Two new members of the genus Sphingobacterium: Sphingobacterium zhuxiongii sp. nov. and Sphingobacterium luzhongxinii sp. nov.

Four strains, designated dk4302T, dk4209, xlx-73T, and xlx-183, were isolated from Tibetan gazelle and red swamp crawfish collected from the Qinghai-Tibet Plateau and Jiangxi Province, PR China. The strains were Gram-stain-negative, aerobic, rod-shaped, non-motile, mucoid, and yellow-pigmented. Strains dk4302T and dk4209 grew at 10-40 °C and pH 6.0-9.0, while strains xlx-73T/xlx-183 grew at 15-40 °C and pH 6.0-10.0. Both strains exhibited growth in the presence of up to 3.5 % (w/v) NaCl. Phylogenetic and phylogenomic analyses based on the 16S rRNA gene sequences and 652 core genes, respectively, revealed that the four strains formed two distinct clusters in the genus Sphingobacterium. Strains dk4302T and dk4209 formed a distinct clade with Sphingobacterium hotanense XH4T and Sphingobacterium humi D1T. The most closely related strains to xlx-73T and xlx-183 were Sphingobacterium nematocida M-SX103T. The DNA G+C contents were 38.9 and 39.8 mol%. The digital DNA-DNA hybridization (dDDH) values between dk4302T and S. humi D1T and S. hotanense XH4T were 19.2 and 21.8 % (19.0 and 21.6 % for strain dk4209), respectively. The corresponding average nucleotide identity (ANI) values were 74.3 and 78.1 % (74.4 and 78.3 % for strain dk4209), respectively. The dDDH values between xlx-73T (xlx-183) and S. nematocida M-SX103T was 24.6 % (25.7 %). The corresponding ANI value was 85.7 % (85.5 % for strain xlx-183). The major fatty acid and respiratory quinone of dk4302T and xlx-73T were iso-C15:0 and MK7. The polar lipids identified in all of the novel strains were phosphatidylethanolamine, phosphoglycolipids, aminophospholipids, and phospholipids. A total of 61/190 (32.1 %) and 82/190 (43.2 %) carbon substrates were metabolized by strains dk4302T and xlx-73T in the Biolog MicroPlates, respectively. Based on the results from this polyphasic taxonomic study, two novel species in the genus Sphingobacteruim are proposed, namely Sphingobacteruim zhuxiongii sp. nov. (type strain dk4302T=CGMCC 1.16795T=JCM 33600T) and Sphingobacteruimluzhongxinii sp. nov. (type strain xlx-73T=GDMCC 1.1712T=JCM 33886T).

Arthrobacter caoxuetaonis sp. nov., Arthrobacter zhangbolii sp. nov. and Arthrobacter gengyunqii sp. nov., isolated from Marmota himalayana faeces from Qinghai-Tibet Plateau.

Six aerobic or facultative anaerobic, motile, Gram-stain-positive, catalase-positive and oxidase-negative strains (zg-Y453T, zg-Y324, zg-Y462T, zg-Y411, zg-Y809T and zg-Y786) were isolated from different faecal samples of Marmota himalayana from the Qinghai-Tibet Plateau. Pale yellow, round, raised and moist colonies appeared 48 h after incubation at 28 °C on brain-heart infusion plates supplemented with 5 % defibrinated sheep blood. According to the 16S rRNA gene sequence alignment, two strain pairs (zg-Y453T/zg-Y324 and zg-Y462T/zg-Y411) shared the highest similarities to Arthrobacter luteolus (99.5 and 99.2 %), and the other one (zg-Y809T/zg-Y786) to Arthrobacter citreus (99.5 %). Results of phylogenetic analysis based on the 16S rRNA gene and genome sequences showed that these six strains represented three separate species within the genus Arthrobacter. The average nucleotide identity and digital DNA-DNA hybridization values between the three novel type strains (zg-Y453T/zg-Y462T/zg-Y809T) and other known species in this genus were all below respective thresholds (70.2-81.5/19.6-24.2 %, 70.6-81.8/19.8-25.0 %, and 70.4-88.2/19.9-35.3 %). Although phylogenetically related, there were obvious chemotaxonomic and phenotypic differences: strain pair zg-Y462T/zg-Y411 had anteiso-C15 : 0 as the only major fatty acid; the three novel species had different dominant quinones, MK-8(H2) in strains zg-Y462T/zg-Y809T (74.8/81.1 %) and MK-8(H2)/MK-9(H2) (43.1/53.0 %) in zg-Y453T; similarly, the ability to reduce nitrate in strains zg-Y453T and zg-Y462T could differentiate them from zg-Y809T. All strains had diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol, but differed slightly in the types of unidentified glycolipids, phospholipids and lipids. Based on the results of these polyphasic taxonomic analyses, three novel species within the genus Arthrobacter are proposed, namely Arthrobacter caoxuetaonis sp. nov. (type strain, zg-Y453T=GDMCC 1.2809T=JCM 35173T), Arthrobacter zhangbolii sp. nov. (type strain, zg-Y462T=GDMCC 1.2880T=JCM 35170T) and Arthrobacter gengyunqii sp. nov. (type strain, zg-Y809T=GDMCC 1.2808T=JCM 35168T).

A novel phytopathogen Erwinia sorbitola sp. nov., isolated from the feces of ruddy shelducks.

The species in the genus Erwinia are Gram-stain-negative, facultatively anaerobic, motile, and rod-shaped. Most species in the genus Erwinia are phytopathogens. Also, Erwinia persicina was involved in several human infections. Based on the reverse microbial etiology principles, it is worth analyzing the pathogenicity of species in this genus. In this study, we isolated and sequenced two species of Erwinia. Phylogenetic, phenotypic, biochemical, and chemotaxonomic analyses were performed to identify its taxonomy position. The virulence tests on plant leaves and pear fruits were used to identify the plant pathogenicity of two species of Erwinia. Bioinformatic methods predicted the possible pathogenic determinants based on the genome sequence. Meanwhile, adhesion, invasion, and cytotoxicity assays on RAW 264.7 cells were applied to identify animal pathogenicity. We isolated two Gram-stain-negative, facultatively anaerobic, motile, and rod-shaped strains from the feces of ruddy shelducks in the Tibet Plateau of China, designated J780T and J316. Distinct phylogenetic, genomic, phenotypic, biochemical, and chemotaxonomic characters of J780T and J316 identified they were novel species and belonged to the genus Erwinia, for which the name Erwinia sorbitola sp. nov. was proposed, the type strain was J780T (= CGMCC 1.17334T = GDMCC 1.1666T = JCM 33839T). Virulence tests showed blight and rot on the leaves and pear fruits confirmed Erwinia sorbitola sp. nov. was a phytopathogen. Predicted gene clusters of motility, biofilm formation, exopolysaccharides, stress survival, siderophores, and Type VI secretion system might be the causes of pathogenicity. In addition, predicted polysaccharide biosynthesis gene clusters on the genome sequence, and the high capacity for adhesion, invasion, and cytotoxicity to animal cells confirmed it has pathogenicity on animals. In conclusion, we isolated and identified a novel phytopathogen Erwinia sorbitola sp. nov. in ruddy shelducks. A predefined pathogen is beneficial for preventing from suffering potential economic losses caused by this new pathogen.

Morphological and genomic characteristics of two novel actinomycetes, Ornithinimicrobium sufpigmenti sp. nov. and Ornithinimicrobium faecis sp. nov. isolated from bat faeces (Rousettus leschenaultia and Taphozous perforates).

Four Gram-staining-positive, aerobic, non-motile, circle-shaped bacteria were isolated from the faeces of bats (Rousettus leschenaultia and Taphozous perforates) collected from Guangxi autonomous region (E106°49'20″, N22°20'54″) and Yunnan province (E102°04'39″, N25°09'10″) of South China. Strains HY006T and HY008 shared highly 16S rRNA gene sequence similarity to those of Ornithinimicrobium pratense W204T (99.3%) and O. flavum CPCC 203535T (97.3%), while the strains HY1745 and HY1793T were closest to the type strains O. ciconiae H23M54T (98.7%), O. cavernae CFH 30183T (98.3%), and O. murale 01-Gi-040T (98.1%). Furthermore, when compared to the other members of the genus Ornithinimicrobium, the digital DNA-DNA hybridization and average nucleotide identity values of the four novel strains were within the ranges of 19.6-33.7% and 70.6-87.4%, respectively, both of which were below the respective recommended cutoff values of 70.0% and 95-96%. Significantly, strain HY006T was resistant to chloramphenicol and linezolid whereas strain HY1793T was resistant to erythromycin, clindamycin (intermediately), and levofloxacin (intermediately). The main cellular fatty acids (>20.0%) of our isolates were iso-C15:0 and iso-C16:0. Strains HY006T and HY1793T contained ornithine as the diagnostic diamino acid, also along with the alanine, glycine and glutamic acid in their cell wall. Based on phylogenetic, chemotaxonomic and phenotypic analyses, these four strains could be classified as two novel species of the genus Ornithinimicrobium, for which the names Ornithinimicrobium sufpigmenti sp. nov. and Ornithinimicrobium faecis sp. nov. are proposed. The type strains are HY006T (=CGMCC 1.16565T =JCM 33397T) and HY1793T (=CGMCC 1.19143T =JCM 34881T), respectively.

Description of Ornithinimicrobium cryptoxanthini sp. nov., a Novel Actinomycete Producing ß-cryptoxanthin Isolated from the Tongtian River Sediments.

Two novel Gram-stain-positive, aerobic, non-motile, and yellow-pigmented, irregular rod-shaped bacteria (JY.X269 and JY.X270T) were isolated from the near-surface sediments of river in Qinghai Province, P. R. China (32°37'13″N, 96°05'37″E) in July 2019. Both strains were shown to grow at 15-35 °C and pH 7.0-10.0, and in the presence of 0-6.0% (w/v) NaCl. The 16S rRNA gene sequence analysis showed that the isolates were closely related to Ornithinimicrobium cavernae CFH 30183 T (98.6-98.8% 16S rRNA gene sequence similarity), O. ciconiae H23M54T (98.5-98.6%) and O. murale 01-Gi-040T (98.3-98.5%). The phylogenetic and phylogenomic trees based on the 16S rRNA gene and 537 core gene sequences, respectively, revealed that the two strains formed a distinct cluster with the above three species. The digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between our two isolates (JY.X269 and JY.X270T) and other Ornithinimicrobium species were within the ranges of 19.0-23.9% and 70.8-80.4%, respectively, all below the respective recommended 70.0% and 95-96% cutoff point. Furthermore, the major cellular fatty acids (> 10.0%) of strains JY.X269 and JY.X270T were iso-C15:0, iso-C16:0, and summed feature 9. Strain JY.X270T contained MK-8(H4) and ornithine as the predominant menaquinone and diagnostic diamino acid component within the cell wall teichoic acids. ß-cryptoxanthin (C40H56O) can be extracted from strain JY.X270T, and its content is 6.3 µg/ml. Based on results from the phylogenetic, chemotaxonomic, and phenotypic analyses, the two strains could be classified as a novel species of the genus Ornithinimicrobium, for which the name Ornithinimicrobium cryptoxanthini sp. nov. is proposed (type strain JY.X270T = CGMCC 1.19147T = JCM 34882T).

Nocardioides ochotonae sp. nov., Nocardioides campestrisoli sp. nov. and Nocardioides pantholopis sp. nov., isolated from the Qinghai-Tibet Plateau.

Six Gram-stain-positive, aerobic and irregular-rod-shaped actinobacteria (ZJ1313T, ZJ1307, MC1495T, Y192, 603T and X2025) were isolated from the Qinghai-Tibet Plateau of China and were characterized using a polyphasic taxonomic method. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the six new strains formed three distinct clusters within the genus Nocardioides, and strains ZJ1313T and ZJ1307 were most closely related to N. solisilvae JCM 31492T (16S rRNA gene sequence similarity, 98.0 %), MC1495T and Y192 to N. houyundeii 78T (98.5 %), and 603T and X2025 to N. dokdonensis JCM 14815T (97.6 %). The digital DNA-DNA hybridization values of strains ZJ1313T, MC1495T and 603T among each other and with type strains of their closest relatives were all below the 70 % cut-off point, but values within each pair of new strains were all higher than the threshold. The major fatty acids of these strains were iso-C16 : 0, C17 : 1 ω8c or C18 : 1 ω9c. MK-8(H4) was the predominant respiratory menaquinone and ʟʟ-2,6-diaminopimelic acid was the diagnostic diamino acid. All the strains shared diphosphatidylglycerol (predominant), phosphatidylglycerol, phosphatidylcholine and phosphatidylinositol as the common polar lipids, with minor difference in the types of unidentified phospholipids, glycolipids and lipids. The G+C contents based on genomic DNA of strains ZJ1313T, MC1495T and 603T were 72.5, 72.1 and 73.2 mol%, respectively. The above results suggested that strain pairs ZJ1313T/ZJ1307, MC1495T/Y192 and 603T/X2025 represent three new species of genus Nocardioides, for which the names Nocardioides ochotonae sp. nov. (ZJ1313T=GDMCC 4.177T=KCTC 49537T=JCM 34185T), Nocardioides campestrisoli sp. nov. (MC1495T=GDMCC 4.176T=KCTC 49536T=JCM 34307T) and Nocardioides pantholopis sp. nov. (603T=CGMCC 4.7510T=DSM 106494T) are proposed accordingly.

Arthrobacter sunyaminii sp. nov. and Arthrobacter jiangjiafuii sp. nov., new members in the genus Arthrobacter.

Four novel bacterial strains (zg-ZUI122T/zg-ZUI10 and zg-ZUI227T/zg-ZUI100) were isolated from the intestinal contents of Marmota himalayana and characterized using a polyphasic approach. Cells were Gram-stain- and catalase-positive, urease- and oxidase-negative. Strains grew optimally at 28-30 °C, pH 7.0, with 0.5 % NaCl (w/v). A comparative analysis of 16S rRNA gene sequences revealed that strain pairs zg-ZUI122T/zg-ZUI10 and zg-ZUI227T/zg-ZUI100 belonged to the genus Arthrobacter and were most closely related to Arthrobacter citreus DSM 20133T, with similarities of 99.6 and 99.5 %, respectively. This was further confirmed by phylogenetic analyses based on the 16S rRNA gene and genome sequences. The digital DNA-DNA hybridization and average nucleotide identity values between the two new type strains (zg-ZUI122T and zg-ZUI227T) and other species in the genus Arthrobacter were 20.0-24.4/77.2-83.4% and 19.9-25.1/77.1-83.4%, all below the thresholds. The major cellular fatty acids detected in the two novel species included iso-C15 : 0 and anteiso-C15 : 0; the predominant polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol. MK-8(H2) (77.3%) was the predominant respiratory quinone detected in strain zg-ZUI122T, while MK-8(H2) (53.7%) and MK-9(H2) (46.3%) were detected in strain zg-ZUI227T. The shared cell-wall amino acids detected in the two novel species were alanine, glutamic acid and lysine; the shared whole cell wall sugars consisted of galactose, mannose and ribose. All these analyses concluded that these four strains represent two different novel species in the genus Arthrobacter, for which the names Arthrobacter sunyaminii sp. nov. (zg-ZUI122T = GDMCC 1.2502T = KCTC 49677T) and Arthrobacter jiangjiafuii sp. nov. (zg-ZUI227T = GDMCC 1.2500T = KCTC 49676T) are proposed.

Investigation of the Genomic and Pathogenic Features of the Potentially Zoonotic Streptococcus parasuis.

Recently, Streptococcus suis reference strains of serotype 20, 22, and 26 were reclassified as Streptococcus parasuis. The public health significance of S. parasuis is underestimated due to the lack of clinical isolates. In the present study, we first reported two sporadic S. parasuis infections in humans, after using full-length 16S rRNA and housekeeping genes' phylogeny and ANI values of genome sequence comparisons to determine the species of their isolates BS26 and BS27. Compared to highly pathogenic S. suis strain P1/7, S. parasuis strains BS26 and BS27 possessed a delayed capacity to initiate lethal infection, which may attribute to the later production of higher level of pro-inflammatory cytokines. Differed to S. suis strain P1/7, S. parasuis strains did not induce significant inflammatory response in the brain of mice. Histopathological changes in liver and lungs were widely present in mice infected with S. parasuis strains. Our data indicated that the pathogenic mechanism of S. parasuis may be different from that of S. suis. Three lineages in the core-genome phylogenetic tree and ten types of cps gene cluster were found in 13 S. parasuis genomes, indicating high heterogeneity of this species. The similarity of CPS structure and antibiotic-resistant genes relative to S. suis indicated the evolutionary affinity between the two species. Our data suggested S. parasuis is a potential zoonotic pathogen and poses severe threat to health of susceptible people. Further study on the epidemiology and public health significance of S. parasuis is urgently necessary.

Enorma shizhengliae sp. nov. and Eggerthella guodeyinii sp. nov., two new members of the family Coriobacteriaceae.

Four obligatory anaerobic, Gram-stain-positive, non-motile and rod-shaped organisms (HF-1365T, HF-1362, HF-1101T and HF-4214) were isolated from faecal samples of healthy Chinese subjects. Results of 16S rRNA gene sequence analyses showed that these isolates belong to the genera Enorma (strains HF-1365T and HF-1362) and Eggerthella (strains HF-1101T and HF-4214), closest to Enorma massiliensis (both 98.6 %) and Eggerthella sinensis (98.0 and 97.8 %), respectively. The whole genome sequences of strains HF-1365T and HF-1101T were 2.3 and 4.2 Mb in size with 61.7 and 66.2 mol% DNA G+C content, respectively. The average nucleotide identity and digital DNA-DNA hybridization values indicated that strains HF-1365T and HF-1101T represent novel species in the genera Enorma and Eggerthella. Major fatty acid constituents (>10 %) of strains HF-1365T and HF-1362 were C12 : 0 (24.7 and 23.9 %), C14 : 0 (21.9 and 20.6 %) and summed feature 1 (C15 : 1iso H/C13 : 0 3OH; 12.8 and 10.8 %); those of strains HF-1101T and HF-4214 were C18 : 1 ω9c (32.4 and 33.1 %) and C16 : 0 (13.9 and 14.0 %). Strain HF-1365T had phospholipid, glycolipid, lipid and phosphoglycolipid without any known quinones, while strain HF-1101T had diphosphatidylglycerol as the major polar lipid and MK-7 (80.7 %) as the predominant quinone. On the basis of their phylogenetic and phenotypic characteristics, strains HF-1365T and HF-1101T represent two distinct species, respectively, in the genera Enorma and Eggerthella, for which the names Enorma shizhengliae sp. nov. (type strain HF-1365T=CGMCC 1.17435T=GDMCC 1.1705T=JCM 33601T) and Eggerthella guodeyinii sp. nov. (type strain HF-1101T=CGMCC 1.17436T=GDMCC 1.1668T=JCM 33773T) are proposed.

Arthrobacter yangruifuii sp. nov. and Arthrobacter zhaoguopingii sp. nov., two new members of the genus Arthrobacter.

Four unknown strains belonging to the genus Arthrobacter were isolated from plateau wildlife on the Qinghai-Tibet Plateau of PR China. Phylogenetic analysis based on 16S rRNA gene sequences showed that the four isolates were separated into two clusters. Cluster I (strains 785T and 208) had the greatest 16S rRNA gene sequence similarity to Arthrobacter citreus (98.6 and 98.7 %, respectively), Arthrobacter luteolus (98.0 and 98.1%, respectively), Arthrobacter gandavensis (97.9 and 98.0 %, respectively) and Arthrobacter koreensis (97.6 and 97.7 %, respectively). Likewise, cluster II (strains J391T and J915) had the highest sequence similarity to Arthrobacter ruber (98.6 and 98.3 %, respectively) and Arthrobacter agilis (98.1 and 97.9  %, respectively). Average nucleotide identity and the digital DNA-DNA hybridization values illustrated that the two type strains, 785T and J391T, represented two separate novel species that are distinct from all currently recognized species in the genus Arthrobacter. These strains had DNA G+C contents of 66.0-66.1 mol% (cluster I) and 68.0 mol% (cluster II). The chemotaxonomic properties of strains 785T and J391T were in line with those of the genus Arthrobacter: anteiso-C15:0 (79.3 and 40.8 %, respectively) as the major cellular fatty acid, MK-8(H2) (65.8 %) or MK-9(H2) (75.6 %) as the predominant respiratory quinone, a polar lipid profile comprising diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, glycolipids and phospholipid, and A3α or A4α as the cell wall peptidoglycan type. On the basis of our results, two novel species in the genus Arthrobacter are proposed, namely Arthrobacter yangruifuii sp. nov. (type strain, 785T=CGMCC 1.16725T=GDMCC 1.1592T=JCM 33491T) and Arthrobacter zhaoguopingii sp. nov. (type strain, J391T=CGMCC 1.17382T=GDMCC 1.1667T=JCM 33841T).

Fudania jinshanensis gen. nov., sp. nov., isolated from faeces of the Tibetan antelope (Pantholops hodgsonii) in China.

Two hitherto unknown bacteria (strains 313T and 352) were recovered from the faeces of Tibetan antelopes on the Tibet-Qinghai Plateau, PR China. Cells were rod-shaped and Gram-stain-positive. The optimal growth conditions were at 37 °C and pH 7. The isolates were closely related to Actinotignum sanguinis (92.6 % 16S rRNA gene sequence similarity), Arcanobacterium haemolyticum (92.5 %), Actinotignum schaalii (92.4 %), Actinobaculum massiliense (92.2 %) and Flaviflexus huanghaiensis (91.6 %). Phylogenetic analyses showed that strains 313T and 352 clustered independently in the vicinity of the genera Actinotignum, Actinobaculum and Flaviflexus, but could not be classified clearly as a member of any of these genera. Phylogenomic analysis also indicated that strains 313T and 352 formed an independent branch in the family Actinomycetaceae. The major cellular fatty acids of the strains were C16 : 0 and C18 : 1ω9c. The polar lipids comprised diphosphatidylglycerol, phosphatidylinositol mannoside, phosphatidylglycerol, phosphatidylinositol and five unidentified components. The peptidoglycan contained lysine, alanine and glutamic acid. The respiratory quinone was absent. The whole-cell sugars included glucose and rhamnose. The DNA G+C content of strain 313T was 60.6 mol%. Based on the low 16S rRNA gene sequence similarities, its taxonomic position in the phylogenetic and phylogenomic trees and its unique lipid pattern, we propose that strains 313T and 352 represent members of a novel species in a new genus, for which the name Fudania jinshanensis gen. nov., sp. nov. is proposed. The type strain is 313T (=CGMCC 4.7453T=DSM 106216T).

Detection of mycobacterial small RNA in the bacterial culture supernatant and plasma of patients with active tuberculosis.

Bacterial small RNA (sRNA) has been shown to play an important role in control of bacteria virulence, stress response and physiological metabolism by post-transcriptional regulation of gene expression. However, there were few reports about bacterial sRNA as a biomarker of infection. To test the potential role of sRNA in indicating infection of Mycobacterium tuberculosis, total RNA were extracted from the filtrated bacterial cultural supernatant. After synthesis of cDNA by reverse transcription, four Mycobacterial sRNAs including ASdes, ASpks, AS1726, and AS1890, which have been experimentally confirmed by Kristine B in the year of 2009, were detected by real time PCR. The specificity was verified by sequencing of the amplified products. Moreover, we demonstrate that the presence of sRNA Asdes in plasma of 55.56% (15/27) TB patients and 25.00% (6/24) normal controls with BCG vaccination (P < 0.05). Our results suggest that bacterial non-coding sRNA can be detected from either bacterial culture supernatants or patient's plasma. Detecting of Mycobacterial sRNA provides a rapid and relatively noninvasive approach for diagnosing disease and could be developed as a biomarker to identify patients with active tuberculosis to help make informed decisions about proper therapies.1.