Each peptide was synthesized with C terminus amidation. Peptides were synthesized and obtained at a purity grade of >95% by HPLC (GenScript Co., Piscataway, NJ 08854, USA). Cationic alpha helical peptides P5, P8, P8.1, P2, and P6.2 were previously designed using a combined rational and computer assisted approach, identifying short putative active regions from AMP databases (Faccone et al., 2014; Maturana et al., 2017). Peptide sequences are: peptide 2: GLLKKWLKKWKEFKRIVGY; peptide 8.1: RIVQRIAKWAKKWYKAGK, peptide 6.2: GLLRKWGKKWKEFLRRVWK; peptide 5: RIVQRIKKWLLKWKKLGY; peptide 8: RIVQRILKWLKKWYKLGK. Omiganan (MBI-226): ILRWPWWPWRRK; Random non-alpha helical peptide: MVVFSVPKFKSTVAKLLSSA.

E. coli C600ΔTOX:GFP strain was obtained from Dr. Weiss, University of Cincinnati (Gamage et al., 2003). E. coli C600ΔTOX:GFP is a lysogenized C600 strain carrying the 933W bacteriophage in which the stx gene was replaced by the gfp sequence. Since this strain does not produce Shiga toxin, it represents a safety option to evaluate bacteriophage infection. E. coli C600ΔTOX:GFP strain was grown in Luria Broth (LB) plus 10 mM CaCl₂ and chloramphenicol (Sigma) (15 μg/ml final concentration) overnight (ON) at 37°C under agitation. The ON culture was diluted to OD 600 nm = 0.1 in LB plus 10 mM CaCl₂ and chloramphenicol (Sigma) (15 μg/ml final concentration). Induction was carried out by adding ciprofloxacin to a final concentration of 40 ng/ml (Ciprax 200, Roemmers). Bacteria were incubated for 6 h at 37°C under agitation and cultures were then centrifuged at 5,000 rpm for 15 min. The bacteriophage-containing supernatant was purified by sucrose ultracentrifugation. Briefly, supernatant containing bacteriophage was ultracentrifugated at 35.000 × g (Beckman XL-70, Rotor J-20), at 4°C during 2 h with 35% sucrose solution. The pellet was resuspended in 1 ml of PBS, filtered with 0.2 μm filters (MC-PES-02S, Microclar) and kept at 4°C until the titration assay was performed.

E. coli strain (ATCC 37197) was grown in LB plus ampicillin (0.05 mg/ml final concentration) overnight at 37°C under agitation at 200 rpm. The culture was diluted 1:100 in LB plus ampicillin (0.05 mg/ml final concentration) and incubated for 2 additional hours at 37°C under agitation. At the end of the incubation, 1,000 μl samples of the E. coli strain were incubated with 100 μl of a suspension containing bacteriophages for 30 min at room temperature. At the end of this incubation, 3 ml of Top Agar (Tryptone 1%; NaCl 0.5%; Agar 0.7%) plus CaCl₂ (10 mM final concentration) was added, and plated on LB-Amp agar plates. Plates were incubated at 37°C and lysis plaques were counted after 24 h.

Bacteriophage ΔTOX:GFP (ϕΔTOX:GFP) was incubated with peptides diluted in 400 μl of PBS at a final concentration of 0.1 μg/ml, 5 μg/ml, 10 μg/ml, or 50 μg /ml for 16 h at 37°C. After incubation, bacteriophage titers were measured as described above. E. coli strain (ATCC 37197), used for titration assay, was incubated with peptides alone as a control. During titration assay, the final concentration of peptides in the bacterial suspension was 1.4, 2.8, and 14 μg/ml, respectively.

The Zeta potential measured in volts (ξ) was calculated using the Smoluchowski equation: where η is the viscosity of the suspension at 20°C, D is the dielectric constant of the solution at 20°C and μ is the electrophoretic mobility of particles (micrometer/s per volt/cm). Bacteriophage was incubated with a solution containing peptide 5 or Omiganan at 5.7 μg/ml final concentration for 30 min at 25°C under gentle agitation. Measurements were conducted in a Nano Particle Analyzer SZ100 (Horiba) at 25°C, each value represents the average of two independents batches, and 100 individual determinations were obtained per batch.

Dynamic light scattering experiments were carried out on an Analyzer SZ100 (Horiba) with a backscattering detection at 173°, using disposable polystyrene cells. The bacteriophage suspensions (with or without peptide) were left equilibrating for 15 min at 25°C. Normalized intensity autocorrelation functions were analyzed using the CONTIN method (Provencher, 1982), yielding a distribution of diffusion coefficients (D). D is used to calculate the hydrodynamic diameter (D_H) through the Stokes-Einstein relationship: (3) where k is the Boltzmann constant, T the absolute temperature, and η the viscosity of the medium. The D_H value was calculated from a set of 15 measurements (~13 runs each) for the bacteriophage in presence of peptide p5 or alone. The D_H of the sample was obtained from the peak with the highest scattered light intensity (i.e., the mode) in light scattering intensity distributions.

The significance of the difference between concentrations was analyzed using Prism 5.0 software (GraphPad Software), and the P-value is indicated by asterisks in the figures. Data correspond to mean ± standard errors of the mean (SEM) for each concentration using triplicates. Statistical differences were determined using the one-way analysis of variance (ANOVA). Comparisons a posteriori between groups were performed using Tukey's Multiple Comparison Test analysis.

Bacteriophages were incubated with 0.1, 5, 10, or 50 μg/ml of each peptide. After incubation, inactivation was evaluated analyzing the bacteriophage capacity to infect E. coli. The seven peptides displayed different inactivation activities against the bacteriophage. Peptides 6.2, 5, and 8 showed the highest inactivation activity, inactivating nearly the 100% of phages in the conditions tested (Figures 1B, 2A,B).

On the other hand, Peptides 2 and 8.1 showed the lowest inactivation activity among the cAMP tested (Figures 1A, 2C).

Random peptide and Omiganan showed no inactivation activity against bacteriophage under the conditions tested (Figure 3).

It is important to notice that, according to the protocol we used, after the incubation of each peptide with phages, the peptide-phage mixture is plated on the E. coli lawn. For that reason a possible antimicrobial activity of these cAMPS on this E. coli strain had to be previously assessed, and no visible activity of the peptides per se was observed. It is worth mentioning that the final peptide concentration affecting the bacterial lawn during the titration assay ranged from1.4 to 14 μg/ml, which is a much lower concentration than the effective minimal inhibitory activity (MIC) previously obtained for each peptide on E. coli. In addition we performed a MIC assay for these peptides at different concentrations between 54 and 3.3 μg/ml on the E. coli C600ΔTOX:GFP strain, and we found no antimicrobial activity below 27 μg/ml for all the peptides tested.

The interaction between peptides and bacteriophages was analyzed using Zeta potential assays. We observed that peptide 5 was able to modify the surface charge of the bacteriophages, by decreasing the net negatively charge exposed by the bacteriophage. The results strongly suggest that positively charged peptides and bacteriophages are interacting through electrostatic charges (Figure 4). Interestingly, the same behavior was found with peptide Omiganan, although this peptide did not show anti-bacteriophage activity.

We used DLS to assess whether the size of the bacteriophage was altered as a consequence of its interaction with the peptides, or inducing any aggregation effect. The results suggest that peptides do not affect the hydrodynamic diameter of the bacteriophage (Figure 5).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.