Eva Staňková

Red-pink pigmented Hymenobacter coccineus sp. nov., Hymenobacter lapidarius sp. nov. and Hymenobacter glacialis sp. nov., isolated from rocks in Antarctica

Four rod-shaped and Gram-stain-negative bacterial strains, CCM 8647, CCM 8649T, CCM 8643T and CCM 8648T, were isolated from rock samples collected on James Ross Island, Antarctica. Extensive biotyping, fatty acid profiling, chemotaxonomy, 16S rRNA gene sequencing and whole-genome sequencing was applied to isolates to clarify their taxonomic position. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that all four isolates belonged to the genus Hymenobacter. Strains CCM 8649T and CCM 8647 were most closely related to Hymenobacter arizonensis OR362-8T (94.4 % 16S rRNA gene sequence similarity), strain CCM 8643T to Hymenobacter terrae DG7AT (96.3 %) and strain CCM 8648T to Hymenobacter glaciei VUG-A130T (96.3 %). The predominant fatty acids of CCM 8649T and CCM 8647 were summed feature 3 (C16 : 1ω7c/C16 : 1ω6c), C16 : 1ω5c and iso-C15 : 0, whereas those of CCM 8643T and CCM 8648T were summed feature 3 (C16 : 1ω7c/C16 : 1ω6c) and C16 : 1ω5c. The quinone systems contained exclusively menaquinone MK-7. The major polyamine was sym-homospermidine. All four strains contained the major polar lipid phosphatidylethanolamine. The G+C content of genomic DNA ranged from 60-63 mol%. Whole-genome sequencing data supported the finding that isolates represented distinct species of the genus Hymenobacter. On the basis of the results obtained, three novel species are proposed for which the names Hymenobacter coccineus sp. nov., Hymenobacter lapidarius sp. nov. and Hymenobacter glacialis sp. nov. are suggested, with the type strains CCM 8649T (=LMG 29441T=P5239T), CCM 8643T (=LMG 29435T=P3150T) and CCM 8648T (=LMG 29440T=P5086T), respectively.

Mucilaginibacter terrae sp. nov., isolated from Antarctic soil

A bacterial strain designated CCM 8645T was isolated from a soil sample collected nearby a mummified seal carcass in the northern part of James Ross Island, Antarctica. The cells were short rods, Gram-stain-negative, non-motile, catalase and oxidase positive, and produced a red-pink pigment on R2A agar. A polyphasic taxonomic approach based on 16S rRNA gene sequencing, extensive biotyping using conventional tests and commercial identification kits and chemotaxonomic analyses were applied to clarify its taxonomic position. Phylogenetic analysis based on the 16S rRNA gene placed strain CCM 8645T in the genus Mucilaginibacter with the closest relative being Mucilaginibacter daejeonensis Jip 10T, exhibiting 96.5 % 16S rRNA pairwise similarity which was clearly below the 97 % threshold value recommended for species demarcation. The major components in fatty acid profiles were Summed feature 3 (C16 : 1ω7c/C16 : 1ω6c), C15 : 0 iso and C17 : 0 iso 3OH. The cellular quinone content was exclusively menaquinone MK-7. The major polyamine was sym-homospermidine and predominant polar lipids were phosphatidylethanolamine and phosphatidylserine. Based on presented results, we propose a novel species for which the name Mucilaginibacter terrae sp. nov. is suggested, with the type strain CCM 8645T (=LMG 29437T).

Pedobacter jamesrossensis sp. nov., Pedobacter lithocola sp. nov., Pedobacter mendelii sp. nov. and Pedobacter petrophilus sp. nov., isolated from the Antarctic environment

A taxonomic study performed on 17 Gram-stain-negative rod-shaped bacterial strains originating from the Antarctic environment is described. Initial phylogenetic analysis using 16S rRNA gene sequencing differentiated the strains into four groups belonging to the genus Pedobacter but they were separated from all hitherto described Pedobacter species. Group I (n=8) was closest to Pedobacter aquatilis (97.8 % 16S rRNA gene sequence similarity). Group II (n=2) and group III (n=4) were closely related (98.8 % 16S rRNA gene sequence similarity) and had Pedobacter jejuensis as their common nearest neighbour. Group IV (n=3) was distantly delineated from the remaining Pedobacter species. Differentiation of the analysed strains into four clusters was further confirmed by repetitive sequence-based PCR fingerprinting, ribotyping, DNA-DNA hybridization and phenotypic traits. Common to representative strains for the four groups were the presence of major menaquinone MK-7, sym-homospermidine as the major polyamine, phosphatidylethanolamine, two unidentified lipids (L2, L5) and an unidentified aminolipid (AL2) as the major polar lipids, presence of an alkali-stable lipid, and C16:1ω7c/C16:1ω6c (summed feature 3), iso-C15:0 and iso-C 17:0 3-OH as the major fatty acids, which corresponded to characteristics of the genus Pedobacter. The obtained results showed that the strains analysed represent four novel species of the genus Pedobacter, for which the names Pedobacter jamesrossensis sp. nov. (type strain CCM 8689T=LMG 29684T), Pedobacter lithocola sp. nov. (CCM 8691T=LMG 29685T), Pedobacter mendelii sp. nov. (CCM 8685T=LMG 29688T) and Pedobacter petrophilus sp. nov. (CCM 8687T=LMG 29686T) are proposed.

Pedobacter psychrophilus sp. nov., isolated from fragmentary rock

Strain P4487AT was isolated during investigation of cultivable bacterial populations of environmental materials sampled at James Ross Island, Antarctica. It revealed Gram-stain-negative short rod-shaped cells producing a pink pigment. Phylogenetic analysis based on 16S rRNA gene sequences allocated strain P4487AT to the genus Pedobacter but showed that the strain represents a distinct intrageneric phylogenetic lineage clearly separated from remaining Pedobacter species. Phylogenetically, strain P4487AT formed a common branch with the Pedobacter arcticus and Pedobacter lignilitoris cluster while the highest value of 94.4 % 16S rRNA gene sequence similarity suggested that Pedobacter lentus is the most closely related species. Biochemical and physiological test results enabled the differentiation of strain P4487AT from all phylogenetically closely related species. Chemotaxonomic analyses of strain P4487AT showed MK-7 as the respiratory menaquinone, sym-homospermidine as the major polyamine, phosphatidylethanolamine and two unidentified lipids as the major polar lipids, presence of sphingolipids, and C16 : 1ω7c/C16 : 1ω6c (summed feature 3), iso-C15 : 0 and iso-C17 : 0 3-OH as the major fatty acids, all of which corresponded with characteristics of the genus Pedobacter. The results showed that strain P4487AT represents a novel species within the genus Pedobacter, for which the name Pedobacter psychrophilus sp. nov. is proposed. The type strain is P4487AT (=CCM 8644T=LMG 29436T).

Rufibacter ruber sp. nov., isolated from fragmentary rock

A red-pigmented, Gram-stain-negative, rod-shaped, aerobic bacterium, designated strain CCM 8646T, was isolated from stone fragments in James Ross Island, Antarctica. Strain CCM 8646T was able to grow from 10 to 40 °C, in the presence of up to 1 % (w/v) NaCl and at pH 7.0-11.0. Analysis of the 16S rRNA gene sequence placed strain CCM 8646T in the genus Rufibacter with the closest relative being Rufibacter roseus H359T (97.07 % 16S rRNA gene sequence similarity). The digital DNA-DNA hybridization values between strain CCM 8646T and R. roseus H359T were low (21.30±2.34 %). The major quinone was menaquinone MK-7. The polar lipids comprised phosphatidylethanolamine, an unknown aminoglycolipid and six unknown polar lipids. The G+C content of strain CCM 8646T was 51.54 mol%. On the basis of phenotypic, chemotaxonomic and genotyping results, strain CCM 8646T is considered to represent a novel species within the genus Rufibacter, for which the name Rufibacter ruber sp. nov. is proposed. The type strain is CCM 8646T (=LMG 29438T).

Isolation of human pathogen Escherichia albertii from faeces of seals (Leptonychotes weddelli) in James Ross Island, Antarctica

A set of nine gram-negative fermenting rods biochemically identified as Escherichia coli was isolated from faeces of seals. These bacteria were characterized by phenotypic classification, 16S rDNA sequence analyses, automated ribotyping, study of whole-cell protein profiles by SDS-PAGE and finally by bacteriocin production. The results of our polyphasic taxonomic study supported the recognition of P4652, P4653 and P4740 isolates as true members of Escherichia albertii species – probably a major enteric human pathogen. To our best knowledge, this is the first evidence showing that E. albertii produces bacteriocin, and colicin D. Obtained data unambiguously showed inconvenience of commercial identification systems to distinguish both Escherichia species due to missing data of E. albertii in the commercial databases. The results of Escherichia isolates taxonomy suggest seals as a novel source of human and animal pathogen, E. albertii in the Antarctic region.

Flavobacterium chryseum sp. nov. and Flavobacterium psychroterrae sp. nov., novel environmental bacteria isolated from Antarctica

A group of rod-shaped, aerobic, Gram-stain-negative, gliding bacteria producing flexirubin-type pigment was isolated from environmental samples collected in Antarctica in 2009-2014. Phylogenetic analysis of the almost complete 16S rRNA gene sequences revealed two separated branches belonging to the genus Flavobacterium. Group I (n=8), represented by strain CCM 8826T, shared the highest sequence similarity to Flavobacterium collinsii 983-08T (98.8 %) and Flavobacterium saccharophilum DSM 1811T (98.4 %), and group II (n=4) represented by strain CCM 8827T shared the highest similarity to Flavobacterium aquidurense WB 1.1-56T (99.6 %). High genetic homogeneity of both groups, separation from each other and from phylogenetically close Flavobacterium species was verified by the rep-PCR fingerprinting method. DNA-DNA hybridization confirmed low genomic relatedness between strain CCM 8826T and F. collinsii 983-08T and F. saccharophilum DSM 1811T (18 and 28 %, respectively) and between strain CCM 8827T and F. aquidurense WB 1.1-56T (27 %). Chemotaxonomic analyses of strains CCM 8826T and CCM 8827T revealed the respiratory quinone to be MK-6, the major identified polar lipid was phosphatidylethanolamine and the predominant polyamine was sym-homospermidine. The common major fatty acids were C15 : 0 iso, C17 : 0 iso 3OH, C15 : 1 iso G, Summed Feature 3 (C16 : 1ω7c/C16 : 1ω6c), C15 : 0 iso 3OH and additionally, C15 : 0 anteiso among group II members. All analyses confirmed that strains of group I and II represent two novel species of the genus Flavobacterium, for which the names Flavobacterium chryseum sp. nov. (type strain CCM 8826T=P3160T=LMG 30615T) and Flavobacterium psychroterrae sp. nov. (type strain CCM 8827T=P3922T=LMG 30616T) are proposed.

Characterization of four Escherichia albertii isolates collected from animals living in Antarctica and Patagonia

Escherichia albertii is a recently discovered species with a limited number of well characterized strains. The aim of this study was to characterize four of the E. albertii strains, which were among 41 identified Escherichia strains isolated from the feces of living animals on James Ross Island, Antarctica, and Isla Magdalena, Patagonia. Sequencing of 16S rDNA, automated ribotyping, and rep-PCR were used to identify the four E. albertii isolates. Phylogenetic analyses based on multi-locus sequence typing showed these isolates to be genetically most similar to the members of E. albertii phylogroup G3. These isolates encoded several virulence factors including those, which are characteristic of E. albertii (cytolethal distending toxin and intimin) as well as bacteriocin determinants that typically have a very low prevalence in E. coli strains (D, E7). Moreover, E. albertii protein extracts caused cell cycle arrest in human cell line A375, probably because of cytolethal distending toxin activity.

Composition of cultivable enteric bacteria from the intestine of Antarctic fish (family Nototheniidae)

In this study, the intestinal bacteria of wild Antarctic fish (family Nototeniidae) were examined with traditional culture-based techniques. The bacterial flora of the gut contents of four marine fish species Notothenia coriiceps, Trematomus bernacchii, Trematomus hansoni and Trematomus newnesi has been investigated in an attempt to describe the allochthonous bacteria inhabiting these Nototheniidae fish. A set of 43 fresh and healthy fish was analysed and intestinal bacteria were retrieved using the dilution plate technique on selective media Endo agar and XLDA agar. A total of 133 different bacterial isolates were obtained and initially characterised by key phenotypical tests. Notothenoid fish gut microbiota showed low species diversity of isolates and intestines were inhabited in average amount of 3 different isolates per fish. The poor bacterial colonization of intestine content showed samples of Trematomus newnesi. Curiously, the Gram-negative non-fermenters, including Pseudomonas sp., Vibrio sp. and Alcaligenes-like isolates represented the majority (59%) of intestine isolates grown on the used selective media for enteric bacteria. Based on preliminary identification, only 54 isolates (41%) were tentatively determined as enteric bacteria. The identification of 54 isolates of Gram-negative fermenting rods to the species level was achieved using biochemical characterization by commercial kits ENTEROtest 24 and Biolog GN2 MicroPlate. Results showed that Enterobacter cloacae phenon and Aeromonas hydrophila were predominant bacterial species in the free-living fish intestine from the group of fermenting Gram-negative rods.