Synergism between a novel chimeric lysin and oxacillin protects against infection by methicillin-resistant Staphylococcus aureus

Abstract

Staphylococcus aureus is the causative agent of several serious infectious diseases. The emergence of antibiotic-resistant S. aureus strains has resulted in significant treatment difficulties, intensifying the need for new antimicrobial agents. Toward this end, we have developed a novel chimeric bacteriophage (phage) lysin that is active against staphylococci, including methicillin-resistant S. aureus (MRSA). The chimeric lysin (called ClyS) was obtained by fusing the N-terminal catalytic domain of the S. aureus Twort phage lysin with the C-terminal cell wall-targeting domain from another S. aureus phage lysin (phiNM3), which displayed Staphylococcus-specific binding. ClyS was expressed in Escherichia coli, and the purified protein lysed MRSA, vancomycin-intermediate strains of S. aureus (VISA), and methicillin-sensitive (MSSA) strains of S. aureus in vitro. In a mouse nasal decolonization model, a 2-log reduction in the viability of MRSA cells was seen 1 h following a single treatment with ClyS. One intraperitoneal dose of ClyS also protected against death by MRSA in a mouse septicemia model. ClyS showed a typical pattern of synergistic interactions with both vancomycin and oxacillin in vitro. More importantly, ClyS and oxacillin at doses that were not protective individually protected synergistically against MRSA septic death in a mouse model. These results strongly support the development of ClyS as an attractive addition to the current treatment options of multidrug-resistant S. aureus infections and would allow for the reinstatement of antibiotics shelved because of mounting resistance.

FIG. 1.

phiNM3 CWT binds specifically to staphylococci. Purified phiNM3 CWT was labeled with FITC and exposed to S. aureus (image pair 1), B. cereus (image pair 2), S. epidermidis (image pair 3), E. coli (image pair 4), S. pyogenes (image pair 5), and a mixed suspension of S. aureus and B. cereus cells (image pair 6). P, phase-contrast image; F, fluorescent image.

FIG. 2.

Activity of ClyS against S. aureus in vitro. S. aureus strain 8325-4 cells were resuspended in 20 mM PB (pH 7.4) and incubated with 250 μg of ClyS, and the OD₆₀₀ (filled circles) was monitored by a spectrophotometer. Control experiments (filled squares) were performed under the same conditions with buffer alone. Cell viability (filled triangles), measured as CFU/ml, was determined by serially diluting and plating the same cell suspensions to TSB agar plates.

FIG. 3.

ClyS causes cell wall disruption and, ultimately, the lysis of 8325-4 cells. (A to C) Thin-section transmission electron micrographs (bars, 200 nm) of S. aureus 3 min after exposure to 250 μg of ClyS. Ultimate lysis results in mostly cell ghosts (D) after the loss of cytoplasmic contents (bar, 500 nm).

FIG. 4.

ClyS exerted specific killing of antibiotic-susceptible and -resistant staphylococci. Log-phase cultures of different bacteria were exposed to 250 μg of ClyS for 15 min. Fold killing was calculated by dividing the number (CFU) of viable bacteria after buffer treatment by the number (CFU) after exposure to ClyS enzyme. Several MSSA and MRSA strains, along with S. epidermidis, S. simulans, and S. sciuri strains were tested for susceptibility to ClyS killing. Control strains (GAS, group A streptococci; GBS, group B streptococci; GCS, group C streptococci; GES, group E streptococci; along with enterococci, other streptococci, bacilli, pseudomonas, and E. coli) also were tested.

FIG. 5.

Effect of hyperimmune rabbit sera on ClyS activity. Ten microliters of either hyperimmune rabbit serum (final ELISA titer of 10,000), preimmune rabbit serum, or PBS was added to 100 μl of ClyS (100 μg). After 15 min, staphylococci were added and the change in OD₆₀₀ was measured for 30 min.

FIG. 6.

Effect of ClyS on nasal colonization by MRSA. Nasal passages of C57BL/6J mice were inoculated with ∼5 × 10⁹ of MRSA strain 191-SM^R. After 24 h, mice were treated by a single intranasal administration of 1 mg of ClyS or 20 mM PB. One hour after treatment, the bacteria from the excised nasal cavities were plated on Spectra MRSA agar to enumerate CFU. Data from three independent experiments were combined (19 mice per treatment group) and analyzed for statistical significance with the Student's t test.

FIG. 7.

ClyS protected mice from death caused by MRSA septicemia. FVB/NJ mice were intraperitoneally injected with ∼5 × 10⁵ CFU of MRSA strain MW2 in 5% mucin. Three hours postinfection, mice received one intraperitoneal injection of 20 mM PB control or 1 mg of ClyS. Mice were monitored for survival for 10 days. The results from three independent experiments were combined (ClyS treatment, n = 16; buffer treatment, n = 14) and the mice survival data plotted with a Kaplan-Meier survival curve.

FIG. 8.

ClyS showed synergistic interaction with vancomycin or oxacillin. Interactions between ClyS and antibiotics were tested by the checkerboard broth microdilution assay. Fractional concentrations of MICs along the inhibitory line for enzyme or antibiotic were plotted on the x/y plot to generate an isobologram. Error bars show standard errors of the means.

FIG. 9.

Synergistic effects of ClyS and oxacillin protected mice from MRSA septicemia-induced death. FVB/NJ mice were intraperitoneally injected with ∼5 × 10⁵ CFU of MRSA strain MW2 in 5% mucin. Three hours postinfection, mice received an i.p. injection of a suboptimal concentration of ClyS (166 μg) or 20 mM PB along with an i.m. injection of oxacillin (10 to 100 μg) or saline control. Mice were monitored for survival for 10 days, and the results of five independent experiments were combined and plotted in a Kaplan-Meier survival curve.