20003. Performance standards for antimicrobial disk susceptibility tests. 309 Approved standard – Eighth Edition M2-A8, ISBN 1-56238-485-6,
CLSI. Wayne, Pa.). Briefly, fresh antibiotic-containing disks (serial dilutions) were used for susceptibility testing. LBG plates were inoculated with B. mallei Tamoxifen purchase ATCC 23344 and disks containing the antibiotic dilutions placed on top of the inoculated agar. The plates were incubated at 37°C for 24–48 h. Zones were measured and the mean diameter was calculated. The interpretation of results was based on the NCCLS zone diameters used for non-Enterobacteriaceae. For the broth dilution method, an inoculum of 105 CFU of washed B. mallei per ml was used, and the test was conducted in LBG for 24 h at 37°C. The interpretation of results was based on the NCCLS MIC breakpoints for non-Enterobacteriaceae and MIC for B. pseudomallei [16]. The inhibition of growth was confirmed by spectrophotometrically measurements and plating of serial dilutions onto LBG plates. Tubes containing bacteria but not
antibiotic were included as a positive growth control. Mice Animal studies were carried out in accordance with the Animal Care and Use Committee’s guidelines as recommended by the National Institutes of Health. Female, 6- to 8-week-old, BALB/c mice were obtained from Harlan Sprague Dawley, Inc. www.selleckchem.com/products/AZD1152-HQPA.html (Indianapolis, Indiana). Animals were provided with rodent feed and water ad libitum and maintained on 12 h light cycle. Challenge with B. mallei and antimicrobial administration Groups of 10 animals were inoculated via intranasal (i.n.) route with 5 × 105 CFUs of B. mallei ATCC 23344, in a total volume of 50 μl in PBS solution given to both nares. Treatment with antibiotic Montelukast Sodium via intraperitoneal route (i.p.) started 24 hours after infection, once a day, for 10 days. Doses of antibiotics used in this study were in the range of high doses used in humans: ceftazidime 100 mg/kg/day and levofloxacin 20 mg/kg/day. The animals were weighed prior to challenge and doses of antibiotics were adjusted accordingly. Levofloxacin
(Levaquin Injection, GlaxoSmithKline) and ceftazidime (Fortaz, Ortho-McNeil, Inc.) were purchased through local UTMB Pharmacy and doses for injection were prepared and stored according to manufacturer’s instructions. Bacterial load determinations Five animals from each group of antibiotic treated animals and survivors from non-treated control animals, were sacrificed and lungs and spleen were harvested for CFU determination. Organs were weighed, homogenized in 5 ml sterile PBS, plated in duplicates on LBG and incubated at 37°C for 2 days prior to CFU determinations. For comparison, spleen weights from healthy non infected but antibiotic treated animals were also evaluated. CFU were expressed as the mean ± SEM.
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