Flavobacterium ammonificans sp. nov. and Flavobacterium ammoniigenes sp. nov., ammonifying bacteria isolated from surface river water.

Three aerobic, Gram-stain-negative, non-motile, rod-shaped bacteria, designated as strains SHINM13T, GENT5T and GENT11 were isolated from surface river water (Saitama Prefecture, Japan). SHINM13T and GENT11 were positive for catalase, whereas GENT5T was negative. Phylogenetic analyses based on the 16S rRNA gene (1341 bp) or 40 marker gene (34,513 bp) sequences revealed that the strains formed distinct phylogenetic lineages within the genus Flavobacterium. The three strains shared 99.3-99.6 % 16S rRNA gene sequence similarity among each other. The average nucleotide identity by orthology (OrthoANI) and digital DNA-DNA hybridization (dDDH) values between strains SHINM13T and GENT11 were 96.56 and 82.1 %, respectively, and those between SHINM13T and GENT5T were 83.46 % and 52.9 %, respectively. The major cellular fatty acids were C15 : 1ω6c, iso-C15 : 0, iso-C15 : 1G, anteiso-C15 : 0 and iso-C15 : 0 3-OH. The major polar lipid was phosphatidylethanolamine. SHINM13T and GENT5T contained menaquinone-6 (MK-6) as the predominant respiratory quinone, and their DNA G+C contents were 34.4 and 35.1 mol%, respectively. Genome sequencing of the three isolates revealed a genome size of 2.26-2.40 Mbp. Furthermore, all three isolates converted dissolved organic nitrogen to ammonium during cell growth. On the basis of the results of phenotypic and phylogenetic analyses, strains SHINM13T and GENT11 and GENT5T represent two distinct novel species in the genus Flavobacterium, for which the names Flavobacterium ammonificans sp. nov. (type strain SHINM13T =JCM 34684T =NCIMB 15379T) and Flavobacterium ammoniigenes sp. nov. (type strain GENT5T =JCM 32249T=NCIMB 15380T) are proposed.

Morbidity and mortality in antiphospholipid syndrome based on cluster analysis: a 10-year longitudinal cohort study.

OBJECTIVE: Using cluster analysis, to identify the subgroup of patients with APS with the poorest prognosis and clarify the characteristics of that subgroup. METHODS: This is a longitudinal retrospective cohort study of APS patients. Using clinical data and the profile of aPL, cluster analysis was performed to classify the patients into subgroups. Events were defined as thrombosis, severe bleeding, and mortality. RESULTS: A total of 168 patients with APS were included. Cluster analysis classified the patients into three subgroups; Cluster A (n = 61): secondary APS, Cluster B (n = 56): accumulation of cardiovascular risks and arterial thrombosis, Cluster C (n = 61): triple positivity of aPL and venous thrombosis. Cluster B showed significantly higher frequency of the events and higher mortality compared with the other clusters (P = 0.0112 for B vs A and P = 0.0471 for B vs C). CONCLUSION: Using cluster analysis, we clarified the characteristics of the APS patients with the poorest prognosis. Risk factors for cardiovascular disease may further increase events in patients with APS.

Cutting Edge: Role of MASP-3 in the Physiological Activation of Factor D of the Alternative Complement Pathway.

The complement system, a part of the innate immune system, can be activated via three different pathways. In the alternative pathway, a factor D (FD) plays essential roles in both the initiation and the amplification loop and circulates as an active form. Mannose-binding lectin-associated serine proteases (MASPs) are key enzymes of the lectin pathway, and MASP-1 and/or MASP-3 are reported to be involved in the activation of FD. In the current study, we generated mice monospecifically deficient for MASP-1 or MASP-3 and found that the sera of the MASP-1-deficient mice lacked lectin pathway activity, but those of the MASP-3-deficient mice lacked alternative pathway activity with a zymogen FD. Furthermore, the results indicate that MASP-3 but not MASP-1 activates the zymogen FD under physiological conditions and MASP-3 circulates predominantly as an active form. Therefore, our study illustrates that, in mice, MASP-3 orchestrates the overall complement reaction through the activation of FD.

Low C4 as a risk factor for severe neuropsychiatric flare in patients with systemic lupus erythematosus.

OBJECTIVE: This study aimed to explore the risk factors for 'severe' neuropsychiatric (NP) flare in patients with systemic lupus erythematosus (SLE). METHODS: This retrospective study comprised newly diagnosed 184 adult SLE patients who visited Hokkaido University Hospital between 2006 and 2017. In this study, severe NP flare was defined as the occurrence of at least one newly developed British Isles Lupus Assessment Group A score in the neurological domain. Overall severe NP flare-free survival was estimated by Kaplan-Meier analysis. Clinical and demographic profiles at SLE diagnosis were assessed as potential risk items in the adjusted multivariate Cox regression model. RESULTS: The median follow-up period was 7.9 years (interquartile range (IQR) 4.6-12.3) years. A total of 28 (15.2%) patients had one or more severe NP flares during the observation period. The median time from patient enrolment date to severe NP flare occurrence was 3.1 years (IQR 0.9-6.3 year). The 2- and 10-year severe NP flare-free survival rates were 92.7% and 86.0%, respectively. Among the manifestations of severe NP flare, psychosis was the most frequent (19.1%). In the multivariate model, low serum levels of C4 (hazard ratio (HR) = 3.67, p = 0.013) and severe NP manifestations at SLE diagnosis (HR = 7.11, p < 0.001) emerged as independent risk factors for developing severe NP flare. CONCLUSION: The first severe NP flare presented early in the course of SLE. Low C4 level and severe NP manifestations at SLE diagnosis could predict the development of severe NP flare.

Fluviibacter phosphoraccumulans gen. nov., sp. nov., a polyphosphate-accumulating bacterium of Fluviibacteraceae fam. nov., isolated from surface river water.

Three aerobic, Gram-stain-negative, non-motile, rod-shaped bacteria, designated as strains SHINM1T, ICHIJ1 and ICHIAU1, were isolated from surface river water (Saitama Prefecture, Japan). Phylogenetic analyses based on 16S rRNA and 40 marker gene sequences revealed that the strains formed a distinct phylogenetic lineage within the order Rhodocyclales. The three strains shared 100 % 16S rRNA gene similarity. Growth occurred at 15-30 °C and pH 6.0-9.5, but not in the presence of ≥1.0 % (w/v) NaCl. The isolates stained positive for intracellular polyphosphate granules. The major cellular fatty acids were C16 : 0, summed feature 2 (C12 : 1 aldehyde and/or iso-C16 : 1 I and/or C14 : 0 3-OH), summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) and summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c). The major polar lipids were phosphatidylethanolamine and an unidentified phospholipid. The predominant quinone system of strain SHINM1T was ubiquinone-8 and its DNA G+C content was 56.7 mol%. Genome sequencing of the three isolates revealed a genome size of 2.29-2.43 Mbp and average nucleotide identity by orthology values of ≥98.9 %. Based on the results of phenotypic and phylogenetic analyses, strains SHINM1T, ICHIJ1 and ICHIAU1 represent a novel species of a new genus, for which the name Fluviibacter phosphoraccumulans gen. nov., sp. nov. is proposed, within a new family, Fluviibacteraceae fam. nov. of the order Rhodocyclales. The type strain is SHINM1T (=JCM 32071T=NCIMB 15105T).

Alistipes communis sp. nov., Alistipes dispar sp. nov. and Alistipes onderdonkii subsp. vulgaris subsp. nov., isolated from human faeces, and creation of Alistipes onderdonkii subsp. onderdonkii subsp. nov.

Three groups of Gram-stain-negative, obligately anaerobic, rod or coccoid-shaped bacteria, which were phylogenetically assigned in the genus Alistipes belonging to the family Rikenellaceae in the phylum Bacteroidetes, were isolated from the faecal samples of healthy Japanese humans. Group I (strains 5CBH24T and 6CPBBH3) showed highest 16S rRNA gene sequence similarity to 'Alistipes obesi' ph8T (99.73 %). Group II (strain 5CPEGH6T) was related to Alistipes shahii WAL 8301T (96.82 %). Ten strains of group III (3BBH6T, 5CPYCFAH4, 5NYCFAH2 and others) were related to Alistipes onderdonkii DSM 19147T (98.96 %). Group I could be differentiated from other strains by the ability to hydrolyse aesculin and the lack of catalase activity. Strain 5CPEGH6T could be differentiated from A. shahii JCM 16773T by the inability to hydrolyse aesculin and the lack of catalase activity, and so on. Phenotypic characteristics of group III were similar to those of A. onderdonkii JCM 16771T. Strains 5CBH24T, 6CPBBH3 and 'A. obesi' ph8T shared 98.8-98.9 % average nucleotide identity (ANI) with each other. In addition, the in silico DNA-DNA hybridization (DDH) values among three strains were 86.7-89.4 %. Strain 5CPEGH6T showed relatively low values (≤ 84.4 % for ANI ; ≤26.2 % for DDH) with other strains. Three strains in the group III (3BBH6T, 5CPYCFAH4 and 5NYCFAH2) shared 97.9-99.9% ANI with each other. These three strains showed 96.9-97.3 % ANI with A. onderdonkii DSM 19147T. The DDH values of strains 3BBH6T, 5CPYCFAH4 and 5NYCFAH2 among themselves were 80.5-99.8 %, while those compared to A. onderdonkii DSM 19147T were 71.0-73.4 %. On the basis of the collected data, three novel species, Alistipes communis sp. nov. (5CBH24T=JCM 32850T=DSM 108979T), Alistipes dispar sp. nov. (5CPEGH6T=JCM 32848T=DSM 108978T) and Alistipes onderdonkii subsp. vulgaris subsp. nov. (3BBH6T=JCM 32839T=DSM 108977T), are proposed.

[Analysis of human microbiome using NGS.]

Recently, next generation sequencers(NGS)became prevalent and enable us possible to comprehensively analyze the entire human microbiome community structures including difficult-to-culture microbes. In this review, we introduce the NGS-based analytical methods of human microbiome, called "meta-16S analysis" and "metagenomic analysis", then show several fundamental data basing these analyses. Furthermore, we also introduce the importance of database improvement used for analysis and the DNA preparation method from human samples.

Complete genome sequences of Bifidobacterium faecale strain JCM19861T, isolated from human feces.

Here, we report the complete genome sequence of the Bifidobacterium faecale strain JCM 19861T (= CU 3-7T = KACC 17904T), isolated from infant feces by Jung-Hye Choi's group in 2014. The B. faecale JCM 19861T genome comprised a circular chromosome of 2,213,206 bp, with a G + C content of 59.0%.

Dimension reduction of microbiome data linked Bifidobacterium and Prevotella to allergic rhinitis.

Dimension reduction has been used to visualise the distribution of multidimensional microbiome data, but the composite variables calculated by the dimension reduction methods have not been widely used to investigate the relationship of the human gut microbiome with lifestyle and disease. In the present study, we applied several dimension reduction methods, including principal component analysis, principal coordinate analysis (PCoA), non-metric multidimensional scaling (NMDS), and non-negative matrix factorization, to a microbiome dataset from 186 subjects with symptoms of  allergic rhinitis (AR) and 106 controls. All the dimension reduction methods supported that the distribution of microbial data points appeared to be continuous rather than discrete. Comparison of the composite variables calculated from the different dimension reduction methods showed that the characteristics of the composite variables differed depending on the distance matrices and the dimension reduction methods. The first composite variables calculated from PCoA and NMDS with the UniFrac distance were strongly associated with AR (FDR adjusted P = 2.4 × 10-4 for PCoA and P = 2.8 × 10-4 for NMDS), and also with the relative abundance of Bifidobacterium and Prevotella. The abundance of Bifidobacterium was also linked to intake of several nutrients, including carbohydrate, saturated fat, and alcohol via composite variables. Notably, the association between the composite variables and AR was much stronger than the association between the relative abundance of individual genera and AR. Our results highlight the usefulness of the dimension reduction methods for investigating the association of microbial composition with lifestyle and disease in clinical research.

A case of tick-borne Yezo virus infection: Concurrent detection in the patient and tick.

A 76-year-old woman infected with Yezo virus (YEZV) developed liver dysfunction and thrombocytopenia following a tick bite. Despite the severity of her elevated liver enzymes and reduced platelet counts, the patient's condition improved spontaneously without any specific treatment. To our knowledge, this represents the first documented case where the YEZV genome was detected simultaneously in a patient's serum and the tick (Ixodes persulcatus) that bit the patient. This dual detection not only supports the hypothesis that YEZV is a tick-borne pathogen but also underscores the importance of awareness and diagnostic readiness for emerging tick-borne diseases, particularly in regions where these ticks are prevalent.

Complete Genome Sequences of Three Limnohabitans sp. (Lhab-A3) Strains, INBF002, TEGF004, and MORI2, Isolated from Two Lakes and a River in Japan.

Freshwater bacterioplankton of the genus Limnohabitans represent a dominant group that has worldwide distribution. Here, we report the complete genome sequences of three Limnohabitans sp. (Lhab-A3 tribe) strains, i.e., INBF002, TEGF004, and MORI2, which were isolated from surface water samples from two shallow eutrophic lakes and a river in Japan.

Complete Genome Sequences of Polynucleobacter sp. Subcluster PnecA Strains SHI2 and SHI8, Isolated from an Oligotrophic-Dystrophic Lake in Japan.

Members of the genus Polynucleobacter belonging to the subcluster PnecA comprise freshwater bacterioplankton with worldwide distribution. Here, we report the complete genome sequences of two Polynucleobacter sp. strains (PnecA), SHI2 and SHI8, isolated from the surface water of an oligotrophic-dystrophic lake in a humid continental climate in Japan.

Complete Genome Sequence of Aurantimicrobium sp. Strain INA4, Isolated from an Oligotrophic Lake in Japan.

The globally distributed freshwater bacterioplankton of the genus Aurantimicrobium belong to the tribe Luna2. Here, we report the complete genome sequence of Aurantimicrobium sp. strain INA4, which was isolated from an oligotrophic lake surface water in Japan.

Complete Genome Sequences of Three Polynucleobacter sp. Subcluster PnecC Strains, KF022, KF023, and KF032, Isolated from a Shallow Eutrophic Lake and a River in Japan.

The genus Polynucleobacter subcluster PnecC consists of bacteria representing the ubiquitous taxon of freshwater bacterioplankton. Here, we report the complete genome sequences of three Polynucleobacter sp. (PnecC) strains, namely, KF022, KF023, and KF032, which were isolated from surface water of a temperate shallow eutrophic lake and its inflow river in Japan.

A comprehensive microbial analysis of pediatric patients with acute appendicitis.

BACKGROUND: Pathogenesis of pediatric acute appendicitis (AA) is yet to be elucidated. Therefore, we performed a comprehensive microbial analysis of saliva, feces, and appendiceal lumen of AA patients using 16S ribosomal RNA (rRNA) gene amplicon sequencing to elucidate the pathogenesis of pediatric AA. METHODS: This study included 33 AA patients and 17 healthy controls (HCs) aged <15 y. Among the AA patients, 18 had simple appendicitis, and 15 had complicated appendicitis. Salivary and fecal samples were obtained from both groups. The contents of the appendiceal lumen were collected from the AA group. All samples were analyzed using 16S rRNA gene amplicon sequencing. RESULTS: The relative abundance of Fusobacterium was significantly higher in the saliva of AA patients as compared to that in HCs (P = 0.011). Bacteroides, Escherichia, Fusobacterium, Coprobacillus, and Flavonifractor were significantly increased in the feces of AA patients, as compared to that in HCs (P = 0.020, 0.010, 0.029, 0.031, and 0.002, respectively). In the appendiceal lumen, Bacteroides, Parvimonas, Fusobacterium, and Alloprevotella were the top bacterial genera with an average relative abundance >5% (16.0%, 9.1%, 7.9%, and 6.0%, respectively). CONCLUSIONS: The relative abundance of Fusobacterium was high in the appendiceal lumen of pediatric AA patients. Moreover, the relative abundance of Fusobacterium was significantly higher in the saliva and feces of pediatric AA patients than in those of healthy children. These results suggest that ectopic colonization of oral Fusobacterium in the appendix might play an important role in the pathogenesis of pediatric AA.

Recovery of microbial DNA by agar-containing solution from extremely low-biomass specimens including skin.

Recovering a sufficient amount of microbial DNA from extremely low-biomass specimens, such as human skin, to investigate the community structure of the microbiome remains challenging. We developed a sampling solution containing agar to increase the abundance of recovered microbial DNA. Quantitative PCR targeting the 16S rRNA gene revealed a significant increase in the amount of microbial DNA recovered from the developed sampling solution compared with conventional solutions from extremely low-biomass skin sites such as the volar forearm and antecubital fossa. In addition, we confirmed that the developed sampling solution reduces the contamination rate of probable non-skin microbes compared to the conventional solutions, indicating that the enhanced recovery of microbial DNA was accompanied by a reduced relative abundance of contaminating microbes in the 16S rRNA gene amplicon sequencing data. In addition, agar was added to each step of the DNA extraction process, which improved the DNA extraction efficiency as a co-precipitant. Enzymatic lysis with agar yielded more microbial DNA than conventional kits, indicating that this method is effective for analyzing microbiomes of low-biomass specimens.

Strain-level detection of Fusobacterium nucleatum in colorectal cancer specimens by targeting the CRISPR-Cas region.

IMPORTANCE: Fusobacterium nucleatum is one of the predominant oral bacteria in humans. However, this bacterium is enriched in colorectal cancer (CRC) tissues and may be involved in CRC development. Our previous research suggested that F. nucleatum is present in CRC tissues originating from the oral cavity using a traditional strain-typing method [arbitrarily primed polymerase chain reaction (AP-PCR)]. First, using whole-genome sequencing, this study confirmed an exemplary similarity between the oral and tumoral strains derived from each patient with CRC. Second, we successfully developed a method to genotype this bacterium at the strain level, targeting the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated system, which is hypervariable (defined as F. nucleatum-strain genotyping PCR). This method can identify F. nucleatum strains in cryopreserved samples and is significantly superior to traditional AP-PCR, which can only be performed on isolates. The new methods have great potential for application in etiological studies of F. nucleatum in CRC.

Complete Genome Sequences of Two Flavobacterium ammonificans Strains and a Flavobacterium ammoniigenes Strain of Ammonifying Bacterioplankton Isolated from Surface River Water.

Flavobacterium ammonificans and Flavobacterium ammoniigenes are ammonifying freshwater bacterioplankton. Here, we report the complete genome sequences of two F. ammonificans strains (SHINM13T and GENT11) and one F. ammoniigenes strain (GENT5T) that were isolated from surface river water in Japan.

Whole-Genome Sequence of Sediminibacterium sp. Strain TEGAF015, Isolated from a Shallow Eutrophic Freshwater Lake in Japan.

The genus Sediminibacterium comprises bacteria representing the ubiquitous taxa of freshwater bacterioplankton. Here, we report the whole-genome sequence of Sediminibacterium sp. strain TEGAF015, isolated from a shallow eutrophic freshwater lake in Japan.

Complete Genome Sequence of Aquiluna sp. Strain KACHI24, Isolated from River Surface Water.

The globally distributed bacterioplankton of the genus Aquiluna belong to the tribe Luna1-A1. Here, we report the complete genome sequence of Aquiluna sp. strain KACHI24, which was isolated from river surface water in Japan.