One mouse ear/group was subjected to histological examination (Additional file

4) and the rest 4 ears/group were subjected to enumeration of staphylococci. Comparison of lysostaphin and LytM185-316 in the mouse model In the last in vivo experiment the CB-5083 staphylococcal strain P1 (106/ear) was used to infect ears of mice with eczema. Twelve hours after inoculation of bacteria the treatment with proteins was started; 100 μg of lysostaphin or BAY 1895344 LytM185-316 in 50 mM glycine pH 8.0 and 10% glycerol buffer was applied to each mouse ear in a volume of 20 μl. In the case of control mice buffer alone was used for the treatment. Ears were treated with proteins or buffer four times every 12 hours. Three hours after the last treatment mice were anesthetized and the ears dissected. The ears were washed with alcohol to remove surface bound bacteria, kept on ice, homogenized and diluted in PBS. One hundred microliter of the homogenate from various dilutions was then transferred to agar plates, containing 7.5% sodium chloride. After incubation at 37°C for 24 hours the colony forming units were counted. 10 mice were used in the control group and in each treatment group. Prior to the in vivo use, staphylococci were cultured

for 24 hours on blood agar plates, re-inoculated and grown on fresh blood agar plates for another 24 hours, harvested, and stored frozen at −20°C after suspending aliquots in phosphate-buffered saline (PBS) supplemented with 5% bovine serum albumin and 10% dimethyl sulphoxide.

Before application check details on ears, staphylococcal suspensions were thawed, bacteria washed in PBS and diluted in PBS to achieve the appropriate concentration of the staphylococci. To determine the CFU, aliquots of staphylococcal suspensions were subjected to dilution, plating on blood agar and enumeration. Acknowledgements We are thankful to Drs Renata Filipek and Elzbieta Nowak for critical reading of the manuscript and fruitful discussions. This work was supported by the European Communities (“Novel non-antibiotic treatment of staphylococcal diseases”, specific RTD program QLRT-2001-01250, Center of Excelence in Bio-Medicine, EC FP7 grant “”Proteins in Health and Disease”" (HEALTH-PROT, Olopatadine GA No 229676), by the Deutsche Forschungsgemeinschaft DFG (“Proteolyse in Prokaryonten: Kontrolle und regulatorisches Prinzip”, BO1733/1-1) and by the Polish Ministry of Education and Science (MEiN, decisions 1789/E-529/SPB/5.PR UE/DZ 600/2002-2005). M.B thanks the European Molecular Biology Organization (EMBO) and the Howard Hughes Medical Institute (HHMI) for Young Investigator support. Electronic supplementary material Additional file 1: Picture of mouse ears untreated (on the left) and treated (on the right) with oxazolone. (TIFF 407 KB) Additional file 2: Stability of LytM185-316 and lysostaphin. Proteins were incubated without (1) or with concentrated, conditioned S.