The 18 clinical isolates
and the two type strains (B. megaterium ATCC14581T and B. frigoritolerans DSM 8801T) were characterized using a standard set of biochemical tests (Weyant et al., 1996). For the production of B. anthracis-specific, d-PGA capsular antigens, the fresh vegetative growth of each isolate was used to inoculate 450 μL of heart infusion broth (Remel) supplemented with 50% heat-inactivated horse serum and 0.8% sodium bicarbonate, and incubated at 35 °C for 3 h. Detection of the d-PGA by the CAP-DFA assay was performed as described previously (De et al., 2002). click here Capsule visualization using India ink was performed as described in Luna et al. (2006), with the following exceptions: (1) cells were grown overnight on SBA at 30 °C, under 5% CO2,
and used to inoculate TSA plates containing 0.8% sodium bicarbonate (bicarbonate agar), which were then incubated under the same conditions; (2) two to three drops of India ink were added directly to the bacterial suspension; and (3) 5 μL of Small molecule library screening the suspension was added to a microscope slide and covered with a coverslip. Cells from both the DFA assay and India ink stain were viewed and photographed under oil immersion at × 1000 (UV and phase contrast, respectively), using a Nikon Eclipse 50i UV microscope and a Nikon Digital Sight DS-1 camera (Melville, NY). To test whether the capsules were covalently attached to the cell surface, the cells were heated at 60 °C for 30 min, and then stained with India ink as just described (Candela & Fouet, 2005). The colony morphology
of the isolates on bicarbonate agar was also noted, as encapsulated cells usually appear as mucoid or shiny colonies. DNA contained in the cell lysates of each isolate was used for all molecular testing (Hoffmaster et al., 2002). 16S rRNA gene sequencing was performed as described previously using the primers 8F and 1492R for amplification (Sacchi et al., 2002). BigDye 3.1 (Applied Biosystems, Foster City, CA) was used for sequencing Ureohydrolase reactions and products were run on an ABI 3130xl (Sacchi et al., 2002). Analysis of the 16S rRNA genes was performed using gcg ver 10.3 (Accelrys, San Diego, CA) to assemble, compare, and align sequences. The 16S rRNA gene sequences were used in blast searches to determine the best similarity to sequences in the NCBI database (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Neighbor-joining analysis was performed using Kimura-2 parameter correction and 1000-step bootstrap in mega 4 (Tamura et al., 2007). The 16S rRNA gene sequences obtained were deposited in the GenBank sequence database under accession numbers GU252108–GU252128. PCR amplification to detect the presence of four B. anthracis capsule genes (capA, capB, capC, and capD) was performed using previously published primers (Hoffmaster et al., 2006; Luna et al., 2006) and carried out in separate, 10-μL reaction volumes containing 1.5 mM MgCl2, 1 × buffer, 200 μM dNTPs, 2.5 U Platinum Taq polymerase (Invitrogen, Foster City, CA), 0.