, 2004, 2006; Klee et al., 2006; Luna et al., 2006; Peak et al., 2007). Over a 3-year period (2005–2007), the Rhode Island Department of Health GPCR Compound Library concentration (RI DOH) Laboratory received 56 clinical isolates for B. anthracis testing from multiple Laboratory Response Network (LRN) sentinel laboratories within the state. All
were initially referred to RI DOH Laboratory as ‘Bacillus spp., unable to rule-out B. anthracis,’ based on the LRN sentinel laboratory protocol and RI DOH Laboratory’s request that all isolates that are nonhemolytic, nonmotile, gram-positive rods, regardless of the colony morphology, be immediately sent for further testing. The RI DOH Laboratory determined that 49 of the 56 isolates submitted were truly nonhemolytic and nonmotile (one hemolytic, six motile), and ruled out B. anthracis for those 49 isolates based on their resistance to gamma phage, lack of amplification of the B. anthracis chromosomal and plasmid PCR targets (Hoffmaster et al., 2002), and negative reactions for the CW-DFA assay. A total of 18 of these isolates did, however, produce positive reactions to the CAP-DFA assay, indicating the production of a B. anthracis-like capsule, likely containing d-PGA capsular antigens. These isolates were forwarded
selleck kinase inhibitor on to the CDC for further phenotypic and molecular characterization. Further characterization of these strains increases our understanding of the genotypic and antigenic diversity of Bacillus
and how it affects our ability to identify B. anthracis and other Bacillus spp. Eighteen clinical Loperamide Bacillus spp. isolates that were originally sent to RI DOH Laboratory were included in this study (Table 1). Each isolate was assigned an identification number, subcultured onto trypticase soy agar (TSA) plates containing 5% SBA (BBL Microbiology Systems, Cockeysville, MD), and incubated overnight at 37 °C. Isolates were stored at −70 °C as spore suspensions in deionized water containing 25% glycerol. The positive and negative control strains used for the CAP-DFA assay included B. anthracis Pasteur (ATCC 4229) (pX01−, pX02+) and B. cereus (ATCC 14579), respectively. For capsule staining using India ink (Remel, Lenexa, KS), B. cereus G9241 was used for an additional, non-B. anthracis capsule-producing (non-d-PGA) positive control (Hoffmaster et al., 2004). For the PCR reactions, DNA in cell lysates of B. anthracis New Hampshire strain (pX01+, pX02+) and B. cereus (ATCC 14579) was used as positive and negative controls, respectively (Plotkin et al., 1960). The two type strains –B. megaterium ATCC 14581T and Brevibacterium frigoritolerans DSM 8801T– were ordered from their respective culture collections [American Type Culture Collection, Manassas, VA, and the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen) (DSMZ), Germany].