Poseidonibacter parvus sp. nov., isolated from a squid.

A Gram-stain-negative, aerobic, short rod-shaped, motile, brownish-coloured bacterium, termed strain LPB0137T, was isolated from a squid. Its cells could grow weakly on marine agar 2216 with 0.04 % 2,3,5-triphenyl tetrazolium chloride (TTC). Each cell of strain LPB0137T has a circular chromosome with a length of 2.87 Mb and 27.7 mol% DNA G+C content. The genome includes 2698 protein-coding genes and six rRNA operons. In 16S rRNA gene sequence trees, strain LPB0137T formed a robust monophyletic clade with Poseidonibacter antarcticus SM1702T with a sequence similarity of 98.3 %. However, the average nucleotide identity and in silico DNA-DNA hybridization values between the two type strains were low (83.9 and 28.1 %, respectively). The overall phenotypic and genomic features of strain LPB0137T supported its assignment to the genus Poseidonibacter. However, the relatively low gene and genome sequence similarity between this strain and other type strains of the genus Poseidonibacter and several enzymatic characteristics indicated the taxonomic novelty of the isolated strain as a new member of the genus Poseidonibacter. Therefore, based on the phylogenetic and phenotypic characteristics of LPB0137T, we proposed a novel species of the genus Poseidonibacter for it, with the name Poseidonibacter parvus sp. nov. The type strain of this new species is thus LPB0137T (=KACC 18888T=JCM 31548T).

Candidatus Sulfurimonas marisnigri sp. nov. and Candidatus Sulfurimonas baltica sp. nov., thiotrophic manganese oxide reducing chemolithoautotrophs of the class Campylobacteria isolated from the pelagic redoxclines of the Black Sea and the Baltic Sea.

Species of the genus Sulfurimonas are reported and isolated from terrestrial habitats and marine sediments and water columns with steep redox gradients. Here we report on the isolation of strains SoZ1 and GD2 from the pelagic redoxcline of the Black Sea and the Baltic Sea, respectively. Both strains are gram-stain-negative and appear as short and slightly curved motile rods. The autecological preferences for growth of strain SoZ1 were 0-25°C (optimum 20°C), pH 6.5-9.0 (optimum pH 7.5-8.0) and salinity 10-40gL-1 (optimum 25gL-1). Preferences for growth of strain GD2 were 0-20°C (optimum 15°C), pH 7.0-8.0 (optimum pH 7.0-7.5) and salinity 5-40gL-1 (optimum 21gL-1). Strain SoZ1 grew chemolithoautotrophically, while strain GD2 also showed heterotrophic growth with short chained fatty acids as carbon source. Both species utilized hydrogen (H2), sulfide (H2S here taken as the sum of H2S, HS- and S2-), elemental sulfur (S0) and thiosulfate (S2O32-) as electron donors and nitrate (NO3-), oxygen (O2) and particulate manganese oxide (MnO2) as electron acceptors. Based on 16S rRNA gene sequence similarity, both strains cluster within the genus Sulfurimonas with Sulfurimonas gotlandica GD1T as the closest cultured relative species with a sequence similarity of 96.74% and 96.41% for strain SoZ1 and strain GD2, respectively. Strains SoZ1 and GD2 share a ribosomal 16S sequence similarity of 99.27% and were demarcated based on average nucleotide identity and average amino acid identity of the whole genome sequence. These calculations have been applied to the whole genus. We propose the names Candidatus Sulfurimonas marisnigri sp. nov. and Candidatus Sulfurimonas baltica sp. nov. for the thiotrophic manganese reducing culture isolates from the Black Sea and Baltic Sea, respectively.

Halarcobacter arenosus sp. nov., isolated from marine sediment.

A Gram-stain-negative, slightly curved, rod-shaped bacterial strain CAU 1517T was isolated from marine sediment in Busan, the Republic of Korea. The taxonomic position of strain CAU 1517T was investigated via a polyphasic approach comprising phenotypic, chemotaxonomic and phylogenetic properties. Strain CAU 1517T grew optimally at 30 °C, pH 7.5 and in the presence of 7% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequencing revealed that strain CAU 1517T belongs to the genus Halarcobacter and is most closely related to Halarcobacter bivalviorum LMG 26154T (96.5% similarity). The average nucleotide identity and digital DNA-DNA hybridization values between strain CAU 1517T and members of genus Halarcobacter ranged from were 76.7-78.0% and 19.5-21.2%, respectively. The strain contained menaquinone-6 (MK-6) as the only respiratory quinone, and C16:0, summed feature 3 (C16:1 ω7c/C16:1 ω6c), and summed feature 8 (C18:1ω7c/C18:1ω6c) as the major fatty acids. The polar lipid pattern consisted of diphosphatidylglycerol, phosphatidylethanolamine, and two unidentified aminophospholipids. The G+C content was 28.2 mol%. Therefore, it has been demonstrated that the isolate represents a novel species of the genus Halarcobacter, for which the name Halarcobacter arenosus sp. nov., is proposed. The type strain is CAU 1517T (=KCTC 72232T =NBRC 113955T).

Loop-mediated isothermal amplification: Development, validation and application of simple and rapid assays for quantitative detection of species of Arcobacteraceae family- and species-specific Aliarcobacter faecis and Aliarcobacter lanthieri.

AIM: The family Arcobacteraceae formerly genus Arcobacter has recently been reclassified into six genera. Among nine species of the genus Aliarcobacter, Aliarcobacter faecis and Aliarcobacter lanthieri have been identified as emerging pathogens potentially cause health risks to humans and animals. This study was designed to develop/optimize, validate and apply Arcobacteraceae family- and two species-specific (A. faecis and A. lanthieri) loop-mediated isothermal amplification (LAMP) assays to rapidly detect and quantify total number of cells in various environmental niches. METHODS AND RESULTS: Three sets of LAMP primers were designed from conserved and variable regions of 16S rRNA (family-specific) and gyrB (species-specific) genes. Optimized Arcobacteraceae family-specific LAMP assay correctly amplified and detected 24 species, whereas species-specific LAMP assays detected A. faecis and A. lanthieri reference strains as well as 91 pure and mixed culture isolates recovered from aquatic and faecal sources. The specificity of LAMP amplification of A. faecis and A. lanthieri was further confirmed by restriction fragment length polymorphism analysis. Assay sensitivities were tested using variable DNA concentrations extracted from simulated target species cells in an autoclaved agricultural water sample by achieving a minimum detection limit of 10 cells mL-1 (10 fg). Direct DNA-based quantitative detection, from agricultural surface water, identified A. faecis (17%) and A. lanthieri (1%) at a low frequency compared to family-level (93%) with the concentration ranging from 2·1 × 101 to 2·2 × 105 cells 100 mL-1 . CONCLUSIONS: Overall, these three DNA-based rapid and cost-effective novel LAMP assays are sensitive and can be completed in less than 40 min. They have potential for on-site quantitative detection of species of family Arcobacteraceae, A. faecis and A. lanthieri in food, environmental and clinical matrices. SIGNIFICANCE AND IMPACT OF THE STUDY: The newly developed LAMP assays are specific, sensitive, accurate with higher reproducibility that have potential to facilitate in a less equipped lab setting and can help in early quantitative detection and rate of prevalence in environmental niches. The assays can be adopted in the diagnostic labs and epidemiological studies.

Real-time quantitative PCR assay development and application for assessment of agricultural surface water and various fecal matter for prevalence of Aliarcobacter faecis and Aliarcobacter lanthieri.

BACKGROUND: Aliarcobacter faecis and Aliarcobacter lanthieri are recently identified as emerging human and animal pathogens. In this paper, we demonstrate the development and optimization of two direct DNA-based quantitative real-time PCR assays using species-specific oligonucleotide primer pairs derived from rpoB and gyrA genes for A. faecis and A. lanthieri, respectively. Initially, the specificity of primers and amplicon size of each target reference strain was verified and confirmed by melt curve analysis. Standard curves were developed with a minimum quantification limit of 100 cells mL- 1 or g- 1 obtained using known quantities of spiked A. faecis and A. lanthieri reference strains in autoclaved agricultural surface water and dairy cow manure samples. RESULTS: Each species-specific qPCR assay was validated and applied to determine the rate of prevalence and quantify the total number of cells of each target species in natural surface waters of an agriculturally-dominant and non-agricultural reference watershed. In addition, the prevalence and densities were determined for human and various animal (e.g., dogs, cats, dairy cow, and poultry) fecal samples. Overall, the prevalence of A. faecis for surface water and feces was 21 and 28%, respectively. The maximum A. faecis concentration for water and feces was 2.3 × 107 cells 100 mL- 1 and 1.2 × 107 cells g- 1, respectively. A. lanthieri was detected at a lower frequency (2%) with a maximum concentration in surface water of 4.2 × 105 cells 100 mL- 1; fecal samples had a prevalence and maximum density of 10% and 2.0 × 106 cells g- 1, respectively. CONCLUSIONS: The results indicate that the occurrence of these species in agricultural surface water is potentially due to fecal contamination of water from livestock, human, or wildlife as both species were detected in fecal samples. The new real-time qPCR assays can facilitate rapid and accurate detection in < 3 h to quantify total numbers of A. faecis and A. lanthieri cells present in various complex environmental samples.

Poseidonibacter antarcticus sp. nov., isolated from Antarctic intertidal sediment.

A Gram-reaction-negative, aerobic, flagellated and coccoid-shaped bacterial strain, designated SM1702T, was isolated from Antarctic intertidal sediment collected off Ardely Island, West Antarctica. The strain grew at 0-30 °C and with 0.5-5.0 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences and single-copy orthologous clusters both showed that strain SM1702T, together with Poseidonibacter lekithochrous, occupied an independent phylogenetic branch, sharing the highest 16S rRNA gene sequence similarity with type strain of the latter (95.6 %). The major fatty acids were summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c), C16 : 0, and summed feature 2 (C14 : 0 3-OH and/or iso-C16 : 1 I). Polar lipids were phosphatidylethanolamine and phosphatidylglycerol. The genomic DNA G+C content of strain SM1702T was 27.1 mol%. Based on the results of the polyphasic characterisation for strain SM1702T, it is identified as the representative of a novel species of Poseidonibacter, for which the name Poseidonibacter antarcticus sp. nov. is proposed. The type strain of Poseidonibacter antarcticus is SM1702T (=MCCC 1K03471T=KCTC 62796T).

Coexistence of two distinct Sulfurospirillum populations respiring tetrachloroethene-genomic and kinetic considerations.

Two anaerobic bacterial consortia, each harboring a distinct Sulfurospirillum population, were derived from a 10 year old consortium, SL2, previously characterized for the stepwise dechlorination of tetrachloroethene (PCE) to cis-dichloroethene (cis-DCE) via accumulation of trichloroethene (TCE). Population SL2-1 dechlorinated PCE to TCE exclusively, while SL2-2 produced cis-DCE from PCE without substantial TCE accumulation. The reasons explaining the long-term coexistence of the populations were investigated. Genome sequencing revealed a novel Sulfurospirillum species, designated 'Candidatus Sulfurospirillum diekertiae', whose genome differed significantly from other Sulfurospirillum spp. (78%-83% ANI). Genome-wise, SL2-1 and SL2-2 populations are almost identical, but differences in their tetrachloroethene reductive dehalogenase sequences explain the distinct dechlorination patterns. An extended series of batch cultures were performed at PCE concentrations of 2-200 µM. A model was developed to determine their dechlorination kinetic parameters. The affinity constant and maximal growth rate differ between the populations: the affinity is 6- to 8-fold higher and the growth rate 5-fold lower for SL2-1 than SL2-2. Mixed cultivation of the enriched populations at 6 and 30 µM PCE showed that a low PCE concentration could be the driving force for both functional diversity of reductive dehalogenases and niche specialization of organohalide-respiring bacteria with overlapping substrate ranges.

Complex interactions between potentially pathogenic, opportunistic, and resident bacteria emerge during infection on a reef-building coral.

Increased bacterial diversity on diseased corals can obscure disease etiology and complicate our understanding of pathogenesis. To untangle microbes that may cause white band disease signs from microbes responding to disease, we inoculated healthy Acropora cervicornis corals with an infectious dose from visibly diseased corals. We sampled these dosed corals and healthy controls over time for sequencing of the bacterial 16S region. Endozoicomonas were associated with healthy fragments from 4/10 colonies, dominating microbiomes before dosing and decreasing over time only in corals that displayed disease signs, suggesting a role in disease resistance. We grouped disease-associated bacteria by when they increased in abundance (primary vs secondary) and whether they originated in the dose (colonizers) or the previously healthy corals (responders). We found that all primary responders increased in all dosed corals regardless of final disease state and are therefore unlikely to cause disease signs. In contrast, primary colonizers in the families Pasteurellaceae and Francisellaceae increased solely in dosed corals that ultimately displayed disease signs, and may be infectious foreign bacteria involved in the development of disease signs. Moving away from a static comparison of diseased and healthy bacterial communities, we provide a framework to identify key players in other coral diseases.

Phylogenetic and Functional Prokaryotic Diversity in the Hoito-Gol Mesothermal Mineral Spring (Eastern Savan Mountains. Buryat Renuhlie).

High-throughput sequencing was used for comparative analysis of microbial communities of the water and mat from the Hoito-Gol mesothermal mineral sulfide spring (Eastern Sayan Mountains, Buryat Republic). Activity of microbial communities was determined. While both spring biotopes were dominated by members of three bacterial phyla, Proteobacteria, Bacteroidetes, and Firmicutes, they differed drastically in the composition of predominant phylotypes (at the genus level). In the water, the organisms wide spread in aquatic'environments were predominant, mostly aerobic chemoorganotrophs of the generaAcinetobacter, Pe- dobacter, and Flavobacterium. In the microbial mat,;the organisms actively involved in the sulfur cycle predominated, including sulfur-reducing bacteria Sulfurospirillum, sulfate-reducing deltaproteobacteria, sulfur- oxidizing chemoautotrophic bacteria, anoxygenic phototrophic bacteria of,the phyla Chloroflexi and Chloro- bi, as well as purple bacteria belonging to the Q-, P--, and y-Proteobacteria. Microbial mats of the spring exhibited higher phylogenetic diversity compared to high-temperature mats containing photosynthetic microorganisms.