Lysostaphin disrupts Staphylococcus aureus and Staphylococcus epidermidis biofilms on artificial surfaces

Abstract

Staphylococci often form biofilms, sessile communities of microcolonies encased in an extracellular matrix that adhere to biomedical implants or damaged tissue. Infections associated with biofilms are difficult to treat, and it is estimated that sessile bacteria in biofilms are 1,000 to 1,500 times more resistant to antibiotics than their planktonic counterparts. This antibiotic resistance of biofilms often leads to the failure of conventional antibiotic therapy and necessitates the removal of infected devices. Lysostaphin is a glycylglycine endopeptidase which specifically cleaves the pentaglycine cross bridges found in the staphylococcal peptidoglycan. Lysostaphin kills Staphylococcus aureus within minutes (MIC at which 90% of the strains are inhibited [MIC(90)], 0.001 to 0.064 microg/ml) and is also effective against Staphylococcus epidermidis at higher concentrations (MIC(90), 12.5 to 64 microg/ml). The activity of lysostaphin against staphylococci present in biofilms compared to those of other antibiotics was, however, never explored. Surprisingly, lysostaphin not only killed S. aureus in biofilms but also disrupted the extracellular matrix of S. aureus biofilms in vitro on plastic and glass surfaces at concentrations as low as 1 microg/ml. Scanning electron microscopy confirmed that lysostaphin eradicated both the sessile cells and the extracellular matrix of the biofilm. This disruption of S. aureus biofilms was specific for lysostaphin-sensitive S. aureus, as biofilms of lysostaphin-resistant S. aureus were not affected. High concentrations of oxacillin (400 microg/ml), vancomycin (800 microg/ml), and clindamycin (800 microg/ml) had no effect on the established S. aureus biofilms in this system, even after 24 h. Higher concentrations of lysostaphin also disrupted S. epidermidis biofilms.

FIG. 1.

Lysostaphin-disrupted biofilms of MRSA strains Col and MBT 5040. Ninety-six-well polystyrene tissue culture wells (four for each sample) were inoculated with ∼10⁸ CFU of S. aureus strain Col or MBT 5040 (as indicated). After we allowed 48 h for biofilm formation, the wells were washed twice and then incubated with (+) or without (−) 50 μg of lysostaphin/ml in PBS for 3 h. Following incubation, the wells were washed again and stained with safranin to visualize biofilms.

FIG. 2.

Lysostaphin did not disrupt biofilms formed by lysostaphin-resistant S. aureus variants. Tissue culture wells (three for each sample) of a microtiter plate were inoculated with ∼10⁸ CFU of S. aureus strain SA113, SA5 LysoR, MBT 5040, or MBT 5040 LysoR (as indicated). After we allowed 48 h for biofilm formation, the wells were washed twice and then incubated with (+) or without (−) 50 μg of lysostaphin/ml in PBS for 3 h. Following incubation, the wells were washed again and stained with safranin to visualize biofilms.

FIG. 3.

SEM revealed that lysostaphin eradicates S. aureus biofilms, removing both the sessile cells and the extracellular matrix. Polycarbonate transwells were inoculated with 5 × 10⁸ CFU of S. aureus ATCC 49521 per ml. Following 48 h of biofilm formation, transwells were washed and then treated with either PBS (A and B) or 100 μg of lysostaphin/ml in PBS (C and D) for 3 h. The treated wells were washed again and then fixed with gluteraldehyde prior to SEM. The results shown are representative of the entire well. Magnifications, ×1,800 (A and C) and ×5,940 (B and D).

FIG. 4.

Lysostaphin caused an immediate and continuous drop in the absorbance of S. aureus biofilms, which continued over time. Polystyrene 96-well tissue culture wells were inoculated, per ml, with ∼10⁸ CFU of S. aureus ATCC 35556 (MIC of lysostaphin, 0.004 μg/ml; MIC of oxacillin, 0.125 μg/ml; and MIC of vancomycin, 1 μg/ml). Following 24 h of biofilm formation, wells were washed twice with PBS and then treated with either lysostaphin (6.25 μg/ml), oxacillin (400 μg/ml), or vancomycin (800 μg/ml) in PBS. The absorbance at 650 nm following treatment was monitored by a microtiter plate reader every 20 min for 3 h and then again at 24 h. The data are the means of results for six samples from three separate experiments ± the standard deviations.

FIG. 5.

Oxacillin or vancomycin had no visible effect on S. aureus biofilms in PBS or bacterial media after incubation for 24 h. Polystyrene 96-well tissue culture wells were inoculated with, per ml, ∼10⁸ CFU of S. aureus ATCC 35556 (MIC of lysostaphin, 0.004 μg/ml; MIC of oxacillin, 0.125 μg/ml; and MIC of vancomycin, 1 μg/ml). Following 24 h of biofilm formation, wells were washed twice with PBS and then treated for 24 h in either PBS (A) (this panel shows one of the plates from the experiment reported in Fig. 4) or TSB plus 0.25% glucose bacterial medium (B) with either no added antibiotic (first column of wells in each plate) or serial twofold dilutions of lysostaphin (first two rows; 0.8 to 200 μg/ml), oxacillin (third and fourth rows; 1.6 μg/ml to 400 μg/ml), or vancomycin (bottom two rows; 3.2 to 800 μg/ml) as indicated on the figure. Following treatment, the wells were washed with PBS and then stained with safranin. The darkly staining wells indicate the presence of biofilm following treatment. Lysostaphin in PBS cleared the biofilm at 0.8 μg/ml (A), while lysostaphin in TSB plus 0.25% glucose cleared the biofilm at 12.5 μg/ml (B).

FIG. 6.

Lysostaphin-disrupted S. epidermidis biofilms. Glass chamber slide wells were inoculated with ∼5 × 10⁷ CFU of either S. aureus strain SA113 as a control (A), S. epidermidis strain Hay (B), S. epidermidis strain ATCC 35984 (C), or S. epidermidis strain SE1175 (D) per ml. Biofilms were allowed to form for 24 h, and then wells were washed twice with PBS. Established biofilms were treated with PBS alone or with lysostaphin in PBS (200 μg/ml) for 3 h. Following treatment, the wells were washed with PBS followed by ddH₂O and then Gram stained. The biofilms in the PBS-treated row (−) stained gram positive, while no gram-positive cells were seen in lysostaphin-treated (+) S. epidermidis wells. Only the residual extracellular glycocalyx in the corners of the wells stained gram negative in the lysostaphin-treated wells. The two enlarged sections reveal the multilayered biofilm of S. epidermidis strain ATCC 35984 (top) and the residual glycocalyx of the same strain with no intact staphylococci following lysostaphin treatment (bottom).