Flavobacterium tructae sp. nov. and Flavobacterium piscis sp. nov., isolated from farmed rainbow trout (Oncorhynchus mykiss).

Four Gram-staining-negative, catalase- and oxidase-positive, pale-orange pigmented bacterial strains (435-08(T), 47B-3-09, 412R-09(T) and 60B-3-09) were isolated from diseased rainbow trout. Analysis of their 16S rRNA gene sequences suggested their adscription to the genus Flavobacterium. Strains formed two phylogenetic groups represented by strains 435-08(T) and 47B-3-09 (group A), and strains 412R-09(T) and 60B-3-09 (group B) displaying 16S rRNA sequence similarities greater than 99.8-99.9% within their respective groups. Strain 435-08(T) exhibited the highest levels of similarity with Flavobacterium aquidurense WB-1.1.56(T) (98.6% sequence similarity) and strain 412R-09(T) with Flavobacterium frigidimaris KUC-1(T) and Flavobacterium aquidurense WB-1.1.56(T) (98.9% and 98.6% sequence similarity, respectively). DNA-DNA hybridization studies showed low levels of relatedness between strain 435-08(T) and strain 412R-09(T) and between both strains and the most closely related species of the genus Flavobacterium. The genomic DNA G+C contents of strains 435-08(T) and 412R-09(T) were 36.2 and 34.3 mol%, respectively. The predominant respiratory quinone of both strains was MK-6 and the major fatty acids were iso-C(15 : 0), C(16 : 1)ω7c and C(15 : 0). The two groups of strains could be distinguished from each other and from related species of the genus Flavobacterium by a number of phenotypic properties. Phylogenetic, genotypic and phenotypic evidence indicated that strains of groups A and B represent two novel species of the genus Flavobacterium, for which the names Flavobacterium tructae sp. nov. (type strain 435-08(T) = CECT 7791(T) = CCUG 60100(T)) and Flavobacterium piscis sp. nov. (type strain 412R-09(T) = CECT 7911(T) = CCUG 60099(T)) are proposed.

Chryseobacterium tructae sp. nov., isolated from rainbow trout (Oncorhynchus mykiss).

Three pale-orange bacteria (strains 1083-08, 1084-08(T) and 1095B-08) were isolated from diseased rainbow trout. The isolates were Gram-staining-negative, catalase- and oxidase-positive, rod-shaped cells. Analyses of their 16S rRNA gene sequences confirmed their adscription to the genus Chryseobacterium. The three isolates shared 100% 16S rRNA gene sequence similarity and 98.5% similarity with Chryseobacterium indologenes CCUG 14556(T), being the closest phylogenetically related species. Genomic DNA-DNA hybridization similarity values between the three isolates were 94-100% and 2-39% between strain 1084-08(T) and the type strains of other related Chryseobacterium species, confirming that the isolates represent a novel species within the genus Chryseobacterium. The DNA G+C content of the species was 33.6-36.1mol%. The predominant respiratory quinone of strain 1084-08(T) was MK-6 and the major fatty acids were iso-C(15:0), iso-C(17:1)ω9c, iso-C(17:0) 3-OH and C(16:1)ω6c. The isolates were distinguished from related Chryseobacterium species by a number of phenotypic properties. Based on the phenotypic, genotypic and phylogenetic findings, it is proposed that the new isolates from rainbow trout be classified as a new species of the genus Chryseobacterium, with the name of Chryseobacterium tructae sp. nov. The type strain is 1084-08(T) (=CECT 7798(T)=CCUG 60111(T)).

Chryseobacterium viscerum sp. nov., isolated from diseased fish.

A taxonomic study was carried out on five Gram-staining-negative, catalase- and oxidase-positive, rod-shaped bacteria isolated from the gills and livers of five diseased rainbow trout. The five novel isolates were designated strains 687B-08(T), 445-08, 452-08, 453B-08 and 967B-08. In phylogenetic analyses based on 16S rRNA gene sequences, the five novel strains appeared almost identical (99.0-100 % sequence similarity) and to belong to the genus Chryseobacterium. Strain 687B-08(T) (the strain selected to represent the five novel isolates) was found to be most closely related to Chryseobacterium oncorhynchi 701B-08(T) (98.9% sequence similarity), Chryseobacterium ureilyticum F-Fue-04IIIaaaa(T) (98.6%), Chryseobacterium indologenes ATCC 29897(T) (98.3%), Chryseobacterium jejuense JS17-8(T) (98.1%) and Chryseobacterium gleum ATCC 35910(T) (98.1%). In DNA-DNA hybridizations, DNA-DNA relatedness values of 99-100% were recorded between the five novel strains. Lower DNA-DNA relatedness values (21-57%) were recorded between strain 687B-08(T) and C. oncorhynchi 701B-08(T), C. ureilyticum F-Fue-04IIIaaaa(T) and the type strains of other closely related, established species of the genus Chryseobacterium. The predominant respiratory quinone of strain 687B-08(T) was MK-6 and the major cellular fatty acids were iso-C(15:0), iso-C(17:1)ω9c, iso-C(17:0) 3-OH and C(16:1)ω6c. The G+C content of the genomic DNA of strain 687B-08(T) was 38.6 mol%. Based on the phenotypic and genotypic evidence, the five novel strains isolated from rainbow trout represent a single, novel species of the genus Chryseobacterium, for which the name Chryseobacterium viscerum sp. nov. is proposed. The type strain is 687B-08(T) ( = CECT 7793(T)  = CCUG 60103(T)).

Bacillus iranensis sp. nov., a moderate halophile from a hypersaline lake.

A Gram-positive, moderately halophilic rod, designated X5BT, was isolated from saline mud of the hypersaline lake Aran-Bidgol in Iran. Strain X5BT was a strictly aerobic, motile bacterium that produced ellipsoidal endospores at a central-subterminal position in non-swollen sporangia. The isolate grew at pH 7.0-10.0 (optimum pH 7.5), at 25-45 °C (optimum 35 °C) and with 2.5-15 % (w/v) NaCl (optimum 5-7.5 %). On the basis of 16S rRNA gene sequences, strain X5BT belonged to the genus Bacillus and showed highest similarity with Bacillus persepolensis HS136T (95.6 % 16S rRNA gene sequence similarity) and Bacillus salarius BH169T (95.5 %). The DNA G+C content was 42.4 mol%. The major cellular fatty acids were anteiso-C15:0 and iso-C15:0 and the polar lipid profile consisted of phosphatidylglycerol, diphosphatidylglycerol, three phospholipids and two glycolipids. The diamino acid found in the cell-wall peptidoglycan was meso-diaminopimelic acid and the isoprenoid quinones were MK-7 (92 %), MK-6 (6 %) and MK-5 (2 %). On the basis of phylogenetic, chemotaxonomic and phenotypic data, a novel species of the genus Bacillus is proposed, with the name Bacillus iranensis sp. nov. The type strain is X5BT (=IBRC 10446T=DSM 23995T).

Chryseobacterium oncorhynchi sp. nov., isolated from rainbow trout (Oncorhynchus mykiss).

Genotypic and phenotypic analyses were performed on five Gram-negative, catalase and oxidase-positive, rod-shaped bacteria isolated from the gill and liver of four rainbow trout. Studies based on comparative 16S rRNA gene sequence analysis showed that the five new isolates shared 99.8-100% sequence similarity and that they belong to the genus Chryseobacterium. The nearest phylogenetic neighbours of the strain 701B-08(T) were Chryseobacterium ureilyticum F-Fue-04IIIaaaa(T) (99.1% 16S rRNA gene sequence similarity) and Chryseobacterium joosteii LMG 18212(T) (98.6%). DNA-DNA hybridization values between the five isolates were 91-99% and ranged from 2 to 53% between strain 701B-08(T) and the type strains of phylogenetically closely related species of Chryseobacterium. Strain 701B-08(T) had a DNA G+C content of 36.3 mol%, the major fatty acids were iso-C(15:0), iso-C(17:1)ω9c, C(16:1)ω6c and iso-C(17:0) 3-OH and the predominant respiratory quinone was MK-6. The novel isolates were distinguished from related Chryseobacterium species by physiological and biochemical tests. The genotypic and phenotypic properties of the isolates from rainbow trout suggest their classification as representatives of a novel species of the genus Chryseobacterium, for which the name Chryseobacterium oncorhynchi sp. nov. is proposed. The type strain is 701B-08(T) (=CECT 7794(T)=CCUG 60105(T)).

Lentibacillus persicus sp. nov., a moderately halophilic species isolated from a saline lake.

A Gram-staining-positive, moderately halophilic bacterium, designated strain Amb31(T), was isolated from water of the hypersaline lake Aran-Bidgol in Iran and characterized taxonomically using a polyphasic approach. Cells were rods, motile and able to produce ellipsoidal endospores at a central position in swollen sporangia. Strain Amb31(T) was facultatively anaerobic and catalase- and oxidase-positive. The strain grew in a complex medium supplemented with 3-25 % (w/v) NaCl (optimum 7.5-10 %). Optimal growth was at 30-35 degrees C and pH 7.5. Phylogenetic analysis based on 16S rRNA gene sequence comparisons showed that strain Amb31(T) belonged to the genus Lentibacillus; it exhibited 16S rRNA gene sequence similarity values of 96.8 and 96.4 % to Lentibacillus salicampi SF-20(T) and Lentibacillus salinarum AHS-1(T), respectively, and values of 95.9-94.7 % to the type strains of other recognized species of Lentibacillus. The cell-wall peptidoglycan of strain Amb31(T) was based on meso-diaminopimelic acid and MK-7 was the respiratory isoprenoid quinone. The major fatty acids were anteiso-C(15 : 0) (44.7 %), iso-C(16 : 0) (21.4 %) and anteiso-C(17 : 0) (15.9 %) and the polar lipid pattern consisted of phosphatidylglycerol, diphosphatidylglycerol, five phospholipids and a glycolipid. The DNA G+C content was 44.1 mol%. All these features confirmed the placement of strain Amb31(T) within the genus Lentibacillus and the strain could be clearly differentiated from strains of the other species of Lentibacillus on the basis of several phenotypic, genotypic and chemotaxonomic features. DNA-DNA relatedness with the type strain of the most closely related strain, L. salicampi DSM 16425(T), was 28 %. Therefore, strain Amb31(T) represents a novel species of the genus Lentibacillus, for which the name Lentibacillus persicus sp. nov. is proposed. The type strain is Amb31(T) (=CCM 7683(T) =CECT 7524(T) =DSM 22530(T) =LMG 25304(T)).

Bacillus halochares sp. nov., a halophilic bacterium isolated from a solar saltern.

A novel halophilic bacterium, designated strain MSS4(T), was isolated from the solar salterns of Mesolongi, Greece. The micro-organism, a motile, Gram-stain-positive, aerobic rod, proliferated at salinities of 1.0-4.0 M NaCl, with optimal growth at 2.5 M NaCl. Endospores were not observed. Strain MSS4(T) showed optimal growth at 37 degrees C and pH 8.0. The G+C content of its DNA was 47.2 mol%. The polar lipid pattern of strain MSS4(T) consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidic acid and phosphatidylethanolamine. It possessed anteiso-C(15 : 0), C(18 : 0), C(16 : 0) and anteiso-C(17 : 0) as the major fatty acids (altogether representing 84.7 % of the total). The predominant isoprenoid quinone was MK-7. The cell-wall peptidoglycan contained meso-diaminopimelic acid. 16S rRNA gene sequence analysis showed that the new isolate has 96.1 % similarity to Bacillus qingdaonensis CM1(T) and Bacillus aidingensis 17-5(T), 95.5 % to Bacillus salarius BH169(T) and lower similarity to other Bacillus species. These results justify the assignment of strain MSS4(T) to a novel species within the genus Bacillus, for which the name Bacillus halochares sp. nov. is proposed. The type strain is MSS4(T) (=LMG 24571(T) =DSM 21373(T)).

Moraxella porci sp. nov., isolated from pigs.

Nine Gram-negative, catalase- and oxidase-positive, coccus-shaped bacteria were isolated from pigs affected by different pathological processes. Phenotypic and genotypic methods were adopted to determine the relationships of these new isolates to recognized species of the genus Moraxella. Analysis of the 16S rRNA gene sequences demonstrated that the clinical isolates represented a new lineage within the genus Moraxella. The isolates were closely related to Moraxella cuniculi and Moraxella pluranimalium with 16S rRNA gene sequence similarities of 98.1 % and 99.1 %, respectively. The isolates displayed DNA-DNA relative binding ratios of 74 % to each other, but distinctly lower levels of DNA-DNA hybridization were observed with phylogenetically closely related moraxellae (<32 %). The new isolates could be distinguished from all other recognized species of the genus Moraxella by physiological and biochemical tests. On the basis of the phenotypic and molecular data, the nine new isolates from pigs represent a novel species within the genus Moraxella, for which the name Moraxella porci sp. nov. is proposed. The type strain is SN9-4M(T) (=CECT 7294(T)=CCUG 54912(T)).

Piscibacillus halophilus sp. nov., a moderately halophilic bacterium from a hypersaline Iranian lake.

A Gram-positive, moderately halophilic bacterium, designated strain HS224(T), was isolated from the hypersaline lake Howz-Soltan in Iran. Cells of strain HS224(T) were rod-shaped, motile and produced oval endospores. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain HS224(T) was affiliated to the genus Piscibacillus, exhibiting 98.5 % sequence similarity to the type strain of Piscibacillus salipiscarius. Strain HS224(T) was also related closely to the type strains of Aquisalibacillus elongatus (98.0 % 16S rRNA gene sequence similarity), Filobacillus milosensis (97.9 %) and Tenuibacillus multivorans (97.0 %). Strain HS224(T) was able to grow at NaCl concentrations of 1-20 % (w/v), with optimum growth occurring at 10 % (w/v) NaCl. The optimum temperature and pH for growth were 35 degrees C and pH 7.5. Major polar lipids were phosphatidylglycerol and diphosphatidylglycerol, the isoprenoid quinone was MK-7 and the peptidoglycan type was A1gamma, with meso-diaminopimelic acid as the diagnostic diamino acid; these characteristics were shared with P. salipiscarius. The major cellular fatty acids of strain HS224(T) were anteiso-C(15 : 0), iso-C(15 : 0), anteiso-C(17 : 0) and iso-C(16 : 0). The G+C content of the DNA was 37.5 mol%. The level of DNA-DNA relatedness between strain HS224(T) and P. salipiscarius JCM 13188(T) was 30.8 %. It is evident from the genotypic, chemotaxonomic and phenotypic data presented that strain HS224(T) represents a novel species of the genus Piscibacillus, for which the name Piscibacillus halophilus sp. nov. is proposed. The type strain is HS224(T) (=CCM 7596(T)=DSM 21633(T)=JCM 15721(T)=LMG 24786(T)).

Thalassobacillus cyri sp. nov., a moderately halophilic Gram-positive bacterium from a hypersaline lake.

A Gram-positive, moderately halophilic bacterium, designated strain HS286(T), was isolated from water of the hypersaline Lake Howz-Soltan in Iran. Cells were strictly aerobic, rod-shaped, motile and able to produce ellipsoidal endospores at a central-subterminal position in swollen sporangia. Isolate HS286(T) grew in a complex medium supplemented with 1-15 % (w/v) NaCl, with optimum growth at 8.0 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence comparisons showed that strain HS286(T) was closely related to Thalassobacillus devorans G-19.1(T) (99.4 % gene sequence similarity). The other closest species were Halobacillus yeomjeoni MSS-402(T) (96.9 %) and other species of the genus Halobacillus (with 96.7-93.5 % similarity). Strain HS286(T) had cell-wall peptidoglycan based on meso-diaminopimelic acid and MK-7 as the respiratory isoprenoid quinone. The major fatty acids were anteiso-C(15 : 0) (43.8 %), iso-C(16 : 0) (21.4 %), iso-C(14 : 0) (9.4 %), anteiso-C(17 : 0) (8.7 %) and iso-C(15 : 0) (7.0 %) and the polar lipid pattern consisted of diphosphatidylglycerol, phosphatidylglycerol, two phospholipids and a glycolipid. The DNA G+C content was 43.0 mol%. All of these features confirmed the placement of isolate HS286(T) within the genus Thalassobacillus. However DNA-DNA hybridization between strain HS286(T) and the only recognized species of the genus Thalassobacillus, T. devorans G-19.1(T), was 27.3 %, showing unequivocally that the novel isolate constituted a new genospecies. Strain HS286(T) could be clearly differentiated from T. devorans and other phylogenetic neighbours on the basis of several phenotypic, genotypic and chemotaxonomic features. Therefore, strain HS286(T) constitutes a novel species, for which the name Thalassobacillus cyri sp. nov. is proposed. The type strain is HS286(T) (=CCM 7597(T)=JCM 15722(T)).

Bacillus persepolensis sp. nov., a moderately halophilic bacterium from a hypersaline lake.

A Gram-positive, moderately halophilic, endospore-forming bacterium, designated strain HS136T, was isolated from the hypersaline lake Howz-Soltan in Iran. Cells were motile rods, producing ellipsoidal endospores at a central-subterminal position in non-swollen sporangia. Strain HS136T, a strictly aerobic bacterium, grew between pH 7.0 and 10.0 (optimal growth at pH 8.0-8.5), between 25 and 45 degrees C (optimal growth at 40 degrees C) and at salinities of 5-20% (w/v) NaCl, growing optimally at 10% (w/v) NaCl. On the basis of 16S rRNA gene sequence analysis, strain HS136T was shown to belong to the genus Bacillus within the phylum Firmicutes and showed closest phylogenetic similarity to Bacillus salarius BH169T (95.2%) and Bacillus qingdaonensis CM1T (94.5%). The DNA G+C content of this new isolate was 37.1 mol%. The major cellular fatty acids of strain HS136T were iso-C15:0, anteiso-C15:0 and anteiso-C17:0, and its polar lipid pattern consisted of phosphatidylglycerol and diphosphatidylglycerol. The isoprenoid quinone was MK-7. The peptidoglycan type is A1gamma, with meso-diaminopimelic acid as the diagnostic diamino acid. On the basis of polyphasic evidence from this study, Bacillus persepolensis sp. nov. is proposed, with strain HS136T (=CCM 7595T=DSM 21632T=JCM 15720T=LMG 25222T) as the type strain.

Moraxella pluranimalium sp. nov., isolated from animal specimens.

Four unusual Gram-negative, catalase-positive, oxidase-positive, coccus-shaped bacteria isolated from one sheep and three pigs were characterized using phenotypic and molecular genetic methods. On the basis of cellular morphology and biochemical criteria, the isolates were tentatively assigned to the genus Moraxella, although the organisms did not appear to correspond to any recognized species. Comparative 16S rRNA gene sequencing studies demonstrated that the isolates represent a novel subline within the genus Moraxella. The most closely related species in phylogenetic terms was Moraxella cuniculi, with 16S rRNA gene sequence similarity of 97.9 % to the type strain CCUG 2154(T), although the DNA-DNA relatedness value was only 29 %. The novel isolates were readily distinguished from all recognized Moraxella species by means of physiological and biochemical tests. On the basis of molecular genetic and phenotypic evidence, therefore, the four isolates represent a novel species of the genus Moraxella, for which the name Moraxella pluranimalium sp. nov. is proposed. The type strain is 248-01(T) (=CECT 7295(T) =CCUG 54913(T)).

Flavobacterium ceti sp. nov., isolated from beaked whales (Ziphius cavirostris).

Three isolates of a Gram-negative, catalase- and oxidase-positive, rod-shaped bacterium, isolated from the lung and liver of two beaked whales, were characterized by phenotypic and molecular genetic methods. Based on cellular morphology and biochemical criteria, the isolates were tentatively assigned to the family Flavobacteriaceae, although they did not appear to correspond to any recognized species. Comparative 16S rRNA gene sequencing showed that the three new isolates shared 100% sequence similarity. The unknown bacterium was phylogenetically closely related to, but distinct from the type strains of Flavobacterium johnsoniae (93.7% sequence similarity), Flavobacterium frigidimaris (93.4%), Flavobacterium aquidurense (93.4%), Flavobacterium hibernum (93.4%) and Flavobacterium degerlachei (93.4%). The novel isolates were readily distinguished from these and other related Flavobacterium species by physiological and biochemical tests. On the basis of phenotypic and phylogenetic evidence, it is proposed that the unknown isolates from whales are classified as a novel species of the genus Flavobacterium, Flavobacterium ceti sp. nov. The type strain is 454-2T (=CECT 7184T=CCUG 52969T).

Aerococcus suis sp. nov., isolated from clinical specimens from swine.

Biochemical and molecular genetic studies were performed for five isolates of unknown Gram-positive, catalase-negative, cocci-shaped micro-organisms obtained from clinical samples from pigs. The micro-organisms were tentatively identified as Aerococcus species on the basis of the results from cellular morphological and biochemical tests. 16S rRNA gene sequencing studies confirmed the provisional identification of the isolates as members of the genus Aerococcus, but the micro-organism did not correspond to any recognized species of this genus. The nearest phylogenetic relatives of these unknown cocci isolated from pigs were Aerococcus viridans (95.9 % 16S rRNA gene sequence similarity) and Aerococcus urinaeequi (95.8 %). The unknown bacterium, however, was distinguishable from these two species and from other animal aerococci by using biochemical tests. On the basis of both phenotypic and phylogenetic findings, the isolates represent a novel species of the genus Aerococcus, for which the name Aerococcus suis sp. nov. is proposed. The type strain is 1821/02(T) (=CECT 7139(T)=CCUG 52530(T)).

Marinobacter lipolyticus sp. nov., a novel moderate halophile with lipolytic activity.

In the course of a screening programme in hypersaline habitats of southern Spain to isolate halophilic bacteria that are able to produce different extracellular enzymes, a novel, moderately halophilic bacterium (strain SM19(T)) that displays lipolytic activity has been isolated and characterized. Strain SM19(T) is a Gram-negative rod that grows optimally in culture media that contain 7.5 % NaCl. The DNA G+C content was 57.0 mol%. According to phenotypic and genotypic data, this strain was assigned to the genus MARINOBACTER: However, 16S rDNA sequence similarity between strain SM19(T) and species of the genus Marinobacter was <96.7 %; this value is sufficiently low to propose its designation as a novel species. In addition, DNA-DNA hybridization with reference strains of close phylogenetic relatives was between 11 and 19 %. On the basis of these data, the inclusion of strain SM19(T) in the genus Marinobacter as a novel species is proposed, with the name Marinobacter lipolyticus sp. nov. The type strain of the novel species is SM19(T) (=DSM 15157(T)=NCIMB 13907(T)=CIP 107627(T)=CCM 7048(T)).

Diversity of moderately halophilic bacteria producing extracellular hydrolytic enzymes.

AIMS: The aim of this study was to determine the diversity of moderately halophilic bacteria with hydrolase activities. METHODS AND RESULTS: Screening bacteria from different hypersaline environments in South Spain led to the isolation of a total of 122 moderately halophilic bacteria able to produce different hydrolases (amylases, DNases, lipases, proteases and pullulanases). These bacteria are able to grow optimally in media with 5-15% salts and in most cases up to 20-25% salts. In contrast to strains belonging to previously described species, that showed very little hydrolase activities, environmental isolates produced a great variety of hydrolases. These strains were identified as members of the genera: Salinivibrio (55 strains), Halomonas (25 strains), Chromohalobacter (two strains), Bacillus-Salibacillus (29 strains), Salinicoccus (two strains) and Marinococcus (one strain), as well as eight non-identified isolates. CONCLUSIONS: Moderately halophilic bacteria are a source of hydrolytic enzymes such as amylases, DNases, lipases, proteases and pullulanases. SIGNIFICANCE AND IMPACT OF THE STUDY: Although most culture collection strains are not able to produce hydrolases, it has been shown that environmental isolates can produce these potentially biotechnological important enzymes.