Chimeric phage lysins act synergistically with lysostaphin to kill mastitis-causing Staphylococcus aureus in murine mammary glands

Abstract

Staphylococci cause bovine mastitis, with Staphylococcus aureus being responsible for the majority of the mastitis-based losses to the dairy industry (up to $2 billion/annum). Treatment is primarily with antibiotics, which are often ineffective and potentially contribute to resistance development. Bacteriophage endolysins (peptidoglycan hydrolases) present a promising source of alternative antimicrobials. Here we evaluated two fusion proteins consisting of the streptococcal λSA2 endolysin endopeptidase domain fused to staphylococcal cell wall binding domains from either lysostaphin (λSA2-E-Lyso-SH3b) or the staphylococcal phage K endolysin, LysK (λSA2-E-LysK-SH3b). We demonstrate killing of 16 different S. aureus mastitis isolates, including penicillin-resistant strains, by both constructs. At 100 μg/ml in processed cow milk, λSA2-E-Lyso-SH3b and λSA2-E-LysK-SH3b reduced the S. aureus bacterial load by 3 and 1 log units within 3 h, respectively, compared to a buffer control. In contrast to λSA2-E-Lyso-SH3b, however, λSA2-E-LysK-SH3b permitted regrowth of the pathogen after 1 h. In a mouse model of mastitis, infusion of 25 μg of λSA2-E-Lyso-SH3b or λSA2-E-LysK-SH3b into mammary glands reduced S. aureus CFU by 0.63 or 0.81 log units, compared to >2 log for lysostaphin. Both chimeras were synergistic with lysostaphin against S. aureus in plate lysis checkerboard assays. When tested in combination in mice, λSA2-E-LysK-SH3b and lysostaphin (12.5 μg each/gland) caused a 3.36-log decrease in CFU. Furthermore, most protein treatments reduced gland wet weights and intramammary tumor necrosis factor alpha (TNF-α) concentrations, which serve as indicators of inflammation. Overall, our animal model results demonstrate the potential of fusion peptidoglycan hydrolases as antimicrobials for the treatment of S. aureus-induced mastitis.

Fig 1

Effect of chimeric lysins on bacterial concentrations of S. aureus in whole cow milk at 37°C. Commercial UHT homogenized whole cow milk was inoculated with 2 × 10⁶ (A) or 2 × 10³ (B) CFU/ml of exponentially growing S. aureus Newbould 305, and λSA2-E-Lyso-SH3b (■) or λSA2-E-LysK-SH3b (●) at a concentration of 100 μg/ml, or buffer (×) as a control, was added after inoculation. Bacterial concentrations were determined by serial dilution plating of samples taken immediately before and after addition of enzyme or buffer and at 1-h intervals thereafter. Error bars represent standard errors of the means from at least three separate experiments.

Fig 2

Isobolograms of the plate lysis checkerboard synergy testing method showing synergy of λSA2-E-Lyso-SH3b with lysostaphin (A) and of λSA2-E-LysK-SH3b with lysostaphin (B) and the additive effect observed when lysostaphin was used on both axes of the plate (C). Lysis zones on checkerboard plates were evaluated by densitometry. For each square of the plate along the inhibitory line, protein amounts (as fractions of the enzymes' MIAs) were entered in an x/y plot. Error bars represent standard errors of the means from three to five independent experiments. The dashed line is the theoretical curve expected for an additive effect.

Fig 3

Effect of protein treatments on intramammary TNF-α concentration. “Snipped only” represents samples from glands that were snipped but not inoculated with bacteria. “Buffer” indicates glands that were inoculated with bacteria and later received DPBS without lysin. The remaining treatments consisted of inoculation of glands with bacteria, followed by administration of lysins in DPBS. Protein concentrations were 25 μg/gland for individually administered proteins (lysostaphin, λSA2-E-Lyso-SH3b, and λSA2-E-LysK-SH3b) and 12.5 μg/gland for each protein in the combination treatment (lysostaphin + λSA2-E-LysK-SH3b). Error bars represent standard deviations from 8 to 15 samples per treatment. Treatment means with different letters are significantly different (P < 0.05).