These tubes were stored on ice and 5 μl of staining solution, consisting of 2.5 mg/ml propidium iodide (Sigma) dissolved in milliQ water, was added in the final propidium iodide concentration of 10 μg/ml. The cells were subjected to FACS analysis [53, 54], on the flow cytometer (BD-LSR, Becton Dickinson). Leakage
of 260 and 280 nm absorbing compounds The find more release of 260 and 280 nm absorbing compounds was determined spectrophotometrically [55]. Briefly, cells suspensions of S. aureus were prepared as for propidium iodide uptake assay. AKBA was added at 64 μg/ml to the MRT67307 bacterial suspension (≈1 × 109 CFU/ml) and incubated for 120 min at 37°C. For the complete release of 260 and 280 nm absorbing compounds, the bacterial suspension (control) was treated with lysozyme (100 μg/ml) at 37°C for 120 min, followed by sonication. Cell supernatants were obtained by centrifugation (10,000 g for 10 min). The absorbance of cell supernatant at 260 and 280 nm was determined using spectrophotometer
(Multiskan Spectrum). Background leakage rates (no compounds added) were used as untreated control. The extent of leakage of 260 and 280 nm absorbing compounds was expressed as percentage of control (suspension treated with lysozyme) measured in supernatants. Statistical analysis All check details experiments were carried out in triplicates in at least three different occasions. Differences between two means were evaluated by the Student’s t -test. The data were analyzed by one-way ANOVA
for comparison of multiple means followed by post bonferroni test using GraphPad Instat2 program JAK inhibitor (GraphPad software Inc. San Diego CA). The chosen level of significance for all statistical tests was P < 0.05. Acknowledgements The authors thankfully acknowledge the Ranbaxy Laboratories Limited, India for providing clinical Isolates. We would like to thank Scientific Faculty members of IIIM Srinagar, for critical reading of the manuscript. This work was funded by the Council of Scientific and Industrial Research, New Delhi, India (research grant no. P-81-101/2010 SRF (A.F.R.). References 1. Tacconelli E, Angelis GD, Cataldo AM, Pozzi E, Cauda R: Does antibiotic exposure increase the risk of methicillin-resistant Staphylococcus aureus (MRSA) isolation? A systematic review and meta-analysis. J Antimicrob Chemother 2008, 61: 26–38.PubMedCrossRef 2. Millar BC, Prendergast BD, Moore JE: Community-associated MRSA (CA-MRSA): an emerging pathogen in infective endocarditis. J Antimicrob Chemother 2008, 61: 1–7.PubMedCrossRef 3. Hiramatsu K: Vancomycin-resistant Staphylococcus aureus : a new model of antibiotic resistance. Lancet Infect Dis 2001, 1: 1470–55.CrossRef 4. Dancer SJ: The effect of antibiotics on methicillin-resistant Staphylococcus aureus . J Antimicrob Chemother 2008, 61: 246–253.PubMedCrossRef 5. Brown MR, Allison DG, Gilbert P: Resistance of bacterial biofilms to antibiotics: a growth-rate related effect? J Antimicrob Chemother 1998, 22: 777–780.CrossRef 6.