Isolation and identification of Wickerhamiella tropicalis from blood culture by MALDI-MS.

Wickerhamiella is a genus of budding yeast that is mainly isolated from environmental samples, and 40 species have been detected. The yeast isolated from human clinical samples usually only contain three species: W. infanticola, W. pararugosa and W. sorbophila. In this study, we isolated W. tropicalis from a blood sample of a six-year-old female with a history of B-cell precursor lymphoblastic leukemia in Japan in 2022. Though the strain was morphologically identified as Candida species by routine microbiological examinations, it was subsequently identified as W. tropicalis by sequencing the internal transcribed spacer (ITS) of ribosomal DNA (rDNA). The isolate had amino acid substitutions in ERG11 and FKS1 associated with azole and echinocandin resistance, respectively, in Candida species and showed intermediate-resistant to fluconazole and micafungin. The patient was successfully treated with micafungin. Furthermore, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) detected three novel peaks that are specific for W. tropicalis, indicating that MALDI-MS analysis is useful for rapid detection of Wickerhamiella species in routine microbiological examinations.

A large-scale genomically predicted protein mass database enables rapid and broad-spectrum identification of bacterial and archaeal isolates by mass spectrometry.

MALDI-TOF MS-based microbial identification relies on reference spectral libraries, which limits the screening of diverse isolates, including uncultured lineages. We present a new strategy for broad-spectrum identification of bacterial and archaeal isolates by MALDI-TOF MS using a large-scale database of protein masses predicted from nearly 200,000 publicly available genomes. We verify the ability of the database to identify microorganisms at the species level and below, achieving correct identification for > 90% of measured spectra. We further demonstrate its utility by identifying uncultured strains from mouse feces with metagenomics, allowing the identification of new strains by customizing the database with metagenome-assembled genomes.

Complete genome sequences of Ruminococcus torques strains JCM 36208 and JCM 36209, isolated from the feces of a healthy Japanese male.

Here, we report the complete genome sequences of two Ruminococcus torques strains (JCM 36208 and JCM 36209) that were newly isolated from the feces of a healthy Japanese male. Both genomes consist of a single circular chromosome with a length of ~2.8 Mbp and a G+C content of 41.8%.

Complete genome sequence of Butyricimonas faecihominis JCM 18676T.

We report a complete genome sequence of Butyricimonas faecihominis JCM 18676T, generated by nanopore sequencing. The genome consists of a single circular chromosome of 4,851,806 bp, with a G + C content of 42.9%, and was predicted to contain 15 rRNA and 61 tRNA genes and encode for 3,946 proteins.

Elucidating colorectal cancer-associated bacteria through profiling of minimally perturbed tissue-associated microbiota.

Sequencing-based interrogation of gut microbiota is a valuable approach for detecting microbes associated with colorectal cancer (CRC); however, such studies are often confounded by the effect of bowel preparation. In this study, we evaluated the viability of identifying CRC-associated mucosal bacteria through centimeter-scale profiling of the microbiota in tumors and adjacent noncancerous tissue from eleven patients who underwent colonic resection without preoperative bowel preparation. High-throughput 16S rRNA gene sequencing revealed that differences between on- and off-tumor microbiota varied considerably among patients. For some patients, phylotypes affiliated with genera previously implicated in colorectal carcinogenesis, as well as genera with less well-understood roles in CRC, were enriched in tumor tissue, whereas for other patients, on- and off-tumor microbiota were very similar. Notably, the enrichment of phylotypes in tumor-associated mucosa was highly localized and no longer apparent even a few centimeters away from the tumor. Through short-term liquid culturing and metagenomics, we further generated more than one-hundred metagenome-assembled genomes, several representing bacteria that were enriched in on-tumor samples. This is one of the first studies to analyze largely unperturbed mucosal microbiota in tissue samples from the resected colons of unprepped CRC patients. Future studies with larger cohorts are expected to clarify the causes and consequences of the observed variability in the emergence of tumor-localized microbiota among patients.

Complete genome sequence of Blautia luti JCM 17040T.

We generated a complete genome sequence of the type strain of Blautia luti (JCM 17040T = DSM 14534T) by Nanopore sequencing. The genome consists of a circular chromosome of 3,741,599 bp with a G + C content of 42.9% and was predicted to contain 3,431 protein-coding sequences.

Complete Genome Sequence of Coprobacter fastidiosus JCM 33896T.

We generated a complete genome sequence of Coprobacter fastidiosus JCM 33896T by nanopore sequencing. The genome consists of a single circular chromosome of 3,444,538 bp with a G+C content of 38.4%. Annotation predicted 15 rRNA genes, 67 tRNA genes and 2,662 protein-coding sequences.

Complete Genome Sequence of Anaerostipes hadrus JCM 17467T.

We report a complete genome sequence of Anaerostipes hadrus JCM 17467T. The genome consists of a circular chromosome of 2,804,089 bp, with a G+C content of 37.3%. The genome was predicted to contain 21 rRNA genes, 65 tRNA genes, and 2,675 protein-coding sequences.

Characterisation of bacteria representing a novel Nitrosomonas clade: Physiology, genomics and distribution of missing ammonia oxidizer.

Members of the genus Nitrosomonas are major ammonia oxidizers that catalyse the first step of nitrification in various ecosystems. To date, six subgenus-level clades have been identified. We have previously isolated novel ammonia oxidizers from an additional clade (unclassified cluster 1) of the genus Nitrosomonas. In this study, we report unique physiological and genomic properties of the strain PY1, compared with representative ammonia-oxidising bacteria (AOB). The apparent half-saturation constant for total ammonia nitrogen and maximum velocity of strain PY1 were 57.9 ± 4.8 µM NH3 + NH4 + and 18.5 ± 1.8 µmol N (mg protein)-1 h-1 , respectively. Phylogenetic analysis based on genomic information revealed that strain PY1 belongs to a novel clade of the Nitrosomonas genus. Although PY1 contained genes to withstand oxidative stress, cell growth of PY1 required catalase to scavenge hydrogen peroxide. Environmental distribution analysis revealed that the novel clade containing PY1-like sequences is predominant in oligotrophic freshwater. Taken together, the strain PY1 had a longer generation time, higher yield and required reactive oxygen species (ROS) scavengers to oxidize ammonia, compared with known AOB. These findings expand our knowledge of the ecophysiology and genomic diversity of ammonia-oxidising Nitrosomonas.

Characterization and Demonstration of Mock Communities as Control Reagents for Accurate Human Microbiome Community Measurements.

Standardization and quality assurance of microbiome community analysis by high-throughput DNA sequencing require widely accessible and well-characterized reference materials. Here, we report on newly developed DNA and whole-cell mock communities to serve as control reagents for human gut microbiota measurements by shotgun metagenomics and 16S rRNA gene amplicon sequencing. The mock communities were formulated as near-even blends of up to 20 bacterial species prevalent in the human gut, span a wide range of genomic guanine-cytosine (GC) contents, and include multiple strains with Gram-positive type cell walls. Through a collaborative study, we carefully characterized the mock communities by shotgun metagenomics, using previously developed standardized protocols for DNA extraction and sequencing library construction. Further, we validated fitness of the mock communities for revealing technically meaningful differences among protocols for DNA extraction and metagenome/16S rRNA gene amplicon library construction. Finally, we used the mock communities to reveal varying performance of metagenome-based taxonomic profilers and the impact of trimming and filtering of sequencing reads on observed species profiles. The latter showed that aggressive preprocessing of reads may result in substantial GC-dependent bias and should thus be carefully evaluated to minimize unintended effects on species abundances. Taken together, the mock communities are expected to support a myriad of applications that rely on well-characterized control reagents, ranging from evaluation and optimization of methods to assessment of reproducibility in interlaboratory studies and routine quality control. IMPORTANCE Application of high-throughput DNA sequencing has greatly accelerated human microbiome research and its translation into new therapeutic and diagnostic capabilities. Microbiome community analyses results can, however, vary considerably across studies or laboratories, and establishment of measurement standards to improve accuracy and reproducibility has become a priority. The here-developed mock communities, which are available from the NITE Biological Resource Center (NBRC) at the National Institute of Technology and Evaluation (NITE, Japan), provide well-characterized control reagents that allow users to judge the accuracy of their measurement results. Widespread and consistent adoption of the mock communities will improve reproducibility and comparability of microbiome community analyses, thereby supporting and accelerating human microbiome research and development.

Sulphur metabolism in colon cancer tissues: a case report and literature review.

Sulphur-containing compounds have been linked to colorectal cancer by factors such as the presence of methyl mercaptan in intestinal gas and long-term dietary intake associated with sulphur-metabolizing microbiota. Therefore, this current case report hypothesized that active sulphur metabolism in colorectal cancer results in the formation of sulphur compounds in the intestine and, thus, examined sulphur metabolites possibly associated with sulphur respiration in colon cancer tissues. The patient was a 73-year-old female that underwent laparoscopic right hemicolectomy for ascending colon cancer. During the surgery, colon cancer tissues and normal intestinal mucosa samples were collected. After optimizing the sample concentrations for homogenization (pre-treatment), the samples were stabilized using a hydroxyphenyl-containing derivative and the relevant metabolites were quantified using liquid chromatography with tandem mass spectrometry. The results showed that cysteine persulfide and cysteine trisulfide levels were higher in colon cancer tissues than in normal mucosal tissues. Thus, sulphur metabolism, possibly sulphur respiration, is enhanced in colon cancer tissues.

Multi-Omics Analysis to Generate Hypotheses for Mild Health Problems in Monkeys.

Certain symptoms associated with mild sickness and lethargy have not been categorized as definitive diseases. Confirming such symptoms in captive monkeys (Macaca fascicularis, known as cynomolgus monkeys) can be difficult; however, it is possible to observe and analyze their feces. In this study, we investigated the relationship between stool state and various omics data by considering objective and quantitative values of stool water content as a phenotype for analysis. By examining the food intake of the monkeys and assessing their stool, urine, and plasma, we attempted to obtain a comprehensive understanding of the health status of individual monkeys and correlate it with the stool condition. Our metabolomics data strongly suggested that many lipid-related metabolites were correlated with the stool water content. The lipidomic analysis revealed the involvement of saturated and oxidized fatty acids, metallomics revealed the contribution of selenium (a bio-essential trace element), and intestinal microbiota analysis revealed the association of several bacterial species with the stool water content. Based on our results, we hypothesize that the redox imbalance causes minor health problems. However, it is not possible to make a definite conclusion using multi-omics alone, and other hypotheses could be proposed.

Sequence-Specific Capture of Oligonucleotide Probes (SCOPE): a Simple and Rapid Microbial rRNA Quantification Method Using a Molecular Weight Cutoff Membrane.

A method named sequence-specific capture of oligonucleotide probes (SCOPE) was developed for quantification of microbial rRNA molecules in a multiplex manner. In this method, a molecular weight cutoff membrane (MWCOM) was used for the separation of fluorescence-labeled oligonucleotide probes hybridized with rRNA from free unhybridized probes. To demonstrate proof of concept, probes targeting bacteria or archaea at different taxonomic levels were prepared and were hybridized with rRNAs. The hybridization stringency was controlled by adjusting reaction temperature and urea concentration in the mixture. Then, the mixture was filtered through the MWCOM. The rRNA and hybridized probes collected on the MWCOM were recovered and quantified using a spectrophotometer and fluorospectrometer, respectively. The method showed high accuracy in detecting specific microbial rRNA in a defined nucleic acid mixture. Furthermore, the method was capable of simultaneous detection and quantification of multiple target rRNAs in a sample with sensitivity up to a single-base mismatch. The SCOPE method was tested and benchmarked against reverse transcription-quantitative PCR (RT-qPCR) for the quantification of Bacteria, Archaea, and some key methanogens in anaerobic sludge samples. It was observed that the SCOPE method produced more reliable and coherent results. Thus, the SCOPE method allows simple and rapid detection and quantification of target microbial rRNAs for environmental microbial population analysis without any need for enzymatic reactions. IMPORTANCE Microorganisms play integral roles in the Earth's ecosystem. Microbial populations and their activities significantly affect the global nutrient cycles. Quantification of key microorganisms provides important information that is required to understand their roles in the environment. Sequence-based analysis of microbial population is a powerful tool, but it provides information only on relative abundance of microorganisms. Hence, the development of a simpler and quick method for the quantification of microorganisms is necessary. To address the shortcomings of a variety of molecular methods reported so far, we developed a simple, rapid, accurate, and multiplexed microbial rRNA quantification method to evaluate the abundance of specific microbial populations in complex ecosystems. This method demonstrated high specificity, reproducibility, and applicability to such samples. The method is useful for quantitative detection of particular microbial members in the environment.

Validation and standardization of DNA extraction and library construction methods for metagenomics-based human fecal microbiome measurements.

BACKGROUND: Validation and standardization of methodologies for microbial community measurements by high-throughput sequencing are needed to support human microbiome research and its industrialization. This study set out to establish standards-based solutions to improve the accuracy and reproducibility of metagenomics-based microbiome profiling of human fecal samples. RESULTS: In the first phase, we performed a head-to-head comparison of a wide range of protocols for DNA extraction and sequencing library construction using defined mock communities, to identify performant protocols and pinpoint sources of inaccuracy in quantification. In the second phase, we validated performant protocols with respect to their variability of measurement results within a single laboratory (that is, intermediate precision) as well as interlaboratory transferability and reproducibility through an industry-based collaborative study. We further ascertained the performance of our recommended protocols in the context of a community-wide interlaboratory study (that is, the MOSAIC Standards Challenge). Finally, we defined performance metrics to provide best practice guidance for improving measurement consistency across methods and laboratories. CONCLUSIONS: The validated protocols and methodological guidance for DNA extraction and library construction provided in this study expand current best practices for metagenomic analyses of human fecal microbiota. Uptake of our protocols and guidelines will improve the accuracy and comparability of metagenomics-based studies of the human microbiome, thereby facilitating development and commercialization of human microbiome-based products. Video Abstract.

Genomic and Physiological Characteristics of a Novel Nitrite-Oxidizing Nitrospira Strain Isolated From a Drinking Water Treatment Plant.

Nitrite-oxidizing bacteria (NOB) catalyze the second step of nitrification, which is an important process of the biogeochemical nitrogen cycle and is exploited extensively as a biological nitrogen removal process. Members of the genus Nitrospira are often identified as the dominant NOB in a diverse range of natural and artificial environments. Additionally, a number of studies examining the distribution, abundance, and characterization of complete ammonia oxidation (comammox) Nitrospira support the ecological importance of the genus Nitrospira. However, niche differentiation between nitrite-oxidizing Nitrospira and comammox Nitrospira remains unknown due to a lack of pure cultures. In this study, we report the isolation, physiology, and genome of a novel nitrite-oxidizing Nitrospira strain isolated from a fixed-bed column at a drinking water treatment plant. Continuous feeding of ammonia led to the enrichment of Nitrospira-like cells, as well as members of ammonia-oxidizing genus Nitrosomonas. Subsequently, a microcolony sorting technique was used to isolate a novel nitrite-oxidizing Nitrospira strain. Sequences of strains showing the growth of microcolonies in microtiter plates were checked. Consequently, the most abundant operational taxonomic unit (OTU) exhibited high sequence similarity with Nitrospira japonica (98%) at the 16S rRNA gene level. The two other Nitrospira OTUs shared over 99% sequence similarities with N. japonica and Nitrospira sp. strain GC86. Only one strain identified as Nitrospira was successfully subcultivated and designated as Nitrospira sp. strain KM1 with high sequence similarity with N. japonica (98%). The half saturation constant for nitrite and the maximum nitrite oxidation rate of strain KM1 were orders of magnitude lower than the published data of other known Nitrospira strains; moreover, strain KM1 was more sensitive to free ammonia compared with previously isolated Nitrospira strains. Therefore, the new Nitrospira strain appears to be better adapted to oligotrophic environments compared with other known non-marine nitrite oxidizers. The complete genome of strain KM1 was 4,509,223 bp in length and contained 4,318 predicted coding sequences. Average nucleotide identities between strain KM1 and known cultured Nitrospira genome sequences are 76.7-78.4%, suggesting at least species-level novelty of the strain in the Nitrospira lineage II. These findings broaden knowledge of the ecophysiological diversity of nitrite-oxidizing Nitrospira.

Complete Genome Sequence of Flavonifractor plautii JCM 32125T.

We report the complete genome sequence of Flavonifractor plautii JCM 32125T (=VPI 0310T). The genome consists of a single circular chromosome of 3,985,392 bp (G+C content, 60.9%) and was predicted to contain 3 complete sets of rRNA genes, 63 tRNA genes, and 3,764 protein-coding sequences.

Complete Genome Sequence of Blautia producta JCM 1471T.

We report a complete genome sequence of Blautia producta JCM 1471T The genome consists of a single circular chromosome of 6,197,116 bp with a G+C content of 45.7%. The genome was annotated as containing 5 complete sets of rRNA genes, 70 tRNA genes, and 5,516 protein-coding sequences.

Physiological and genomic characterization of a new 'Candidatus Nitrotoga' isolate.

Oxidation of nitrite to nitrate is an important process in the global nitrogen cycle. Recent molecular biology-based studies have revealed that the widespread nitrite-oxidizing bacteria (NOB) belonging to the genus 'Candidatus Nitrotoga' may be highly important for the environment. However, the insufficient availability of pure Nitrotoga cultures has limited our understanding of their physiological and genomic characteristics. Here, we isolated the 'Ca. Nitrotoga' sp. strain AM1P, from a previously enriched Nitrotoga culture, using an improved isolation strategy. Although 'Ca. Nitrotoga' have been recognized as cold-adapted NOB, the strain AM1P had a slightly higher optimum growth temperature at 23°C. Strain AM1P showed a pH optimum of 8.3 and was not inhibited even at high nitrite concentrations (20 mM). We obtained the complete genome of the strain and compared the genome profile to five previously sequenced 'Ca. Nitrotoga' strains. Comparative genomics suggested that lactate dehydrogenase may be only encoded in the strain AM1P and closely related genomes. While the growth yield of AM1P did not change, we observed faster growth in the presence of lactate in comparison to purely chemolithoautotrophic growth. The characterization of the new strain AM1P sheds light on the physiological adaptation of this environmentally important, but understudied genus 'Ca. Nitrotoga'.

Complete Genome Sequence of Collinsella aerofaciens JCM 10188T.

We report a complete genome sequence of Collinsella aerofaciens JCM 10188T (=VPI 1003T). The genome consists of a circular chromosome (2,428,218 bp with 60.6% G+C content) and two extrachromosomal elements. The genome was predicted to contain 5 sets of rRNA genes, 58 tRNA genes, and 2,079 protein-encoding sequences.

Complete Genome Sequence of Megamonas funiformis JCM 14723T.

We announce the complete genome sequence of Megamonas funiformis JCM 14723T (YIT 11815T). The genome consists of a circular chromosome (2,522,577 bp, 31.5% G+C content) and a plasmid of 46,189 bp (29.4% G+C content). The genome was predicted to contain 6 rRNA operons, 53 tRNA genes, and 2,440 protein-coding sequences.