Anti-methicillin-resistant Staphylococcus aureus and antibiofilm activity of new peptides produced by a Brevibacillus strain

Bacterial cultivation

This study was approved by the Institutional Biosafety Committee (WU-IBC-65-003; 26 January 2022). Brevibacillus sp. SPR19 (GenBank accession number: MZ298491) was a soil bacterium isolated from a botanical garden in Nakhon Si Thammarat, Thailand, and deposited at Thailand Institute of Science and Technological Research (TISTR), Pathum Thani, Thailand with the collection number of TISTR 10170 (Songnaka et al., 2022b). It was cultured on Mueller-Hinton (MH) agar (Titan Biotech Ltd., Rajasthan, India) and incubated at 30 °C for 24 h. Tested bacteria (S. aureus TISTR 517 and MRSA isolate 142, 1096, and 2468 from Maharaj Nakhon Si Thammarat Hospital, Nakhon Si Thammarat, Thailand) were streaked on MH agar and incubated at 37 °C for 24 h (Memmert GmbH+ Co., Schwabach, Germany).

Purification of anti-MRSA substances

A colony of SPR19 was suspended in 0.9% NaCl solution, and its optical density (OD) at 625 nm was adjusted to 0.1. A total of 4 mL of cell suspension was added into 196 mL of Luria-Bertani (LB) broth (Titan Biotech Ltd., Rajasthan, India) before shaking at 30 °C, 150 rpm for 24 h. The cell-free supernatant (CFS) was prepared by centrifugation of cell culture at 10,000 × g and 4 °C for 15 min. The active substances in CFS were separated by ammonium sulfate precipitation, cation exchange chromatography (CIEX), and reversed-phase chromatography (RPC) as detailed in our previous study (Songnaka et al., 2022a). The purified samples were evaporated by a refrigerated speed vacuum concentrator. The dried substance was dissolved in deionized water and prepared in a two-fold serial dilution. A total of 100 μL of sample solutions was used to determine the antimicrobial activity by agar well diffusion method, using MRSA isolate 2468 as a tested bacterium. The purification balance sheet was recorded by measuring the dried weight of the anti-MRSA substance and calculating its antimicrobial activity in each step of purification by using the following equation:

Antimicrobial activity (arbitrary unit; AU) = ${\displaystyle \frac{2^{\mathrm{n}}\times 1,000}{\mathrm{V}}}$ where n is the reciprocal of the highest two-fold dilution, exhibiting a clear zone, and V is the sample volume in µL (Yang et al., 2018).

Evaluation of anti-MRSA activity

Broth microdilution assay

The assay was conducted in accordance with the guideline of the Clinical and Laboratory Standard Institute (CLSI). The cell suspension of tested strains (S. aureus TISTR 517 and MRSA isolate 142, 1096, and 2468) was prepared by adjusting the turbidity at the OD 625 nm to 0.1 (1 × 10⁸ CFU/mL). The cell suspension was prepared in a 20-fold dilution using Cation-Adjusted Mueller-Hinton broth (CAHMB) as a diluent. A total of 10 µL of the diluted bacterial preparation and 100 µL of pure substances (0.25–64 µg/mL) were loaded into the 96-well plates. The plates were incubated at 37 °C for 24 h. Control wells were performed for broth (CAMHB) and inoculum. Vancomycin was tested as a control drug. The experiment was performed in triplicate for each tested strain. Minimum inhibition concentration (MIC) was described as the lowest concentration of pure substances that showed no visible bacterial growth. A total of 10 µL from the samples in the above wells was spread on the MH agar and incubated at 37 °C for 24 h to measure the minimum bactericidal concentration (MBC). MBC was the lowest concentration of the substances that showed no colony on MH agar after 24 h incubation (CLSI, 2018).

Peptide sequencing

The purified peptides were dissolved in 0.1% formic acid and 1% acetonitrile (ACN) and then subjected to a high-resolution liquid chromatography-tandem mass spectrometer (LC-MS/MS) (Thermo Fisher Scientific Inc., Waltham, MA, USA). A total of 3 µL of the samples was loaded on a C18 column. The LC elution condition and MS/MS operational parameters were adopted from our previous study (Songnaka et al., 2022a). Peak Studio X (Bioinformatics Solutions Inc., Waterloo, CA, USA) was used for peptide identification (Krobthong & Yingchutrakul, 2020). The acceptable de novo peptide sequences were obtained by scoring the sequences with average local confidence (ALC) above 80%. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (Perez-Riverol et al., 2022) with the dataset identifier PXD042035. The mass, pI, net charge, and hydrophobicity of the peptides were estimated by HeliQuest (Gautier et al., 2008).

Circular dichroism (CD) spectropolarimetry

The peptide solutions (0.60 mg/mL) were prepared by deionized water or 50 mM SDS. The circular dichroism (CD) spectra were recorded by a J-815 spectropolarimeter (JASCO Corporation, Tokyo, Japan) at 25 °C in the wavelength between 190 and 260 nm. The samples were put in a 1 mm cuvette cell, and the parameter settings were as follows; a bandwidth of 1.0 nm, a scanning speed of 100 nm/min, and a data interval of 0.1 nm. The corresponding diluent was used to subtract its signal from the sample spectrum. The experiment was performed in triplicates, and the estimation of the peptide secondary structure was analyzed by the CONTINLL program (Sreerama & Woody, 2000).

Polyacrylamide gel electrophoresis and gel overlay assay

The purified peptides (5 μg) were analyzed on a 15% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). The gel was soaked in a Tris-glycine buffer pH 8.3 and ran at a constant voltage of 100 V. The protein bands in a one-half gel were detected by silver staining, and a protein marker was also loaded in the gel for comparison of band position. Another half gel was immersed in a fixing solution that contained 5% acetic acid and 25% methanol for 30 min and washed with distilled water for 3 h. The gel was poured with 18 mL of soft MH agar containing MRSA isolate 2468 to determine the inhibition zone after incubation at 37 °C for 24 h (Songnaka et al., 2022a).

Stability tests for the active peptide

The stability of the anti-MRSA peptide in response to temperature, pH, and proteolytic enzyme was determined by incubating the sample solutions (80 µg/mL) in the following conditions. Heat treatment was conducted at 25, 50, 75, and 100 °C for 1 h. The sensitivity to acid and basic solutions was performed at different pH of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 for 2 h at 37 °C. The pH was subsequently adjusted to 7.0 before further evaluation. Sensitivity to proteolysis was tested by incubating the purified peptides with trypsin, α-chymotrypsin, and proteinase K (1.0 mg/mL) (Vivantis Technologies Sdn. Bhd., Selangor Darul Ehsan, Malaysia) at 37 °C for 2 h. The antimicrobial activity of all treated samples was determined by agar well diffusion against S. aureus TISTR 517 and MRSA isolate 2468. The residual activity was calculated as a percentage based on the proportion of the inhibition zone of treated and untreated samples. The untreated peptides and fresh medium were used as the negative and blank controls, respectively (Baindara et al., 2016). Each experiment was conducted in triplicates, and the data were presented as mean ± SD. The result analysis was done by Student’s t-test, and the p-value of less than 0.05 accounted for statistical significance.

Scanning electron microscopy (SEM)

The cell suspension of S. aureus TISTR 517 and MRSA isolate 2468 was prepared in 0.9% NaCl, and adjusted the OD at 625 nm to 0.1. The purified peptide (1× MIC) was introduced into the cells and incubated at 37 °C for 1, 3, and 16 h. The untreated cells were used as a negative control. Furthermore, the cells were obtained by centrifugation at 10,000× g and 4 °C for 5 min and gently put on the surface of a glass slide. The cell-fixing was done by adding 2.5% glutaraldehyde in 0.1 M phosphate buffer at pH 7.2 and incubating at 4 °C for 24 h. Cells on slides were later washed with 0.1 M phosphate buffer pH 7.2, and 1% osmium tetroxide (OsO₄) in distilled water was added for 1 h as the post-fixation. The dehydration process employed a stepwise gradient with increasing ethanol concentrations (20–100%) for 15 min in each step. A critical point dryer (Quorum Technologies Ltd., Lewes, UK) was used to dry the fixed cells on the slides and covered them with gold (Cressington Scientific Instrument Ltd., Watford, UK). The cells were captured under 50,000× magnification of a scanning electron microscope (Songnaka et al., 2022a).

Time-killing kinetic assay

The overnight culture of S. aureus TISTR 517 and MRSA isolate 2468 were prepared with CAMHB and standardized at OD 625 nm to 0.1. Various peptide concentrations (2×, 10×, and 20× MIC) were also prepared. A total of 50 µL of different peptide concentrations was added to 50 µL of diluted cell suspension (20-fold dilution). The mixture was incubated at 37 °C for 0, 1, 2, 3, 6, 12, 18, and 24 h, respectively. At each point, 10 µL of the incubated mixture was used to prepare a 10-fold serial dilution using CAMHB as a diluent. Furthermore, 10 µL from the serially diluted sample was plated on a MH agar and incubated at 37 °C for 24 h. Cell colonies were counted, and the result was reported as colony-forming units (CFU)/mL. Experiments were done in triplicates, and the significant difference (p-value < 0.05, 0.01, and 0.001) were determined by one-way ANOVA for multiple comparisons between the treated and untreated samples (Yasir, Dutta & Willcox, 2019).

Cell membrane permeability assay

The sytox green uptake was used to assess the integrity of the cell membrane of S. aureus TISTR 517 and MRSA isolate 2468. Phosphate buffered saline (PBS) supplemented with 0.2% CAMHB (PBS-CAMHB) was used to prepare a bacterial suspension from a 16 h culture. The cell culture was centrifuged at 10,000× g and 4 °C for 15 min, and then the pellets were washed three times with PBS-CAMHB. The cell turbidity was adjusted at OD 625 nm equal to 0.1, and 100 µL of the standardized cell was added into a black 96-well microplate. The cells were mixed with the sytox green solution (5 μM) (Thermo Fisher Scientific Inc., Waltham, MA, USA) and incubated in a dark place for 15 min. A total of 100 µL of peptide solution were mixed to obtain the final concentrations between 2 and 128 µg/mL. The fluorescence was quantified at excitation and emission wavelength of 504 and 523 nm, respectively. Triton X-100 (1%) (AppliChem GmbH, Darmstadt, Germany) and non-treated samples were used as the positive and negative controls, while PBS-CAMHB only was used as a blank control (Yasir, Dutta & Willcox, 2019). Three independent experiments were performed. The statistically significant differences (p-value < 0.05) were evaluated by two-way ANOVA, using post hoc Tukey’s test for multiple comparisons between treatments and non-treatments.

Inhibition of biofilm formation

The overnight culture of S. aureus TISTR 517 and MRSA isolate 2468 was prepared in soybean-casein digest (SCD) broth. The cells were standardized using the same medium to achieve 1 × 10⁸ CFU/mL. The cell suspension was mixed with peptide at various concentrations (0.5–256 µg/mL) in a 96-well plate. The plates were then incubated at 37 °C for 48 h. Then, the planktonic bacteria cells were removed from the plates by washing them with PBS. Thereafter, 100 µL of methanol was added to the plate to fix the biofilm-associated cells. The plates were dried and stained with 0.1% crystal violet for 15 min before rinsing twice with PBS. A total of 100 μL of 0.33% acetic acid was finally added to resolubilize the stained crystal violet. The plates were incubated at 37 °C for 30 min, and the supernatants were transferred to a new 96-well plate. The absorbance at 570 nm was measured by a microplate reader (Thermo Fisher Scientific Inc., Waltham, MA, USA) to determine the biofilm formation. Vancomycin and untreated samples were used as the positive and negative controls, whereas SCD media without cells was a blank control (Garrison et al., 2015; Zhu et al., 2020). One-way ANOVA was used to analyze the significant differences among treated samples compared to the untreated controls at various p-values. Dose-response curves of P2 and vancomycin on biofilm inhibition were constructed using GraphPad Prism software version 9.5.1 for Windows (GraphPad Software, Boston, MA, USA). The data (logarithm of the peptide concentration and biofilm formation) were fitted by a nonlinear regression with four parameter logistic, and the half maximal inhibitory concentrations (IC₅₀) with their 95% confidence interval (CI) were calculated.

Biofilm eradication assay

A single colony of S. aureus TISTR 517 and MRSA isolate 2468 was inoculated into SCD broth and incubated at 37 °C overnight. The bacteria were standardized to achieve 1 × 10⁸ CFU/mL, and 100 µL of cell suspension was transferred to a 96-well microplate. The plates were incubated at 37 °C for 48 h for biofilm formation. The free bacteria cells were removed from the plates by washing them with PBS. Peptide solutions at the concentrations (0.5–256 µg/mL) were added to the microplate and incubated at 37 °C for 24 h. Subsequently, PBS was used to rewash the plate, and 100 µL of PBS was loaded into each well. The plate was sonicated for 30 min at room temperature, and the samples were taken and made a ten-fold serial dilution. A total of 10 µL of the diluted samples was plated on MH agar for colony count. Vancomycin and untreated samples were used as the positive and negative controls. Only SCD media without bacteria was used as the blank (Yuan et al., 2019). The significant difference was evaluated by one-way ANOVA (p-value < 0.05) compared to untreated controls.

Hemolytic assay

The Ethics Committee in Human Research at Walailak University has approved this study (WUEC-22-242-01; 15 August 2022). The written consent was obtained from a subject involved in the research. Whole blood (5 mL) was collected from a human volunteer and centrifuged at 700× g for 8 min. The pellets were washed 3 times with PBS before diluting to obtain 0.5% erythrocyte suspension. A total of 50 µL of erythrocytes was added to a 96-well plate, and the purified peptide in PBS (50 μL) was mixed to achieve the final peptide concentration of 1, 2, 4, 8, 16, 32, 64, 128, 256, and 512 µg/mL. The mixtures were then incubated at 37 °C for 1 h. They were further centrifuged at 1,000× g for 10 min, and the supernatant was loaded into a microplate before measuring the absorbance at 540 nm. The positive and negative controls were 1% Triton X-100 and untreated samples, respectively, while PBS was used as a blank control. This assay was carried out in three independent experiments. The percent hemolysis was calculated by the following equation:

$$\mathrm{\%}\mathrm{H}\mathrm{e}\mathrm{m}\mathrm{o}\mathrm{l}\mathrm{y}\mathrm{s}\mathrm{i}\mathrm{s}={\displaystyle \frac{[{\mathrm{A}}_{\mathrm{s}\mathrm{a}\mathrm{m}}-{\mathrm{A}}_{\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k}}]}{[{\mathrm{A}}_{\mathrm{p}\mathrm{o}\mathrm{s}}-{\mathrm{A}}_{\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k}}]}\times 100}$$where A_sam is the sample absorbance, A_pos and A_blank are the absorbance of positive and blank controls, respectively (Oddo & Hansen, 2017). The therapeutic index (TI) of hemolysis was measured as a ratio between the highest peptide concentration that exhibited 10% hemolysis and its geometric mean MIC value (Sengkhui, Klubthawee & Aunpad, 2023).

Isolation of anti-MRSA substances

The CFS of SPR19 was pooled, and the anti-MRSA substances were purified by a three-stepwise method; salt-precipitation, CIEX, and RPC consecutively. There was no inhibition zone against MRSA isolate 2468 found from the precipitate after ammonium sulfate precipitation at a 25% saturation level. The substances after salt precipitation at 50% saturation exhibited the inhibition zone. It was shown that the active substances were purified 2.13-fold with a percent yield of 80.00 in this step (Table 1). The active fractions were subjected to CIEX, by which the partially purified substances had a purification fold of 4.26 and a percent yield of 28.00. The final step of RPC could fractionate five anti-MRSA substances (BrSPR19-P1 to BrSPR19-P5; referred to henceforth as P1 to P5) (Fig. 1) with a purification fold between 4.81 and 7.92, and a percent recovery in the range from 2.00 and 7.20.

Antimicrobial activity by microdilution assay

The pure substances (P1–P5) from RPC were collected to evaluate for the MIC and MBC values against S. aureus TISTR 517 and MRSA isolates. P2 had the highest inhibitory activity with a MIC of 2 µg/mL against all tested bacteria and was followed by P3 (4 µg/mL), P4 and P5 (8 µg/mL), and P1 (32 µg/mL) (Table 2). Furthermore, P2 had the highest killing activity against all tested bacteria with MBC between 4 and 8 µg/mL and was followed by P3 and P4 (16 µg/mL), P5 (16–32 µg/mL), and P1 (64 µg/mL). Vancomycin had MIC and MBC values of 2 µg/mL against all tested strains. P2 was the most active substance from SPR19 as it had the lowest MIC and MBC values. Its inhibitory activity was the same as vancomycin, although its bactericidal activity differed among tested pathogens. Then, P2 was selected for further investigation. The result from the agar well diffusion assay found that the P2 activity was dependent on its concentration. P2 at the 1× MIC level did not exhibit inhibition zones in all tested bacteria, but the larger inhibition zones were significantly observed when incubated with increasing P2 concentrations (5×, 10×, 20×, and 40× MIC) (Table 3; Fig. S1). Vancomycin (30 μg or 150× MIC) showed a significantly large inhibition zone from 20.73 ± 0.23 to 23.90 ± 0.10 mm. It is evident that P2 could serve as an anti-MRSA agent and should be further studied for stability, mode of action, and toxicity.

Characterization of anti-MRSA peptides

Five anti-MRSA substances from RPC were subjected to a LC-MS/MS to fragment the daughter ions of the b- and y-ion series (Table S1). A de novo algorithm derived the peptide sequences based on the mass difference between two fragment ions and the confidence scores of each amino acid (Fig. 2). The result revealed that the anti-MRSA peptides from SPR19 had a length between 12 and 16 residues. The presence of non-polar amino acids (valine, leucine, and phenylalanine) contributed to peptide hydrophobicity. The positively (lysine) and negatively (aspartate and glutamate) charged residues dominated the hydrophilic structures and charges of the peptides. The predicted physiochemical properties of these peptides were as follows; mass (1,437.89 to 1,837.01 Da), isoelectric point (pI 4.25 to 10.18), net charge at pH 7.4 (−3 to +4), and hydrophobicity (0.12 to 0.96) (Table 4). The most occurring amino acid in P1 was glutamic acid (25.00%), whereas valine was abundant in P2 (41.67%), P3 (35.71%), P4 (23.08%), and P5 (23.08%). This result reflects that all peptides are amphiphilic, which suggests potential anti-MRSA activities against tested pathogens.

Determination of peptide secondary structure

The circular dichroism (CD) spectra of all peptides were measured by a spectropolarimeter. The result revealed that all peptides (P1-P5) in the water had similar CD spectra with a negative band between 190 and 230 nm (Fig. 3A). The random coil (48.5–50.9%) conformations were predicted to be the main secondary structure (Table 5). SDS is generally used to mimic the negatively charged and hydrophobic part of bacterial membranes (Krenev et al., 2020; Wu et al., 2020). When SDS was incubated with the peptides, only P2–P5 had spectral changes with a positive band between 190 and 205 nm and a negative band between 205 and 240 nm. The great magnitude at 195 and 220 nm was observed in the positive and negative bands, respectively (Fig. 3B). It was estimated that the proportion of α-helix structure was increased (2.5–5.1%) and that of turn conformation was decreased (5.8–8.2%) in the mixture of peptides (P2–P5) and SDS (Table 5). The structural change of P2–P5 might result in higher anti-MRSA activity when compared to that of P1. In addition, the CD spectra of P1 did not change in the presence of SDS, implying that the P1 had an alternative action on antimicrobial activity.

SDS-PAGE and gel overlay assay of peptide

The 15% SDS-PAGE gel showed the peptide bands of P1–P5 with an estimated size between 5 and 10 kDa (Fig. S2A). After the gel was overlaid with soft agar containing MRSA isolate 2468, it displayed an inhibition zone at the same location of peptides on the stained gel (Fig. S2B). It can be deduced that all purified peptides exhibited anti-MRSA activity.

Stability test of the purified peptide (P2)

P2 was the most active peptide from SPR19. It was further used to determine its stability. The antimicrobial activity of P2 against the tested bacteria (S. aureus TISTR 517 and MRSA 2468) was constant, although the temperature was raised to 100 °C for 1 h (p-value > 0.05). The remaining activity was between 98.41 ± 0.55% and 100.90 ± 1.35%, indicating that this peptide was thermostable (Table 6). The addition of proteolytic enzymes (α-chymotrypsin, trypsin, and proteinase K) to the peptide unaffected the P2 function by which the residual activity was 97.54 ± 0.48% to 100.46 ± 1.37% (p-value > 0.05). It suggested that P2 was stable to proteolytic enzymes. However, the P2 activity against S. aureus TISTR 517 significantly decreased when incubated under a pH less than 5 (p-value < 0.05), but it was stable at a pH between 5 and 10. There was a fluctuation in the antimicrobial activity for MRSA isolate 2468 at pH between 3 and 10, but it was not significantly different (p-value > 0.05). Collectively, P2 activity against tested bacteria was tolerant in the pH range from 3 to 10 with the activity of more than 90%.

Effect of the anti-MRSA peptide on the bacterial cells and membrane

The SEM micrograph revealed that both S. aureus TISTR 517 and MRSA isolate 2468 in the untreated condition appeared to be unbroken, firm, and round cells with smooth surfaces throughout the study period (Figs. 4A and 4B). When incubated with P2 at 1× MIC value for 1 h, these tested cells had a deflated and irregular shape. The cells were more damaged under a longer duration of incubation (3 and 16 h), by which the cell surface was rough and deformed, resulting in cell collapse and lysis. In addition, the complex formations between sytox green dye (a fluorescent dye) and nucleic acids generally imply that the cellular membrane permeability after being treated with AMPs. Before the addition of the tested substances, the fluorescence intensity showed no significant difference compared to the non-treatment conditions. The overall intensities for P2 (1× to 64× MIC) and Triton X-100 (1%) in both bacteria were 0.95 ± 0.19 to 1.18 ± 0.14 and 1.05 ± 0.06 to 1.17 ± 0.12 folds, respectively, in comparison to the non-treatment conditions. There was an increase in fluorescence immediately after incubation with P2. At the end of the study (1,440 min or 24 h), the intensity in S. aureus TISTR 517 increased by 1.36 ± 0.02 to 5.80 ± 0.76 folds when treated with P2 at concentrations ranging from 1× to 64× MIC (Fig. 5A). Similarly, an increased fluorescence was observed in MRSA isolate 2468 by which the intensity was changed from 1.53 ± 0.14 to 9.11 ± 0.22 folds when incubated with P2 at concentrations between 1× and 64× MIC (Fig. 5B). The intensity rapidly increased within 30 and 150 min after introducing P2 (4× to 64× MIC) in S. aureus TISTR 517 and MRSA isolate 2468, respectively, indicating that membrane disruption occurred during this time. However, the intensity remained constant thereafter, suggesting the complete binding of dye and nucleic acid. Furthermore, the incubation of sytox green dye and P2 peptide (64× MIC) did not result in an increase in fluorescence signal (Figs. 5A and 5B). The higher intensity was found in MRSA isolate 2468, implying that P2 could alter membrane permeability in MRSA isolate 2468 more than S. aureus TISTR 517. Interestingly, the fluorescence intensity was significantly increased when the tested strains were treated with P2 at concentrations of more than 2× MIC (p-value < 0.05). The result showed that the uptake of sytox green dye was dependent on the peptide concentration. Triton X-100 (1%) was used as a positive control, and the fluorescence intensity was elevated from 1.17 ± 0.12 to 16.31 ± 1.54 and 1.05 ± 0.06 to 17.49 ± 0.36 folds for S. aureus TISTR 517 and MRSA isolate 2468, respectively, when compared to the non-treatment. It was demonstrated that the fluorescence of the Triton X-100 treated sample exhibited a time-dependent pattern. This intensity was unchanged significantly since 960 min or 16 h. Furthermore, the fluorescence level in the treatment of MRSA isolate 2468 with Triton X-100 was higher than that of S. aureus TISTR 517, suggesting this resistant bacterium was more sensitive to membrane disruption.

Bacterial killing kinetics of anti-MRSA peptide

The bacterial growth in the absence of P2 increased between 3 and 18 h of the incubation period. It reached the stationary phase after 18 h with an increased growth of 2.64 and 2.80 log₁₀ CFU/mL at 24 h for S. aureus TISTR 517 and MRSA isolate 2468, respectively, when compared with the initial inoculum (Figs. 6A and 6B). The growing rate constant and the killing half-time were calculated based on the linear regression between log₁₀ of viable cells and time. After the addition of P2 to S. aureus TISTR 517 for 3 h, the growth rates were 0.04 (95% CI [0.01–0.07]), −0.06 (95% CI [−0.01 to −0.12]), and −0.13 (95% CI [−0.07 to −0.18]) h⁻¹ for 1×, 5×, and 10× MIC, respectively (Fig. 6A). The decrease in growth rates was observed between 3 and 12 h in which the rates were −0.04 (95% CI [−0.09 to 0.00]), −0.42 (95% CI [−0.34 to −0.49]), and −0.52 (95% CI [−0.44 to −0.60]) h⁻¹, and the calculated killing half-times were 75.82 (95% CI [59.50–101.79]), 10.68 (95% CI [8.18–13.90]), and 9.84 h (95% CI [6.62–14.41]) for 1×, 5×, and 10× MIC, respectively. Similarly, the growth rates of MRSA isolate 2468 in the first 3 h were 0.13 (95% CI [0.09–0.18]), −0.04 (95% CI [−0.13 to 0.05]), and −0.12 (95% CI [−0.01 to −0.22]) h⁻¹ after incubation with P2 at 1×, 5×, and 10× MIC, respectively (Fig. 6B). The rates between 3 and 12 h were 0.03 (95% CI [0.00–0.06]), −0.31 (95% CI [−0.24 to −0.38]), and −0.52 (95% CI [−0.45 to −0.60]) h⁻¹, and the predicted killing half-times were 82.22 (95% CI [51.39–172.18]), 11.61 (95% CI [9.35–14.40]), and 9.73 h (95% CI [6.52–14.33]) when treating with P2 at 1×, 5×, and 10× MIC levels, respectively. P2 at all concentrations significantly inhibited cell growth during 6–24 h compared to the non-treatment at the corresponding time (p-value < 0.05). P2 at 5× and 10× MIC showed complete bacterial killing at 18 and 12 h of incubation, respectively. It indicates that P2 activity depends on the incubation time and peptide concentration.

Biofilm inhibition and eradication

The biofilm formation of tested bacteria can be inhibited by AMPs, which could be measured by a crystal violet assay in a 96-well microplate. The result showed that the biofilm of S. aureus TISTR 517 was significantly decreased (72.82 ± 8.91% and 74.78 ± 2.64%) when the cells were pre-incubated with P2 and vancomycin, respectively, at 2 μg/mL or 1× MIC value (p-value < 0.05) (Fig. 7A). Interestingly, higher concentrations of P2 and vancomycin (4 μg/mL or 2× MIC level) significantly inhibited biofilm formation of MRSA isolate 2468 (64.59 ± 4.90% and 68.25 ± 4.21%, respectively) (p-value < 0.01), suggesting that the biofilm-forming capacity was an important virulence factor for this resistant strain (Fig. 7B). The biofilm production of both bacteria was significantly decreased, although it did not totally diminish when the concentration of both tested substances increased (8-256 μg/mL) (p-value < 0.01 or < 0.001). The Hill equation was used to quantify the half-maximal inhibitory concentrations (IC₅₀) of substances on antibiofilm activity. The IC₅₀ of P2 and vancomycin was 2.92 (95% CI [2.45–3.49]) and 4.84 (95% CI [4.08–5.74]) µg/mL, respectively for S. aureus TISTR 517 (Figs. S3A and S3B). The higher concentration of P2 and vancomycin at 4.84 (95% CI [4.26–5.51]) and 5.91 (95% CI [5.20–6.70]) µg/mL, respectively, were used for half-maximal inhibition of biofilms from MRSA isolate 2468 (Figs. S3C and S3D). It indicated that P2 had more potency than vancomycin in biofilm inhibition. This could be explained by the result that P2 killed planktonic cells more than vancomycin before the survived persistent cells formed biofilm. In addition, the biofilm of the resistant strain was more persistent than the sensitive bacteria. Treatment of preformed S. aureus TISTR 517 and MRSA isolate 2468 biofilms with P2 revealed that the cell reduction was a function of peptide concentration (Figs. 8A and 8B). P2 at the concentration of 4 µg/mL significantly decreased both mature biofilms compared to untreated samples (p-value < 0.05), whereas it completely killed these persistent cells at 128 µg/mL. Similarly, vancomycin at 64 µg/mL significantly reduced the persistent cells in the biofilms of MRSA isolate 2468, and the drug at 128 µg/mL entirely eradicated both biofilms. Although P2 could significantly reduce the number of persistent cells at a lower concentration than vancomycin, it completely eradicated these cells at the same vancomycin concentration. This might result from the high concentration of peptide or drug (128 μg/mL) that killed almost all cells in the biofilm environment. It indicated that P2 and vancomycin required a high concentration (64× MIC) to destroy mature biofilms, while those substances at low concentrations (2× and 1× MIC, respectively) killed planktonic cells of S. aureus TISTR 517 and MRSA isolate 2468.

In vitro hemolytic assay

Hemolysis is used to monitor the toxicity of AMPs on the mammalian cell membrane. Generally, the hemolytic activity of less than 10% was safe for human use (Amin & Dannenfelser, 2006). In this study, the P2 peptide had a percentage of hemolysis below 10% (0.84 ± 1.42% to 9.92 ± 1.04%) in concentrations between 1 and 64 µg/mL. In contrast, the increased activity (16.62 ± 0.85% to 72.03 ± 5.61%) was found when incubated with higher concentrations (128–512 µg/mL) (Fig. 9). Although the degree of hemolysis depended on the P2 concentration, it had a low hemolytic effect at the inhibitory concentrations. The index threshold of P2 was estimated to be 32, indicating that the peptide was highly selective towards bacterial cells compared to erythrocyte cells.