Birgit Huber

Brucella microti sp. nov., isolated from the common vole Microtus arvalis

Two Gram-negative, non-motile, non-spore-forming, coccoid bacteria (strains CCM 4915(T) and CCM 4916), isolated from clinical specimens of the common vole Microtus arvalis during an epizootic in the Czech Republic in 2001, were subjected to a polyphasic taxonomic study. On the basis of 16S rRNA (rrs) and recA gene sequence similarities, both isolates were allocated to the genus Brucella. Affiliation to Brucella was confirmed by DNA-DNA hybridization studies. Both strains reacted equally with Brucella M-monospecific antiserum and were lysed by the bacteriophages Tb, Wb, F1 and F25. Biochemical profiling revealed a high degree of enzyme activity and metabolic capabilities not observed in other Brucella species. The omp2a and omp2b genes of isolates CCM 4915(T) and CCM 4916 were indistinguishable. Whereas omp2a was identical to omp2a of brucellae from certain pinniped marine mammals, omp2b clustered with omp2b of terrestrial brucellae. Analysis of the bp26 gene downstream region identified strains CCM 4915(T) and CCM 4916 as Brucella of terrestrial origin. Both strains harboured five to six copies of the insertion element IS711, displaying a unique banding pattern as determined by Southern blotting. In comparative multilocus VNTR (variable-number tandem-repeat) analysis (MLVA) with 296 different genotypes, the two isolates grouped together, but formed a separate cluster within the genus Brucella. Multilocus sequence typing (MLST) analysis using nine different loci also placed the two isolates separately from other brucellae. In the IS711-based AMOS PCR, a 1900 bp fragment was generated with the Brucella ovis-specific primers, revealing that the insertion element had integrated between a putative membrane protein and cboL, encoding a methyltransferase, an integration site not observed in other brucellae. Isolates CCM 4915(T) and CCM 4916 could be clearly distinguished from all known Brucella species and their biovars by means of both their phenotypic and molecular properties, and therefore represent a novel species within the genus Brucella, for which the name Brucella microti sp. nov. with the type strain CCM 4915(T) (=BCCN 07-01(T)=CAPM 6434(T)) is proposed.

Steroidobacter denitrificans gen. nov., sp. nov., a steroidal hormone-degrading gammaproteobacterium

A denitrifying bacterium, designated strain FS(T), was isolated from anoxic digested sludge on oestradiol [17beta-oestra-1,3,5(10)-triene-3,17-diol] or testosterone (17beta-hydroxyandrost-4-en-3-one) as the sole source of carbon and energy with nitrate as the electron acceptor. Strain FS(T) represents the first known bacterium to grow anaerobically on both oestradiol (C-18) and testosterone (C-19). Steroidal hormones were degraded completely by nitrate reduction to dinitrogen monoxide, which was further reduced to dinitrogen in stationary-phase cultures. Gram-negative cells were slightly curved rods, 0.3-0.5 x 0.6-1.6 microm in size, motile, non-fermentative, non-spore-forming and catalase- and oxidase-positive, showing optimal growth at pH 7.0, 28 degrees C and 0.1% (w/v) NaCl. Beside steroidal hormones, the bacterium utilized only a narrow range of organic substrates with nitrate as the electron acceptor, including several fatty acids and glutamate. No aerobic or anaerobic growth occurred on liquid or solid complex media. Phylogenetic analysis of the 16S rRNA gene sequence showed that strain FS(T) has no known close relatives and represents a distinct lineage within the Gammaproteobacteria. Together with the genera Nevskia, Hydrocarboniphaga, Solimonas and Sinobacter (less than 88% 16S rRNA gene sequence similarity to strain FS(T)), it forms a phylogenetic cluster separated from the families Chromatiaceae, Ectothiorhodospiraceae and Xanthomonadaceae. The quinone system of strain FS(T) consisted exclusively of ubiquinone Q-8. The dominant polar lipids were diphosphatidylglycerol and phosphatidylethanolamine. Spermidine in combination with putrescine and traces of sym-homospermidine were the basic polyamines. The major fatty acids detected in testosterone- or heptanoate-grown cells were C(15:0) and C(17:1)omega8c, minor hydroxylated fatty acids were C(11:0) 3-OH and C(12:0) 3-OH. The G+C content of the DNA was 61.9 mol%. Based on the high 16S rRNA gene sequence divergence and different phenotypic properties from previously described gammaproteobacteria in combination with chemotaxonomic data, strain FS(T) is considered to represent a new genus and species, for which the name Steroidobacter denitrificans gen. nov., sp. nov. is proposed. The type strain of Steroidobacter denitrificans is FS(T) (=DSM 18526(T) =JCM 14622(T)).

Description of Francisella hispaniensis sp. nov., isolated from human blood, reclassification of Francisella novicida (Larson et al. 1955) Olsufiev et al. 1959 as Francisella tularensis subsp. novicida comb. nov. and emended description of the genus Francisella.

Strain FhSp1T, isolated from human blood in Spain in 2003, was studied for its taxonomic allocation. By 16S rRNA and recA gene sequencing, the strain was shown to belong to the genus Francisella. In the 16S rRNA gene sequence, Francisella sp. FhSp1T shared similarity of more than 99% with strains of Francisella tularensis subspecies and Francisella novicida U112T, 98% with Francisella piscicida GM2212T and 98.4% with Francisella philomiragia ATCC 25015T. In the recA gene sequence, Francisella sp. FhSp1T exhibited 91.6-91.7% similarity to strains of F. tularensis subspecies, 91.2% to F. novicida U112T and 84% to F. philomiragia ATCC 25017. The genus affiliation was supported by a quinone system typical of Francisella (Q-8 as the major component), a complex polar lipid profile similar to that of F. tularensis with the major components diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and an unknown aminophospholipid (APL4) and a fatty acid profile consisting mainly of C10:0 (17.2%), C14:0 (11.2%), C16:0 (13.1%), C18:0 3-OH (14.2%) and C18:1omega9c (7.1%). DNA-DNA hybridization, which showed unambiguously that FhSp1T represents a novel species, and the results of biochemical tests allowed genotypic and phenotypic differentiation of the isolate from all hitherto-described Francisella species. A multiplex PCR developed in the course of this study discriminated FhSp1T from representatives of all other Francisella species and subspecies, clades A.I and A.II of F. tularensis subsp. tularensis and F. tularensis subsp. holarctica biovar japonica and also between these representatives of the genus. Therefore, we propose the name Francisella hispaniensis sp. nov., with the type strain FhSp1T (=FnSp1T =FSC454T =F62T =DSM 22475T =CCUG 58020T). Furthermore, we formally propose the transfer of the species Francisella novicida to the species Francisella tularensis as Francisella tularensis subsp. novicida comb. nov. (type strain ATCC 15482T =CCUG 33449T =CIP 56.12T). We also present an emended description of the genus Francisella.

Development of a PCR assay for typing and subtyping of Brucella species.

In the course of this study, examinations were carried out to develop a PCR-based test which allows discrimination of Brucella species and biovars not targeted by the currently established gel-based PCR assays. Appropriate primers were designed based on specific deletions and insertions in the different Brucella genomes as determined by RAPD-PCR and whole-genome comparisons. After testing the specificity of the primers with a set of 22 Brucella reference strains of all species and biovars, they were used to supplement the existing PCR assays resulting in a 19-primer multiplex PCR. In addition to the commonly used PCR assays, the developed assay specifically identified B. neotomae, B. pinnipedialis, B. ceti, and B. microti. Furthermore, it differentiated B. abortus biovars 1, 2, 4 from biovars 3, 5, 6, 9, as well as between B. suis biovar 1, biovars 3, 4, and biovars 2 and 5. When tested in the multiplex assay, all Brucella type and reference strains and the majority of 118 field strains examined could be accurately identified by their respective banding patterns according to their previous typing. B. canis strains were subdivided into 2 groups, one exhibiting a unique pattern and the other one a banding pattern shared with B. suis biovars 3 and 4. Species of the closely related genus Ochrobactrum and several other clinically relevant bacteria showed no amplification product. Hence, the developed PCR assay is useful for rapid identification of Brucella at the species and at the biovar level.

Objections to the transfer of Francisella novicida to the subspecies rank of Francisella tularensis – response to Johansson et al.

IP: 54.70.40.11 On: Tue, 11 Dec 2018 06:22:18 Huber, B. E., Escudero, R., Busse, H. J., Seibold, E., Scholz, H. C., Anda, P., Kämpfer, P. & Splettstoesser, W. D. (2010). Description of Francisella hispaniensis sp. nov., isolated from human blood, reclassification of Francisella novicida (Larson et al. 1955) Olsufiev et al. 1959 as Francisella tularensis subsp. novicida comb. nov., and emended description of the genus Francisella. Int J Syst Evol Microbiol 60, 1887– 1896.

Streptomyces specialis sp. nov.

A Gram-positive, non-endospore-forming bacterium (GW41-1564(T)) was isolated from soil. Comparison of 16S rRNA gene sequences showed that strain GW41-1564(T) is a member of the genus Streptomyces, exhibiting highest similarities with Streptomyces hainanensis YIM 47672(T) (97.8 %) and Streptomyces cacaoi subsp. cacaoi NBRC 12748(T) (97.5 %). Strain GW41-1564(T) could be distinguished from any other Streptomyces species with validly published names by sequence similarity values less than 97.5 %. Strain GW41-1564(T) exhibited an unusual quinone system, with the predominant compounds MK-10(H(4)) and MK-10(H(6)) and smaller amounts of MK-9(H(4)) and MK-9(H(6)). The type strain of the most closely related species, S. hainanensis YIM 47672(T), also contained an unusual quinone system composed of MK-9(H(6)) and MK-9(H(8)) in addition to MK-9(H(4)) and MK-10(H(0)), whereas the type strain of the second most closely related species, S. cacaoi NBRC 12748(T), contained a quinone system, composed of MK-9(H(6)) and MK-9(H(8)), typical of Streptomyces. The polar lipid profile of GW41-1564(T) consisted of the predominant compound diphosphatidylglycerol, moderate amounts of phosphatidylethanolamine, phosphatidylglycerol and phosphatidylinositol and minor to trace amounts of two phosphatidylinositol mannosides and several unknown lipids, and the major fatty acids were iso-C(16 : 0,) anteiso-C(17 : 1)omega9c and anteiso-C(17 : 0). The results of physiological and biochemical tests allowed further phenotypic differentiation of strain GW41-1564(T) from the related species S. hainanensis. Strain GW41-1564(T) clearly merits species status, and we propose the name Streptomyces specialis sp. nov., with the type strain GW41-1564(T) (=DSM 41924(T) =CCM 7499(T)).

Ochrobactrum rhizosphaerae sp. nov. and Ochrobactrum thiophenivorans sp. nov., isolated from the environment

Two Gram-negative, rod-shaped, non-spore-forming bacteria, PR17(T) and DSM 7216(T), isolated from the potato rhizosphere and an industrial environment, respectively, were studied for their taxonomic allocation. By rrs (16S rRNA) gene sequencing, these strains were shown to belong to the Alphaproteobacteria, most closely related to Ochrobactrum pseudogrignonense (98.4 and 99.3 % similarity to the type strain, respectively). Chemotaxonomic data (major ubiquinone Q-10; major polyamines spermidine, sym-homospermidine and putrescine; major polar lipids phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylglycerol and phosphatidylcholine and the Ochrobactrum-specific unidentified aminolipid AL2; major fatty acids C(18 : 1)omega7c and C(19 : 0) cyclo omega8c) supported the genus affiliation. The results of DNA-DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of the isolates from all hitherto-described Ochrobactrum species. Hence, both isolates represent novel species of the genus Ochrobactrum, for which the names Ochrobactrum rhizosphaerae sp. nov. (type strain PR17(T) =CCUG 55411(T) =CCM 7493(T) =DSM 19824(T)) and Ochrobactrum thiophenivorans sp. nov. (type strain DSM 7216(T) =CCUG 55412(T) =CCM 7492(T)) are proposed.

Ochrobactrum pituitosum sp. nov., isolated from an industrial environment.

Strain CCUG 50899, a Gram-negative, rod-shaped, non-spore-forming, motile bacterium isolated from industrial environment in Sweden and tentatively assigned to the species Ochrobactrum anthropi, was studied in order to clarify its taxonomic status. 16S rRNA gene sequence similarities placed the strain in the genus Ochrobactrum, sharing highest similarity with the type strains of Ochrobactrum rhizosphaerae (99.3 %), Ochrobactrum thiophenivorans (98.7 %), Ochrobactrum pseudogrignonense (98.6 %) and Ochrobactrum grignonense (98.5 %). The fatty acid profile of [O. anthropi] CCUG 50899 (major fatty acids C(18 : 1)omega7c and C(19 : 0) cyclo omega8c and presence of C(18 : 1) 2-OH), the polar lipid profile (diphosphatidylglycerol, phosphatidylglycerol, phosphatidylmonomethylethanolamine, phosphatidylethanolamine, two unknown aminolipids and an unknown phospholipid), the presence of the quinone system ubiquinone Q-10 and a polyamine pattern with the major compounds putrescine and spermidine and moderate amounts of sym-homospermidine supported its affiliation to the genus Ochrobactrum. DNA-DNA reassociation experiments with the type strains of its closest relatives O. rhizosphaerae, O. pseudogrignonense, O. thiophenivorans and O. grignonense demonstrated that [O. anthropi] CCUG 50899 should be placed in a novel species, which is distinguishable from related species by a set of biochemical traits. Based on these data, reclassification of [O. anthropi] CCUG 50899 as the type strain of a novel species appears to be justified. Hence, we describe a novel species to accommodate this strain, for which we propose the name Ochrobactrum pituitosum sp. nov. The type strain is CCUG 50899(T) (=DSM 22207(T)).

Pseudochrobactrum lubricantis sp. nov., isolated from a metal-working fluid

A Gram-negative, rod-shaped, oxidase-positive, non-spore-forming, non-motile bacterium (KSS 7.8(T)) was isolated from a water-mixed metal-working fluid. On the basis of 16S rRNA gene and recA sequence similarities, the isolate was clearly grouped in the genus Pseudochrobactrum. This allocation was confirmed by fatty acid data (major fatty acids: C(18 : 2)omega7c and C(19 : 0) cyclo omega8c), polar lipid profile (major components: phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and phosphatidylcholine, plus moderate amounts of phosphatidylmonomethylethanolamine and unknown aminolipid AL1), quinone system (ubiquinone Q-10) and polyamine pattern (spermidine and putrescine predominant). DNA-DNA pairing with the most closely related Pseudochrobactrum species showed values ranging from 24.2 to 45.7 %, and physiological and biochemical data clearly differentiated this isolate from described Pseudochrobactrum species. This organism represents a novel species of the genus Pseudochrobactrum, for which the name Pseudochrobactrum lubricantis sp. nov. is proposed, with the type strain KSS 7.8(T) (=CCUG 56963(T)=CCM 7581(T)).

Paenochrobactrum gallinarii gen. nov., sp. nov., isolated from air of a duck barn, and reclassification of Pseudochrobactrum glaciei as Paenochrobactrum glaciei comb. nov.

A Gram-negative, rod-shaped, oxidase-positive, non-spore-forming, non-motile bacterium (Sa25(T)) was isolated from air of a duck barn. 16S rRNA gene and recA sequence analyses clearly placed the isolate in the vicinity of the Brucella-Ochrobactrum-Pseudochrobactrum group, with the closest relative being Pseudochrobactrum glaciei KMM 3858(T). This allocation was confirmed by analyses of the quinone system (ubiquinone Q-10), fatty acid data (major fatty acids C(18 : 1)omega7c and C(19 : 0) cyclo omega8c) and polar lipid profile (major components diphosphatidylglycerol, phosphatidylmonomethylethanolamine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine and unknown aminolipid AL1; moderate amounts of three unknown polar lipids, L1-L3, an unknown aminolipid and an unknown aminophospholipid APL2). The polyamine pattern of Sa25(T) exhibited the major compound putrescine and moderate amounts of spermidine; a similar polyamine pattern with the major compound putrescine was also detected in Pseudochrobactrum glaciei KMM 3858(T). DNA-DNA hybridization of strain Sa25(T) with Pseudochrobactrum glaciei KMM 3858(T) and the type strains of the other Pseudochrobactrum species showed values ranging from 50.3 to 24.8 %, and physiological and biochemical data clearly differentiated this isolate from the described Pseudochrobactrum species. Since Sa25(T) and Pseudochrobactrum glaciei KMM 3858(T) form a distinct lineage in the 16S rRNA gene sequence-based phylogenetic tree, and this separate position is supported by unique characteristics of their polyamine patterns and polar lipid profiles, we propose the novel genus Paenochrobactrum gen. nov., with the type species Paenochrobactrum gallinarii sp. nov. (type strain Sa25(T) =CCUG 57736(T) =CCM 7656(T)) and the reclassification of Pseudochrobactrum glaciei as Paenochrobactrum glaciei comb. nov. (type strain Pi26(T) =KMM 3858(T) =NRIC 0733(T) =JCM 15115(T)).