adjusted so the bacteria to macrophage infection ratio was 1:1. Wells were washed twice, and then incubated with 8 mg/ml of gentamicin for 1 hr at 37uC. After washing twice, as described, fresh complete medium containing 2 mg/ml of gentamicin was added, and cells were incubated for an additional 3 or 9 hrs. The infected macrophages that were not supplied with the 2 mg/ml gentamicin were water lysed for bacterial CFU enumeration, and the bacterial CFUs (defined time point, 0 hr) were compared among the 6 strains to determine their infection capabilities. The bacterial CFUs obtained at 0, 3, and 9 hrs post-infection were then calculated for bacterial macrophage survival assay with the initial bacterial counts (t = 0) as 100%. After water lysis, the released bacteria were placed on BHI agar plates and incubated for 48 hrs at 27uC, and the bacterial CFUs were enumerated. Mouse studies Pathogen-free female BALB/c mice (National Cancer Institute, Frederick Cancer Research Facility) of 7–9 wks of age were used throughout this study. All mice were maintained at Montana State University Animal Resource Center under pathogen-free condi- tions in individually ventilated cages under HEPA-filtered barrier conditions and were fed sterile food and water ad libitum. All animal care and procedures were in accordance with institutional policies for animal health and well-being. To assess the virulence of the newly constructed P1-derived strains, groups of BALB/c mice (5–7 individuals/group) were orally gavaged with 1.06109 CFUs of P1-pF1, -pHF, -pSA, -pSF1, -pSM, and -pY in 200 ml sterile phosphate buffered saline (sPBS). Mice were monitored for mortality for 4 wks post-administration, and experiment was repeated twice. For tissue colonization assay, mice were sacrificed at 4 days post-administration. Spleen, PP, and liver were excised under aseptic conditions for determination of bacterial burden as previously described [17,34]. Antimicrobial susceptibility tests The recombinant Salmonella strains of P1-pF1, -pHF, -pSA, - pSF1, -pSM with control -pY were determined for MIC under aerated conditions. They were subjected to various concentrations of erythromycin and PMB in polypropylene microtiter plates (Costar Corp., Cambridge, MA) at 37uC in LB medium. MIC was calculated according to the method as previously described [35]. The KIM6+/pF1, pHF, pSA, pSF1, pSM, and control pY were determined for erythromycin and PMB MICs using the procedure as previously described with slight modifications [36]. Y. pestis strains KIM6+/pF1, pHF, pSA, pSF1, pSM, and pY were grown in BHI broth at 27uC for 18 hrs without shaking, since we found that shaking would result in serious cell agglutination for all these 6 strains (data not shown). The cell cultures were then used for evaluating in MIC assays. In the preliminary experiment, we found that by using the cell density of 105 CFU/ml [36] we were unable to detect any bacterial growth after 48 hrs incubation at 27uC in 96-well polypropylene microtiter plates, even though no antibiotics were added in the medium. Thus, we raised the cell density by 100-fold (final cell concentration, 107 CFU/ml) for the References 1. Williamson ED (2009) Plague. Vaccine 27 Suppl 4: D56–60. 2. Alvarez ML, Cardineau GA (2010) Prevention of bubonic and pneumonic plague using plant-derived vaccines. Biotechnol Adv 28: 184–196. 3. Russell P, Eley SM, Hibbs SE, Manchee RJ, Stagg AJ, et al. (1995) A comparison of plague vaccine, USP and EV76 vaccine induced protection against Yersinia pestis in a murine model. Vaccine 13: 1551–1556. 4. Perry RD, Fetherston JD (2011) Yersiniabactin iron uptake: mechanisms and role in Yersinia pestis pathogenesis. Microbes Infect 13: 808–817. 5. Prentice MB, Rahalison L (2007) Plague. Lancet 369: 1196–1207. caf Runs as a Double-Edged Sword during Infection MIC determination. At this cell density, the MICs of erythromycin and PMB for these recombinant strains were determined at 24 hrs post-incubation at 27uC. At the end of the assays, the OD600 of the bacterial suspensions were measured by using a multichannel plate reader BioScreen C. The MICs were determined as the lowest concentrations of erythromycin and PMB, which did not result in measurable growth at the end of the experimental period [37]. Each sample was run in triplicate, and experiments were repeated three times. The recombinant KIM6+ strains were further subjected to analysis of susceptibility to two additional antimicrobial reagents, hydrogen peroxide (H2O2) and ox bile salt (Sigma-Aldrich (St. Louis, MO). Human cells produce hydrogen peroxide as a first line of defense against bacteria pathogens [38], and the secreted bile serves as the humoral barrier, interfering with normal function of the bacterial membrane and damaging the bacterial DNA [39]. The sensitivity to H2O2 was conducted according to the previously described method [40]. H2O2 (final concentration, 2.5 mM) was added to bacteria (final cell density, 105 CFU/ml) resuspended in sPBS. The mixture was allowed to incubate at 27uC for 1 hr, and the cells were briefly spun down to remove the H2O2. After a series of dilutions, the cells were placed on BHI agar for incubation at 27uC for 48 hrs, and the CFUs were enumerated. A parallel set of cultures not exposed to H2O2 served as a control. The bile sensitivity assay was performed by adding bile salt to BHI agar (final concentration, 1%) [41], and the log phase bacteria were placed onto the plates. Cells were incubated at 27uC for 48 hrs, and the CFUs were enumerated. The bacteria grown up on BHI agar without bile were used as a control. Three independent experiments were done for both hydrogen peroxide and bile assays. Statistical analysis The Tukey Kramer multiple comparisons test was used for assessing differences among experimental parameters. The Kaplan-Meier method (GraphPad Prism, GraphPad Software, Inc., La Jolla, CA) was applied to obtain the mouse survival fractions following infection with recombinant Salmonella strains. Using the Mantel-Haenszel log rank test, the P-values for statistical differences between inactivated Salmonella strains and control Salmonella strain-dosed mice were discerned at the 95% confidence interval. Acknowledgments We appreciate Dr. Jerod Skyberg from Montana State University for his critical reviews of this manuscript. We appreciate Dr. Robert D. Perry from University of Kentucky for providing us with strain Y. pestis KIM6+. The authors have declared no conflict of interests. Author Contributions Conceived and designed the experiments: XY. Performed the experiments: LC TL SJ TT XY. Analyzed the data: XY. Contributed reagents/ materials/analysis tools: RA XY. Wrote the paper: XY. 6. Sebbane F, Jarrett C, Gardner D, Long D, Hinnebusch BJ (2009) The Yersinia pestis caf1M1A1 fimbrial capsule operon promotes transmission by flea bite in a mouse model of bubonic plague. Infect Immun 77: 1222–1229. 7. Davis KJ, Fritz DL, Pitt ML, Welkos SL, Worsham PL, et al. (1996) Pathology of experimental pneumonic plague produced by fraction 1-positive and fraction 1- negative Yersinia pestis in African green monkeys (Cercopithecus aethiops). Arch Pathol Lab Med 120: 156–163. PLoS ONE | www.plosone.org 9 April 2012 | Volume 7 | Issue 4 | e36283