Joong-Su Kim

Application of rpoB and Zinc Protease Gene for Use in Molecular Discrimination of Fusobacterium nucleatum Subspecies

ABSTRACT Fusobacterium nucleatum is classified into five subspecies that inhabit the human oral cavity (F. nucleatum subsp. nucleatum, F. nucleatum subsp. polymorphum, F. nucleatum subsp. fusiforme, F. nucleatum subsp. vincentii, and F. nucleatum subsp. animalis) based on several phenotypic characteristics and DNA-DNA hybridization patterns. However, the methods for detecting or discriminating the clinical isolates of F. nucleatum at the subspecies levels are laborious, expensive, and time-consuming. Therefore, in this study, the nucleotide sequences of the RNA polymerase β-subunit gene (rpoB) and zinc protease gene were analyzed to discriminate the subspecies of F. nucleatum. The partial sequences of rpoB (approximately 2,419 bp), the zinc protease gene (878 bp), and 16S rRNA genes (approximately 1,500 bp) of the type strains of five subspecies, 28 clinical isolates of F. nucleatum, and 10 strains of F. periodonticum (as a control group) were determined and analyzed. The phylogenetic data showed that the rpoB and zinc protease gene sequences clearly delineated the subspecies of F. nucleatum and provided higher resolution than the 16S rRNA gene sequences in this respect. According to the phylogenetic analysis of rpoB and the zinc protease gene, F. nucleatum subsp. vincentii and F. nucleatum subsp. fusiforme might be classified into a single subspecies. Five clinical isolates could be delineated as a new subspecies of F. nucleatum. The results suggest that rpoB and the zinc protease gene are efficient targets for the discrimination and taxonomic analysis of the subspecies of F. nucleatum.

Description of Lysinibacillus sinduriensis sp. nov., and transfer of Bacillus massiliensis and Bacillus odysseyi to the genus Lysinibacillus as Lysinibacillus massiliensis comb. nov. and Lysinibacillus odysseyi comb. nov. with emended description of the genus Lysinibacillus.

A Gram-positive, rod-shaped, endospore-forming bacterium, designated strain BLB-1(T), was isolated from samples of tidal flat sediment from the Yellow Sea. 16S rRNA gene sequence analysis demonstrated that the isolate belonged to the Bacillus rRNA group 2 and was closely related to Bacillus massiliensis CIP 108446(T) (97.4%), Bacillus odysseyi ATCC PTA-4993(T) (96.7%), Lysinibacillus fusiformis DSM 2898(T) (96.2%) and Lysinibacillus boronitolerans DSM 17140(T) (95.9%). Sequence similarities with related species in other genera, including Caryophanon, Sporosarcina and Solibacillus, were <96.1%. Chemotaxonomic data supported the affiliation of strain BLB-1(T) with the genus Lysinibacillus. The major menaquinone was MK-7, the cell-wall sugars were glucose and xylose, the cell-wall peptidoglycan type was A4α (L-Lys-D-Asp), the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and several unknown phospholipids, and the major fatty acids were anteiso-C(15:0) (35.6%), iso-C(15:0) (25.6%) and anteiso-C(17:0) (16.5%). The most closely related species, Bacillus massiliensis and Bacillus odysseyi, were also assigned to this genus based on phylogenetic analysis and phenotypic data. The results of DNA-DNA hybridizations and phenotypic tests supported the differentiation of all three taxa from species of the genus Lysinibacillus with validly published names. Thus, strain BLB-1(T) ( = KCTC 13296(T)  = JCM 15800(T)) represents a novel species, for which the name Lysinibacillus sinduriensis sp. nov. is proposed. It is also proposed that Bacillus massiliensis CIP 108446(T) ( =4400831(T) = CCUG49529(T)  =KCTC 13178(T)) and Bacillus odysseyi NBRC 100172(T) ( =34hs-1(T)  =ATCC PTA-4993(T)  =NRRL B-30641(T)  =DSM 18869(T)  =CIP 108263(T)  =KCTC 3961(T)) be transferred to the genus Lysinibacillus as Lysinibacillus massiliensis comb. nov. and Lysinibacillus odysseyi comb. nov., respectively.

Bacillus manliponensis sp. nov., a new member of the Bacillus cereus group isolated from foreshore tidal flat sediment

A Gram-positive, endospore-forming, new Bacillus species, strain BL4-6T, was isolated from tidal flat sediment of the Yellow Sea. Strain BL4-6T is a straight rod, with motility by peritrichate flagella. The cell wall contains meso-diaminopimelic acid, and the major respiratory quinone is menaquinone-7. The major fatty acids are iso-C15:0 and summed feature 3 (containing C16:1 ω7c/iso-C15:0 2OH, and/or iso-C15:0 2OH/C16:1 ω7c). Cells are catalase-positive and oxidase-negative. The G+C content of the genomic DNA is 38.0 mol%. Based on a comparative 16S rRNA gene sequence analysis, the isolate belongs to the genus Bacillus, forms a clade with the Bacillus cereus group, and is closely related to Bacillus mycoides (98.5%), Bacillus cereus (98.5%), Bacillus anthracis (98.4%), Bacillus thuringiensis (98.4%), Bacillus weihenstephanensis (98.1%), and Bacillus pseudomycoides (97.5%). The isolate showed less than 85% similarity of the gyrA gene sequence and below 95% similarity of the rpoB gene sequence to the members of this group. DNA-DNA relatedness between strain BL4-6T and B. cereus group was found to be in a range of 22.8–42.3%, and thus BL4-6T represents a unique species. On the basis of these studies, strain BL4-6T (=KCTC 13319T =JCM 15802T) is proposed to represent the type strain of a novel species, Bacillus manliponensis sp. nov.

Peptoniphilus rhinitidis sp. nov., isolated from specimens of chronic rhinosinusitis

Chronic rhinosinusitis (CRS) is an inflammatory disorder of the nasal cavity and paranasal sinus related to bacterial infection. A previous study suggested that a specific bacterial group may have an important role in the course of CRS. In this study, bacteria isolated from CRS patients were characterized. A total of 15 strains were identified as Gram-positive anaerobic cocci (GPAC), which were able to utilize peptone as a sole carbon source. Sequencing of the 16S ribosomal RNA gene revealed that the isolates were closely related to members of the genus Peptoniphilus (>97% similarity) within the Clostridiales Family XI. Incertae Sedis. Genotypic and phenotypic characterization suggests that these isolates represent a novel species of the genus Peptoniphilus associated with CRS. The type strain of Peptoniphilus rhinitidis is 1-13T (=KCTC 5985T=JCM 17448T).

Bacteroides koreensis sp. nov. and Bacteroides kribbi sp. nov., two new members of the genus Bacteroides

Three bacterial isolates from human faeces, YS-aM39T, R2F3-3-3T and R2F3-5-1, were characterized as Gram-negative, strictly anaerobic, non-spore-forming, non-motile, and rod-shaped. Isolate YS-aM39T formed a distinct line of descent, showing greatest 16S rRNA gene sequence relatedness with R2F3-3-3T (97.5 %), R2F3-5-1 (97.5 %), Bacteroides ovatus (98.8 %) and Bacteroides xylanisolvens (97.2 %). Isolates R2F3-3-3T and R2F3-5-1 also formed a distinct line of descent, sharing greatest 16S rRNA gene sequence relatedness with B. ovatus (98.2 %) and B. xylanisolvens (97.2 %). The DNA G+C content of YS-aM39T was 44.8 mol%, that of R2F3-3-3T was 42.4 mol% and that of R2F3-5-1 was 42.6 mol%. The respiratory quinone of all three isolates was menaquinone MK-10. Polar lipid analysis identified phosphatidylethanolamine as the major lipid. The predominant fatty acids in all three isolates were anteiso-C15 : 0, iso-C15 : 0, C16 : 0 3-OH and iso-C17 : 0 3-OH. The major end products of glucose fermentation were acetic acid, lactic acid and formic acid. DNA-DNA hybridization data indicated that two isolates, YS-aM39T and R2F3-3-3T, represent a species distinct from B. ovatus and B. xylanisolvens. Finally, in this study, the two isolates represented two new species in the genus Bacteroides, for which we propose the names Bacteroides koreensis sp. nov. (type strain, YS-aM39T=KCTC 15520T=JCM 31393T) and Bacteroides kribbi sp. nov. (type strain, R2F3-3-3T=KCTC 15460T=JCM 31391T).