- Volume 53, Issue 5, 2003

A moderately thermophilic bacterium is described, strain N2-214T, that was isolated from an enrichment culture, growing on caprolactone, obtained from a sample from a water-treatment sludge aerobic digester operating at temperatures around 60 °C. The organism was aerobic, Gram-negative, oxidase- and catalase-positive, with a polar flagellum, and capable of growth at temperatures as high as 61 °C. The major fatty acids of strain N2-214T were C16 : 0, C18 : 1 and cyclo-C19 : 0. The phylogenetic relationships of the strain, derived from 16S rRNA gene sequence comparisons, demonstrated it to be a member of the β-subclass of the Proteobacteria. The highest 16S rDNA sequence similarity of isolate N2-214T was to Azoarcus buckelii (91·9 %), Thauera aromatica (92 %) and Hydrogenophilus thermoluteolus (92·7 %). On the basis of phylogenetic analyses and physiological and chemotaxonomic characteristics, it is proposed that isolate N2-214T (=DSM 15129T=LMG 21637T) represents a new genus and species, Tepidiphilus margaritifer gen. nov., sp. nov.

Fourteen sucrose-positive Aeromonas jandaei-like isolates from fresh water and reared European eels were subjected to a polyphasic study to determine their taxonomic position. Numerical taxonomy was used to analyse phenotypic data obtained for these isolates and 43 type and reference strains representative of recognized Aeromonas species. The A. jandaei cluster (phenon 1) was defined at 81·6 % similarity (S J); this included the A. jandaei-like isolates, the sucrose-positive strain Aeromonas veronii biogroup sobria CECT 4910 and nearly all A. jandaei reference strains used in the study. Four other reference strains of A. veronii biogroup sobria and the type strain of Aeromonas ichthiosmia were peripheral to the A. jandaei cluster. The supra-group ‘A. jandaei–A.veronii biogroup sobria–A. ichthiosmia’ was linked at 80·7 % similarity (S J) and was clearly segregated from the phenotypic core of the A. veronii biogroup sobria species, which was related to the reference strain Popoff 224 (CECT 4835). DNA relatedness between strains grouped in the A. jandaei cluster (phenon 1) and A. jandaei CECT 4228T ranged from 70 to 100 %, but was below 50 % when DNAs from A. veronii biogroup sobria CECT 4835, A. veronii biogroup veronii CECT 4257T and A. ichthiosmia CECT 4486T were used. In addition, DNA relatedness between peripheral A. veronii biogroup sobria strains and the species A. jandaei (CECT 4228T), A. veronii (CECT 4257T, CECT 4835) and A. ichthiosmia (CECT 4486T) was always below 54 %, as it was between the species A. ichthiosmia (CECT 4486T) and A. veronii (CECT 4257T, CECT 4835). Emendation of A. jandaei is proposed; this taxon now includes sucrose-positive clinical and environmental strains as well as environmental isolates that are pathogenic for fish and humans. Other new traits for this species are the ability to grow at 4–42 °C, acid production from glycerol but not from lactose, d-melibiose or d-raffinose, the use of d-gluconate, l-glutamate or l-proline but not l-lactate, l-alanine, l-arabinose or l-arginine, hydrolytic activity against casein, elastin, starch and lecithin and the inability to lyse arbutin. The DNA G+C content of A. jandaei is also reported for the first time; it ranges from 58·1 to 61·1 mol%. On the other hand, the DNA relatedness data support the classification of peripheral reference strains of A. veronii biogroup sobria outside this taxon, indicating that biogroup sobria requires further revision.

Earlier investigations have shown that ‘Haemophilus somnus’, ‘Haemophilus agni’ and ‘Histophilus ovis’ represent the same species. In the present investigation, the taxonomic position of this species is explored further by sequencing the 16S rRNA and rpoB genes of strains that were investigated previously by DNA–DNA hybridization. These results clearly support the allocation of this species to a novel genus within the family Pasteurellaceae. The phenotypic separation of Histophilus somni gen. nov., sp. nov. from other members of the family can, for most strains, be based on capnophilia, yellowish pigmentation and indole production. However, due to phenotypic variation, the use of a species-specific PCR test based on the 16S rRNA gene is included in the species description. This is justified by the high sequence similarity of the 16S rRNA gene within the species and the fact that the highest sequence similarity to any other taxon within the family is 93·4 %. The type strain, 8025T=ATCC 43625T=CCUG 36157T, was isolated in the USA from a bovine brain with lesions of thromboembolic meningoencephalitis.

The 16S rDNA sequences of [Aquaspirillum] dispar LMG 4329T and Microvirgula aerodenitrificans SGLY2T (=LMG 18919T) were found to be very similar (>99 %). DNA–DNA hybridizations between the two strains revealed a high level of DNA–DNA binding (84 %), showing that they represent a single species. M. aerodenitrificans and [A.] dispar were also phenotypically very similar. It is concluded that [A.] dispar and M. aerodenitrificans are subjective synonyms. As [A.] dispar was wrongly assigned to the genus Aquaspirillum, we propose that strains of [A.] dispar must be reclassified in the genus Microvirgula. The name Microvirgula aerodenitrificans must be retained for the unified taxon since it is the type of the genus Microvirgula.

Strains of fluorescent pseudomonads, isolated from the phyllosphere of grasses, were analysed by a polyphasic approach in order to clarify their interspecific position. Classification on the basis of ribotyping revealed six genotypes; four of these, which could be differentiated clearly from each other and from Pseudomonas species with validly published names on the basis of phenotypic features, were chosen for detailed phylogenetic analysis. DNA–DNA hybridization studies among representative strains of the four genotypes and closely related Pseudomonas species, determined by comparison of 16S rDNA sequences, showed that three of the studied ribotypes represented novel species. Two of them were related to mainly saprophytic fluorescent pseudomonads and could be easily distinguished by a negative arginine dihydrolase reaction. One ribotype, also characterized by a negative arginine dihydrolase reaction, was closely related to potentially plant-pathogenic fluorescent pseudomonads and differed in certain phenotypic features from its phylogenetic neighbours. As a consequence of the phenotypic and phylogenetic analyses, Pseudomonas trivialis sp. nov. (type strain: P 513/19T=DSM 14937T=LMG 21464T), Pseudomonas poae sp. nov. (type strain: P 527/13T=DSM 14936T=LMG 21465T) and Pseudomonas congelans sp. nov. (type strain: P 538/23T=DSM 14939T=LMG 21466T) are proposed.

Two marine bacterial strains, KMM 3823T and KMM 3836, isolated from a sipuncula (Phascolosoma japonicum), a common inhabitant of Troitsa Bay in the Gulf of Peter the Great (Sea of Japan), were studied. Comparative 16S rRNA gene sequence-based phylogenetic analysis placed these bacteria into a separate branch of the ‘Gammaproteobacteria’ within members of the genus Shewanella. KMM 3823T showed the highest similarity (96·6 %) with Shewanella fidelis. The DNA G+C contents of the two strains studied were 43·0 mol%. The level of DNA homology between these two strains was conspecific (93 %), indicating that they represent a single genospecies. These organisms were greenish-brown, Gram-negative, polarly flagellated, facultatively anaerobic, mesophilic (temperature range 4–30 °C), neutrophilic, haemolytic and were able to degrade elastin, gelatin and DNA. They were susceptible to ampicillin, carbenicillin, gentamicin and kanamycin. The predominant fatty acids were characteristic for shewanellas: 13 : 0-i, 15 : 0-i and 16 : 1(n-7); up to 6·7 % of eicosapentaenoic fatty acid, 20 : 5(n-3), was produced during growth at 28 °C. Phylogenetic evidence, confirmed by DNA hybridization and phenotypic characteristics revealed that the two bacteria studied constitute a new species, Shewanella waksmanii sp. nov., the type strain of which is KMM 3823T (=CIP 107701T=ATCC BAA-643T).

A terephthalate-assimilating bacterium was isolated from activated sludge collected from a domestic wastewater treatment plant in Japan by enrichment with terephthalate as sole carbon source. The isolate, designated strain T7T, was a Gram-negative, short rod-shaped micro-organism. A phylogenetic study based on 16S rRNA gene sequences indicated that strain T7T should be placed in the genus Delftia. A DNA–DNA hybridization value of 69 % was determined between strain T7T and Delftia acidovorans ATCC 15668T. Major cellular fatty acids of strain T7T were C16 : 0, C16 : 1 and C18 : 1. Substantial amounts of cyclopropanoic acid (C17 : 0), 3-OH C10 : 0, C12 : 0, C15 : 0 and C14 : 0 were also detected. The total DNA G+C content of strain T7T was 66·2 mol%. Strain T7T could utilize the following compounds as carbon sources: acetamide, β-alanine, citrate, d-fructose, glycerol, isobutyrate, isophthalate, d(−)-mannitol, maleate, malonate, phenylacetate, propionate, protocatechuate, terephthalate, d-tryptophan and l-tryptophan. Comparisons of phenotypic and genotypic characteristics with other known species belonging to the genus Delftia suggest that strain T7T represents a novel species, for which the name Delftia tsuruhatensis sp. nov. is proposed; strain T7T is the type strain (=IFO 16741T=ATCC BAA-554T).

In this study, the taxonomic positions of 19 Vibrio isolates disclosed in a previous study were evaluated. Phylogenetic analysis based on 16S rDNA sequences partitioned these isolates into groups that were closely related (98·8–99·1 % similarity) to Vibrio pelagius and Vibrio xuii, respectively. DNA–DNA hybridization experiments further showed that these groups had <70 % similarity to other Vibrio species. Two novel Vibrio species are proposed to accommodate these groups: Vibrio fortis sp. nov. (type strain, LMG 21557T=CAIM 629T) and Vibrio hepatarius sp. nov. (type strain, LMG 20362T=CAIM 693T). The DNA G+C content of both novel species is 45·6 mol%. Useful phenotypic features for discriminating V. fortis and V. hepatarius from other Vibrio species include production of indole and acetoin, utilization of cellobiose, fermentation of amygdalin, melibiose and mannitol, β-galactosidase and tryptophan deaminase activities and fatty acid composition.

The taxonomic status of seven glucose-non-acidifying, non-proteolytic Acinetobacter strains characterized by forming small colonies on agar media was studied. With one exception, all strains were from human specimens. They could be distinguished from all described Acinetobacter (genomic) species by their ability to grow on ethanol and acetate as sole sources of carbon but not on 22 other substrates tested including dl-lactate or dl-4-aminobutyrate. DNA–DNA hybridization studies, 16S rRNA gene sequence analysis, amplified rDNA restriction analysis and DNA polymorphism analysis by AFLP showed that these strains represent a hitherto unknown species of the genus Acinetobacter, for which the name Acinetobacter parvus (type strain LMG 21765T=LUH 4616T=NIPH 384T=CCM 7030T) is proposed.

Three strains were isolated from cultured aquatic organisms. They were Gram-negative, oxidase-positive, motile, fermentative, arginine dihydrolase-positive, lysine and ornithine decarboxylase-negative and sensitive to vibriostatic agent O/129. These strains differ from other related Vibrio species by several phenotypic features, which include acetoin and indole production and utilization of amygdalin and d-mannitol. Comparison of 16S rDNA sequences showed a close relationship to the recently described species Vibrio kanaloae (96·6 %) and Vibrio pomeroyi (96·4 %) and to Vibrio furnissii (96·6 %), but DNA–DNA hybridization experiments showed that the three isolates form a tight novel species with ⩽30 % DNA–DNA similarity to its closest phylogenetic neighbours. Vibrio pacinii sp. nov. is proposed, with LMG 19999T (=CAIM 530T=STD3-1057T; DNA G+C content, 44·9 mol%) as the type strain.

Twenty-nine rhizobial isolates from root nodules of Astragalus and Lespedeza spp. growing in the Loess Plateau of China were characterized by numerical taxonomy, RFLP and sequencing of PCR-amplified 16S rRNA genes, measurement of DNA G+C content, DNA–DNA relatedness and cross-nodulation with selected legume species. Based on the results of numerical taxonomy, the isolates formed two clusters (1 and 2) with some single isolates at a similarity level of 82 %. Cluster 1 contained six isolates from Astragalus and Lespedeza spp. Cluster 2 consisted of nine isolates from Astragalus spp. DNA relatedness was greater than 80 % among isolates within cluster 2. Phylogenetic analysis based on 16S rRNA gene sequences showed that CCBAU 7190BT, representing cluster 2, was closely related to Rhizobium galegae and Rhizobium huautlense. DNA–DNA relatedness between CCBAU 7190BT and reference strains of R. galegae, R. huautlense and other related species ranged from 0 to 48·6 %. The cluster 2 isolates could also be differentiated phenotypically from related species. Based on these data, a novel species, Rhizobium loessense sp. nov., is proposed for cluster 2, with the type strain CCBAU 7190BT (=AS1.3401T=LMG 21975T).

A novel moderately thermophilic, hydrogenotrophic, sulfate-reducing bacterium, strain 6NT (=DSM 15269T=CIP 107713T), was isolated from matrixes of Alvinella and Riftia originating from deep-sea hydrothermal-vent samples collected on the 13°N East-Pacific Rise at a depth of approximately 2600 m. It was a Gram-negative, non-sporulating, curved rod, motile with one polar flagellum, that did not possess desulfoviridin. It grew at temperatures ranging from 30 to 60 °C, with an optimum at 45 °C, in the presence of 0–5 % NaCl (optimum 2 %). Strain 6NT utilized only H2/CO2 and formate as electron donors with acetate as carbon source. Sulfate, sulfite, thiosulfate and elemental sulfur were used as terminal electron acceptors during hydrogen oxidation. The G+C content of DNA was 34·4 mol%. Strain 6NT grouped with members of the family Desulfohalobiaceae in the δ-subclass of the Proteobacteria. Its closest phylogenetic relative was Desulfonatronovibrio hydrogenovorans, with only 90 % similarity between the sequences of the genes encoding 16S rRNA. Because of significant phylogenetic differences from all sulfate-reducing bacteria described so far in the domain Bacteria, this novel thermophile is proposed to be assigned to a new genus and species, Desulfonauticus submarinus gen. nov., sp. nov.

The taxonomic positions of three representative strains of Vibrio hollisae (LMG 17719T, LMG 21416 and LMG 21538) were investigated by means of 16S rDNA sequences and phenotypic data. V. hollisae strains (GenBank/EMBL accession nos AJ514909–AJ514911) shared 99·5 % 16S rDNA sequence similarity, but had only 94·6 % similarity to their closest phylogenetic neighbour, Enterovibrio norvegicus. 16S rDNA sequence similarity of V. hollisae and Vibrio cholerae was only 91 %. These results suggest that V. hollisae should be placed into a novel genus, for which the name Grimontia gen. nov. is proposed.

Two Gram-negative, motile, non-spore-forming and moderately halophilic rods (strains SW-47T and SW-49) were isolated from sea water of the East Sea in Korea and subjected to a polyphasic taxonomic study. The two strains grew optimally between 30 and 37 °C, and grew at 4 and 44 °C but not at temperatures above 45 °C. They grew optimally in the presence of 2–5 % (w/v) NaCl, but did not grow in the absence of NaCl. Strains SW-47T and SW-49 had ubiquinone-8 (Q-8) as the predominant respiratory lipoquinone and C16 : 1 ω7c and/or iso-C15 : 0 2OH, C16 : 0 and C18 : 1 ω7c as the major fatty acids, which are consistent with the corresponding data for Alteromonas macleodii. The DNA G+C contents of strains SW-47T and SW-49 were 45 and 44 mol%, respectively. Strains SW-47T and SW-49 showed a high level of 16S rDNA sequence similarity (99·9 %) and a mean level of DNA–DNA relatedness of 96·5 %. Phylogenetic analyses based on 16S rDNA sequences showed that the two strains form a coherent cluster with A. macleodii. Strains SW-47T and SW-49 exhibited levels of 16S rDNA sequence similarity of 99·3 and 99·1 %, respectively, with A. macleodii DSM 6062T and of less than 89·4 % with other species used in the phylogenetic analyses. Alteromonas fuliginea CIP 105339T was found to be more closely related to the genus Pseudoalteromonas than to the genus Alteromonas. On the basis of phenotypic properties and phylogenetic and genomic data, strains SW-47T and SW-49 represent a new species of the genus Alteromonas, for which the name Alteromonas marina (type strain SW-47T=KCCM 41638T=JCM 11804T) is proposed.

Two bacterial strains associated with the white-rot fungus Phanerochaete sordida were subjected to taxonomic investigation. The isolates, designated KCTC 12081T and KCTC 12082, were Gram-negative, non-motile, non-spore-forming and ovoid to rod-shaped. The strains contained major amounts of hexadecanoic acid, cyclo-heptadecanoic acid and ω-7-cis-octadecenoic acid in their cell envelopes. Strain KCTC 12081T contained ubiquinone-8 as the major isoprenoid quinone and the G+C content of its genomic DNA was 61·3 mol%. Morphological and chemotaxonomic properties of the strains were consistent with classification in the genus Burkholderia. In a comparison of 16S rDNA sequence, KCTC 12081T shared 100 % similarity with KCTC 12082 and both strains formed a distinct phylogenetic lineage within the genus Burkholderia. The two strains were also differentiated from other species of this genus by fatty acid composition and phenotypic properties. DNA–DNA relatedness data further supported the separation of the new isolates from closely related species. It is therefore proposed that strains KCTC 12081T (=JCM 11778T) and KCTC 12082 be recognized as a novel species, for which the name Burkholderia sordidicola sp. nov. is proposed.

Four strains of thermophilic, endospore-forming, sulfate-reducing bacteria were enriched and isolated from hot solfataric fields in the Krafla area of north-east Iceland, using methanol and sulfite as substrates. Morphologically, these strains resembled thermophilic Desulfotomaculum species. The strains grew with alcohols, including methanol, with glucose and fructose as electron donors, and with sulfate, sulfite or thiosulfate as electron acceptors. For all four strains, the optimum temperature and pH for growth were 60 °C and pH 7·3, respectively; no added NaCl was required. Phylogenetic analysis based on partial 16S rRNA gene sequence comparisons showed high levels of similarity of the novel strains (>92 %) with Desulfotomaculum kuznetsovii and Desulfotomaculum luciae. However, DNA–DNA hybridization studies with D. kuznetsovii revealed that the four strains belonged to one novel species. A representative of this group of isolates, strain V21T, is proposed as the type strain of a novel species of the spore-forming, sulfate-reducing genus Desulfotomaculum, namely Desulfotomaculum solfataricum (type strain V21T=DSM 14956T=CIP 107984T).

Two Arthrobacter strains, CA15-8T and CA15-9, were isolated from soil, under alkali conditions, and characterized phenotypically, chemotaxonomically and genetically. These alkalitolerant organisms grew over a wide pH range (pH 7·0–12·0), with an optimum at pH 7·0–8·0. The mean G+C content of the DNA of these strains was 63±2 mol%. The strains contained MK-8(H2) and MK-9(H2) as the main respiratory quinones. The cell-wall peptidoglycan was Lys-Thr-Ala2 and the whole-cell sugar was rhamnose. The major cellular fatty acids of the isolates were anteiso-C15 : 0 and iso-C15 : 0. A phylogenetic analysis, based on 16S rDNA sequence data, revealed that strains CA15-8T and CA15-9 formed an evolutionary lineage distinct from other Arthrobacter species. On the basis of morphological, physiological and chemotaxonomic characteristics, 16S rDNA sequence comparisons and DNA–DNA hybridization data, a novel species of Arthrobacter is proposed, namely Arthrobacter koreensis (type strain CA15-8T=KCTC 9922T=IFO 16787T).

Two Gram-positive or Gram-variable, endospore-forming, moderately halophilic rods (strains TF-11T and TF-12T) were isolated from a tidal flat of the Yellow Sea in Korea and were subjected to a polyphasic taxonomic study. Strains TF-11T and TF-12T had cell-wall peptidoglycan based on meso-diaminopimelic acid. The predominant menaquinone found in the two strains was MK-7. The cellular fatty acid profiles of both strains contained large amounts of branched and saturated fatty acids. The major fatty acids were anteiso-C15 : 0 and iso-C15 : 0. The DNA G+C contents of strains TF-11T and TF-12T were respectively 49 and 38 mol%. Phylogenetic analysis based on 16S rDNA sequences showed that strains TF-11T and TF-12T fell within the radiation of the cluster comprising Bacillus species. The level of 16S rDNA sequence similarity between strains TF-11T and TF-12T was 98·3 %. Strains TF-11T and TF-12T exhibited levels of 16S rDNA sequence similarity of less than 96·0 and 96·3 %, respectively, to Bacillus species. The mean level of DNA–DNA relatedness between the two strains was approximately 7 %. On the basis of phenotypic and phylogenetic data and genomic distinctiveness, strains TF-11T and TF-12T should be placed in the genus Bacillus as two distinct novel species, for which the names Bacillus marisflavi sp. nov. (type strain TF-11T=KCCM 41588T=JCM 11544T) and Bacillus aquimaris sp. nov. (type strain TF-12T=KCCM 41589T=JCM 11545T) are proposed.

A comparison of Mycobacterium tuberculosis complex isolates from seals (pinnipeds) in Australia, Argentina, Uruguay, Great Britain and New Zealand was undertaken to determine their relationships to each other and their taxonomic position within the complex. Isolates from 30 cases of tuberculosis in six species of pinniped and seven related isolates were compared to representative and standard strains of the M. tuberculosis complex. The seal isolates could be distinguished from other members of the M. tuberculosis complex, including the recently defined ‘Mycobacterium canettii’ and ‘Mycobacterium caprae’, on the basis of host preference and phenotypic and genetic tests. Pinnipeds appear to be the natural host for this ‘seal bacillus’, although the organism is also pathogenic in guinea pigs, rabbits, humans, Brazilian tapir (Tapirus terrestris) and, possibly, cattle. Infection caused by the seal bacillus is predominantly associated with granulomatous lesions in the peripheral lymph nodes, lungs, pleura, spleen and peritoneum. Cases of disseminated disease have been found. As with other members of the M. tuberculosis complex, aerosols are the most likely route of transmission. The name Mycobacterium pinnipedii sp. nov. is proposed for this novel member of the M. tuberculosis complex (the type strain is 6482T=ATCC BAA-688T=NCTC 13288T).

Seven closely related xylanolytic, thermophilic bacilli were isolated from mud and water samples from the Gonen and Diyadin hot springs, respectively located in the Turkish provinces of Balikesir and Agri. On the basis of morphology and biochemical characteristics, one of the isolates, designated strain G2T, was studied further. Strain G2T is a xylanolytic, sporulating, Gram-positive, rod-shaped bacterium. The isolate is a thermophilic (optimum temperature for growth, 55–60 °C), facultative anaerobe that grows on a wide range of carbon sources, including glucose, starch, xylose and mannitol. It expressed a high level of xylose isomerase activity on xylose and also on glucose. 16S rRNA gene sequence analysis showed that this isolate resembled Anoxybacillus flavithermus DSM 2641T (>97 % similarity), but 16S–23S rDNA internally transcribed spacer polymorphism PCR showed variation between DSM 2641T and isolate G2T. However, it is also known that analysis of 16S rRNA gene sequences may be insufficient to distinguish between some species. In DNA–DNA hybridization, thermophilic isolate G2T showed relatedness of 53·4 % to A. flavithermus and about 45·0 % to Anoxybacillus pushchinoensis, indicating that it is distinct at the species level. On the basis of the evidence presented, it is proposed that strain G2T (=NCIMB 13933T=NCCB 100040T) be designated as the type strain of Anoxybacillus gonensis sp. nov.