baumannii ATCC 17978, for practical simulation of the bactericidal effect of ϕAB2 on MDRAB in a hospital environment. A. baumannii M3237was purchased from the Bioresource Collection and Research Center INK1197 of Taiwan (BCRC 80276). A. baumannii M3237 is a MDRAB clinical isolate from the

Buddhist Tzu-Chi General Hospital and was maintained and grown in LB or agar at 37°C. Phage preparation ϕAB2 was A1155463 isolated from the raw sewage of a local hospital [35]. A high-titer stock of phage ϕAB2 (109–1010 plaque-forming units (PFU)/ml) was prepared via plate lysis and elution. ϕAB2 was propagated and assayed in triplicate using the double-agar-layer method as previously described [45]. Phage adsorption assay A. baumannii M3237 was infected with phage ϕAB2 at a multiplicity of infection (MOI; phage concentration/bacterial concentration) of 0.001 and incubated at room temperature. The bacterial host A. baumannii ATCC 17978 was also evaluated for comparison. Samples (100 μl) were taken at 2-min intervals for 10 min, diluted in 0.9 ml of cold LB, centrifuged (12,000 × g, 5 min), and supernatants containing unadsorbed phages were titrated. Effect of temperature on ϕAB2 stability ϕAB2 stock (1010 PFU/ml) was diluted to 108 PFU/ml with distilled water. The mixed phage solution was subsequently

divided into 1 ml vials and stored at −20°C, 4°C, or 25°C. At various time points up to 360 days, solution from one vial at each temperature was inoculated for plaque assay. Used www.selleckchem.com/products/YM155.html vials were discarded. To assess the effect of refreezing on phage survival, a vial with 500 ml of a 108 PFU/ml phage solution was stored at −20°C and inoculated for plaque assays at various time points, after which the solution was stored at −20°C again until the next sampling time. Effect of pH on ϕAB2 stability The stability of ϕAB2 at different pH values was determined by mixing 1010 PFU/ml of ϕAB2 suspension with sterile water at different pH values (pH 2, 4, 7, or 11) to obtain a 100 ml phage solution with a final phage concentration of 108 PFU/ml.

The pH was adjusted with 1 N HCl or KOH. After phage solutions were prepared, the initial concentration was determined within 5 min, and then stored at 25°C until used. Effect Farnesyltransferase of chloroform concentration on ϕAB2 stability Briefly, phage solutions (108 PFU/ml) were exposed to 0.5% or 2% chloroform. The first sample was inoculated within 5 min to determine the initial concentration, and the solution was then inoculated for plaque assays at different storage times up to 360 days. Stability of ϕAB2 on glass slides Aliquots of 100 μl of a 109 PFU/ml ϕAB2 suspension were spiked on the surface of sterilized glass slides (108 PFU/13.8 cm2 surface), and incubated in a biosafety hood at room temperature for 30 min until completely dry. At various time points, a spiked glass slide was placed into a conical tube with 20 ml of peptone and gently vortexed for 30 s. ϕAB2 recovered in the eluant was enumerated by plaque assay.