Blood was collected by cardiac puncture and their entire lungs have been removed aseptically. The lungs had been homogenized in two ml of sterile 0. 9% saline, along with the homogenates and blood were serially diluted ten fold with sterile saline. one hundred uL with the diluents of lung homog enates too as blood was spread onto BAP supple mented with 5% sheep blood, along with the plates had been incubated at 37 C for 24 h. The numbers of CFU have been determined by counting the numbers of single colonies that appeared around the plates showing alpha hemolysis. Efficacy as assessed by bacterial density, Determination of bacterial loads in blood and lungs Blood was obtained at 0 hours, 1, 2, three, four, five, and six hours post antibiotic therapy immediately after AMRI SP 1 infection by cardiac puncture under ether anesthesia and exsanguinated at these chosen in tervals.
The blood from every infected mice was diluted with sterile saline in 1,1 ratio and 100 ul of this diluted sample was plated on Columbia BAP supplemented with 5% sheep blood. In the previously mentioned time points post infection, bacterial loads within the lungs of SP infected mice have been determined. For determination from the numbers of CFU inside the lungs, NU6027 CDK inhibitor lung tissues had been dissected and homogenized in Hanks balanced salt resolution with out supplements by utilizing a tissue homogenizer. The resulting homogenates of each and every sample had been then plated in ten fold serial dilutions on BAP, followed by incubation at 37 C for determination of your bacterial loads, as not too long ago described in detail. Pharmacokinetic and pharmacodynamic studies Pharmacokinetic and pharmacodynamic research were conducted for AMP and AZM in mice.
Concentra tion in sera was determined following administration via the tail vein a single intravenous selleck inhibitor dose of AMP at 200 mg kg physique weight and AZM at 50 mg kg physique weight. This dosage of ampicillin and azithromycin produces concen trations similar to those achieved in humans just after an oral dose of 500 mg, showing concentrations in pulmonary tis sues of mice that were above MIC for the organism for 48 to 72 h right after injection. The drugs have been administered through the tail vein inside a volume of one hundred uL per dose, 18 h right after intranasal challenge with AMRI SP1. At 0, 1, 2, three, four, five and 6 hours following a single dose of AMP or AZM or each in mixture, blood samples have been ob tained from the mice in groups of 3 by cardiac puncture in the course of ether anesthesia.
Just after blood collec tion, samples have been centrifuged at 5000 ? g at four C plus the serum was collected and stored at 80 C until it was analyzed. Antibiotic concentrations in serum have been de termined by the agar nicely diffusion method by using Ba cillus subtilis ATCC 12432 because the bioassay reference strain. The zone diameter obtained were plotted against known antibiotic concentration comprising a appropriate range on a semi log graph paper to acquire a standard curve which was used to extrapolate the antibiotic con centration in serum samples at various time points as stated prior to.