Identification and characterization of a novel α-haemolytic streptococci, Streptococcus parapneumoniae sp. nov., which caused bacteremia with pyelonephritis.

OBJECTIVES: We report a case of bacteremia with pyelonephritis in an adult male with an underlying disease caused by α-hemolytic streptococci. α-Hemolytic streptococci were isolated from blood, but it was challenging to identify its species. This study aimed to characterize the causative bacterium SP4011 and to elucidate its species. METHODS: The whole-genome sequence and biochemical characteristics of SP4011 were determined. Based on the genome sequence, phylogenetic analysis was performed with standard strains of each species of α-hemolytic streptococci. Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values were calculated. RESULTS: SP4011 showed optochin susceptibility and bile solubility, but did not react with pneumococcal omni antiserum. Phylogenetic analysis of the whole-genome sequence showed that SP4011 clustered with S. pneumoniae and S. pseodopneumoniae and was most closely related to S. pseodopneumoniae. Genomic analysis revealed that ANI and dDDH values between SP4011 and S. pseodopneumoniae were 94.0 % and 56.0 %, respectively, and between SP4011 and S. pneumoniae were 93.3 % and 52.2 %, respectively. Biochemical characteristics also showed differences between SP4011 and S. pseodopneumoniae and between SP4011 and S. pneumoniae. These results indicate that SP4011 is a novel species. CONCLUSION: Our findings indicate that SP4011 is a novel species of the genus Streptococcus. SP4011 has biochemical characteristics similar to S. pneumoniae, making it challenging to differentiate and requiring careful clinical diagnosis. This isolate was proposed to be a novel species, Streptococcus parapneumoniae sp. nov. The strain type is SP4011T (= JCM 36068T = KCTC 21228T).

Taxonogenomic analysis of the Xanthomonas translucens complex leads to the descriptions of Xanthomonas cerealis sp. nov. and Xanthomonas graminis sp. nov.

The pathovar-based taxonomy of the Xanthomonas translucens group is very confusing due to an overlap of plant host ranges and level of host specificity. Here, whole-genome sequence-based parameters (digital DNA-DNA hybridization and blast-based average nucleotide identity), phylogenomic, biochemical and phenotypical data were used to taxonomically analyse the 11 known pathovars of the X. translucens complex. This polyphasic approach taxonomically assigned the 11 pathovars of X. translucens complex into three distinct species, two of which are new: X. translucens, X. cerealis sp. nov. and X. graminis sp. nov. X. translucens consists of three pathovars: pv. translucens (=pv. hordei), pv. pistaciae strain A ICMP 16316PT and pv. undulosa (=pv. secalis). X. cerealis sp. nov. encompasses the pv. cerealis strain LMG 679PT and pv. pistaciae strain B ICMP 16317PT with genome similarity of 92.7% (dDDH) and 99.0% (ANIb) suggesting taxonomically similar genotypes. The other new species, X. graminis sp. nov., consists of the remaining five designated pathovars (pv. graminis, pv. arrhenatheri, pv. poae, pv. phleipratensis and pv. phlei) with highly variable dDDH and ANIb values ranging from 74.5 to 93.0% and from 96.7 to 99.2%, respectively, an indication of a very divergent taxonomic group. Only strains of pvs. phlei and phleipratensis showed the highest genomic similarities of 93.0% (dDDH) and 99.2% (ANIb), suggesting synonymic pathovars as both infect the same plant hosts. The dDDH and ANI data were corroborated by phylogenomics clustering. The fatty acid contents were similar but the type strain of X. graminis sp. nov. exhibited 20% less C15 : 0 iso and 40% more C17 : 0 iso fatty acids than the other species. Based on phenotypic, biochemical and whole-genome sequence data, we propose two new species, Xanthomonas cerealis sp. nov. and Xanthomonas graminis sp. nov. with type strains LMG 679T (=NCPPB 1944T) and LMG 726T (=NCPPB 2700T), respectively.

Stenotrophomonas riyadhensis sp. nov., isolated from a hospital floor swab.

During the analysis of a collection of Pseudomonas strains linked to an outbreak in an intensive care unit at King Faisal Specialist Hospital and Research Center in 2019, one isolate (CFS3442T) was identified phenotypically as Pseudomonas aeruginosa. However, whole-genome sequencing revealed its true identity as a member of the genus Stenotrophomonas, distinct from both P. aeruginosa and Stenotrophomonas maltophilia. The isolate demonstrated: (i) a significant phylogenetic distance from P. aeruginosa; (ii) considerable genomic differences from several S. maltophilia reference strains and other Stenotrophomonas species; and (iii) unique phenotypic characteristics. Based on the combined geno- and phenotypic data, we propose that this isolate represents a novel species within the genus Stenotrophomonas, for which the name Stenotrophomonas riyadhensis sp. nov. is proposed. The type strain is CFS3442T (=NCTC 14921T=LMG 33162T).

TaqMan Real-Time PCR Assay for Specific Detection and Differentiation of Xanthomonas translucens pv. undulosa from Other Pathovars Targeting a Recombination Mediator Gene, recF.

Bacterial leaf streak and black chaff diseases of wheat caused by Xanthomonas translucens pv. undulosa is becoming a major constraint to growers and trade since it is seedborne. Molecular tools for specific detection/differentiation of pv. undulosa are lacking. We report the development of a TaqMan real-time PCR for specific detection/identification of pv. undulosa targeting the recombination mediator gene (recF). Analysis of the complete recF (1,117 bp) sequences identified the gene as a reliable phylogenetic marker for identification of pv. undulosa, differentiating it from the other pathovars; recF-based sequence homology values among the 11 pathovars correlated well with genome-based DNA-DNA hybridization values. The discriminatory power of recF to differentiate pv. undulosa from the other pathovars is due to nucleotide polymorphic positions. We used these nucleotide polymorphisms to develop a TaqMan PCR for specific detection of pv. undulosa. The specificity of the assay was validated using 67 bacterial and fungal/oomycete strains. The selected primers and the double-quenched FAM-labeled TaqMan probe were specific for the detection of 11 pv. undulosa/secalis strains. The 56 strains of other X. translucens pathovars (n = 39) and non-Xanthomonas spp. (n = 17) did not exhibit any detectable fluorescence. Also, greenhouse-inoculated and naturally infected wheat leaf samples showed positive reactions for the presence of pv. undulosa DNA but not healthy control plants. The TaqMan assay reliably detected as low as 1-pg DNA amount and 10 colony forming units of the target pathogen per reaction. This TaqMan assay could be useful to regulatory agencies with economic benefits to wheat growers.

Roseateles albus sp. nov., Roseateles koreensis sp. nov. and Janthinobacterium fluminis sp. nov., isolated from freshwater at Jucheon River, and emended description of Roseateles aquaticus comb. nov.

Three Gram-stain-negative, facultatively anaerobic, rod-shaped, catalase-positive, oxidase-negative bacterial strains were designated as hw1T, hw8T and hw3T. Strains hw1T, hw8T and hw3T grew at 15-28 °C (optimum, 25 °C), 15-35 °C (optimum, 30 °C) and 4-28 °C (optimum, 20 °C), respectively, and at pH 7.0-12.0 (optimum, pH 9.0), pH 6.0-11.0 (optimum, pH 9.0) and 5.0-12.0 (optimum, pH 7.0), respectively. Additionally, strains hw1T and hw8T only grew when the NaCl concentration was 0 %, while strain hw3T grew at between 0 and 0.5 % (w/v; optimum, 0 %). The average nucleotide identity (ANI) values between strains hw1T, hw8T and the Roseateles type strains ranged from 73.8 to 84.2 %, while the digital DNA-DNA hybridization (dDDH) values ranged from 19.7 to 27.5 %. The ANI values between strain hw3T and the Janthinobacterium type strains ranged from 78.7 to 80.7 %, while dDDH values ranged from 22.3 to 23.0 %. The draft genomes of strains hw1T, hw8T and hw3T consisted of 5.5, 4.4 and 5.9 Mbp, with DNA G+C contents of 61.7, 61.8 and 66.0 mol%, respectively. The results of the dDDH, ANI, phylogenetic, biochemical and physiological analyses indicated that the novel strains were distinct from other members of their genera. Thus, we proposed the names Roseateles albus sp. nov. (type strain hw1T= KACC 22887T= TBRC 16613T), Roseateles koreensis sp. nov. (type strain hw8T= KACC 22885T= TBRC 16614T) and Janthinobacterium fluminis sp. nov. (type strain hw3T= KACC 22886T= TBRC 16615T).

Sphingomonas oryzagri sp. nov., isolated from a rice field.

A Gram-stain-negative, aerobic, short rod-shaped and motile novel bacterial strain, designated MAHUQ-71T, was isolated from the soil of a rice field. The colonies were observed to be milky yellow-coloured, smooth, spherical and 0.1-0.4 mm in diameter when grown on Reasoner's 2A agar medium for 2 days. Strain MAHUQ-71T was found to be able to grow at 15-37 °C, pH 5.0-10.0 and with 0-3.0 % NaCl (w/v). The strain was found to be positive for the catalase test, but negative for the oxidase test. The strain was positive for hydrolysis of aesculin and Tween 20. According to the 16S rRNA gene sequence comparisons, the isolate was identified as a member of the genus Sphingomonas and to be closely related to Sphingomonas chungangi MAH-6T (98.5 % sequence similarity), Sphingomonas polyaromaticivorans B2-7T (98.4 %) and Sphingomonas oligoaromativorans SY-6T (96.6 %). Strain MAHUQ-71T has a draft genome size of 4 255 278 bp (10 contigs), annotated with 4098 protein-coding genes, 47 tRNA and three rRNA genes. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain MAHUQ-71T and the closest type strain S. chungangi MAH-6T were in the range of 85.6 and 30.6 %, respectively. The genomic DNA G+C content was determined to be 66.7 mol%. The predominant isoprenoid quinone was ubiquinone 10. The major fatty acids were identified as summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c), C16 : 0 and C14 : 0 2OH. The main polar lipids were phosphatidylcholine, phosphatidylethanolamine, diphosphatidylglycerol and sphingoglycolipid. On the basis of dDDH and ANI values, as well as the results of genotypic, chemotaxonomic and physiological analyses, strain MAHUQ-71T represents a novel species within the genus Sphingomonas, for which the name Sphingomonas oryzagri sp. nov. is proposed, with MAHUQ-71T (=KACC 22252T=CGMCC 1.19065T) as the type strain.

Sphingobium agri sp. nov., isolated from rhizospheric soil of eggplant.

A Gram-stain-negative, aerobic, short rod-shaped and motile bacterial strain, designated MAH-33T, was isolated from rhizospheric soil of eggplant. The colonies were observed to be yellow-coloured, smooth, spherical and 0.1-0.3 mm in diameter when grown on TSA agar medium for 2 days. Strain MAH-33T was found to be able to grow at 10-40 °C, at pH 5.0-10.0 and at 0-3.0 % NaCl (w/v). The strain was found to be positive for both oxidase and catalase tests. The strain was positive for hydrolysis of tyrosine and aesculin. According to the 16S rRNA gene sequence comparisons, the isolate was identified as a member of the genus Sphingobium and to be closely related to Sphingobium quisquiliarum P25T (98.4 % similarity), Sphingobium mellinum WI4T (97.8 %), Sphingobium fuliginis TKPT (97.3 %) and Sphingobium herbicidovorans NBRC 16415T (96.9 %). The novel strain MAH-33T has a draft genome size of 3 908 768 bp (28 contigs), annotated with 3689 protein-coding genes, 45 tRNA and three rRNA genes. The average nucleotide identity and digital DNA-DNA hybridization values between strain MAH-33T and closely related type strains were in the range of 79.8-81.6 % and 23.2-24.5 %, respectively. The genomic DNA G+C content was determined to be 62.2 %. The predominant isoprenoid quinone was ubiquinone 10. The major fatty acids were identified as C16 : 0 and summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c). The polar lipids identified in strain MAH-33T were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, sphingoglycolipid, phosphatidylcholine; one unknown phospholipid and one unknown lipid. On the basis of digital DNA-DNA hybridization, ANI value, genotypic analysis, chemotaxonomic and physiological data, strain MAH-33T represents a novel species within the genus Sphingobium, for which the name Sphingobium agri sp. nov. is proposed, with MAH-33T (=KACC 19973T = CGMCC 1.16609T) as the type strain.

Streptomyces longhuiensis sp. nov, a novel species isolated from soil of Lilium brownii in Hunan Province, PR China.

An actinobacterium strain, designated BH-MK-02T, was isolated from the soil of Lilium brownii. The taxonomic position was determined using a polyphasic approach. Strain BH-MK-02T grew well on International Streptomyces Project series media and formed well-developed, branched substrate hyphae and aerial mycelium that differentiated into straight spore chains with a wrinkled surface. The diagnostic diamino acid was ll-diaminopimelic acid. The major menaquinones were MK-9(H4), MK-9(H6) and MK-9(H8). The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol mannosides, phosphatidylglycerol and unidentified lipid spots. The predominant fatty acids were anteiso-C15 : 0, iso-C16 : 0, C16 : 0 and C16 : 1 ω7c/C16 : 1 ω6c. The phenotypic characteristics of strain BH-MK-02T indicated that it belonged to the genus Streptomyces. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain BH-MK-02T was most closely related to Streptomyces aureus CGMCC 4.1833T (99.7 %). However, the average nucleotide identity and digital DNA-DNA hybridization values between the whole-genome sequences of strain BH-MK-02T and S. aureus CGMCC 4.1833T were 78.1 and 23.2 %, respectively, below the 96.7 and 70 % cut-off points respectively recommended for delineating Streptomyces species. Furthermore, the novel isolate could be distinguished from S. aureus CGMCC 4.1833T by morphological, physiological and biochemical characteristics. Based on all these data, strain BH-MK-02T (=MCCC 1K06237T=JCM 34789T) clearly represents a novel species within the genus Streptomyces, for which the name Streptomyces longhuiensis sp. nov. is proposed.

Solitalea agri sp. nov., a new member of the genus Solitalea isolated from rhizospheric soil of a jujube tree.

A Gram-stain negative, aerobic, rod-shaped and creamy pink-coloured bacterium, designated MAHUQ-68T, was isolated from rhizospheric soil of a jujube tree. Colonies grew at 10-40 °C (optimum, 28 °C), pH 6.0-9.0 (optimum pH, 7.0) and in the presence of 0-1.5 % NaCl (optimum 0-0.5 %). Positive for both catalase and oxidase activity. Strain MAHUQ-68T hydrolysed casein, starch, aesculin and l-tyrosine. Based on the results of phylogenetic analysis using 16S rRNA gene and genome sequences, strain MAHUQ-68T clustered together within the genus Solitalea. The closest members were Solitalea longa HR-AVT (98.8 % sequence similarity), Solitalea canadensis DSM 3403T (96.9 %) and Solitalea koreensis R2A36-4T (94.0 %). The genome of strain MAHUQ-68 T was 4 250 173 bp long with 68 scaffolds and 3 570 protein-coding genes. The genomic DNA G+C content of the type strain was 38.0 mol%. The average nucleotide identity and in silico DNA-DNA hybridization values between strain MAHUQ-68T and its closest relatives were 72.0-81.4% and 19.8-24.3 %, respectively. The major cellular fatty acids were iso-C15 : 0 and summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c). The main respiratory quinone was menaquinone-7. The polar lipids comprised phosphatidylethanolamine, an unidentified aminolipid and four unidentified lipids. Based on these data, strain MAHUQ-68T represents a novel species in the genus Solitalea, for which the name Solitalea agri sp. nov. is proposed. The type strain is MAHUQ-68T (=KACC 22249T=CGMCC 1.19062T).

Massilia agrisoli sp. nov., isolated from rhizospheric soil of banana.

A Gram-stain-negative, aerobic, short rod-shaped and motile novel bacterial strain, designated MAHUQ-52T, was isolated from the rhizospheric soil of a banana plant. Colonies grew at 10-35 °C (optimum, 28 °C), pH 6.0-9.5 (optimum, pH 7.0-7.5), and in the presence of 0-1.0 % NaCl (optimum 0 %). The strain was positive for catalase and oxidase tests, as well as hydrolysis of gelatin, casein, starch and Tween 20. Based on the results of phylogenetic analysis using 16S rRNA gene and genome sequences, strain MAHUQ-52T clustered together within the genus Massilia. Strain MAHUQ-52T was closely related to Massilia soli R798T (98.6 %) and Massilia polaris RP-1-19T (98.3 %). The novel strain MAHUQ-52T has a draft genome size of 4 677 454 bp (25 contigs), annotated with 4193 protein-coding genes, 64 tRNA and 19 rRNA genes. The genomic DNA G+C content was 63.0 %. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain MAHUQ-52T and closely related type strains were ≤88.4 and 35.8 %, respectively. The only respiratory quinone was ubiquinone-8. The major fatty acids were identified as C16 : 0 and summed feature 3 (C15 : 0 iso 2-OH and/or C16 : 1 ω7c). Strain MAHUQ-52T contained phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol as the major polar lipids. On the basis of dDDH and ANI values, as well as genotypic, chemotaxonomic and physiological data, strain MAHUQ-52T represents a novel species within the genus Massilia, for which the name Massilia agrisoli sp. nov. is proposed, with MAHUQ-52T (=KACC 21999T=CGMCC 1.18577T) as the type strain.

Isolation and characterization of Pinibacter soli sp. nov., and in silico genome mining of Pinibacter for biosynthetic gene cluster prediction.

A Gram-stain-negative, aerobic, rod-shaped, non-motile and non-flagellated novel bacterial strain, designated MAH-24T, was isolated from the rhizospheric soil of a pine garden. The colonies were observed to be orange-coloured, smooth, spherical and 0.4-0.8 mm in diameter when grown on Reasoner's 2A agar medium for 2 days. Strain MAH-24T was found to be able to grow at 10-35 °C, at pH 6.0-9.0 and in the presence of 0-1.0 % NaCl (w/v). The strain was found to be positive for the catalase and oxidase tests. The strain was positive for hydrolysis of aesculin and l-tyrosine. According to the 16S rRNA gene sequence comparisons, the isolate was identified as a member of the genus Pinibacter and to be closely related to Pinibacter aurantiacus MAH-26T (99.2 % sequence similarity). The novel strain MAH-24T has a draft genome size of 5 918 133 bp (13 contigs), annotated with 4613 protein-coding genes, 47 tRNA and three rRNA genes. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain MAH-24T and the closest type strain P. aurantiacus MAH-26T were in the range of 85.3 and 29.9 %, respectively. In silico genome mining revealed that both novel strain MAH-24T and P. aurantiacus MAH-26T have a significant potential for the production of novel natural products in the future. The genomic DNA G+C content was determined to be 41.0 mol%. The predominant isoprenoid quinone was menaquinone-7. The major fatty acids were identified as C15:0 iso, C15:1 iso G and C17:0 iso 3OH. On the basis of dDDH, ANI, genotypic, chemotaxonomic and physiological data, strain MAH-24T represents a novel species within the genus Pinibacter, for which the name Pinibacter soli sp. nov. is proposed, with MAH-24T (=KACC 19747T=CGMCC 1.13659T) as the type strain.

Vibrio chanodichtyis sp. nov., isolated from the intestine of swordfish Chanodichthys dabryi.

A Gram-stain-negative, facultatively anaerobic, motile, curved-rod-shaped flagellated bacterium, designated DSL-7T, was isolated from the intestine of Chanodichthys dabryi in the Yangtze river, PR China. The strain grew optimally in tryptone soy broth medium at 37 °C, pH 7.0 and with 1 % (w/v) NaCl. Strain DSL-7T showed less than 96.2 % 16S rRNA gene sequence similarity to type strains of the genus Vibrio. Phylogenetic analysis based on genomes indicated that strain DSL-7T belonged to the genus Vibrio and formed a subclade with Vibrio mimicus NCTC 11435T, Vibrio metoecus OP3HT, Vibrio cholerae ATCC 14035T, Vibrio albensis ATCC14547T, Vibrio paracholerae OP3HEDC-792T and Vibrio tarriae 2521-89T. The average nucleotide identity (ANI) and in digital DNA-DNA hybridization (dDDH) values between DSL-7T and closely related type strains were below the accepted threshold to delineate a new species of 95 and 70 %, respectively. The major cellular fatty acids were summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C16 : 0, summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) and C14 : 0. The genomic DNA G+C content was 47.6 mol%. Based on the phenotypic, chemotaxonomic, phylogenetic and genomic data, strain DSL-7T represents a novel species of the genus Vibrio, for which the name Vibrio chanodichtyis sp. nov. is proposed, with strain DSL-7T (=KCTC 92851T=CCTCC AB 2022396T) as the type strain.

Legionella maioricensis sp. nov., a new species isolated from the hot water distribution systems of a hospital and a shopping center during routine sampling.

Two Legionella-like strains isolated from hot water distribution systems in 2012 have been characterized phenotypically, biochemically and genomically in terms of DNA relatedness. Both strains, HCPI-6T and EUR-108, exhibited biochemical phenotypic profiles typical of Legionella species. Cells were Gram-negative motile rods which grew on BCYEα agar but not on blood agar and displayed phenotypic characteristics typical of the family Legionellaceae, including a requirement for l-cysteine and testing catalase positive. Both strains were negative for oxidase, urease, nitrate reduction and hippurate negative, and non-fermentative. The major ubiquinone was Q12 (59.4 % HCPI-6T) and the dominant fatty acids were C16 : 1 ω7c (28.4 % HCPI-6T, ≈16 % EUR-108), C16 : 0 iso (≈22.5 % and ≈13 %) and C15 : 0 anteiso (19.5 % and ≈23.5 %, respectively). The percent G+C content of genomic DNA was determined to be 39.3 mol %. The 16S rRNA gene, mip sequence and comparative genome sequence-based analyses (average nucleotide identity, ANI; digital DNA-DNA hybridization, dDDH; and phylogenomic treeing) demonstrated that the strains represent a new species of the genus Legionella. The analysis based on the 16S rRNA gene sequences showed that the sequence similarities for both strains ranged from 98.8-90.1 % to other members of the genus. The core genome-based phylogenomic tree (protein-concatemer tree based on concatenation of 418 proteins present in single copy) revealed that these two strains clearly form a separate cluster within the genus Legionella. ANI and dDDH values confirmed the distinctiveness of the strains. Based on the genomic, genotypic and phenotypic findings from a polyphasic study, the isolates are considered to represent a single novel species, for which the name Legionella maioricensis sp. nov. is proposed. The type strain is HCPI-6T (=CCUG 75071T=CECT 30569T).

Helicobacter kumamotonensis sp. nov., isolated from human clinical specimens.

A Gram-stain-negative, spiral bacterium (PAGU 1991T) was isolated from the blood of a patient with diffuse large B-cell lymphoma. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolate was very closely related to Helicobacter equorum LMG 23362T (99.1 % similarity), originally isolated from a faecal sample from a healthy horse. PAGU 1991T was also very closely related to PAGU 1750 in our strain library (=CCUG 41437) with 99.7 % similarity. Additional phylogenetic analyses based on the 23S rRNA gene sequence and GyrA amino acid sequence further supported the close relationship between the two human isolates (PAGU 1991T and PAGU 1750) and the horse strain. However, a phylogenetic analysis based on 16S rRNA showed that the two human isolates formed a lineage that was distinct from the horse strain (less than 99.2 % similarity). In silico whole-genome comparisons based on digital DNA-DNA hybridization, average nucleotide identity based on blast and orthologous average nucleotide identity using usearch between the two human isolates and the type strain of H. equorum showed values of less than 52.40, 93.47, and 93.50 %, respectively, whereas those between the two human isolates were 75.8, 97.2, and 97.2 %, respectively. These data clearly demonstrated that the two human isolates formed a single species, distinct from H. equorum. Morphologically, the human isolates could be distinguished by the type of flagella; the human isolates showed a bipolar sheathed flagellum, whereas that of H. equorum was monopolar. Biochemically, the human isolate was characterized by growth at 42 °C under microaerobic conditions and nitrate reduction unability. We conclude that the two human isolates, obtained from geographically and temporally distinct sources, were a novel species, for which we propose the name Helicobacter kumamotonensis sp. nov., with the type strain PAGU 1991T (=GTC 16810T=CCUG 75774T).

Pseudomonas quebecensis sp. nov., a bacterium isolated from root-zone soil of a native legume, Amphicarpaea bracteata (L.) Fernald, in Quebec, Canada.

Four bacterial strains (S1Bt3, S1Bt7, S1Bt30 and S1Bt42T) isolated from soil collected from the rhizosphere of a native legume, Amphicarpaea bracteata, were investigated using a polyphasic approach. Colonies were fluorescent, white-yellowish, circular and convex with regular margins on King's B medium. Cells were Gram-reaction-negative, aerobic, non-spore-forming rods. Oxidase- and catalase-positive. The optimal growth temperature of the strains was 37 °C. Phylogenetic analysis of the 16S rRNA gene sequences placed the strains within the genus Pseudomonas. Analysis of the 16S rRNA-rpoD-gyrB concatenated sequences clustered the strains and well separated from Pseudomonas rhodesiae CIP 104664T and Pseudomonas grimontii CFM 97-514T with the type strains of the closest species. Phylogenomic analysis of 92 up-to-date bacterial core gene and matrix-assisted laser desorption/ionization-time-of-flight MS biotyper data confirmed the distinct clustering pattern of these four strains. Digital DNA-DNA hybridization (41.7 %-31.2 %) and average nucleotide identity (91.1 %-87.0 %) values relative to closest validly published Pseudomonas species were below the species delineation thresholds of 70 and 96 %, respectively. Fatty acid composition results validated the taxonomic position of the novel strains in the genus Pseudomonas. Phenotypic characteristics from carbon utilization tests differentiated the novel strains from closely related Pseudomonas species. In silico prediction of secondary metabolite biosynthesis gene clusters in the whole-genome sequences of the four strains revealed the presence of 11 clusters involved in the production of siderophore, redox-cofactor, betalactone, terpene, arylpolyene and nonribosomal peptides. Based on phenotypic and genotypic data, strains S1Bt3, S1Bt7, S1Bt30 and S1Bt42T represent a novel species for which the name Pseudomonas quebecensis sp. nov. is proposed. The type strain is S1Bt42T (=DOAB 746T=LMG 32141T=CECT 30251T). The genomic DNA G+C content is 60.95 mol%.

Phylogenomics-based reclassifications in the genus Psychrobacter including emended descriptions of Psychrobacter pacificensis, Psychrobacter proteolyticus and Psychrobacter submarinus.

The taxonomic status of 43 Psychrobacter species was examined based upon the genome sequences of their type strains. Three groups of type strains were found to be conspecific, Psychrobacter salsus Shivaji et al. (Syst Appl Microbiol 27:628-635, 2004. 10.1078/0723202042369956) and Psychrobacter submarinus Romanenko et al. (Int J Syst Evol Microbiol 52:1291-1297, 2002. 10.1099/00207713-52-4-1291); Psychrobacter oceani Matsuyama et al. (Int J Syst Evol Microbiol 65:1450-1455, 2015. 10.1099/ijs.0.000118) and Psychrobacter pacificensis Maruyama et al. (Int J Syst Evol Microbiol 50:835-846, 2000. 10.1099/00207713-50-2-835); and Psychrobacter proteolyticus Denner et al. (Syst Appl Microbiol 24:44-53, 2001. 10.1078/0723-2020-00006), Psychrobacter marincola Romanenko et al. (Int J Syst Evol Microbiol 52:1291-1297, 2002. 10.1099/00207713-52-4-1291) and Psychrobacter adeliensis Shivaji et al. (Syst Appl Microbiol 27:628-635, 2004. 10.1078/0723202042369956). For all three groups, the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values are > 97.69% and > 80.2%, respectively. This conclusion is supported by similarities in morphology, growth properties, and fatty acid compositions. Based on this evidence, we propose the reclassification of Psychrobacter salsus Shivaji et al. (Syst Appl Microbiol 27:628-635, 2004. 10.1078/0723202042369956) as a later heterotypic synonym of Psychrobacter submarinus Romanenko et al. (Int J Syst Evol Microbiol 52:1291-1297, 2002. 10.1099/00207713-52-4-1291); Psychrobacter oceani Matsuyama et al. (Int J Syst Evol Microbiol 65:1450-1455, 2015. 10.1099/ijs.0.000118) as a later heterotypic synonym of Psychrobacter pacificensis Maruyama et al. (Int J Syst Evol Microbiol 50:835-846, 2000. 10.1099/00207713-50-2-835), and Psychrobacter marincola Romanenko et al. (Int J Syst Evol Microbiol 52:1291-1297, 2002. 10.1099/00207713-52-4-1291) and Psychrobacter adeliensis Shivaji et al. (Syst Appl Microbiol 27:628-635, 2004. 10.1078/0723202042369956) as later heterotypic synonyms of Psychrobacter proteolyticus Denner et al. (Syst Appl Microbiol 24:44-53, 2001. 10.1078/0723-2020-00006).

Description of Flavobacterium agricola sp. nov., an auxin producing bacterium isolated from paddy field.

An auxin-producing bacterial strain, CC-SYL302T, was isolated from paddy soil in Taiwan and identified using a polyphasic taxonomic approach. The cells were observed to be aerobic, non-motile, non-spore-forming rods, and tested positive for catalase and oxidase. Produced carotenoid but flexirubin-type pigments were absent. Optimal growth of strain CC-SYL302T was observed at 25 °C, pH 7.0, and with 2% (w/v) NaCl present. Based on analysis of 16S rRNA gene sequences, it was determined that strain CC-SYL302T belongs to the genus Flavobacterium of the Flavobacteriaceae family. The closest known relatives of this strain are F. tangerinum YIM 102701-2 T (with 93.3% similarity) and F. cucumis R2A45-3 T (with 93.1% similarity). Digital DNA-DNA hybridization (dDDH) values were calculated to assess the genetic distance between strain CC-SYL302T and its closest relatives, with mean values of 21.3% for F. tangerinum and 20.4% for F. cucumis. Strain CC-SYL302T exhibited the highest orthologous average nucleotide identity (OrthoANI) values with members of the Flavobacterium genus, ranging from 67.2 to 72.1% (n = 22). The dominating cellular fatty acids (> 5%) included iso-C14:0, iso-C15:0, iso-C16:0, iso-C15:0 3-OH, iso-C17:0 3-OH, C16:1 ω6c/C16:1 ω7c and C16:0 10-methyl/iso-C17:1 ω9c. The polar lipid profile consisted of phosphatidylethanolamine, an unidentified aminolipid, an unidentified aminophospholipid, and nine unidentified polar lipids. The genome (2.7 Mb) contained 33.6% GC content, and the major polyamines were putrescine and sym-homospermidine. Strain CC-SYL302T exhibits distinct phylogenetic, phenotypic, and chemotaxonomic characteristics, as well as unique results in comparative analysis of 16S rRNA gene sequence, OrthoANI, dDDH, and phylogenomic placement. Therefore, it is proposed that this strain represents a new species of the Flavobacterium genus, for which the name Flavobacterium agricola sp. nov. is proposed. The type strain is CC-SYL302T (= BCRC 81320 T = JCM 34764 T).

Phylogenomic Insights on the Xanthomonas translucens Complex, and Development of a TaqMan Real-Time Assay for Specific Detection of pv. translucens on Barley.

The reemergence and spread of Xanthomonas translucens, the causal agent of bacterial leaf streak in cereal crops and wilt in turfgrass and forage species, is a concern to growers in the United States and Canada. The pathogen is seedborne and listed as an A2 quarantine organism by EPPO, making it a major constraint to international trade and exchange of germplasm. The pathovar concept of the X. translucens group is confusing due to overlapping of plant host ranges and specificity. Here, comparative genomics, phylogenomics, and 81 up-to-date bacterial core gene set (ubcg2) were used to assign the pathovars of X. translucens into three genetically and taxonomically distinct clusters. The study also showed that whole genome-based digital DNA-DNA hybridization unambiguously can differentiate the pvs. translucens and undulosa. Orthologous gene and proteome matrix analyses suggest that the cluster consisting of graminis, poae, arrhenatheri, phlei, and phleipratensis is very divergent. Whole-genome data were exploited to develop the first pathovar-specific TaqMan real-time PCR tool for detection of pv. translucens on barley. Specificity of the TaqMan assay was validated using 62 Xanthomonas and non-Xanthomonas strains as well as growth chamber-inoculated and naturally infected barley leaves. Sensitivity levels of 0.1 pg (purified DNA) and 23 CFUs per reaction (direct culture) compared favorably with other previously reported real-time PCR assays. The phylogenomics data reported here suggest that the clusters could constitute novel taxonomic units or new species. Finally, the pathovar-specific diagnostic tool will have significant benefits to growers and facilitate international exchange of barley germplasm and trade.

First Report of Bacterial Leaf Streak of Barley (Hordeum vulgare) Caused by Xanthomonas translucens pv. translucens in British Columbia, Canada.

Bacterial leaf streak (BLS) of barley is caused by the Gram-negative bacterial pathogen Xanthomonas translucens (Sapkota et al. 2020). In 2021, we observed multiple hill plots with BLS symptomatic plants in a barley stripe rust nursery in Vancouver, BC, Canada. We collected 29 leaf samples showing typical BLS symptoms (e.g. necrotic lesions; Fig. S1) and stored at 4 oC until bacterial isolation. Samples were surface-sterilized in 10% NaOCl for 20 sec and rinsed twice. About 1 cm2 of leaf tissue containing BLS characteristic lesions was macerated in 200 µL sterile H2O on a petri dish, incubated for 15 min, and 10 µl of the homogenates was streaked onto Wilbrink's - Boric Acid - Cephalexin (WBC) agar medium. Plates were incubated at 28-30 oC for 48 hrs. Four single colonies were obtained: BC10-1-2a (USask BC10-2a), BC10-1-2b (USask BC10-2b), UBC026 and UBC028. Colonies were grown in WBC broth and gDNA was extracted using E.Z.N.A. Bacterial DNA Kit (Omega Bio-Tek) or DNeasy Plant Pro Kit® (Qiagen) following manufacturer protocols. Genus-level identification was achieved using 16S rRNA sequencing with 27F/1492R primers (Lane 1991) of UBC026 (1,399 bp; NCBI # OP327375) and UBC028 (1,415 bp; NCBI #OP327376). Complete 16S rRNA sequences (1,533bp) of BC10-2a and BC10-2b (1,533 bp) were extracted from the draft whole-genome sequences (WGS) generated in this study. The 16S rRNA sequence homology values of 99.0-100% were recorded between the 4 strains. BLAST analyses of the 16S rRNA sequences to GenBank entries exhibited 99.5-100% similarity values (100% coverage) with the pathotype strains of Xtt DSM 18974T (LT604072) and X. translucens pv. undulosa (Xtu) CFBP 2055 (CP074361). Whole genomes of BC10-2a (JANUQY01) and BC10-2b (JANUQZ01) were sequenced (150-bp; reads 33.1 million; mean coverage 2125x) using NovaSeq Illumina, assembled (Unicycler v0.4.8; Wick et al. 2017) and analyzed to identify the strains to the species-level (Tambong et al. 2021). WGS of strains USask BC10-2a and USask BC10-2b exhibited genome-based DNA-DNA hybridization (dDDH; Meier-Kolthoff et al. 2013) and BLAST-based average nucleotide identity (ANIb; Richter et al. 2015) of 100%. The two strains also showed dDDH and ANIb of 90.4% (species-leel cut-off of 70%) and 98.780% and 98.80% (cut-off of 96%), respectively, with Xtt DSM 18974T (LT604072). In contrast, the WGS of BC10-2a and BC10-2b exhibited only 78.2% dDDH homology values with Xtu CFBP 2055T, suggesting that the strains are genetically more similar to Xtt. The assignment of these strains to Xtt is corroborated by phylogenomic analysis (Fig. S2; Meier-Kolthoff and Göker 2019) that showed the two strains clustering together (100% bootstrap) with the type strain DSM 18974T. These data suggest that these strains are taxonomically members of Xtt. Identification was also confirmed to the genus-level by LAMP assay using published X. translucens primers (Langlois et al. 2017). Pathovar-level identification was confirmed using a cbsA and S8.pep multiplex PCR diagnostic assay (Roman-Reyna et al. 2022). Koch's postulates were verified by greenhouse inoculation via leaf infiltration of UBC026 and UBC028 on 21-day old barley plants (line HB522) using an inoculum of 108 CFU ml-1 followed by re-isolation of the bacteria on WBC. The inoculated plants showed typical BLS symptoms similar to those observed in the field (Fig. S1). Water-inoculated plants had no symptoms. To our knowledge, this is the first published report of BLS of barley in British Columbia.

Campylobacter majalis sp. nov. and Campylobacter suis sp. nov., novel Campylobacter species isolated from porcine gastrointestinal mucosa.

Strains LMG 7974T and LMG 8286T represent single, novel Campylobacter lineages with Campylobacter pinnipediorum and Campylobacter mucosalis as nearest phylogenomic neighbours, respectively. The results of average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) analyses of LMG 7974T, LMG 8286T and type strains of species of the genus Campylobacter confirmed that these strains represent novel species of the genus Campylobacter. The 16S rRNA gene sequences of both strains showed highest identity towards C. mucosalis (97.84 and 98.74 %, respectively). Strains LMG 7974T and LMG 8286T shared 72.5 and 73.7% ANI, respectively, with their nearest phylogenomic neighbours and less than 21 % dDDH. The draft genome sizes of LMG 7974T and LMG 8286T are 1 945429 bp and 1 708214 bp in length with percentage DNA G+C contents of 33.8 and 37.2 %, respectively. Anomalous biochemical characteristics and low MALDI-TOF mass spectrometry log scores supported their designation as representing novel species of the genus Campylobacte. We therefore propose to classify strain LMG 7974T (=CCUG 20705T) as the type strain of the novel species Campylobacter majalis sp. nov. and strain LMG 8286T (=CCUG 24193T, NCTC 11879T) as the type strain of the novel species Campylobacter suis sp. nov.