Characterization of Bifidobacterium kashiwanohense that utilizes both milk- and plant-derived oligosaccharides.

Bifidobacteria are prominent members of the human gut microbiota throughout life. The ability to utilize milk- and plant-derived carbohydrates is important for bifidobacterial colonization of the infant and adult gut. The Bifidobacterium catenulatum subspecies kashiwanohense (B. kashiwanohense) was originally isolated from infant feces. However, only a few strains have been described, and the characteristics of this subspecies have been poorly investigated. Here, we characterized genotypes and phenotypes of 23 B. kashiwanohense-associated strains, including 12 newly sequenced isolates. Genome-based analysis clarified the phylogenetic relationship between these strains, revealing that only 13 strains are genuine B. kashiwanohense. We defined specific marker sequences and investigated the worldwide prevalence of B. kashiwanohense based on metagenome data. This revealed that not only infants but also adults and weaning children harbor this subspecies in the gut. Most B. kashiwanohense strains utilize long-chain xylans and possess genes for extracellular xylanase (GH10), arabinofuranosidase and xylosidase (GH43), and ABC transporters that contribute to the utilization of xylan-derived oligosaccharides. We also confirmed that B. kashiwanohense strains utilize short- and long-chain human milk oligosaccharides and possess genes for fucosidase (GH95 and GH29) and specific ABC transporter substrate-binding proteins that contribute to the utilization of a wide range of human milk oligosaccharides. Collectively, we found that B. kashiwanohense strains utilize both plant- and milk-derived carbohydrates and identified key genetic factors that allow them to assimilate various carbohydrates.

Direct measurement of stool consistency by texture analyzer and calculation of reference value in Belgian general population.

Stool consistency is evaluated mainly in reference to indirect indicators such as water content or the appearance of stool forms using Bristol Stool Form Scale (BSFS). Methods of measurement are limited. We thus aimed to develop a simple protocol for direct measurement of stool consistency using the TA.XTExpress Texture Analyser (Stable Micro Systems Ltd.). We developed a protocol which enables mechanical quantification of the gram-force against a cylindrical probe (ø 6 mm) pushed into the stool surface at 2.0 mm/s to 5 mm depth. The consistency of 252 stools collected from 40 healthy Belgians was evaluated by the direct method and by the indirect indicators (water content and BSFS) for comparison. The log-transformed stool consistency values measured by the texture analyzer had a negative linear correlation with the stool water contents (rrm = - 0.781) with homoscedastic variance, suggesting the appropriateness of the new protocol. They showed a similar correlation with the BSFS, but with a large variance in the consistency values of normal stool forms. This correlation was much smaller for BSFS scored by subjects (rrm = - 0.587) than by experts (rrm = - 0.789), collectively indicating BSFS as a rough indicator of stool consistency susceptible to subjective bias despite its effectiveness in clinical use. The optimized direct method using the texture analyzer enables the accurate quantification of stool consistency, which facilitates understanding of the intestinal environment and function and thus may enhance the value of the stool as a predictor of human health.

Bifidobacterium jacchi sp. nov., isolated from the faeces of a baby common marmoset (Callithrix jacchus).

A novel Bifidobacterium strain, MRM 9.3T, was isolated from a faecal sample of a baby common marmoset (Callithrixjacchus). Cells were Gram-stain-positive, non-motile, non-sporulating, non-haemolytic, facultatively anaerobic and fructose 6-phosphate phosphoketolase-positive. Phylogenetic analyses based on 16S rRNA genes as well as multilocus sequences (representing hsp60, rpoB, clpC, dnaJ and dnaG genes) and the core genomes revealed that strain MRM 9.3T exhibited phylogenetic relatedness to Bifidobacterium myosotis DSM 100196T. Comparative analysis of 16S rRNA gene sequences confirmed the phylogenetic results showing the highest gene sequence identity with strain B.ifidobacterium myosotis DSM 100196T (95.6 %). The average nucleotide identity, amino acid average identity and in silico DNA-DNA hybridization values between MRM 9.3T and DSM 100196T were 79.9, 72.1 and 28.5 %, respectively. Phenotypic and genotypic features clearly showed that the strain MRM 9.3T represents a novel species, for which the name Bifidobacterium jacchi sp. nov. is proposed. The type strain is MRM 9.3T (=DSM 103362T =JCM 31788T).

Long-term colonization exceeding six years from early infancy of Bifidobacterium longum subsp. longum in human gut.

BACKGROUND: The importance of the gut microbiota at the early stage of life and their longitudinal effect on host health have recently been well investigated. In particular, Bifidobacterium longum subsp. longum, a common component of infant gut microbiota, appears in the gut shortly after birth and can be detected there throughout an individual's lifespan. However, it remains unclear whether this species colonizes in the gut over the long term from early infancy. Here, we investigated the long-term colonization of B. longum subsp. longum by comparing the genotypes of isolates obtained at different time points from individual subjects. Strains were isolated over time from the feces of 12 subjects followed from early infancy (the first six months of life) up to childhood (approximately six years of age). We also considered whether the strains were transmitted from their mothers' perinatal samples (prenatal feces and postnatal breast milk). RESULTS: Intra-species diversity of B. longum subsp. longum was observed in some subjects' fecal samples collected in early infancy and childhood, as well as in the prenatal fecal samples of their mothers. Among the highlighted strains, several were confirmed to colonize and persist in single individuals from as early as 90 days of age for more than six years; these were classified as long-term colonizers. One of the long-term colonizers was also detected from the corresponding mother's postnatal breast milk. Quantitative polymerase chain reaction data suggested that these long-term colonizers persisted in the subjects' gut despite the existence of the other predominant species of Bifidobacterium. CONCLUSIONS: Our results showed that several strains belonging to B. longum subsp. longum colonized in the human gut from early infancy through more than six years, confirming the existence of long-term colonizers from this period. Moreover, the results suggested that these strains persisted in the subjects' gut while co-existing with the other predominant bifidobacterial species. Our findings also suggested the importance of microbial-strain colonization in early infancy relative to their succession and showed the possibility that probiotics targeting infants might have longitudinal effects. TRIAL REGISTRATION: TRN: ISRCTN25216339 . Date of registration: 11/03/2016. Prospectively registered.

Bifidobacterium catulorum sp. nov., a novel taxon from the faeces of the baby common marmoset (Callithrix jacchus).

In our previous study based on hsp60 PCR-restriction fragment length polymorphism and 16S rRNA gene sequencing, we stated that the bifidobacterial strains isolated from the individual faecal samples of five baby common marmosets constituted different phylogenetically isolated groups of the genus Bifidobacterium. In that study, we also proposed that these isolated groups potentially represented novel species of the genus Bifidobacterium. Out of them, Bifidobacterium aesculapii, Bifidobacterium myosotis, Bifidobacterium tissieri and Bifidobacterium hapali, have been described recently. Another strain, designated MRM 8.19T, has been classified as member of the genus Bifidobacterium on the basis of positive results for fructose-6-phosphate phosphoketolase activity and analysis of partial 16S rRNA, hsp60, clpC, dnaJ, dnaG and rpoB gene sequences. Analysis of 16S rRNA and hsp60 gene sequences revealed that strain MRM 8.19T was related to B. tissieri DSM 100201T (95.8 %) and to Bifidobacterium bifidum ATCC 29521T (93.7 %), respectively. The DNA G+C composition was 63.7 mol% and the peptidoglycan structure was l-Orn(Lys)-l-Ser. Based on the phylogenetic, genotypic and phenotypic data reported, strain MRM 8.19T represents a novel taxon within the genus Bifidobacterium for which the name Bifidobacterium catulorum sp. nov. (type strain MRM 8.19T=DSM 103154T=JCM 31794T) is proposed.

Comprehensive analysis of the fecal microbiota of healthy Japanese adults reveals a new bacterial lineage associated with a phenotype characterized by a high frequency of bowel movements and a lean body type.

BACKGROUND: In Japan, a variety of traditional dietary habits and daily routines have developed in many regions. The effects of these behaviors, and the regional differences in the composition of the gut microbiota, are yet to be sufficiently studied. To characterize the Japanese gut microbiota and identify the factors shaping its composition, we conducted 16S metagenomics analysis of fecal samples collected from healthy Japanese adults residing in various regions of Japan. Each participant also completed a 94-question lifestyle questionnaire. RESULTS: We collected fecal samples from 516 healthy Japanese adults (325 females, 191 males; age, 21-88). Heatmap and biplot analyses based on the bacterial family composition of the fecal microbiota showed that subjects' region of residence or gender were not strongly correlated with the general composition of the fecal microbiota. Although clustering analysis for the whole cohort did not reveal any distinct clusters, two enterotype-like clusters were observed in the male, but not the female, subjects. In the whole subject population, the scores for bowel movement frequency were significantly correlated with the abundances of Christensenellaceae, Mogibacteriaceae, and Rikenellaceae in the fecal microbiota (P < 0.001). These three bacterial families were also significantly more abundant (P < 0.05 or 0.01) in lean subjects (body mass index (BMI) < 25) than in obese subjects (BMI > 30), which is consistent with previously published results. However, a previously reported correlation between BMI and bowel movement frequency was not observed. In addition, the abundances of these three families were positively correlated with each other and comprised a correlative network with 14 other bacterial families. CONCLUSIONS: The present study showed that the composition of the fecal microbiota of healthy Japanese adults at the national level was not strongly correlated with subjects' area of residence or gender. In addition, enterotype partitioning was ambiguous in this cohort of healthy Japanese adults. Finally, the results implied that the abundances of Christensenellaceae, Mogibacteriaceae, and Rikenellaceae, along with several other bacterial components that together comprised a correlative network, contributed to a phenotype characterized by a high frequency of bowel movements and a lean body type.

Bifidobacterium myosotis sp. nov., Bifidobacterium tissieri sp. nov. and Bifidobacterium hapali sp. nov., isolated from faeces of baby common marmosets (Callithrix jacchus L.).

In a previous study on bifidobacterial distribution in New World monkeys, six strains belonging to the Bifidobacteriaceae were isolated from faecal samples of baby common marmosets (Callithrix jacchus L.). All the isolates were Gram-positive-staining, anaerobic, asporogenous and fructose-6-phosphate phosphoketolase-positive. Comparative analysis of 16S rRNA gene sequences revealed relatively low levels of similarity (maximum identity 96 %) to members of the genus Bifidobacterium, and placed the isolates in three independent clusters: strains of cluster I (MRM_5.9T and MRM_5.10) and cluster III (MRM_5.18T and MRM_9.02) respectively showed 96.4 and 96.7 % 16S rRNA gene sequence similarity to Bifidobacterium callitrichos DSM 23973T, while strains of cluster II (MRM_8.14T and MRM_9.14) showed 95.4 % similarity to Bifidobacterium stellenboschense DSM 23968T. Phylogenetic analysis of partial hsp60 and clpC gene sequences supported an independent phylogenetic position of each cluster from each other and from the related type strains B. callitrichos DSM 23973T and B. stellenboschense DSM 23968T. Clusters I, II and III respectively showed DNA G+C contents of 64.9-65.1, 56.4-56.7 and 63.1-63.7 mol%. The major cellular fatty acids of MRM_5.9T were C14 : 0, C16 : 0 and C18 : 1ω9c dimethylacetal, while C16 : 0 was prominent in strains MRM_5.18T and MRM_8.14T, followed by C18 : 1ω9c and C14 : 0. Biochemical profiles and growth parameters were recorded for all the isolates. Based on the data provided, the clusters represent three novel species, for which the names Bifidobacterium myosotis sp. nov. (type strain MRM_5.9T = DSM 100196T = JCM 30796T), Bifidobacterium hapali sp. nov. (type strain MRM_8.14T = DSM 100202T = JCM 30799T) and Bifidobacterium tissieri sp. nov. (type strain MRM_5.18T = DSM 100201T = JCM 30798T) are proposed.

Subspeciation of Bifidobacterium longum by multilocus approaches and amplified fragment length polymorphism: Description of B. longum subsp. suillum subsp. nov., isolated from the faeces of piglets.

The species Bifidobacterium longum is currently divided into three subspecies, B. longum subsp. longum, B. longum subsp. infantis and B. longum subsp. suis. This classification was based on an assessment of accumulated information on the species' phenotypic and genotypic features. The three subspecies of B. longum were investigated using genotypic identification [amplified-fragment length polymorphism (AFLP), multilocus sequence analysis (MLSA) and multilocus sequence typing (MLST)]. By using the AFLP and the MLSA methods, we allocated 25 strains of B. longum into three major clusters corresponding to the three subspecies; the cluster comprising the strains of B. longum subsp. suis was further divided into two subclusters differentiable by the ability to produce urease. By using the MLST method, the 25 strains of B. longum were divided into eight groups: four major groups corresponding to the results obtained by AFLP and MLSA, plus four minor disparate groups. The results of AFLP, MLSA and MLST analyses were consistent and revealed a novel subspeciation of B. longum, which comprised three known subspecies and a novel subspecies of urease-negative B. longum, for which the name B. longum subsp. suillum subsp. nov. is proposed, with type strain Su 851(T)=DSM 28597(T)=JCM 19995(T).

Isolation and identification of cultivable Bifidobacterium spp. from the faeces of 5 baby common marmosets (Callithrix jacchus L.).

Ninety-two bifidobacterial strains were obtained from the faeces of 5 baby common marmosets, three known species Bifidobacterium aesculapii, Bifidobacterium callithricos and Bifidobacterium reuteri and 4 novel putative bifidobacterial species were retrieved. The occurrence of bifidobacteria in non-human primate babies is described for the first time.

Pyrosequencing analysis of the microbial diversity of airag, khoormog and tarag, traditional fermented dairy products of mongolia.

Here, we used pyrosequencing to obtain a detailed analysis of the microbial diversities of traditional fermented dairy products of Mongolia. From 22 Airag (fermented mare's milk), 5 Khoormog (fermented camel's milk) and 26 Tarag (fermented milk of cows, goats and yaks) samples collected in the Mongolian provinces of Arhangai, Bulgan, Dundgobi, Tov, Uburhangai and Umnugobi, we obtained a total of 81 operational taxonomic units, which were assigned to 15 families, 21 genera and 41 species in 3 phyla. The genus Lactobacillus is a core bacterial component of Mongolian fermented milks, and Lactobacillus helveticus, Lactobacillus kefiranofaciens and Lactobacillus delbrueckii were the predominant species of lactic acid bacteria (LAB) in the Airag, Khoormog and Tarag samples, respectively. By using this pyrosequencing approach, we successfully detected most LAB species that have been isolated as well as seven LAB species that have not been found in our previous culture-based study. A subsequent analysis of the principal components of the samples revealed that L. delbrueckii, L. helveticus, L. kefiranofaciens and Streptococcus thermophilus were the main factors influencing the microbial diversity of these Mongolian traditional fermented dairy products and that this diversity correlated with the animal species from which the milk was sourced.

Lactobacillus saniviri sp. nov. and Lactobacillus senioris sp. nov., isolated from human faeces.

Two Gram-stain-positive strains, YIT 12363(T) and YIT 12364(T), were isolated from human faeces. They were rod-shaped, non-motile, asporogenous, facultatively anaerobic and did not exhibit catalase activity. Comparative analyses of 16S rRNA, pheS and rpoA gene sequences demonstrated that the novel strains were members of the genus Lactobacillus. On the basis of 16S rRNA gene sequence similarity, the type strains of Lactobacillus casei (95.3% similarity), Lactobacillus paracasei subsp. paracasei (95.6%), Lactobacillus paracasei subsp. tolerans (95.3%) and Lactobacillus rhamnosus (95.4%) were the closest neighbours to strain YIT 12363(T). For strain YIT 12364(T), the highest similarity values were observed with the type strains of Lactobacillus diolivorans, Lactobacillus parafarraginis and Lactobacillus rapi (95.8, 96.0 and 96.0%, respectively). Phenotypic and genotypic features demonstrated that these strains each represent a separate novel species of the genus Lactobacillus, and the names Lactobacillus saniviri sp. nov. (type strain YIT 12363(T)=JCM 17471(T)=DSM 24301(T)) and Lactobacillus senioris sp. nov. (type strain YIT 12364(T)=JCM 17472(T)=DSM 24302(T)), respectively, are proposed.

Lactobacillus delbrueckii subsp. sunkii subsp. nov., isolated from sunki, a traditional Japanese pickle.

Although four strains of bacteria isolated from sunki, a traditional Japanese, non-salted pickle, were initially identified as Lactobacillus delbrueckii, the molecular and phenotypic characteristics of the strains did not match those of any of the four recognized subspecies of L. delbrueckii. Together, the results of phenotypic characterization, DNA-DNA hybridizations (in which the relatedness values between the novel strains and type strains of the recognized subspecies of L. delbrueckii were all >88.7%) and 16S rRNA gene sequence, amplified fragment length polymorphism (AFLP) and whole-cell MALDI-TOF/MS spectral pattern analyses indicated that the four novel strains represented a single, novel subspecies, for which the name Lactobacillus delbrueckii subsp. sunkii subsp. nov. is proposed. The type strain is YIT 11221(T) (=JCM 17838(T) =DSM 24966(T)).

Lactic acid bacteria isolated from ethnic preserved meat products of the Western Himalayas.

We used culture- and molecular-biology-based methods to investigate the diversity of lactic acid bacteria (LAB) in the ethnic chevon (goat) meat products chartayshya, jamma and arjia of the Western Himalayas. In six chartayshya, six jamma and four arjia samples, LAB were the predominant microbial component involved in the fermentation of these samples, and the total LAB population in arjia (7.8 ± 0.1 log cfu g(-1); mean ± SD) was significantly higher (P < 0.05) than in chartayshya (6.9 ± 0.1 log cfu g(-1)) and jamma (7.5 ± 0.1 log cfu g(-1)). We identified 53 LAB samples by 16S rRNA and phenylalanyl-tRNA synthase (pheS) genes sequencing. The LAB isolates were identified as Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus hirae, Leuconostoc citreum, Leuconostoc mesenteroides, Pediococcus pentosaceus, and Weissella cibaria. These results revealed that there is a high level of diversity of LAB in the Himalayan ethnic preserved meat products.

The role of urease activity on biofilm formation by Staphylococcus sp. T-02 isolated from the toilet bowl.

Urolith, which consists of dirty yellow-colored attachments on the toilet bowl, is associated with a variety of odorous chemicals, including ammonia, and causes disadvantages in daily life. Although largely it is derived from microorganisms, little is known about the microbial processes underlying the formation of urolith. In order to gain insight into the types and the activities of microorganisms present in urolith, culturable bacteria were isolated, identified, and physiologically characterized. One of the isolates exhibited higher ability to produce ammonia when it was grown in artificial urine medium. Phylogenetic and physiological analyses indicated that this strain (T-02) belonged to a new group of Staphylococcus species, showing combined phenotypes as between S. lentus and S. xylosus. T-02 exhibited high urease activity and was capable of growing in the urinary condition by forming robust biofilms. The results of this study indicate that T-02 has successfully adapted itself to the environment of urolith.