Bacterial Monks

MATERIALS AND METHODS
Bacterial strains and culture conditions
Two S. aureus strains were used in the present study; S. aureus 8325-4 (SigB-) and SH1000 representing a SigB+.strain. Overnight cultures were grown in Luria Broth (LB) at 37°Cwith shaking at 150 rpm. Exposure to antibiotics was carried out as detailed below.

Antibiotics

Ciprofloxacin were purchased from Sigma-Aldrich CO. 10 mg/ml stock solution of antibiotic were prepared freshly with 0.1N HCl and stored at -20°C. During the experiment we diluted with sterile water 1:10 and 1:100 depending on the different drug concentration.

Determination of minimal inhibitory concentration (MIC)

The MIC of Ciprofloxacin for both strains were estimated by using the broth microdilution method recommended by the Clinical and Laboratory Standards Institutes (CLSI) guidelines

Then 1 ml from these bacterial solutions were added to 1 ml of MHII containing different concentration of ciprofloxacin 0.5x, 1x, 5x, 10x, 20x, 30x, 50x,75x and 100x MIC to each well in 12-well plate. Colony counts (CFU/ml) were determined at different time points (T0, T3, T24) by using appropriate dilutions of each culture. Using spot-plating method (32) 10µl was spotted on LB agar plates. However after 24 hour exposure the bacterial cell from dilutions of ≤ 1x102 were washed twice with sterile PBS prior to plating in triplicate. These were then incubated overnight at 37°C. The experiment for each different density was repeated on three independent occasions and plating was carried out in