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Laboratoire de Bactériologie – Virologie, Hôpital de la Timone, CNRS UMR 6020, IFR48, 264 rue Saint-Pierre, 13385 Marseille, Cedex 05, France
Correspondence
Véronique Roux
vroux91{at}hotmail.com
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain 4400831T is AY677116.
Transmission electron micrographs of cells of strain 4400831T are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Aquino de Muro et al., 1992; Woodburn et al., 1995). Recently, based on 16S rRNA gene sequence comparisons, two novel species, Bacillus fusiformis (Priest et al., 1988) and Bacillus silvestris (Rheims et al., 1999), were included in the B. sphaericus-like group. Nakamura (2000) also proposed four novel potential species and the reclassification of earlier isolates based on the same technique. The B. sphaericus-like group consists of seven genetically distinct groups. The recognized species B. fusiformis and B. sphaericus were placed in groups 2 and 3, respectively, whereas B. silvestris appeared to be in a separate cluster. In 2002, the same author (Nakamura et al., 2002) described two novel species, Bacillus pycnus and Bacillus neidei, belonging to groups 6 and 7, respectively. Most of the strains included in the B. sphaericus-like group were isolated from soil, marine environments, mud or different types of insects, with some strains being pathogenic for their host (Nakamura, 2000). Usually, Bacillus spp. are considered as contaminants when they are isolated from biological samples but, in immunocompromised patients, they can cause significant disease. Bacteraemia due to B. sphaericus has been described in children receiving antineoplasic treatment or bone-marrow transplantation (Castagnola et al., 2001). In this paper, we describe a novel species of the genus Bacillus that was isolated from human cerebrospinal fluid (CSF).
Strain isolation and characterization
A 54-year-old man with cerebellar syndrome and diarrhoea episodes was diagnosed with Whipple's disease in January 2004 by PCR amplification of Tropheryma whipplei DNA from the CSF. The patient was treated with ceftriaxone, in association with trimethoprim-sulfamethoxazole. The treatment with trimethoprim-sulfamethoxazole was continued for 6 months. After 3 months of antibiotic therapy, a sample of CSF was taken and cell culture was performed. The presence of a Gram-negative-staining bacillus was detected in the supernatant using a Gram-stain kit (Color Gram 2; bioMérieux), according to the manufacturer's instructions. Subcultivation was carried out using sheep-blood agar (bioMérieux). The isolate, strain 4400831T, could not be identified using a phenotypic approach, but was identified as a strain of Bacillus at the molecular biology laboratory of Timone Hospital (Marseille, France), based on 16S rRNA gene sequence comparison. The antimicrobial susceptibility of the novel isolate was determined according to the NCCLS criteria and is presented in the species description.
After growth for 24 h on sheep-blood agar at 30 °C, surface colonies of strain 4400831T were circular, white–greyish, smooth and shiny and approximately 1–2 mm in diameter. Growth also occurred in trypticase soy broth (TSB; Becton Dickinson). The ability of the strain to grow at different temperatures (25, 30, 37, 45 and 50 °C) was investigated using sheep-blood agar. Growth occurred at 25–45 °C, with optimum growth at 30–37 °C. Growth also occurred in the presence of air, 5 % CO2 and a microaerophilic atmosphere that was created using a GENbag microaer (bioMérieux), but did not occur in an anaerobic atmosphere created using a GENbag anaer (bioMérieux), similar to other representatives of the B. sphaericus-like group (Reva et al., 2001).
The size and ultrastructure of the cells and spores were determined by electron microscopy. Cells were grown in TSB for 24 h at 30 °C and stained with 1 % (w/v) phosphotungstic acid. The samples were examined using a Morgagni 268D (Philips) electron microscope at an operating voltage of 60 kV. The cells were rods of 1.5–4 µm in length and 0.3–0.5 µm in width, occurring singly or in groups, and had peritrichous flagella (Supplementary Fig. S1 in IJSEM Online). Catalase activity was determined by using an ID color catalase test kit (bioMérieux) and oxidase activity was assayed by application of cells to moistened discs impregnated with dimethyl-p-phenylene diamine (bioMérieux). The novel isolate was catalase- and oxidase-positive. Tolerance of high NaCl concentrations was tested in TSB supplemented with 2, 5, 7 and 10 % (w/v) NaCl. The bacterium was able to grow in 5 % NaCl, but not in 7 %. This observation differed from that for B. silvestris (Table 1). The bacterium was motile, as determined by light microscopy.
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Preparation of cellular fatty acids and determination of their composition were carried out according to the methods of the Sherlock Microbial Identification System (MIDI Inc.). The major fatty acids were iso-C15 : 0 (48 %), anteiso-C15 : 0 (15.3 %) and iso-C16 : 0 (13.5 %). Smaller amounts of anteiso-C17 : 0 (5.6 %), iso-C17 : 0 (3.7 %), C15 : 0 (3.2 %), iso-C14 : 0 (3.1 %), C16 : 1
7c alcohol (2.6 %), C16 : 0 (1.5 %), C16 : 111c (0.7 %) and C18 : 19c (0.7 %) were also present. The predominance of iso-C15 : 0 was similar to other members of the B. sphaericus-like group; the composition and amounts of other fatty acids are characteristic for each group of Nakamura (2000) and for B. silvestris (Rheims et al., 1999).
Bacterial DNA was extracted using a FastDNA kit (BIO 101), as described by the manufacturer. PCR of the 16S rRNA gene was performed using the universal primer pair fD1 and rp2 (Weisburg et al., 1991). The PCR products were purified using MultiScreen PCR (Millipore) and sequencing was carried out using a DNA sequencing kit (BigDye Terminator Cycle sequencing version 2.0 ready reactions; PE Biosystems), according to the manufacturer's instructions. The sequence products were purified and electrophoresis was performed using a 3100 Genetic Analyzer (Applied Biosystems). Gene sequences were aligned using the multisequence alignment program CLUSTAL X (1.8). Phylogenetic relationships with closely related species were determined using MEGA version 2.1 (Kumar et al., 2001). Distance matrices were determined following the assumptions described by Kimura (1980) and were used to elaborate dendrograms using the neighbour-joining method (Saitou & Nei, 1987). The maximum-parsimony algorithm was also used to infer phylogenetic relationships. A bootstrap analysis was performed to investigate the stability of the trees obtained. Bootstrap values were obtained for a consensus tree based on 100 randomly generated trees. The tree organization was the same with the two methods. The phylogenetic analysis showed that the novel isolate is related to Bacillus odysseyi, which was isolated from the Mars Odyssey spacecraft (La Duc et al., 2004), and B. silvestris (Fig. 1). This cluster branched between the cluster including representatives of groups 1, 2 (B. fusiformis) and 3 (B. sphaericus) and the cluster including groups 6 (B. pycnus) and 7 (B. neidei). The percentage similarity between 16S rRNA gene sequences was determined using NALIGN in the PC/GENE software package (IntelliGenetics). The sequence similarity between B. odysseyi and strain 4400831T was 96.2 %; lower sequence similarities were obtained with all other recognized species of the genus Bacillus.
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Description of Bacillus massiliensis sp. nov.
Bacillus massiliensis (mas.si.li.en'sis. L. masc. adj. massiliensis of Massilia, the ancient Greek and Roman name for Marseille, France, where the type strain was isolated).
Cells are aerobic, endospore-forming, motile, Gram-negative-staining straight rods, with peritrichous flagella. Growth occurs on sheep-blood agar and in TSB. After growth for 24 h on sheep-blood agar, colonies are circular, white–greyish, smooth, shiny, and approximately 1–2 mm in diameter. The temperature range for growth is 25–45 °C, with optimum growth occurring at 30–37 °C. After growth for 24 h in TSB, rods are 1.5–4x0.3–0.5 µm. Grows in 5 % NaCl in TSB. Catalase- and oxidase-positive. Voges–Proskauer test is positive. Using the API 20E strip, activities of arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase and urease are positive, and citrate utilization is positive. Gelatin is not liquefied and ONPG hydrolysis, hydrogen sulfide production and indole production are negative. D-Glucose, D-mannitol, inositol, D-sorbitol, L-rhamnose, sucrose, D-melibiose, amygdalin and L-arabinose are not fermented. No sugar is fermented in the API 50 CH strips, using CHB/E suspension medium. Acetate, pyruvate and methyl pyruvate are oxidized, but 
-hydroxybutyrate, 
-hydroxybutyrate, L-alanine, glycyl L-glutamate, adenosine, 2'-deoxyadenosine, inosine, AMP and UMP are not. The fatty acid profile is characterized by the predominance of iso-C15 : 0 (48 %), followed by anteiso-C15 : 0 (15.3 %) and iso-C16 : 0 (13.5 %). Smaller amounts of anteiso-C17 : 0 (5.6 %), iso-C17 : 0 (3.7 %), C15 : 0 (3.2 %), iso-C14 : 0 (3.1 %), C16 : 17c alcohol (2.6 %), C16 : 0 (1.5 %), C16 : 111c (0.7 %) and C18 : 19c (0.7 %) are also present. Sensitive to amoxycillin, augmentin, ceftriaxone, ticarcillin, claventin, imipinem, ciprofloxacin, gentamicin (10 IU), amikacin, tobramycin, trimethoprim-sulfamethoxazole, rifampicin, ofloxacin, colistin, piperacillin-tazobactam, isepamycin, chloramphenicol, tetracycline and streptomycin, moderately sensitive to cefepime and cefpirome, and resistant to ceftazidime, fosfomycin and aztreonam.
The type strain, 4400831T(=CIP 108446T=CCUG 49529T), was isolated from human CSF.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Aquino de Muro, M., Mitchell, W. J. & Priest, F. G. (1992). Differentiation of mosquito-pathogenic strains of Bacillus sphaericus from non-toxic varieties by ribosomal RNA gene restriction patterns. J Gen Microbiol 138, 1159–1166.[Medline]
Castagnola, E., Fioredda, F., Barretta, M. A., Pescetto, L., Garaventa, A., Lanino, E., Micalizzi, C., Giacchino, R. & Dini, G. (2001). Bacillus sphaericus bacteraemia in children with cancer: case reports and literature review. J Hosp Infect 48, 142–145.[CrossRef][Medline]
Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111–120.[CrossRef][Medline]
Kumar, S., Tamura, K., Jakobsen, I. B. & Nei, M. (2001). MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17, 1244–1245.
La Duc, M. T., Satomi, M. & Venkateswaran, K. (2004). Bacillus odysseyi sp. nov., a round-spore-forming bacillus isolated from the Mars Odyssey spacecraft. Int J Syst Evol Microbiol 54, 195–201.
Nakamura, L. K. (2000). Phylogeny of Bacillus sphaericus-like organisms. Int J Syst Evol Microbiol 50, 1715–1722.[Abstract]
Nakamura, L. K., Shida, O., Takagi, H. & Komagata, K. (2002). Bacillus pycnus sp. nov. and Bacillus neidei sp. nov., round-spored bacteria from soil. Int J Syst Evol Microbiol 52, 501–505.[Abstract]
Priest, F. G., Goodfellow, M. & Todd, C. (1988). A numerical classification of the genus Bacillus. J Gen Microbiol 234, 1847–1882.
Reva, O. N., Sorokulova, I. B. & Smirnov, V. V. (2001). Simplified technique for identification of the aerobic spore-forming bacteria by phenotype. Int J Syst Evol Microbiol 51, 1361–1371.[Abstract]
Rheims, H., Frühling, A., Schumann, P., Rohde, M. & Stackebrandt, E. (1999). Bacillus silvestris sp. nov., a new member of the genus Bacillus that contains lysine in its cell wall. Int J Syst Bacteriol 49, 795–802.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Weisburg, W. G., Barns, S. M., Pelletier, D. A. & Lane, D. J. (1991). 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173, 697–703.
Woodburn, M. A., Yousten, A. A. & Hilu, K. H. (1995). Random amplified polymorphic DNA fingerprinting of mosquito-pathogenic and nonpathogenic strains of Bacillus sphaericus. Int J Syst Bacteriol 45, 212–217.
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