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Laboratoire de Bactériologie–Virologie, Hôpital de la Timone, CNRS UMR 6020, IFR48, Boulevard Jean Moulin, 13385 Marseille Cedex 05, France
Correspondence
Didier Raoult
didier.raoult{at}medecine.univ-mrs.fr
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence of P. massiliensis strain 2301065T, P. sanguinis strain 2301083T and P. timonensis strain 2301032T are respectively AY323608, AY323609 and AY323610 (clone 1), AY323611 (clone 2) and AY323612 (clone 3).
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TOPABSTRACTMAIN TEXTREFERENCES |
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, identification and phylogenetic analysis of many bacteria have been performed and have allowed for the classification of non-cultivable organisms and organisms with ambiguous biochemical profiles. Based on phylogenetic studies using the 16S rRNA gene, the so-called rRNA group 3 bacilli were separated to form the novel genus Paenibacillus (Ash et al., 1993); several novel species of Paenibacillus have recently been described (Aguilera et al., 2001; Elo et al., 2001; Meehan et al., 2001; Dasman et al., 2002; Takeda et al., 2002; Yoon et al., 2003).
Paenibacillus species have been isolated from human biological samples and the possibility that these bacteria may cause human infection has been considered. Prosthetic hip infection and endophthalmitis caused by Paenibacillus alvei (Antonello & Weinstein, 1989; Reboli et al., 1989), brain abscess, catheter-associated infection and wound infection due to Paenibacillus macerans were reported (Bert et al., 1995; Barrero et al., 1996). Pseudobacteraemia due to Paenibacillus may also result from contamination of gloves, alcohol swabs, intravenous catheters or blood culture media (CDC, 1974; Berger, 1983; York, 1990; Noskin et al., 2001; Teng et al., 2003).
The data presented in this paper describe three novel Paenibacillus species, two of which are related closely to Paenibacillus macerans (Ash et al., 1993; Heyndrickx et al., 1996); the third can be grouped with Paenibacillus illinoisensis (Shida et al., 1997). These species were initially isolated from blood culture samples of patients suffering from carcinoma, chronic interstitial nephropathy or leukaemia.
Isolation and characterization of strains
Reference strains were obtained from the Pasteur Institute (Paris, France).
Paenibacillus macerans strain CIP 66.19T, Paenibacillus pabuli strain CIP 103119T and Paenibacillus polymyxa strain CIP 66.22T, and clinical strains 2301083T (=CIP 107938T=CCUG 48214T), 2301065T (=CIP 107939T=CCUG 48215T) and 2301032T (=CIP 108005T=CCUG 48216T), were isolated on Columbia blood agar plates (Oxoid) from blood samples in the bacteriology laboratory of the Timone hospital.
In October 1999, cervical adenopathy was detected in a 49-year-old man. Diagnosis of epidermoid carcinoma of the oropharynx was established and chemotherapy and radiotherapy were performed. In November 2002, at the time of hospitalization for chemotherapy, the patient was afebrile, but a blood sample was collected from which Gram-positive rods were isolated (strain 2301083T).
In March 2001, acute lymphoblastic leukaemia was diagnosed in a 13-year-old boy who received chemotherapy. In August 2001, allograft with compatible stem cells of umbilical blood was performed. At this time, cutaneous and hepatic graft versus host disease was noted. In October 2002, he was hospitalized with fever (38·5 °C) and neutropenia. Blood samples were obtained and Gram-positive rods (strain 2301065T) were isolated from one of three blood samples.
In October 2002, strain 2301032T was isolated from a 75-year-old woman who presented with chronic interstitial nephropathy that required haemodialysis from February 1989. At the time of the bacterial isolation, she was apyretic, but presented with loose cough and sputum.
All of the isolated bacteria were Gram-positive rods; their size was determined by transmission electron microscopy using cells from a 48 h culture. Bacterial suspension was pre-fixed in 5 % (v/v) glutaraldehyde in phosphate buffer (Gibco) for at least 1 h at room temperature, washed in the same buffer and stained with 1 % (w/v) phosphotungstic acid. Sporulation was studied by the same method. After the pre-fixation step, specimens were post-fixed with 4 % (w/v) osmium tetroxide for 1 h, dehydrated by using a graded series of acetone, transferred to 100 % acetone and embedded in Epon 812 (Fluka) substitute. Thin sections were cut with a diamond knife on a Leica Ultracut VCT microtome and stained with uranyl acetate and lead citrate. The grids were examined on a MORGANI 268 D (Philips) transmission electron microscope at an operating voltage of 60 kV. Bacteria of strain 2301065T were 0·5 µm wide and 2·0–4·0 µm long. They formed translucent, flat colonies of beige colour after 24 h incubation at 30 °C. Cells of strain 2301083T were 0·5 µm wide and 2·0–3·0 µm long, forming greyish colonies that were translucent, shiny, 1 mm in diameter after 24 h incubation at 30 °C, circular and regularly shaped. Isolated bacteria of strain 2301032T were rods of 0·5 µm width and 2·0 µm length. Colonies were greyish, translucent, shiny, 1·5 mm in diameter, regularly shaped and low convex. Presence of spores was studied after 8 days growth at room temperature and was reported for the three isolates. Terminal or subterminal ellipsoidal spores were observed in swollen sporangia.
Growth temperatures tested were 30, 37, 44 and 50 °C, the optimum being 30–37 °C. Catalase activity was determined with the ID colour catalase test kit (bioMérieux). Oxidase activity was assayed by applying the cells to moistened discs that were impregnated with dimethyl-p-phenylene diamine (bioMérieux). Mobility was determined by light microscopy. An anaerobic atmosphere was created by using GENbag anaer (bioMérieux). Acid production from 49 carbon sources was tested at 30 °C under anaerobic conditions with an API 50CH kit combined with API 50CHB/E medium (bioMérieux). Other tests were completed with API 20 E (bioMérieux). Tolerance to NaCl was measured in trypticase soy broth that contained 1–5 % (w/v) NaCl. All three strains were motile by means of peritrichous flagella and facultatively anaerobic. With the exception of strain 2301083T, the isolates grew at 50 °C. None of the strains showed haemolytic activity on Columbia blood agar. ADH, LDC, ODC, CIT, H2S, URE, TDA and IND tests in API 20 E strips were negative for all isolates tested. The three strains grew in the presence of 3 % NaCl, but strain 2301065T also grew in the presence of 5 % NaCl. Diagnostic traits are specified in Table 1.
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and compared with values that are available for closely related Paenibacillus species.
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). PCR products were purified by using MultiScreen PCR (Millipore) and sequencing reactions were carried out by using a DNA sequencing kit (BigDye Terminator Cycle Sequencing v2.0 Ready Reactions; PE Biosystems) as described by the manufacturer. Sequence products were purified and electrophoresis was performed with a 3100 Genetic Analyzer (Applied Biosystems). PCR products were cloned in pGEM-T Easy Vector (Promega) as described by the manufacturer. Ten clones were cultivated in LB medium (USB) overnight and plasmid purification was performed by using the Wizard Plus SV Minipreps DNA purification system (Promega). Determined sequences were compared with those available in GenBank by using the BLAST program through the NCBI server.
16S rRNA gene sequences were aligned by using the multisequence alignment program CLUSTALX (1.8). Phylogenetic relationships between representatives of the genus Paenibacillus were determined by using MEGA version 2.1 (Kumar et al., 2001). Distance matrices were determined following the assumptions described by Kimura (1980). These matrices were used to elaborate dendrograms by using the neighbour-joining method (Saitou & Nei, 1987). 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. Percentage similarities between 16S rRNA gene sequences were determined by using NALIGN in the PC/GENE software package (IntelliGenetics); similarities between existing Paenibacillus species and strains 2301065T, 2301032T and 2301083T were 87·6–94·4, 88·5–95·4 and 87·5–96·0 %, respectively.
It was impossible to determine the 3' end of the 2301032T 16S rRNA gene sequence. We therefore cloned the PCR product and found that several sequences were present. Direct sequencing was impossible, due to mutations and insertions. For this isolate, at least three different sequences were characterized. Recently, whole genomes of Bacillus species, which are phylogenetically close to the genus Paenibacillus, were sequenced. In the genomes of Bacillus subtilis, Bacillus cereus and Bacillus anthracis, respectively 10, 13 and 11 genes that encode 16S rRNA were described (Kunst et al., 1997; Ivanova et al., 2003; Read et al., 2003). The high number of 16S rRNA genes in the genomes of Paenibacillus species could explain the high diversity in the sequences of this gene for the same isolate. In fact, we observed this phenomenon for several representatives of the genus Paenibacillus (V. Roux, unpublished data).
Phylogenetic analysis, based on 16S rRNA gene sequence comparison, showed that isolate 2301065T could be grouped with Paenibacillus illinoisensis, but the bootstrap value at the node was low (33 %) (Fig. 1). Isolates 2301083T and 2301032T were grouped with Paenibacillus macerans in the same cluster. Bootstrap values at the different nodes were high (82–100 %). The 16S rRNA gene sequences for strains 2301065T, 2301083T and 2301032T were found to contain the Paenibacillus-specific 22 bp sequence in variable region V5 (Ash et al., 1993).
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Description of Paenibacillus massiliensis sp. nov.
Paenibacillus massiliensis (mas.si.li.en'sis. L. masc. adj. massiliensis of Massilia, the old Greek and Roman name for Marseille, where the type strain was isolated).
Gram-positive, facultatively anaerobic rods (0·5 µm wide, 2·0–4·0 µm long). Ellipsoidal endospores are formed in swollen sporangia. The organism grows on routine media and forms translucent, beige-coloured, flat colonies after incubation for 24 h at 30 °C. Bacteria are motile by means of peritrichous flagella. Catalase-positive, but oxidase-negative. Optimal growth occurs at 30–37 °C, but growth at 50 °C is not inhibited. Growth occurs in the presence of 5 % (w/v) NaCl. Nitrate is reduced, but gelatin is not liquefied. Acid is produced from glycerol, L-arabinose, ribose, galactose, glucose, fructose, mannose, mannitol, amygdalin, arbutin, aesculin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, raffinose, starch, glycogen and gentiobiose. Acid is not produced from erythritol, D-arabinose, D-xylose, L-xylose, adonitol, methyl 
-D-xyloside, sorbose, rhamnose, dulcitol, inositol, sorbitol, methyl 
-D-mannoside, methyl -D-glucoside, N-acetylglucosamine, inulin, melezitose, xylitol, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate or 5-ketogluconate.
The type strain, which was isolated from blood culture, is strain 2301065T (=CIP 107939T=CCUG 48215T).
Description of Paenibacillus sanguinis sp. nov.
Paenibacillus sanguinis (san'gui.nis. L. masc. gen. n. sanguinis of blood, referring to the fact that the type strain was isolated from a blood sample).
Gram-positive, facultatively anaerobic rods (0·5 µm wide, 2·0–3·0 µm long). Ellipsoidal endospores are formed in swollen sporangia. Colonies are greyish, translucent, shiny, circular, 1 mm in diameter after incubation for 24 h at 30 °C and regularly shaped. Bacteria are motile by means of peritrichous flagella. Catalase- and oxidase-negative. Optimal growth occurs at 30–37 °C and growth is inhibited at 50 °C. Growth does not occur in the presence of 5 % (w/v) NaCl. Nitrate is not reduced and gelatin is not liquefied. Acid is produced from L-arabinose, D-xylose, methyl -D-xyloside, galactose, fructose, mannitol, methyl -D-glucoside, N-acetylglucosamine, amygdalin, arbutin, aesculin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, inulin, melezitose and raffinose. Acid is not produced from glycerol, erythritol, D-arabinose, ribose, L-xylose, adonitol, glucose, mannose, sorbose, rhamnose, dulcitol, inositol, sorbitol, methyl -D-mannoside, starch, glycogen, xylitol, gentiobiose, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate or 5-ketogluconate.
The type strain, which was isolated from blood culture, is strain 2301083T (=CIP 107938T=CCUG 48214T).
Description of Paenibacillus timonensis sp. nov.
Paenibacillus timonensis (ti.mo.nen'sis. N.L. masc. adj. timonensis from the name Hôpital de la Timone, where the type strain was isolated).
Gram-positive, facultatively anaerobic rods (0·5 µm wide, 2·0 µm long). Ellipsoidal endospores are formed in swollen sporangia. Colonies are greyish, translucent, shiny, 1·5 mm in diameter, regularly shaped and low convex after incubation for 24 h at 30 °C. Bacteria are motile by means of peritrichous flagella. Catalase-positive, but oxidase-negative. Optimal growth occurs at 30–37 °C, but growth is not inhibited at 50 °C. Growth does not occur in the presence of 5 % (w/v) NaCl. Nitrate is reduced weakly, but gelatin is not liquefied. Acid is produced from L-arabinose, D-xylose, galactose, glucose, fructose, mannose, methyl -D-glucoside, N-acetylglucosamine, amygdalin, arbutin, aesculin, salicin, cellobiose, maltose, lactose, melibiose, trehalose, starch, glycogen, gentiobiose and gluconate. Acid is not produced from glycerol, erythritol, D-arabinose, ribose, D-xylose, L-xylose, adonitol, mannose, sorbose, rhamnose, dulcitol, inositol, mannitol, sorbitol, methyl -D-mannoside, sucrose, inulin, melezitose, raffinose, xylitol, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, 2-ketogluconate or 5-ketogluconate.
The type strain, which was isolated from blood culture, is strain 2301032T (=CIP 108005T=CCUG 48216T).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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