Role of the Pre-neck Appendage Protein (Dpo7) from Phage vB_SepiS-phiIPLA7 as an Anti-biofilm Agent in Staphylococcal Species

Abstract

Staphylococcus epidermidis and Staphylococcus aureus are important causative agents of hospital-acquired infections and bacteremia, likely due to their ability to form biofilms. The production of a dense exopolysaccharide (EPS) matrix enclosing the cells slows the penetration of antibiotic down, resulting in therapy failure. The EPS depolymerase (Dpo7) derived from bacteriophage vB_SepiS-phiIPLA7, was overexpressed in Escherichia coli and characterized. A dose dependent but time independent response was observed after treatment of staphylococcal 24 h-biofilms with Dpo7. Maximum removal (>90%) of biofilm-attached cells was obtained with 0.15 μM of Dpo7 in all polysaccharide producer strains but Dpo7 failed to eliminate polysaccharide-independent biofilm formed by S. aureus V329. Moreover, the pre-treatment of polystyrene surfaces with Dpo7 reduced the biofilm biomass by 53-85% in the 67% of the tested strains. This study supports the use of phage-encoded EPS depolymerases to prevent and disperse staphylococcal biofilms, thereby making bacteria more susceptible to the action of antimicrobials.

Keywords: S. aureus; S. epidermidis; biofilm; biofilm matrix; exopolysaccharide depolymerase.

FIGURE 1

Purification of the recombinant exopolysaccharide depolymerase Dpo7 from Escherichia coli BL21(DE3) pLys pET21a-dpo7. L: Standard molecular weight marker in kDa (Low Range Molecular Weight SDS-PAGE Standards, BioRad); Fraction eluted from nickel affinity chromatography containing purified Dpo7. Bands indicated with a black arrow were identified by mass spectrometry.

FIGURE 2

Activity of Dpo7 against 24 h-biofilms of Staphylococcus epidermidis F12 in PBS buffer at 37°C. Biofilm disruption was determined using (A) different concentrations of Dpo7 (μM) for 3 h at 37°C and (B) 0.15 μM of Dpo7 throughout time ranging 30 min to 6 h. Total attached biomass was measured by crystal violet staining after treatment and expressed as A₅₉₅ units (▲); Adhered cultivable bacteria (●) and supernatant cultivable bacteria (■) are expressed as Log (CFU/well). Each value corresponds to the mean ± standard deviation of three independent experiments.

FIGURE 3

Temperature (A) and pH (B) stability of Dpo7. Temperature stability was tested by incubation of Dpo7 (0.15 μM) for 30 min and pH stability after maintenance at room temperature for 1 h. C: control biofilm of S. epidermidis F12 without Dpo7 treatment; NT: activity of Dpo7 at pH 7.4 without temperature treatment. Values represent the mean ± standard deviation of three independent experiments. Bars having an asterisk are statistically significant different from the control and bars with a lower case ‘a’ indicate a statistically significant difference between the biofilm treatment with standard Dpo7 and the activity after thermal or pH treatment (ANOVA; P < 0.05).

FIGURE 4

Maneval’s staining of extracellular material in exponential-phase cultures of S. epidermidis F12 (A) before treatment and (B) after treatment with 0.15 μM of Dpo7 for 3 h at 37°C. A magnification of the figure allows for the comparison of the presence/absence of polysaccharide matrix represented by a white halo surrounding the cell.

FIGURE 5

Removal of 24 h S. epidermidis and S. aureus biofilms after addition of Dpo7 (0.15 μM) for 3 h at 37°C. (A) Adhered cultivable bacteria and (B) crystal violet staining of control biofilms (black) and treated biofilms (white). Means and standard deviations were calculated from three biological replicates. Bars having an asterisk are significantly different from the control (ANOVA; P < 0.05).

FIGURE 6

Biofilm growth inhibition in presence of Dpo7 (0.15 μM). (A) Planktonic growth of the cultures was determined by absorbance measurement at 600 nm after 24 h of incubation at 37°C. (B) Percentage of biofilm inhibition was calculated by crystal violet staining of adhered cells after growth of 24 h biofilms. Black bars represent the control values and white bars represent the values of wells treated with Dpo7. Means and standard deviations were calculated from three biological replicates. Bars having an asterisk are significantly different from the control (ANOVA; P < 0.05).