Maki Kitahara

Molecular Analysis of Fecal Microbiota in Elderly Individuals Using 16S rDNA Library and T‐RFLP

Fecal microbiota in six elderly individuals were characterized by the 16S rDNA libraries and terminal restriction fragment length polymorphism (T‐RFLP) analysis. Random clones of 16S rRNA gene sequences were isolated after PCR amplification with universal primer sets from total genomic DNA extracted from feces of three elderly individuals. These clones were partially sequenced (about 500 bp). T‐RFLP analysis was performed using 16S rDNA amplified from six subjects. The lengths of the terminal restriction fragment (T‐RF) were analyzed after digestion by HhaI and MspI. Among 240 clones obtained, approximately 46% belonged to 27 known species. About 54% of the other clones were 56 novel “phylotypes” (at least 98% homology of clone sequence). These libraries included 83 species or phylotypes. In addition, about 13% (30 phylotypes) of these phylotypes were newly discovered in these libraries. A large number of species that are not yet known exist in the feces of elderly individuals. 16S rDNA libraries and T‐RFLP analysis revealed that the majority of bacteria were Bacteroides and relatives, Clostridium rRNA cluster IV, IX, Clostridium rRNA subcluster XIVa, and “Gammaproteobacteria”. The proportion of Clostridium rRNA subcluster XIVa was lower than in healthy adults. In addition, although Ruminococcus obeum and its closely related phylotypes were detected in high frequency in healthy young subjects, hardly any were detected in our elderly individuals. “Gammaproteobacteria” were detected at high frequency.

Unification of Bifidobacterium infantis and Bifidobacterium suis as Bifidobacterium longum.

The relationships between Bifidobacterium infantis, Bifidobacterium longum and Bifidobacterium suis were examined by means of carbohydrate fermentation, DNA-DNA hybridization, ribotyping and random amplified polymorphic DNA-PCR (RAPD-PCR). The levels of DNA-DNA hybridization among the strains of B. infantis, B. longum and B. suis used in this study were 67-81% under optimal conditions (42 degrees C) and 63-85% under stringent conditions (52 degrees C). Although the strains showed varied carbohydrate-fermentation patterns, the three species were divided into three types, namely the infantis type, the longum type and the suis type, by ribotyping and RAPD-PCR. On the basis of these results, strains of B. infantis, B. longum and B. suis were recognized as distinct groups within a single species. It is concluded that B. infantis and B. suis should be unified as B. longum, the latter species being divided into three biotypes, the infantis type, the longum type and the suis type, by molecular methods.

Clostridium hiranonis sp. nov., a human intestinal bacterium with bile acid 7alpha-dehydroxylating activity

The Clostridium-like organisms TO-931T and HD-17, isolated from human faeces, have high levels of bile acid 7alpha-dehydroxylating activity. Sequencing of their 16S rDNA demonstrated that they belong to cluster XI of the genus Clostridium and that they represent a new and distinct line of descent. Clostridium bifermentans and Clostridium sordellii in cluster XI also possess bile acid 7alpha-dehydroxylating activity. DNA-DNA hybridization experiments with the isolates, TO-931T and HD-17, and C bifermentans and C. sordellii revealed that the isolates are a single species distinct from C. bifermentans and C sordellii. On the basis of phylogenetic analysis, using 16S rDNA sequences, and DNA-DNA hybridization analysis, it is concluded that strains TO-931T and HD-17 are members of a new species of the genus Clostridium, for which the name Clostridium hiranonis is proposed. The type strain is strain TO-931T (= JCM 10541T = DSM 13275T).

Slackia equolifaciens sp. nov., a human intestinal bacterium capable of producing equol

An equol-producing bacterium, strain DZE(T), which was isolated from human faeces, was characterized by morphological, biochemical and molecular methods. The isolate was Gram-positive, obligately anaerobic, non-spore-forming, asaccharolytic and rod-shaped. 16S rRNA gene sequence analysis showed 92.8, 91.0, 91.1 and 90.6% similarities with Slackia faecicanis, Slackia exigua, Slackia heliotrinireducens and Slackia isoflavoniconvertens, respectively. Based on these data, we propose a novel species of the genus Slackia, Slackia equolifaciens sp. nov. The major cellular fatty acids are C14:0, C18:1omega9c and C18:1omega9c DMA (dimethyl acetal). The DNA G+C content of the strain is 60.8 mol%. The type strain of S. equolifaciens sp. nov. is DZET (=JCM 16059T =CCUG 58231T).

Assignment of Eubacterium sp. VPI 12708 and related strains with high bile acid 7alpha-dehydroxylating activity to Clostridium scindens and proposal of Clostridium hylemonae sp. nov., isolated from human faeces.

Unknown Eubacterium-like organisms VPI 12708 and five strains (Y-1113, I-10, M-18, TH-82 and 36S) had high bile acid 7alpha-dehydroxylating activity; the unknown Clostridium-like organisms TN-271T and TN-272 also had the same activity. Analysis of their 16S rDNA sequences demonstrated that all strains belong to cluster XIVa of the genus Clostridium (Collins et al., 1994). Strain VPI 12708 and five other strains (Y-1113, I-10, M-18, TH-82 and 36S) formed a single cluster and strains TN-271T and TN-272 formed another single cluster. Clostridium scindens JCM 6567T was the most closely related species for two clusters in the phylogenetic tree. Values for DNA-DNA similarities among C. scindens JCM 6567T, strain VPI 12708 and the other five strains were greater than 70%, showing that these micro-organisms were a single species. Therefore, we identified strain VPI 12708 and the five other strains as C. scindens. In addition, DNA-DNA similarities among C. scindens JCM 6567T, strain TN-271T and strain N-272 revealed that strains TN-271T and TN-272 were distinct from C. scindens JCM 6567T. On the basis of phylogenetic analysis and DNA-DNA similarity data, it was concluded that strains TN-271T and TN-272 are members of a new species of the genus Clostridium, for which the name Clostridium hylemonae is proposed. The type strain is strain TN-271T (= JCM 10539T).

Lactobacillus florum sp. nov., a fructophilic species isolated from flowers.

Three strains of fructophilic lactic acid bacteria were isolated from flowers in South Africa. The isolates formed a subcluster in the Lactobacillus buchneri phylogenetic group, closely related to Lactobacillus fructivorans, Lactobacillus homohiochii, Lactobacillus lindneri and Lactobacillus sanfranciscensis according to phylogenetic analysis based on the 16S rRNA gene sequences. Levels of DNA-DNA relatedness indicated that the three strains belonged to the same taxon and formed a genetically distinct group, well separated from their phylogenetic relatives. The three strains produced acids from only two of the 49 carbohydrates tested, i.e. D-glucose and D-fructose. D-Fructose was more rapidly fermented than D-glucose. Good growth was recorded on d-fructose or D-glucose in the presence of external electron acceptors. However, delayed growth was recorded on d-glucose without electron acceptors. The novel strains produced lactic acid, ethanol and acetic acid from D-glucose at a ratio of 1 : 0.8 : 0.2. These characteristics were distinct from other species of the genus Lactobacillus. Based on the data provided, the three isolates represent a fructophilic and novel species of the genus Lactobacillus, for which the name Lactobacillus florum sp. nov. is proposed. The type strain is F9-1(T) (=JCM 16035(T)=DSM 22689(T)=NRIC 0771(T)).

Genotypic and phenotypic characterization of lactic acid bacteria isolated from Italian ryegrass silage

Twenty-three lactic acid bacteria (LAB) isolated from three cultivars (Akiaoba, Nagahahikari and Tachiwase) of Italian ryegrass (Lolium multiflorum Lam.) silage were precisely characterized by a combination of phenotypic tests, genotypic 16S ribosomal DNA sequencing and rapid PCR-based analyses, focusing on their useful phenotypes for silage preparation as inoculants. We successfully identified both at the species and subspecies levels: phenotypically novel Lactococcus lactis subsp. lactis, Lactobacillus brevis, Lactobacillus coryniformis subsp. torquens, Lactobacillus curvatus, Lactobacillus plantarum subsp. plantarum, Lactobacillus sakei subsp. carnosus, Leuconostoc mesenteroides subsp. dextranicum and Pediococcus parvulus. This is the first report to elucidate the presence of Lactobacillus coryniformis ssp. torquens and Leuconostoc mesenteroides subsp. dextranicum in Italian ryegrass silages. Physiological and biochemical tests revealed that phenotypic characteristics are different among the different strains of the same species and subspecies, and that the isolates show unique and diverse phenotypes related to fermentation factors, such as available carbohydrates, optimal growth pH and temperature. These results suggest that, for various well-preserved silage preparations, the isolates may be useful in producing novel inoculants corresponding to their optimally climatic and ecological niches.

Lactococcus fujiensis sp. nov., a lactic acid bacterium isolated from vegetable matter

Three strains of lactic acid bacteria, designated NJ 317(T), NJ 414 and NJ 415, were isolated from the outer leaves of Chinese cabbages (Brassica rapa L. var. glabra Regel) and characterized taxonomically. The strains were gram-reaction-positive, catalase-negative, facultatively anaerobic cocci that did not produce gas from glucose and formed L-lactic acid. The major fatty acids were C(18 : 1)ω9c, C(16 : 0), C(14 : 0) and summed feature 10. Morphological, physiological and phylogenetic data indicated that the strains belonged to the genus Lactococcus. These strains shared similar phenotypic characteristics and exhibited DNA relatedness values >96.6 % to each other, indicating that they represent a single species. The DNA G+C contents of the three strains were 42.1-42.5 mol%. 16S rRNA gene sequences of the novel strains were determined and aligned with those of other species of the genus Lactococcus. On the basis of phylogenetic analysis the three strains grouped with other members of the genus Lactococcus. Lactococcus lactis and Lactococcus garvieae were the most closely related species, sharing a sequence similarity value of 94.4 % with the three strains. Ribotyping patterns, however, revealed that these strains were well-separated from reference strains of species of the genus Lactococcus and DNA-DNA hybridization studies indicated that the novel strains had low levels (<20.2 %) of DNA relatedness with reference strains of L. lactis, L. garvieae and other type strains of previously described species, showing that they represent a different species. Based on this evidence, strains NJ 317(T), NJ 414 and NJ 415 represent a novel species of the genus Lactococcus, for which the name Lactococcus fujiensis sp. nov. is proposed. The type strain is NJ 317(T) ( = JCM16395(T)  = CGMCC 1.10453(T)).

Weissella oryzae sp. nov., isolated from fermented rice grains.

A taxonomic study was conducted on two Gram-reaction-positive, catalase-negative, irregular short-rod-shaped or coccoid lactic acid bacteria, designated strains SG25(T) and SG23, that were isolated from grains of fermented Japanese rice (Oryza sativa L. subsp. japonica). A phylogenetic analysis based on 16S rRNA gene sequence data clearly showed that the strains belonged to the genus Weissella and were most closely related to Weissella soli LMG 20113(T) (with a sequence similarity of 96.9 % for each novel strain). The peptidoglycan of each strain contained the amino acids glutamic acid, lysine, serine and alanine in a molar ratio of 1.0 : 1.2 : 0.5 : 3.0, respectively. On the basis of the unusual phenotypic characteristics of the novel strains and the low levels of DNA-DNA relatedness recorded between each novel strain and Weissella soli JCM 12536(T), strains SG25(T) and SG23 represent a single novel species in the genus Weissella, for which the name Weissella oryzae sp. nov. is proposed. The type strain is SG25(T) ( = JCM 18191(T)  = DSM 25784(T)).

Lactobacillus nasuensis sp. nov., a lactic acid bacterium isolated from silage, and emended description of the genus Lactobacillus.

Two strains of lactic acid bacteria, designated SU 18(T) and SU 83, were isolated from silage prepared with Sudan grass [Sorghum sudanense (Piper) Stapf.]. The isolates were Gram-stain-positive, catalase-negative, facultatively anaerobic rods that did not produce gas from glucose. The isolates exhibited ≥93.5 % DNA-DNA relatedness to each other and shared the same phenotypic characteristics, which indicated that they belonged to a single species. The DNA G+C content was 58.5-59.2 mol%. On the basis of 16S rRNA gene sequence analysis, the isolates were placed in the genus Lactobacillus. Their closest phylogenetic neighbours were Lactobacillus manihotivorans JCM 12514(T) and Lactobacillus camelliae JCM 13995(T) (95.9 and 96.8 % 16S rRNA gene sequence similarity, respectively, with strain SU 18(T)). Ribotyping revealed that strain SU 18(T) was well separated from L. manihotivorans JCM 12514(T) and L. camelliae JCM 13995(T). Strain SU 18(T) exhibited ≤23.7 % DNA-DNA relatedness with its closest phylogenetic neighbours. The isolates represent a novel species in the genus Lactobacillus, for which the name Lactobacillus nasuensis sp. nov. is proposed. The type strain is SU 18(T) ( = JCM 17158(T)  = CGMCC 1.10801(T)). The description of the genus Lactobacillus is also amended.