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1 USF Center for Biological Defense, College of Public Health, University of South Florida, 3602 Spectrum Blvd, Tampa, FL 33612-9401, USA
2 University of Oklahoma, Department of Botany and Microbiology, 770 Van Vleet Oval, Norman, OK 73019-6131, USA
3 Florida Department of Health, Bureau of Laboratories, 3602 Spectrum Blvd, Tampa, FL 33612-9401, USA
Correspondence
K. K. Peak
kpeak1{at}health.usf.edu
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Present address: Esoteric Testing, Pathology Department, Tampa General Hospital, Tampa, FL 33601, USA. 
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain CBD 119T is DQ374637.
Supplementary tables are available with the online version of this paper.
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) are compiled as Supplementary Table S1 (available in IJSEM Online). After the presence of B. anthracis was ruled out, the powder and Bacillus sp. were released to the Center for Biological Defense (CBD) for further study. Colonies consistent with Bacillus sp. CBD 119T were repeatedly isolated by touching a sterile swab pre-moistened in 1x PBS to the loose powder then rolling the swab across the surface of a BA plate, incubated as before. Reisolations of strain CBD 119T consistently tested positive by PCR for the signature chromosomal element BA3 and the pX02 plasmid but negative for the pX01 plasmid. Additionally, the isolates were repeatedly ruled out as B. anthracis based on negative results for gamma phage susceptibility and direct fluorescent antibody (DFA) testing for cell wall and capsule. Failure to grow on mannitol egg yolk polymyxin B agar indicated that Bacillus sp. CBD 119T was not a member of the B. cereus group (Luna et al., 2005). Luna et al. (2006) confirmed the presence of a large plasmid that carries 10 genes (acpA, capA, capB, capC, capR, capD, IS1627, ORF 48, ORF 61 and repA) and the capsule-promoter sequence present on pX02 in strain CBD 119T. Gene sequences from the isolate shared 100 % nucleotide identity to B. anthracis plasmid pX02 for nine of the genes and 99.9 and 99.7 % identity to capD and capsule promoter, respectively. Although some of these plasmid genes have been reported in B. cereus and Bacillus thuringiensis (Pannucci et al., 2002), the CBD was the first to report pX02 capsule genes in Bacillus species that are not in the B. cereus group.
When the isolate was not recognized by three commercial systems, API (bioMérieux), OmniLog (Biolog) and RiboPrinter (DuPont-Qualicon), conventional biochemical and physiological tests were conducted to identify and characterize strain CBD 119T. Sporulation was induced on nutrient agar (NA) (Becton Dickinson) containing 5 mg MnSO4 l–1, grown at 30 °C for 48–72 h. Morphology, Gram-stain reaction and growth at pH 5.7 and 6.8 were tested with cells grown at 30 °C in 100 ml nutrient broth (NB) (Becton Dickinson) in 250 ml flasks aerated by shaking at 125 r.p.m. for 6, 24 and 30 h. Cell morphology, endospore characterization and swelling of the sporangium, presence of parasporal bodies, capsule production and motility were observed in wet mounts by using phase-contrast microscopy at x1000 under oil. Cells observed for motility were grown to the exponential phase in aerated tryptic soy broth (TSB) (Becton Dickinson) for 3–6 h at 30 °C. Growth of cells at varied temperatures was tested in 3 ml TSB in 13x100 mm tubes for 72 h in water baths set to 30, 35, 40, 45, 50 and 55 °C and examined for turbidity at 24 h intervals. Growth of cells at varied pH was tested in the same manner in TSB adjusted to pH 4.5, 5.5, 6.0, 6.5, 7.0, 8.0 and 9.0, incubated at 30 °C. The pH range was also evaluated in cells grown in aerated NB, set at 0.5 intervals of pH 4.5–9.0, incubated at 30 °C and examined for turbidity at 24 and 48 h. Growth dynamics of the cells were studied in 100 ml buffered TSB at pH 5.9 (25 mM MES), pH 7.1 (25 mM MOPS) and pH 7.5 (25 mM MOPS) in 250 ml flasks aerated at 125 r.p.m. and incubated at 30 °C for 24 h. Estimates of cell doubling times were made from plots of optical density against time and from viable cell counts for generation times. Salt tolerance was tested on NA plates supplemented with varied concentrations of NaCl incubated at 30 °C for 7 days. Physiological tests performed on cells grown in commercial medium (REMEL) at 30 °C were as follows: casein and starch hydrolysis, incubated 14 days; growth on mannitol egg yolk polymyxin agar, incubated 48 h; growth in Methyl red Voges–Proskauer (MRVP) broth for final pH, incubated 7 days; and growth on Sabouraud's 4 % glucose agar, pH 5.6, incubated 72 h. Gelatin hydrolysis was tested in 12 % nutrient gelatin (REMEL), grown at 30 °C for 2 weeks. Oxidase reaction was tested with Kovács' phenylenediamine redox dye reagent (Becton Dickinson). Anaerobic growth of the cells was studied for 1 week at 30 °C in the Mitsubishi Pack-Anaero anaerobic gas generating system with the following pre-reduced media: fluid thioglycollate medium with glucose and indicator (REMEL), marine agar plates (Becton Dickinson) and anaerobic agar (Claus & Berkeley, 1986), inoculated in the molten state. Acid production from 49 carbohydrates or carbohydrate derivatives was tested using the API 50CH panel with API CHB/E medium in combination with 11 biochemical tests from the API 20E kit. Following the manufacturer's instructions, the API panels were incubated at 30 °C for 48 h and evaluated by using the API LAB Plus Identification Program V.3.3.3/3.0 (bioMérieux).
Susceptibility studies were performed using the Sensititre (TREK Diagnostics) instrument-based system for MIC determination with ampicillin, chloramphenicol, ciprofloxacin, clarithromycin, clindamycin, erythromycin, gatifloxicin, gentamicin, levofloxacin, moxifloxacin, oxacillin, penicillin, quinopristin/dalfopristin, rifampicin, streptomycin, tetracycline and vancomycin. The Sensititre platform uses a 96-well format with a panel of precision-dosed antimicrobial dilutions to automate the classical microbroth dilution method. The microtitre plates were incubated at 30 °C in the Sensititre ARIS component. The MIC value for each antimicrobial was determined by the instrument and confirmed manually. Amoxicillin and ceftriaxone were tested by Etest (AB Biodisk) following the standardized protocol (CLSI, 2005); the MIC values were evaluated following the manufacturer's instructions. The breakpoints for the Bacillus species (CLSI, 2006b) were used for all antimicrobials except clarithromycin, gatifloxacin, moxifloxacin, oxacillin and quinopristin/dalfopristin for which the breakpoints for the Staphylococcus species were used (CLSI, 2006a). Results are given in the species description.
Amplification of 16S rRNA gene sequence used universal eubacterial primers 27F and 1492R (Herrick et al., 1993) to give a product of approximately 1500 bp in length. The amplified product was purified with Montage PCR Centrifugal Filter Devices (Millipore). Sequencing was performed by the Oklahoma Medical Research Foundation DNA Sequencing facility by using an ABI capillary sequencer with primers 27F, 907R, 926F and 704F (Johnson, 1994). Sequencher software (Windows V.4.2, Gene Codes) was used to assemble the fragments. The sequencing data was analysed by comparison of the consensus sequences with sequences available in the GenBank database using the Basic Local Alignment Search Tool in a nucleotide-to-nucleotide (BLASTN 2.2.16) search. 16S rRNA gene sequences from type strains of selected Bacillus species were obtained from GenBank and aligned using CLUSTAL_X (Thompson et al., 1997).
When 1495 bp of 16S rRNA gene sequences were compared to sequences available in the GenBank database, strains having greater than 98.5 % sequence similarity (Keswani & Whitman, 2001) to strain CBD 119T were Bacillus luciferensis LMG 18422T (AJ419629) (99.3 %; gaps=1/1488) and three unpublished Bacillus spp., KJ2C12 (AY514023) (99.3 %; gaps=3/1465), GPTSA100-1 (DQ854980) (99.1 %; gaps=1/1447) and L105 (DQ248043) (98.9 %; gaps=1/1435). Unpublished strains Bacillus spp. KJ2C12, GPTSA100-1 and L105 are cited in GenBank as an antagonist to Phytophthora blight of pepper, an isolate from a warm spring in Assam, India and an isolate from human impact zones in Kartchner Caverns, Arizona, respectively. Strain CBD 119T had only 95 % sequence similarity to each of the three facultative or obligate alkaliphiles closely related to B. luciferensis (Logan et al., 2002), Bacillus halmapalus (X76447), Bacillus horikoshii (AB043865) and Bacillus cohnii (X76437).
The hypervariable regions, V1–V3, in the 16S rRNA gene sequence of the Bacillus species correspond to nt 70–344 of rrnE (NC_000964.2, GeneID: 2914197) from B. subtilis subsp. subtilis strain 168 (Blackwood et al., 2004; Goto et al., 2000). The nucleotides of V1–V3 were identified in the sequence of strain CBD 119T by alignment with conserved regions at positions 47–69, 345–365 and 491–510 of the B. subtilis subsp. subtilis rrnE. GenBank BL2SEQ program was used to make the pairwise alignments of 460 bp in the hypervariable region of strain CBD 119T to those of closely related strains, resulting in 99.3 % similarity to Bacillus sp. KJ2C12 with 3 nt differences, 99.1 % to Bacillus sp. GTPSA100-1 with 4 nt differences and 98.2 % to B. luciferensis LMG 18422T with 8 nt differences. Thus, both Bacillus spp. KJ2C12 and GTPSA100-1 ranked above the B. luciferensis type strain as the best match to strain CBD 119T when the comparison was limited to 460 bp of the V1–V3 hypervariable regions.
Phylogenetic trees were constructed using neighbour-joining (NJ), maximum-parsimony (MP) and maximum-likelihood (ML) (PAUP v. 4.0) methods (data not all shown). The data were consistent in essential aspects whether based on 1340 bp of 16S rRNA gene sequence or 414 bp from the hypervariable region. Strain CBD 119T was consistently in the same clade as B. luciferensis LMG 18422T in MP and ML trees. In an NJ tree (Fig. 1) based on 1340 bp comparison, the B. luciferensis cluster, as defined by 100 % bootstrap support (Felsenstein, 1985), was divided into two monophyletic branches; strain CBD 119T groups with Bacillus spp. KJ2C12 and GPTSA100-1, while B. luciferensis LMG 18422T groups with Bacillus sp. L105. The NJ tree (not shown) based on the comparison of 414 bp also supported Bacillus sp. KJ2C12 as the closest relative to strain CBD 119T. An abbreviated DNA distance matrix (DNADIST, PHYLIP v. 3.6; Felsenstein, 2004) is presented in Supplementary Table S2 (available in IJSEM Online). The matrix shows the nearest neighbours based on the number of substitutions per nucleotide over a sequence length of approximately 1340 bp for all strains. By this measurement, strain CBD 119T had equal relatedness to both B. luciferensis LMG 18422T and Bacillus sp. KJ2C12. The DNA distance matrix, like the MP and ML trees, did not support a closer association of strain CBD 119T with Bacillus sp. KJ2C12 than with B. luciferensis LMG 18422T. Keswani & Whitman (2001) studied the relationship of 16S rRNA gene sequence similarity (S) to DNA–DNA hybridization (D). Among 40 Bacillus species, for an S of 0.991, D could be expected to be <0.70 about 99 % of the time. Thus, having an S of 99.3 % does not present a high probability that strain CBD 119T belongs to the same species as either Bacillus sp. KJ2C12 or B. luciferensis LMG 18422T.
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Cellular fatty acid composition of strain CBD 119T and B. luciferensis JCM 12212T was determined by Microbial ID system (MIDI) using the Sherlock Microbial Identification System library RTSB50 5.00. Strains were grown on TSA at 28 °C for 24 h. The major cellular fatty acids measured in strain CBD 119T and B. luciferensis JCM 12212T were anteiso-C15 : 0 and iso-C15 : 0, and each had 
7 iso or anteiso forms, consistent with the Bacillus species (Kaneda, 1967; Welch, 1991). Total cellular fatty acids are compared (see Supplementary Table S3 available in IJSEM Online). The isolate and type strain were misidentified as Brevibacillus, SI of 0.655 and 0.706, respectively. In proprietary dendrograms scaled to Euclidean distance (ED), an ED of 
10 links strains of the same species, an ED of approximately six links subspecies, and Bacillus isolates of the same strain link at approximately three ED (MIDI). Strain CBD 119T linked with B. luciferensis JCM 12212T at 10.5 ED, indicating the two are likely to be different but closely related species.
Capsules were observed in India ink mounts by phase-contrast microscopy at x1000 under oil (Luna et al., 2006). Strain CBD 119T produced thin but omnipresent capsules on TSA grown overnight at 35 °C with or without 5 % CO2 atmosphere. B. luciferensis JCM 12212T failed to produce capsules under all conditions tested. Strain CBD 119T was persistently negative in DFA capsule tests, suggesting that while the complete operon is present (Luna et al., 2006) the capsule does not manifest the epitope recognized by the test antibody. Alternatively, the observed capsule may be the product of genes other than those on the pX02-like plasmid. The chemical nature of the CBD 119T capsule has not been determined.
Phenotypically, strain CBD 119T was inconsistent with B. luciferensis JCM 12212T for 18 of 96 phenotypic traits evaluated (data not all shown), including motility, degree of chain formation and endospore-driven swelling. Strain CBD 119T had a more narrow temperature range for robust growth and an acidic pH optimum, suggesting that the isolate and B. luciferensis occupy different niches. Differential traits for strain CBD 119T and the type strain are compared in Table 1. The G+C DNA contents of strain CBD 119T and B. luciferensis JCM 12212T were determined by the Identification Service of the DSMZ following the methods of Cashion et al. (1977), Mesbah et al. (1989) and Tamaoka & Komagata (1984); for the percentage of DNA–DNA hybridization, DSMZ followed Cashion et al. (1977) and De Ley et al. (1970). DNA base composition of B. luciferensis JCM 12212T (=LMG 18422T) was 36.8 mol% G+C, whereas strain CBD 119T was 37.3 mol%. Logan et al. (2002) reported 33.0 mol% DNA G+C content of LMG 18422T by using a different HPLC system, column, running conditions and solvent system. Two measurements of DNA–DNA relatedness indicated very low-level affinity of 12.9 and 17.9 % between strain CBD 119T and B. luciferensis JCM 12212T. Based on the recommended threshold value of 70 % DNA–DNA relatedness between strains of the same bacterial species (Wayne et al., 1987; Stackebrandt et al., 2002), strain CBD 119T does not belong to the species B. luciferensis. Having <20 % DNA–DNA relatedness value with B. luciferensis JCM 12212T, the only species with validly published name to which the isolate had >97 % 16S rRNA sequence similarity, a new species designation for CBD 119T is justified.
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Colonies grown at 30 °C for 24 h on TSA with 5 % sheep blood are approximately 3 mm in diameter, cream to pearly grey, non-haemolytic, like sintered glass in opacity, smooth, shiny, becoming dull with a fine crepe texture with age, round, entire, raised to low convex and becoming crater form with age. Cells grown in NB, pH 5.8, 30 °C, aerated, are non-motile, round-ended rods (0.8–1x2–12 µm) with rudimentary branching and pronounced chaining; are initially long and flexed (3 h), becoming grossly pleomorphic with distended forms in torturous whorls flanked by ghost cells (23 h); stain Gram-positive to Gram-variable at 6 h. Cells in TSB, pH 5.9, 30 °C, aerated, at 6 h are pairs or short chains of bifurcating rods (1–1.2x7–15 µm) with branches of 2–15 µm in length (Fig. 2a, b); branches originate at one or less often, both poles and occasionally from the sidewalls. Colonies grown at 30 °C for 24 h on NA, pH 6.8, and TSA, pH 6.0 and 7.2, produced only rare rudimentary branching. Endospores are ellipsoidal and usually subterminal in unswollen to overtly swollen cells (Fig. 2c). No parasporal bodies or crystals were observed. Thin, omnipresent capsule is produced; genes (acpA, capA, capB, capC, capR, capD, IS1627, ORF 48, ORF 61 and repA) and capsule promoter with 99.7 % sequence similarity to the capsule operon of B. anthracis are carried on a large plasmid. Catalase- and oxidase-positive, does not grow anaerobically in the presence of glucose. Reduces nitrate to nitrite, weakly Voges–Proskauer positive. 
-Galactosidase, arginine dihydrolase, indole, ornithine, lysine decarboxylase and citrate are not utilized. Hydrolyses casein, gelatin and aesculin, but not starch or urea. Optimum pH is near 6; grows well on Sabouraud's agar at pH 5.6 but not at 4.5; alkaline range is to 8.5. Respective doubling times (optical density against time) and generation times at 30 °C in aerated, buffered TSB are 30 and 32 min at pH 5.9, 46 and 38–49 min at pH 7.1, and 52 and 48 min at pH 7.5. Autolysis is incipient as early as 4 h and extensive at 24 h in TSB at pH 7.1 and 7.5. Does not grow with 5 % NaCl or at 45 °C. Fatty acids anteiso-C15 : 0 (46.66 %), iso-C15 : 0 (28.11 %), iso-C16 : 0 (8.21 %) and iso-C14 : 0 (4.47 %) predominate. The DNA G+C content is 37.3 mol%. Acid produced in API 50CH panels from amygdalin, arbutin, D-cellobiose, D-fructose, gentiobiose, gluconate, D-glucose, N-acetyl-D-glucosamine, glycerol, glycogen, maltose, D-mannitol, D-mannose, salicin, starch, sucrose, trehalose. Acid not produced from adonitol, D-arabinose, L-arabinose, D-arabitol, L-arabitol, dulcitol, erythritol, D-fucose, L-fucose, D-galactose, 2- or 5-keto-D-gluconate, methyl 
-D-glucoside, inositol, inulin, lactose, L-lyxose, methyl -D-mannoside, D-melezitose, melibiose, D-raffinose, L-raffinose, L-rhamnose, D-ribose, D-sorbitol, sorbose, D-tagatose, D-turanose, xylitol, D-xylose, L-xylose or -methyl-xyloside. Resistant to penicillin, oxacillin, ampicillin, amoxicillin and ceftriaxone; susceptible to chloramphenicol, ciprofloxacin, clarithromycin, clindamycin, erythromycin, gatifloxicin, gentamicin, levofloxacin, moxifloxacin, quinopristin/dalfopristin, rifampicin, streptomycin, tetracycline and vancomycin.
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ACKNOWLEDGEMENTS |
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