All in vitro experiments have been carried out in Mueller Hinton broth. Brain heart infusion broth was utilised as the medium for pneumococcal cultures prior to experiments with mouse. All experimental samples were placed on Columbia BAP supplemented with 5% sheep blood. In vitro susceptibility tests Minimum inhibitory concentrations and min imal bactericidal concentrations were deter mined by tube dilution approach in MHB supplemented with 5% sheep blood. The tubes contained two fold dilu tions of antibiotics and a final bacterial density of 105 CFU ml. The tubes have been incubated for 18 h at 37 C. The MIC was defined as the lowest concentration of antibiotic at which no turbidity was visible for the naked eye. For figuring out MBC, 0. 01 ml aliquots from tubes with no visible development were plated onto BAP supple mented with 5% sheep blood and incubated overnight at 37 C.
The MBC was defined selleck chemical as the lowest concentration of antibiotic that killed 99. 9% in the original inoculums. Likewise disk agar diffusion test was performed utilizing Mueller Hinton agar supplemented with 5% sheep blood. The disk content material of every single drug, the amount of an tibiotics tested for MIC and MBC for the clinical isolate and for the good quality control strain ATCC 49619 was carried out as per CLSI breakpoints for pneumonia. Modal values from 3 separate determinations selleck chemical molecule library were taken as the operating values. Lung infection model Male Balb C mice have been obtained from regis tered animal suppliers to the Department. Institutional Animal Ethical Committee reviewed and ap proved the methodology for use of these animals.
All an imals have been maintained and utilized in accordance with recommendations in the IAEC and have been supplied with meals and water ad libitum. Following overnight incuba tion on BHI broth supplemented with 5% sheep blood, freshly grown colonies were suspended in fresh BHI broth supplemented with 10% filtered horse serum to an optical density of 0. 12 at 550 nm. Experimental pneumo nia was induced in the animals having a penicillin and macrolide resistant strain of S. pneumoniae AMRI SP 1. Mice were anesthetized lightly by intravenous injection of ketamine hydrochlor ide at 1 mg kg of body weight through the tail vein, and one hundred ul of a bacterial suspen sion was inoculated by means of the nares in to the lungs of every mouse. The benefit of intra nasal inoculation will be to mimic oropharyngeal aspiration, properly infects upper and decrease respiratory tract and is very straightforward. To investigate the transform in colony type ing units inside the lungs and blood, animals were sacrificed under ether anesthesia, from 18th 24th h post infection.