aureus 43300(106 CFU/ml) intranasally Group 2: Mice were administered S. aureus 43300, left for a period of 48 hours to allow
nasal colonisation followed by intranasal administration of selleck chemical 50 μl of phage (107 PFU/ml) given twice (at an interval of 24 hours). Group 3: Mice were administered S. aureus 43300, left for a period of 48 hours to allow nasal colonisation followed by intranasal administration of 50 μl of mupirocin (5 mg/kg dissolved in water; given once) the next day. Group 4: Mice were administered S. aureus 43300, left for a period of 48 hours to allow nasal colonisation followed by intranasal administration of phage as well as mupirocin (5 mg/kg) the next day. The parameters used to monitor colonization included a) Bacterial load (CFU/ml) in nares b) Phage counts in nares c) Nasal myeloperoxidase (MPO) levels and e) Histopathological examination Nasal bacterial OSI-027 research buy load Four mice from each of group were taken and sacrificed on day 2, 5, 7, 10, 12 post treatment by selleck chemicals cervical dislocation. The nasal region was wiped
externally with 70% ethanol, nose was removed along with nasal bone. The entire nasal tissue was excised using sterile scissors and homogenized. The homogenates were plated quantitatively on nutrient agar containing 20 μg/ml of ampicillin to select S. aureus 43300 after overnight incubation at 37°C. Nasal homogenates were also processed to determine the phage titer by modified double layer agar method . Myeloperoxidase (MPO) estimation Mice from each group (same groups as those categorized for phage protection studies with 20 animals per group) were killed
and their nasal tissue was excised and homogenised in 50 mM PBS (pH 7.4). Nasal samples were processed for MPO determination as per the method of Greenberger et al. . The absorbance was read immediately at 490 nm over a period of 4 minutes. MPO was calculated as the change in optical density (O.D) x dilution factor (D.F). Histopathological examination Extent of injury caused by S. aureus and healing of the colonized mouse nose following therapy with phage or antibiotic was assessed on the basis of histopathological analysis of the injured and recovered nose according to the method of Brans et al. . The sections were picked Digestive enzyme on separate slides, stained with hematoxylin and eosin (Hi-Media, Mumbai) and the slides then examined under a microscope to evaluate the extent of damage. Statistical methods The data is expressed as mean ± standard deviation of replicated values where indicated. The statistical significance of differences between groups was determined by Student’s t-test (two groups),one-way ANOVA followed by a Tukey test using Sigma Stat, Graph pad prism (Graph pad software, San Diego, CA). p value of less than 0.05 and 0.01 was considered statistically significant for a confidence interval of 95% and 99% respectively. Results The nasal epithelial cells were isolated from mouse nasal tissue and cultured at 37°C in presence of 5% CO2.