All buffers were 50 TPX-0005 mM with pH adjusted to 8.0 and data were collected after 60 min of reaction. Conductivity reflects both ion concentration and mobility. We reasoned that ionic strength was more likely than conductivity to influence protein activity, and therefore varied

conductivity systematically by changing the concentration of sodium chloride between 0 and 500 mM. Lysostaphin and LytM185-316 activities were again dependent on the ionic strength in the expected manner, but conductivity was more directly correlated with ionic strength in this experiment (Figure 7). Figure 7 The effect of ionic strength of reaction buffer on lytic activity of lysostaphin and LytM 185-316 . Lysis was done in standard conditions (see Material and Methods) in 20 mM glycine buffer pH 8.0 Tideglusib mouse supplemented with 0 to 500 mM NaCl. Conductivity of the reaction was measured at room temperature after addition of S. aureus cells. Presented results were collected after 60 min of lysis reaction at 37°C. The influence of ionic strength could also be demonstrated in a different way that was more directly related to the in vivo experiments. The low lytic efficiency of lysostaphin in glycine

buffer could be overcome by addition Oligomycin A clinical trial of 25 to 100% of serum. Conversely, the addition of 25% or more serum to optimal reaction conditions for LytM185-316 (50 mM glycine-NaOH) completely abolished the activity of enzyme (data not shown). The analysis of MIC and MBC for lysostaphin and LytM185-316 confirmed the above conclusions. The MIC for lysostaphin was around 0.0015-0.003 μg/ml, but inhibition of bacterial growth was not observed even with 5 μg/ml of LytM185-316. The MBC of lysostaphin was approximately 0.15 μg/ml in CASO broth and glycine buffer in agreement with previous data [36]. LytM185-316 had an MBC around 0.3 μg/ml in the low ionic strength glycine buffer, but did not exhibit bactericidal activity in CAMH or CASO broth growth media which have conductivity 18 mS/cm. Discussion Lysostaphin treatment of S. aureus infection has been reported earlier.

In a cotton rat model, S. aureus nasal colonization has been eradicated of by this enzyme [26]. In the mouse, S. aureus systemic infections have been successfully treated [25] and biofilms have been effectively eliminated from a catheterized jugular vein [24]. The chronic dermatitis model of staphylococcal infection reported in this paper differs significantly from the earlier models and therefore represents an independent confirmation for the efficacy of lysostaphin. The lack of efficacy of the LytM185-316 treatment was initially surprising in light of previously observed comparable activity of lysostaphin and LytM185-316, though in experiments carried out in low salt buffers. As a result of this work, we now know that LytM185-316 differs from lysostaphin in several ways that could all explain the outcome of the mouse experiments.