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, 57 (6), 2743-50

Novel Bacteriophage Lysin With Broad Lytic Activity Protects Against Mixed Infection by Streptococcus Pyogenes and Methicillin-Resistant Staphylococcus Aureus

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Novel Bacteriophage Lysin With Broad Lytic Activity Protects Against Mixed Infection by Streptococcus Pyogenes and Methicillin-Resistant Staphylococcus Aureus

Daniel B Gilmer et al. Antimicrob Agents Chemother.

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus pyogenes (group A streptococcus [GrAS]) cause serious and sometimes fatal human diseases. They are among the many Gram-positive pathogens for which resistance to leading antibiotics has emerged. As a result, alternative therapies need to be developed to combat these pathogens. We have identified a novel bacteriophage lysin (PlySs2), derived from a Streptococcus suis phage, with broad lytic activity against MRSA, vancomycin-intermediate S. aureus (VISA), Streptococcus suis, Listeria, Staphylococcus simulans, Staphylococcus epidermidis, Streptococcus equi, Streptococcus agalactiae (group B streptococcus [GBS]), S. pyogenes, Streptococcus sanguinis, group G streptococci (GGS), group E streptococci (GES), and Streptococcus pneumoniae. PlySs2 has an N-terminal cysteine-histidine aminopeptidase (CHAP) catalytic domain and a C-terminal SH3b binding domain. It is stable at 50 °C for 30 min, 37 °C for >24 h, 4°C for 15 days, and -80 °C for >7 months; it maintained full activity after 10 freeze-thaw cycles. PlySs2 at 128 μg/ml in vitro reduced MRSA and S. pyogenes growth by 5 logs and 3 logs within 1 h, respectively, and exhibited a MIC of 16 μg/ml for MRSA. A single, 2-mg dose of PlySs2 protected 92% (22/24) of the mice in a bacteremia model of mixed MRSA and S. pyogenes infection. Serially increasing exposure of MRSA and S. pyogenes to PlySs2 or mupirocin resulted in no observed resistance to PlySs2 and resistance to mupirocin. To date, no other lysin has shown such notable broad lytic activity, stability, and efficacy against multiple, leading, human bacterial pathogens; as such, PlySs2 has all the characteristics to be an effective therapeutic.

Figures

Fig 1
Fig 1
PlySs2 displayed activity against various species. Multiple strains of staphylococci (including MRSA, MSSA, and VISA), streptococci, enterococci, Listeria, bacilli, and lactobacilli were tested for susceptibility to PlySs2 activity. Escherichia and Pseudomonas were tested as Gram-negative controls. Log-phase cultures were exposed to 32 μg/ml PlySs2 for 30 min in PB (for 60-min readings) (see also Fig. S4 in the supplemental material). The final OD600 of the treated samples was divided by the final OD600 of the untreated samples to generate the normalized values. Complete lysis registered a ratio of ∼0.02. ST, serotype.
Fig 2
Fig 2
PlySs2 was bactericidal across multiple species of bacteria. Log-phase bacteria were treated in 96-well plates with 128 μg/ml PlySs2 in buffer A for 60 min, then serially diluted and plated onto BHI agar for CFU enumeration. The log kill was calculated by comparing the difference between vehicle-treated and PlySs2-treated CFU results.
Fig 3
Fig 3
MRSA, MSSA, and GrAS did not acquire resistance to PlySs2 in vitro. MRSA strain MW2, MSSA strain 8325, and GrAS strain MGAS 5005 were exposed to 1/32× to 4× the MIC of PlySs2 and mupirocin (S. aureus strains) over 8 days. The daily MICs of PlySs2 were compared to the starting MIC of PlySs2 for each strain of bacteria to ascertain resistance. None developed resistance to PlySs2. Both MW2 and 8325 developed resistance to the positive control, mupirocin.
Fig 4
Fig 4
PlySs2 protected mice from death caused by mixed MRSA and GrAS infection. FVB/NJ mice were injected i.p. with 5% mucin containing the pathogen of interest. Three hours postinfection, mice received one i.p. injection of either 20 mM phosphate buffer (control) or lysin treatment. (A) Survival data for the MRSA infection. Mice were infected with ∼5 × 105 CFU of MRSA strain MW2 and treated with either 1 mg of ClyS, 1 mg of PlyC, or 1 mg of PlySs2. (B) Survival data for the GrAS infection. Mice were infected with ∼1 × 107 GrAS strain MGAS 5005 and treated with either 1 mg of ClyS, 1 mg of PlyC, or 2 mg of PlySs2. (C) Survival data for the mixed MRSA and GrAS infection. Mice were infected with a combination of both bacteria from the above inoculums at the same concentrations. Mice were treated with either 1 mg of ClyS, 1 mg of PlyC, a combination of 1 mg of ClyS plus 1 mg of PlyC, or 2 mg of PlySs2. In all tests (A to C), mice were monitored for survival over 10 days. The results from 4 independent experiments were combined, and the data are plotted as a Kaplan-Meier survival curve; bars indicate standard errors. All the PlySs2 treatment groups (A to C) showed statistically significant differences (P < 0.0001) compared to the nonspecific single lysin or buffer controls, based on the log rank (Mantel-Cox) test.

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