Intragenic Antimicrobial Peptide Hs02 Hampers the Proliferation of Single- and Dual-Species Biofilms of P. aeruginosa and S. aureus: A Promising Agent for Mitigation of Biofilm-Associated Infections

Abstract

Pseudomonas aeruginosa and Staphylococcus aureus are two major pathogens involved in a large variety of infections. Their co-occurrence in the same site of infection has been frequently reported and is linked to enhanced virulence and difficulty of treatment. Herein, the antimicrobial and antibiofilm activities of an intragenic antimicrobial peptide (IAP), named Hs02, which was uncovered from the human unconventional myosin 1H protein, were investigated against several P. aeruginosa and S. aureus strains, including multidrug-resistant (MDR) isolates. The antibiofilm activity was evaluated on single- and dual-species biofilms of P. aeruginosa and S. aureus. Moreover, the effect of peptide Hs02 on the membrane fluidity of the strains was assessed through Laurdan generalized polarization (GP). Minimum inhibitory concentration (MIC) values of peptide Hs02 ranged from 2 to 16 μg/mL against all strains and MDR isolates. Though Hs02 was not able to hamper biofilm formation by some strains at sub-MIC values, it clearly affected 24 h preformed biofilms, especially by reducing the viability of the bacterial cells within the single- and dual-species biofilms, as shown by confocal laser scanning microscopy (CLSM) and atomic force microscopy (AFM) images. Laurdan GP values showed that Hs02 induces membrane rigidification in both P. aeruginosa and S. aureus. Peptide Hs02 can potentially be a lead for further improvement as an antibiofilm agent.

Keywords: Hs02; Pseudomonas aeruginosa; Staphylococcus aureus; intragenic antimicrobial peptide; polymicrobial biofilms.

Figure 1

Biomass quantification through the crystal violet assay of biofilms formed by two P. aeruginosa and two methicillin-resistant S. aureus (MRSA) isolates in presence of peptide Hs02. Biofilms were formed in the presence of different concentrations (ranging from the MIC to 0.25× MIC) of peptide Hs02. Two independent experiments were performed in triplicate. Error bars represent SD. Statistically significant differences in comparison to the control (p < 0.05) are marked with an asterisk (*). Abs: Absorbance.

Figure 2

Effect of peptide Hs02 on the proliferation of 24-h preformed biofilms. The OD₆₀₀ of planktonic phases of biofilms was measured and used to infer the biofilm proliferation. Pa4 and SA007 biofilms were treated with 8× MIC (32 µg/mL in both cases) of peptide Hs02; the dual-species biofilm of Pa4+SA007 was also treated with the same concentration. PA002 and Sa3 biofilms were treated with 8× MIC; that is, 128 µg/mL and 64 µg/mL, respectively, while the dual-species biofilm PA002+Sa3 was treated with 128 µg/mL. Pa3 and Sa1 biofilms were treated with 8× MIC; that is, 40 and 80 µg/mL, respectively, while the dual-species biofilm Pa3+Sa1 was treated with 80 µg/mL. Two independent experiments were performed in triplicate. Error bars represent SD. Statistically significant differences in comparison to the control are highlighted for p < 0.001 (***) or for 0.01 ≤ p < 0.05 (*). OD₆₀₀: optical density at 600 nm.

Figure 3

Confocal laser scanning microscopy (CLSM) images of single- and dual-species multidrug-resistant (MDR) P. aeruginosa and methicillin-resistant S. aureus (MRSA) biofilms. Biofilms were grown for 24 h and then treated for further 24 h with peptide Hs02 (1–6). Control images: no antimicrobial added. 1, 2, and 3: 8× MIC peptide Hs02 (32 µg/mL); 4: 8× MIC (128 µg/mL); 5: 8× MIC (64 µg/mL). 6: 128 µg/mL.

Figure 4

AFM images of the dual-species biofilm of PA002+Sa3. Biofilms were grown for 24 h and then treated for further 24 h with no peptide Hs02 (A) or with 128 µg/mL of the peptide (B). Scale bars correspond to 5 µm. Some features discussed in the text are indicated as follows: 1: undamaged PA002 cells; 2: undamaged Sa3 cells; 3: damaged PA002 cells.