Ornithinibacillus contaminans sp. nov., an endospore-forming species

The genus Ornithinibacillus was proposed by Mayr et al. (2006) and comprises two species, Ornithinibacillus bavariensis from pasteurized milk in Bavaria, Germany, and Ornithinibacillus californiensis from coastal surface sediments in California, USA. The genus is characterized by having l-ornithine as the diagnostic diamino acid in the peptidoglycan (which is a major differentiating feature from the genus Oceanobacillus) and MK-7 as the characteristic menaquinone.

Strain CCUG 53201^T was isolated as a contaminant from a human blood sample of a 75-year-old woman in Sweden. The strain was isolated on blood agar at 37 °C and maintained on tryptone soy agar (TSA; Oxoid) at 30 °C. Gram-staining was performed as described by Gerhardt et al. (1994). Cell morphology was examined with a Zeiss light microscope at ×1000 magnification, using cells harvested from TSA after 24 h at 30 °C. Cells of strain CCUG 53201^T were Gram-stain-positive, strictly aerobic, non-motile rods (0.8–1.0×2.0–3.0 μm) that formed spherical endospores in a terminal position.

The 16S rRNA gene was analysed as described by Kämpfer et al. (2003). Amplification of the 16S rRNA gene was performed with the primer pair 27F (5′-GAGTTTGATCMTGGCTCAG-3′) and MR 1492R (5′-ACGGYTACCTTGTTACGACTT-3′) (Lane, 1991) and sequencing was performed using standard sequencing primers for the 16S rRNA gene. The 16S rRNA gene sequence of strain CCUG 53201^T was a continuous stretch of 1254 bp. Phylogenetic analysis was performed using arb software (version December 2007; Ludwig et al., 2004) and the corresponding silva SSURef 95 database (version July 2008; Pruesse et al., 2007). Trees were constructed using the maximum-likelihood method with fastDNAml (Olsen et al., 1994) and topologies were tested without a conservation filter. Distances were calculated using mega (version 4.0; Tamura et al., 2007) using the distance options according to the Kimura-2 model. Maximum-likelihood (Fig. 1⇓) and neighbour-joining (not shown) trees showed that strain CCUG 53201^T clustered consistently within the genus Ornithinibacillus. The closest relatives of strain CCUG 53201^T were O. californiensis MB-9^T (98.7 % 16S rRNA gene sequence similarity) and O. bavariensis WSBC 24001^T (97.9 %).

The peptidoglycan of strain CCUG 53201^T was isolated after disintegrating cells with glass beads in a Vibrogen cell mill (D-72408; Johanna Otto, Bodelshausen, Germany) and subsequent trypsin digestion according to the method of Schleifer & Seidl (1985). The peptidoglycan structure was analysed using the methods of Schleifer (1985). The peptidoglycan contained the amino acids ornithine, alanine, glutamic acid and aspartic acid. The peptide l-Orn–d-Asp, which is stable under the conditions for peptidoglycan hydrolysis (4 M HCl, 100 °C, 16 h), was detected. The partial hydrolysate (4 M HCl, 100 °C, 0.75 h) contained the peptides l-Ala–d-Glu and d-Ala–l-Orn–d-Asp. From these results, it was concluded that strain CCUG 53201^T showed the peptidoglycan type A4β l-Orn–d-Asp (Schleifer & Kandler, 1972), which was in agreement with its affiliation to the genus Ornithinibacillus (Mayr et al., 2006). Polyamines, quinones and polar lipids were extracted from cells of strain CCUG 53201^T after cultivation on PYE medium (0.3 % peptone from casein, 0.3 % yeast extract; pH 7.2) at 28 °C and analysed as reported elsewhere (Altenburger et al., 1996, 1997; Tindall, 1990a, b). HPLC analysis of polyamines and quinones was carried out using the apparatus reported by Stolz et al. (2007). The polyamine pattern of strain CCUG 53201^T was composed of the major amines spermidine and spermine [6.5 and 2.3 μmol (g dry weight)⁻¹, respectively] and small amounts of putrescine and cadaverine [<0.3 μmol (g dry weight)⁻¹]. This polyamine pattern is in agreement with those of other mesophilic bacilli, although the relative proportion of spermine is usually lower. In contrast, thermophilic bacilli have been shown to exhibit major amounts of spermine and only moderate amounts of spermidine (Hamana et al., 1989, 1993; Meier-Stauffer et al., 1996). The quinone system of strain CCUG 53201^T consisted of menaquinones MK-7 (93 %) and MK-6 (7 %). This trait is shared by the majority of bacilli, including members of the genus Ornithinibacillus (Mayr et al., 2006).

For the detection of polar lipids, spray reagents were applied to separate plates. Molybdatophosphoric acid was used to detect total lipids, ninhydrin and molybdenum blue were used in succession, ninhydrin first to detect lipids with an amino group and molybdenum blue second to detect lipids with a phosphate group, and α-naphthol was used to detect lipids with a sugar moiety. In the polar lipid profile (Fig. 2⇓), diphosphatidylglycerol and an unidentified phospholipid (PL1) predominated. Moderate amounts of phosphatidylglycerol and another two unidentified phospholipids (PL2, PL3) were found and minor to trace amounts of three unidentified glycolipids, a fourth phospholipid, an aminolipid and a polar lipid were detected. This polar lipid profile shared several of the major characteristics reported for Ornithinibacillus species, including the major compound diphosphatidylglycerol and the presence of phosphatidylglycerol and an unidentified phospholipid (PL3) that exhibited chromatographic behaviour similar to that of PL10 of O. bavariensis WSBC 24001^T (Mayr et al., 2006). The presence of PL1 and PL2 has not been shown before for Ornithinibacillus species, but it appeared that the diphosphatidylglycerol spot may have previously overlaid the PL1 spot because of poor chromatographic separation. However, the presence of two unidentified phospholipids with chromatographic behaviour similar to that of PL1 and PL2 has been reported for representatives of genera closely related to the genus Ornithinibacillus, such as Virgibacillus and Gracilibacillus (Wainø et al., 1999).

Fatty acid analysis was performed according to Kämpfer & Kroppenstedt (1996). The fatty acid profile of strain CCUG 53201^T contained anteiso- and iso-branched fatty acids and was very similar to those of type strains of species of the genus Ornithinibacillus (Table 1⇓).

Results from comparative physiological characterization of strain CCUG 53201^T and the type strains of O. californiensis and O. bavariensis are given in Table 2⇓. All of the strains were inactive in most biochemical tests and showed only a few positive results after prolonged incubation. DNA–DNA hybridization was performed according to the method of Ziemke et al. (1998). DNA–DNA relatedness between strain CCUG 53201^T and O. bavariensis DSM 15681^T and O. californiensis DSM 16628^T was 40 and 31 %, respectively.

The peptidoglycan type, quinone system and fatty acid and polar lipid profiles, as well as 16S rRNA gene sequence analysis, showed that strain CCUG 53201^T is affiliated with the genus Ornithinibacillus. On the basis of the observed phenotypic differences, DNA–DNA relatedness and the differences in the 16S rRNA gene sequences, we propose a novel species, Ornithinibacillus contaminans sp. nov.