly decreased in presence of ANP. The same was observed with the RT5 (Figure 2B) strain in the presence of CNP 10^-6 M. In the case of C. acnes it appeared that both NUPs were capable of modulating the growth dynamics, although these effects were generally moderate. At 33°C, the effects of both NUPs were virtually as marginal as observed at 37°C (data not shown).

Analysis of the mean biofilm thickness and biofilm biomass density of S. aureus MFP03 revealed the crucial significance of cultivation conditions in observing the effects of ANP and CNP (Figure 3). It is interesting to note that the biggest impact was observed at low concentrations of both ANP and CNP, i.e., 10^-8 M. In the absence of treatment and in aerobic conditions at 37°C, the biomass density and the average thickness of the biofilm formed by S. aureus MFP03 were 27.66 ± 1.46 μm³/μm² and 30.13 ± 1.69 μm, respectively. At all tested concentrations, ANP inhibited S. aureus MFP03 biofilm growth. Adding ANP 10^-8 M decreased the average biomass density of the biofilms to 18 ± 5.9 μm³/μm² (Figure 3A) and decreased the average thickness of the biofilms to 25.6 ± 1.9 μm (Figure 3B). CNP significantly inhibited S. aureus biofilms only at the lowest concentration (10^-8 M) where it decreased the biomass density to 17.7 ± 4 μm³/μm² and the average biofilm thickness to 22 ± 0.5 μm. The association of the two NUPs (ANP and CNP; 10^-6 M each) had a slight synergistic inhibitory effect on biofilm parameters. Individually, ANP 10^-6 M decreased the average biomass density and thickness of the biofilms to 24.8 ± 0.9 μm³/μm²) and 27.1 ± 1 μm, respectively, while CNP 10^-6 M alone reduced these values to 24.9 ± 3.2 μm³/μm² and 25.8 ± 4.7 μm, respectively. Combining the two NUPs decreased the biomass density and thickness to 19.9 ± 0.7 μm³/μm² and to 22.11 ± 0.5 μm, respectively. At 33°C in aerobic conditions, no effect of the peptides was observed except in terms of mean thickness, where an increase was observed in the presence of CNP 10^-8 M. More precisely, at 33°C in aerobic conditions the average biomass density was only 14.07 ± 0.85 μm³/μm², and the average biofilm thickness fell to 14.1 ± 0.9 μm. Then, at 33°C and in aerobic conditions, S. aureus MFP03 showed reduced biofilm formation activity in comparison to that observed aerobically at 37°C. When ANP or CNP were added, the biofilm biomass density varied from 14.21 ± 1 μm³/μm² to 15.9 ± 0.84 μm³/μm² after exposure to CNP 10^-8 M. The average thickness varied from 13.74 ± 1.3 μm to 16.3 ± 1.3 μm with CNP 10^-8 M. Then, at 33°C the weak inhibitory effect of both NUPs observed at 37°C disappeared and a tendency to stimulate emerged.

When S. aureus was grown in anaerobic conditions at 33°C, we observed that the biofilms reached a biomass density of 16.7 ± 0.58 μm³/μm² and an average thickness of 17.3 ± 0.66 μm. These biofilms appeared denser and thicker than biofilms grown at 33°C in an aerobic atmosphere but remained less developed at 37°C. High concentrations (10^-6 M) of both peptides did not significantly affect biofilm formation in these growth conditions (anaerobic, 33°C). In contrast, we observed that peptides used at 10^-7 M and 10^-8 M stimulated biofilm growth (Figure 3). In the presence of ANP 10^-8 M, biofilm biomass density reached 22.8 ± 3.1 μm³/μm² and an average thickness of 23.2 ± 3.6 μm. In the same conditions, CNP increased the average biomass density and thickness up to 21.1 ± 1.7 μm³/μm² and 21.2 ± 1.7 μm, respectively. At a concentration of 10^-7 M the effect of CNP on the biofilm density and thickness was more limited. Exposure of S. aureus to both peptides in association didn’t bring any advantage and didn’t have any additional effects. Moreover, changing the TSB medium to RCM led to the disappearance of all significant stimulatory effects of NUPs (Figure 3).

When the same experiment was performed on C. acnes, we observed that both strains were sensitive to NUPs. Of course, as RT4 and RT5 strains are unable to grow in the presence of oxygen, the effect of NUPs was only investigated in anaerobic conditions. At 37°C the biofilm formation activity of C. acnes RT4 was inhibited by both NUPs alone or in association (Figures 4A,C). In control conditions the biomass density and average thickness of the biofilm formed by C. acnes RT4 were 20.1 ± 0.4 μm³/μm² and 23.2 ± 0.5 μm, respectively. ANP maximally reduced RT4 biofilms when 10^-7 Ì: density and thickness decreased to 12.5 ± 2.6 μm³/μm² and 15.9 ± 2.5 μm, respectively. When ANP was used at a concentration of 10^-8 M we observed the same effect. CNP was more efficient at a concentration of 10^-7 M, with thickness and density biofilms values that reached only 51 ± 12 and 49 ± 13% of the control, respectively. Combining ANP and CNP had a limited effect on C. acnes RT4 biofilm formation. NUPs also exerted an inhibitory effect on the biofilm formation activity of C. acnes RT5 when grown at 37°C. In control conditions at 37°C, C. acnes RT5 had strong biofilm formation activity (26.9 μm average thickness and 23.3 μm³/μm² biomass density) (Figures 4B,D). At all tested concentrations ANP inhibited C. acnes RT5 biofilm density and thickness to a level of 67–79% and 75.5–79.5% of the control, respectively. The maximal effect of CNP was noted at 10^-6 M, with a decrease in biofilm thickness and density to a level of 42.3 ± 17 and 49.2 ± 16% of the control, respectively. CNP 10^-7 M had virtually the same effect. In contrast, combining ANP and CNP had no synergistic effect.

When the incubation temperature of the two C. acnes strains was reduced to 33°C, their biofilm formation activity and sensitivity to ANP and CNP differed. The RT4 strain produced thinner biofilms of about 8.9 ± 0.49 μm³/μm² biomass density and 9.3 ± 0.5 μm thickness. Moreover, no inhibitory effect of ANP and CNP was observed and even a marginal stimulation of the biomass density was noted (Figures 4A,C), especially in presence of ANP. Indeed, ANP 10^-7 M increased RT4 strain biofilm biomass density and average thickness to 121.1 ± 24 and 108.3 ± 19% of the control. The biofilm formation activity of C. acnes RT5 at 33°C was also decreased but the effect was more marginal than that of the RT4 strain, with an average thickness and biomass density of 20.7 ± 0.9 μm³/μm² and 21 ± 1 μm, respectively (Figures 4B,D). The inhibitory effect of ANP and CNP was preserved though it was weaker. The strongest effect of ANP was observed at a concentration of 10^-8 M with a decrease in biofilm density and thickness to 77 ± 12 and 77 ± 13% of the control, respectively. CNP exerted the strongest inhibitory effect when it was used at a concentration of 10^-7 M, leading to a biofilms thickness and biomass density of 68.5 ± 11 and 67 ± 1% of the control, respectively. This shows that the effect of NUPs on C. acnes is generally inhibitory but is also strain-specific and can be modulated by its microenvironment. This can be correlated to the behavior and ecological role of C. acnes strains in different human skin niches. On the other hand, the general lack of additive or synergistic effect of ANP and CNP suggests that the two peptides have competing effects on bacteria.

First, we analyzed how the co-existence of S. aureus MFP03 and C. acnes strains in mixed biofilms produced on glass fiber filters can affect their growth at 37°C. We observed that in biofilms, C. acnes forms very solid microcolonies, and these aggregates were impossible to disperse as single cells even by sonication or enzymatic treatment (data not shown). Therefore, we decided to measure cultivable bacteria present in the biofilm by direct counting of S. aureus CFU from dispersed biofilms and to determine the metabolic activity present in the biofilms by MTT staining, allowing us to estimate indirectly the evolution of the C. acnes biomass.

An average amount of 3.2 × 10⁷ ± 4.1 × 10⁶ S. aureus CFU was distributed on the filters. After 72 h of culture at 37°C in monospecies biofilms, the number of cultivable S. aureus MFP03 recovered from the monospecies biofilms reached a mean of 6.1 × 10⁸ ± 1.2 × 10⁸ CFU. In the presence of C. acnes, S. aureus grew more effectively. More precisely, using the C. acnes RT4 strain or the C. acnes RT5 strain (8.75 × 10⁹ ± 3.9 × 10⁹ CFU) in co-cultures, the amount of S. aureus MFP03 CFU recovered in the presence of the RT4 and RT5 strains of C. acnes was 2.62 × 10⁹± 1.3 × 10⁹ CFU and 8.75 × 10⁹± 3.9 × 10⁹, respectively (Figure 5A). The MTT staining of biofilms showed that the average staining intensity of monospecies S. aureus MFP03 and C. acnes biofilms were virtually equal (Figure 5B). However, it is interesting to note that in the case of mixed biofilms, the MTT values were far more than the values for the sum of monospecies biofilms. In addition, the CFU counts showed that S. aureus grew better in mixed biofilms. These results suggest that the growth of C. acnes was reduced in the presence of S. aureus MFP03.

To simulate better the skin microenvironment where C. acnes and S. aureus can coexist, we decided to study mixed biofilms at 33°C as the skin surface is not at 37°C and much closer to 33°C (Ariyaratnam and Rood, 1990; Boutcher et al., 1995). Also, to analyze and model the probable effects of colonization, we studied different types of mixed biofilms. We used only strain RT5 because in experiments with CLSM of monospecies biofilms, it demonstrated an inhibitory effect when exposed to ANP and CNP at 33°C, whereas S. aureus MFP03 was insensitive to NUPs at this temperature. We observed that in the case of simultaneously grown mixed biofilms and in mixed biofilms for which pre-formed S. aureus MFP03 biofilm was first formed, there was no significant difference between the CFU amount of S. aureus MFP03 in monospecies and mixed biofilms. In the case of mixed pre-formed C. acnes biofilms, the amount of S. aureus MFP03 CFU recovered from monospecies biofilms was higher compared to mixed biofilms (7.6 × 10⁹ ± 3.8 × 10⁸ and 4.6 × 10⁹± 3 × 10⁸ CFU, respectively). Likewise, in the case of C. acnes biofilms, the amount of S. aureus CFU in monospecies biofilms was higher compared to other binary biofilms studied: 7.6 × 10⁹± 3.8 × 10⁸ compared to 4.8 × 10⁹± 1.3 × 10⁹, 4.5 × 10⁹± 7.2 × 10⁸, and 4.2 × 10⁹± 6.8 × 10⁸ CFU for simultaneously grown biofilms in aerobic and anaerobically grown S. aureus biofilms, respectively. That should be explained by the cell death level in long-term incubated monospecies S. aureus MFP03 biofilms. Indeed, in the case of simultaneously grown biofilms, both monospecies and mixed biofilms were incubated for 3 days, whereas in the case of pre-formed biofilms S. aureus MFP03 biofilms needed to be grown for 4 days (with a replacing of filters with biofilms on a fresh medium).

The MTT staining of monospecies and mixed biofilms at 33°C showed that for each type of mixed biofilm, the MTT staining value was less than the sum of the intensities obtained independently for each monospecies biofilm, suggesting that C. acnes RT5 grew less in mixed biofilms than in monospecies biofilms. Nevertheless, this inhibitory effect was not as pronounced as in studies realized at 37°C, especially in the case of preformed C. acnes RT5 biofilms. More precisely, the OD₅₉₅ of formazan extracts in that case were 1.92 ± 0.04 and 1.7 ± 0.21 for monospecies S. aureus MFP03 and C. acnes RT5 biofilms, respectively, but reached 2.95 ± 0.035 for the binary biofilms, i.e., 81.6% of the algebraic sum of the two monospecies biofilms. These data suggest that C. acnes in mature biofilms can tolerate the negative effects of S. aureus MFP03, and that mature biofilms of C. acnes are not favorable for S. aureus MFP03 adhesion.

To investigate the potential effects of NUPs on mixed biofilms we used liquid RCM and grew biofilms simultaneously in equilibrium with planktonic cultures. We decided to test concentrations of the NUPs that had the strongest effect on monospecies biofilms in experiments with CLSM, i.e., 10^-8 M for ANP and 10^-7 M for CNP.

In the presence of NUPs, the number of S. aureus MFP03 CFU obtained from monospecies biofilms was decreased (Figure 6A) ranging from 1.76 × 10⁹± 1.1 × 10⁸ CFU in control studies to 1.64 × 10⁹± 2 × 10⁸ CFU with ANP and only 7.58 × 10⁸± 2.2 × 10⁸ CFU with CNP. This suggests that, if cells must adhere to the surface from a planktonic state in the presence of the peptides, NUPs should interfere with this process. This could be an explanation for the decrease in cultivable bacteria obtained from experiments in static conditions on filters, since biofilm production for CLSM experiments cells requires an initial adhesion step.

In mixed biofilms, the number of S. aureus MFP03 CFU measured was 2.15-fold lower than monospecies biofilms (8.1 × 10⁸± 3 × 10⁸ CFU, Figure 6A). Adding ANP further reduced S. aureus development in mixed biofilms (3.3-fold reduction with an average of 5 × 10⁸± 4 × 10⁷ CFU recovered from the biofilms). In contrast, adding CNP did not significantly change the ratio between planktonic and biofilm bacteria in monospecies and binary biofilms although in binary biofilms the number of S. aureus MFP03 was 2.33-fold lower than in mixed ones (3.25 × 10⁸± 4.1 × 10⁷ CFU).

It is interesting to note that the S. aureus CFU count results were correlated to MTT values (Figure 6B). MTT staining, corresponding to the metabolic activity in biofilms, was reduced in mixed biofilms in comparison to S. aureus MFP03 monospecies biofilms grown in the presence of ANP and CNP (-17.4 and -25.4%). The same was observed with C. acnes RT5, where an 18.7% reduction of metabolic activity was observed between monospecies and binary biofilms in the presence of ANP or CNP. However, the more striking result was that the MTT value of mixed biofilms was of the same range in control studies (OD = 0.65 ± 0.08) and in the presence of ANP (0.61 ± 0.1) or CNP (0.72 ± 0.13). These data suggest that S. aureus MFP03 is the dominant component of dual-species biofilms but, more importantly, considering the absence of variation of MTT values in binary biofilms and the decrease of cultivable S. aureus MFP03, that the balance shifted to C. acnes RT5 in presence of NUPs.

Considering these results and the hypothesis, we decided to go further by investigating by confocal microscopy-mixed biofilms labeled with a FISH probe for C. acnes. In control studies, we exposed monospecies biofilms of S. aureus with this probe for C. acnes and we observed no false hybridization. Then, we calculated the percentage of hybridization for each repeat in mixed biofilms and, in parallel, we stained with the FISH-probe monospecies C. acnes RT5 biofilms (data not shown). Coefficients varied from 0.2 to 0.99. DAPI staining was used as an indicator of total biomass in binary biofilms. These values were used to calculate C. acnes biomass in mixed biofilms. NUPs were tested at the same concentration as described above.

Using simultaneously formed biofilms, in control studies the DAPI labeling illustrating the total amount of bacteria was 2.93 ± 1.2 μm³/μm². In the presence of ANP and CNP it was 2.87 ± 1.6 and 3.06 ± 1.1 μm³/μm², respectively, showing no significant change in total biomass density (Figure 7). However, in all types of mixed biofilms, adding ANP or CNP increased C. acnes RT5 biomass. Indeed, the biomass of C. acnes RT5 rose from 0.36 ± 0.18 μm³/μm² in the control studies to 0.85 ± 0.53 and 0.54 ± 0.28 μm³/μm² in the presence of ANP and CNP, i.e., +236 and +150%, respectively. Taking into account C. acnes biomass in monospecies biofilms, these results demonstrated that the proportion of C. acnes RT5 in binary biofilms in the presence of ANP and CNP increased to 31 and 18%, respectively, whereas it was only of 12% in the control biofilms (Figures 7A–C, 8A). These results are coherent with CFU counts and MTT measurements.

As noted in simultaneously formed biofilms, using binary biofilms grown in S. aureus MFP03 pre-formed biofilms, we observed that ANP increased C. acnes RT5 biomass. In control studies it was an increase of 2.9 ± 1.1 μm³/μm² (Figure 7D) and reached 3.1 ± 1.6 μm³/μm² after exposure to ANP (Figure 7E). Conversely, the biomass of C. acnes measured after exposure to CNP decreased to 0.8 ± 0.4 μm³/μm² (Figure 7F). This stark reduction of C. acnes biomass in the presence of CNP can be attributed to the strong reduction of the S. aureus MFP03 CFU count previously observed, suggesting that in these conditions CNP should exert a strong inhibitory effect on biofilm formation. However, it is interesting to note that the calculated relative percentage of C. acnes produced on pre-formed S. aureus biofilms evolved from 21% in control studies to 40.3 and 36.3% in the presence of ANP and CNP (Figure 8B). This suggests that S. aureus biofilm is favorable for C. acnes adhesion and that the decrease in C. acnes biomass in biofilms observed with CNP results from the strong reduction of S. aureus development provoked by this NUP.

This hypothesis is coherent with results from studies realized on mixed biofilms based on pre-formed C. acnes RT5 biofilms showing and increase in total biofilm biomass from 2.7 ± 1.3 to 2.9 ± 0.5 and 3.4 ± 0.7 μm³/μm² under the effect of ANP and CNP, respectively. This increase can be essentially attributed to a rise of the specific C. acnes RT5 biomass, which evolved from 55% in control studies to 68.2 and 74.5% after exposure to ANP and CNP, respectively (Figures 7G–I, 8C). Although, particularly in the case of S. aureus, the differences in growth kinetics of the two bacterial species could impact their survival in studies requiring long-term cultivation, these results suggest that the surface of C. acnes biofilms cannot be favorable for S. aureus adhesion and development.