Antibiotics were used as follows: erythromycin (1 μg mL−1), chloramphenicol (10 μg mL−1), and streptomycin (200 μg mL−1). Overnight cultures grown in brain heart infusion broth (BHI) were added at a 1 : 250 (v/v) dilution to fresh BHI and incubated at 37 °C with Imatinib cell line aeration. CFU mL−1 were determined
by plating dilutions of culture aliquots on BHI agar. Competitive indices of mixed bacterial cultures during stationary phase were performed as previously described (Zambrano et al., 1993; Finkel et al., 2000; Bruno & Freitag, 2010) (Fig. 1a). Aliquots of 12-day-old cultures were stored at −80 °C. For each experiment, an aliquot of frozen cells was thawed and 50 μL was added to 12.5 mL of BHI and grown overnight at 37 °C. One hundred and twenty-five microliters of the overnight 12-day-old culture was added to 12.5 mL of a 1-day-old culture at a ratio of 1 : 100 and incubated at 37 °C for 10 days. Twelve-day-old and 1-day-old cultures were distinguished based on chloramphenicol
resistance of the 1-day-old cultures containing the site-specific integration vector pPL2 that conferred chloramphenicol resistance without influencing bacterial growth (Lauer et al., 2002). NVP-AUY922 order Every 24 h, an aliquot of the mixed culture was removed, diluted, and plated onto BHI agar to enumerate bacterial CFUs. One hundred and fifty of the resulting colonies were then patched onto BHI agar containing chloramphenicol, selecting for the original 1-day-old chloramphenicol resistant bacteria; this was found to be the most reliable method for clearly distinguishing drug-resistant colonies. The competitive index (CI) value was determined as follows: CI = (test strain CFU)/(reference strain CFU). Mid-log L. monocytogenes were washed and diluted in PBS to a final concentration of 1 × 105 CFU mL−1. Seven- to 8-week-old ND4 Swiss Webster mice (Harlan Laboratories, Inc., Madison, WI) were Chlormezanone infected via tail vein with
2 × 104 CFU. Forty-eight hours post infection homogenized tissue dilutions were plated on BHI agar to determine CFU per organ. For CI experiments, mice were infected via tail vein with a 1 : 1 mixture of a reference and test strains. The reference strain was DP-L3903, a wild type strain with a Tn917-LTV3 insertion that confers erythromycin resistance and has been confirmed to have no effect on L. monocytogenes virulence [(Auerbuch et al., 2001) and Fig. 5b]. Strains were grown to mid-log phase and mixed together in PBS. Two hundred microliters of 2 × 104 CFU mixed bacterial suspension were used for infection. After 48 h, livers and spleens were harvested and homogenized. The CI value for each organ was determined as previously described (Auerbuch et al., 2001). Statistical analysis was performed using Prism software (graphpad v.2.0). Where appropriate, a Student’s t-test was used to identify statistically significant differences. In all cases, a P-value <0.05 was considered significant.