Average Nucleotide Identity Analysis Research Articles

Methanomethylophilus alvi gen. nov., sp. nov., a Novel Hydrogenotrophic Methyl-Reducing Methanogenic Archaea of the Order Methanomassiliicoccales Isolated from the Human Gut and Proposal of the Novel Family Methanomethylophilaceae fam. nov.

The methanogenic strain Mx-05T was isolated from the human fecal microbiome. A phylogenetic analysis based on the 16S rRNA gene and protein marker genes indicated that the strain is affiliated with the order Methanomassiliicoccales. It shares 86.9% 16S rRNA gene sequence identity with Methanomassiliicoccus luminyensis, the only member of this order previously isolated. The cells of Mx-05T were non-motile cocci, with a diameter range of 0.4-0.7 μm. They grew anaerobically and reduced methanol, monomethylamine, dimethylamine, and trimethylamine into methane, using H2 as an electron donor. H2/CO2, formate, ethanol, and acetate were not used as energy sources. The growth of Mx-05T required an unknown medium factor(s) provided by Eggerthella lenta and present in rumen fluid. Mx-05T grew between 30 °C and 40 °C (optimum 37 °C), over a pH range of 6.9-8.3 (optimum pH 7.5), and between 0.02 and 0.34 mol.L-1 NaCl (optimum 0.12 mol.L-1 NaCl). The genome is 1.67 Mbp with a G+C content of 55.5 mol%. Genome sequence annotation confirmed the absence of the methyl branch of the H4MPT Wood-Ljungdahl pathway, as described for other Methanomassiliicoccales members. Based on an average nucleotide identity analysis, we propose strain Mx-05T as being a novel representative of the order Methanomassiliicoccales, within the novel family Methanomethylophilaceae, for which the name Methanomethylophilus alvi gen. nov, sp. nov. is proposed. The type strain is Mx-05T (JCM 31474T).

Genomic analyses of Bacteroides fragilis: subdivisions I and II represent distinct species.

Introduction. Bacteroides fragilis is a Gram-negative anaerobe that is a member of the human gastrointestinal microbiota and is frequently found as an extra-intestinal opportunistic pathogen. B. fragilis comprises two distinct groups - divisions I and II - characterized by the presence/absence of genes [cepA and ccrA (cfiA), respectively] that confer resistance to β-lactam antibiotics by either serine or metallo-β-lactamase production. No large-scale analyses of publicly available B. fragilis sequence data have been undertaken, and the resistome of the species remains poorly defined.Hypothesis/Gap Statement. Reclassification of divisions I and II B. fragilis as two distinct species has been proposed but additional evidence is required.Aims. To investigate the genomic diversity of GenBank B. fragilis genomes and establish the prevalence of division I and II strains among publicly available B. fragilis genomes, and to generate further evidence to demonstrate that B. fragilis division I and II strains represent distinct genomospecies.Methodology. High-quality (n=377) genomes listed as B. fragilis in GenBank were included in pangenome and functional analyses. Genome data were also subject to resistome profiling using The Comprehensive Antibiotic Resistance Database.Results. Average nucleotide identity and phylogenetic analyses showed B. fragilis divisions I and II represent distinct species: B. fragilis sensu stricto (n=275 genomes) and B. fragilis A (n=102 genomes; Genome Taxonomy Database designation), respectively. Exploration of the pangenome of B. fragilis sensu stricto and B. fragilis A revealed separation of the two species at the core and accessory gene levels.Conclusion. The findings indicate that B. fragilis A, previously referred to as division II B. fragilis, is an individual species and distinct from B. fragilis sensu stricto. The B. fragilis pangenome analysis supported previous genomic, phylogenetic and resistome screening analyses collectively reinforcing that divisions I and II are two separate species. In addition, it was confirmed that differences in the accessory genes of B. fragilis divisions I and II are primarily associated with carbohydrate metabolism and suggest that differences other than antimicrobial resistance could also be used to distinguish between these two species.

Tenacibaculum bernardetii sp. nov., isolated from Atlantic salmon (Salmo salar L.) cultured in Chile.

Strain PVT-9aT, a novel Gram-stain-negative, aerobic, non-spore-forming, motile-by-gliding and rod-shaped bacterium, was isolated from a skin lesion of Atlantic salmon (Salmo salar L.) during a tenacibaculosis outbreak that occurred in 2016 at a Chilean fish farm. Phylogenetic analysis based on 16S rRNA gene sequencing confirmed that strain PVT-9aT belonged to the genus Tenacibaculum, being related to the closest type strains Tenacibaculum haliotis KCTC 52419T (98.49 % sequence similarity), Tenacibaculum aestuariivivum JDTF-79T (97.36 %), Tenacibaculum insulae JDTF-31T (97.29 %) and Tenacibaculum ovolyticum IFO 15947T (97.15 %). The genome size of strain PVT-9aT was 2.73 Mb with a DNA G+C content 31.09 mol%. Average nucleotide identity analysis among 30 Tenacibaculum species rendered the most similar strains as follows: T. haliotis KCTC 52419T (87.91 %), T. ovolyticum IFO 15947T (82.47 %), Tenacibaculum dicentrarchi 35/09T (81.08 %), Tenacibaculum finnmarkense gv finnmarkense TNO006T (80.91 %) and T. finnmarkense gv ulcerans TNO010T (80.96 %). Menaquinone MK-6 was the predominant respiratory quinone. The predominant cell fatty acids (>10 %) were iso-C15 : 0, iso-C15 : 1 G and iso-C15 : 0 3-OH. Phenotypic, chemotaxonomic and genomic data supported the assignment of strain PVT-9aT (=DSM 115155T=RGM 3472T) as representing a novel species of Tenacibaculum, for which the name Tenacibaculum bernardetii sp. nov. is proposed.

Marinobacter iranensis sp. nov., a slightly halophilic bacterium from a hypersaline lake.

A novel halophilic bacterium, strain 71-iT, was isolated from Inche-Broun hypersaline lake in Golestan province, in the north of Iran. It was a Gram-stain-negative, non-endospore forming, rod-shaped bacterium. It grew at 4-40 °C (optimum 30 °C), pH 6.0-11.0 (optimum pH 7.5) and with 0.5-15 % (w/v) NaCl [optimum 3 % (w/v) NaCl]. The results of phylogenetic analyses based on the 16S rRNA gene sequence comparison indicated its affiliation to the genus Marinobacter and the low percentage of identity with the most closely related species (97.5 %), indicated its placement as a novel species within this genus. Digital DNA-DNA hybridization (dDDH) values and average nucleotide identity (ANI) analyses of this strain against closely related species confirmed its condition of novel taxon. On the other hand, the percentage of the average amino acid identity (AAI) affiliated strain 71-iT within the genus Marinobacter. The DNA G+C content of this isolate was 57.7 mol%. The major fatty acids were C16 : 0 and C16 : 1ω7c and/or C16 : 1 ω6c. Ubiquinone-9 was the major isoprenoid quinone and diphosphatidylglycerol (DPG), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) were the main polar lipids of this strain. On the basis of the phylogenomic and phenotypic (including chemotaxonomic) features, we propose strain 71-iT (= IBRC M 11023T = CECT 30160T = LMG 29252T) as the type strain of a novel species within the genus Marinobacter, with the name Marinobacter iranensis sp. nov. Genomic detections of this strain in various metagenomic databases indicate that it is a relatively abundant species in environments with low salinities (approximately 5 % salinity), but not in hypersaline habitats with high salt concentrations.

Mechanism and evolutionary analysis of Yarrowia lipolytica CA20 capable of producing erythritol with a high yield based on comparative genomics.

Combined mutagenesis is widely applied for the breeding of robust Yarrowia lipolytica used in the production of erythritol. However, the changes of genome after mutagenesis remains unclear. This study aimed to unravel the mechanism involved in the improved erythritol synthesis of CA20 and the evolutionary relationship between different Y. lipolytica by comparative genomics analysis. The results showed that the genome size of Y. lipolytica CA20 was 20,420,510 bp, with a GC content of 48.97%. There were 6330 CDS and 649 ncRNA (non-coding RNA) in CA20 genome. Average nucleotide identity (ANI) analysis showed that CA20 genome possessed high similarity (ANI > 99.50%) with other Y. lipolytica strains, while phylogenetic analysis displayed that CA20 was classified together with Y. lipolytica IBT 446 and Y. lipolytica H222. CA20 shared 5342 core orthologous genes with the 8 strains while harbored 65 specific genes that mainly participated in the substrate and protein transport processes. CA20 contained 166 genes coding for carbohydrate-active enzymes (CAZymes), which was more than that found in other strains (108－137). Notably, 4, 2, and 13 different enzymes belonging to glycoside hydrolases (GHs), glycosyltransferases (GTs), and carbohydrate esterases (CEs), respectively, were only found in CA20. The enzymes involved in the metabolic pathway of erythritol were highly conserved in Y. lipolytica, except for transaldolase (TAL1). In addition, the titer and productivity of erythritol by CA20 were 190.97 g/L and 1.33 g/L/h, respectively, which were significantly higher than that of WT5 wherein 128.61 g/L and 0.92 g/L/h were obtained (P< 0.001). Five frameshift mutation genes and 15 genes harboring nonsynonymous mutation were found in CA20 compared with that of WT5. Most of these genes were involved in the cell division, cell wall synthesis, protein synthesis, and protein homeostasis maintenance. These findings suggested that the genome of Y. lipolytica is conserved during evolution, and the variance of living environment is one important factor leading to genome divergence. The varied number of CAZymes existed in Y. lipolytica is one factor that contributes to the performance difference. The increased synthesis of erythritol by Y. lipolytica CA20 is correlated with the improvement of the stability of cell structure and internal environment. The results of this study provide a basis for the directional breeding of robust strains used in erythritol production.

A Class 4-like Chromosomal Integron Found in Aeromonas sp. Genomospecies paramedia Isolated from Human Feces.

Integrons are genetic elements that store, express and exchange gene cassettes. These elements are characterized by containing a gene that codes for an integrase (intI), a cassette integration site (attI) and a variable region holding the cassettes. Using bioinformatics and molecular biology methods, a functional integron found in Aeromonas sp. 3925, a strain isolated from diarrheal stools, is described. To confirm the integron class, a phylogenetic analysis with amino acid sequences was conducted. The integrase was associated to class 4 integrases; however, it is clearly different from them. Thus, we classified the associated element as a class 4-like integron. We found that the integrase activity is not under the control of the SOS or catabolic repression, since the expression was not increased in the presence of mitomycin or arabinose. The class-4-like integron is located on the chromosome and contains two well-defined gene cassettes: aadA1 that confers resistance to streptomycin and lpt coding for a lipoprotein. It also includes eight Open Reading frames (ORFs) with unknown functions. The strain was characterized through a Multilocus Phylogenetic Analyses (MLPA) of the gyrB, gyrA, rpoD, recA, dnaJ and dnaX genes. The phylogenetic results grouped it into a different clade from the species already reported, making it impossible to assign a species. We resorted to undertaking complete genome sequencing and a phylogenomic analysis. Aeromonas sp. 3925 is related to A. media and A. rivipollensis clusters, but it is clearly different from these species. In silico DNA-DNA hybridization (isDDH) and Average Nucleotide Identity (ANI) analyses suggested that this isolate belongs to the genomospecies paramedia. This paper describes the first class 4-like integron in Aeromonas and contributes to the establishment of genomospecies paramedia.

Genome sequencing and analysis of Bacillus velezensis VJH504 reveal biocontrol mechanism against cucumber Fusarium wilt.

One major issue in reducing cucumber yield is the destructive disease Cucumber (Cucumis sativus L.) wilt disease caused by Fusarium oxysporum f. sp. cucumerinum (Foc). When using the isolate VJH504 isolated from cucumber rhizosphere soil and identified as Bacillus velezensis, the growth of Foc in the double culture experiment was effectively inhibited. Phenotypic, phylogenetic, and genomic analyses were conducted to enhance understanding of its biocontrol mechanism. According to the result of the phenotype analysis, B. velezensis VJH504 could inhibit cucumber fusarium wilt disease both in vitro and in vivo, and significantly promote cucumber seed germination and seedling growth. Additionally, the tests of growth-promoting and biocontrol characteristics revealed the secretion of proteases, amylases, β-1,3-glucanases, cellulases, as well as siderophores and indole-3-acetic acid by B. velezensis VJH504. Using the PacBio Sequel II system, we applied the complete genome sequencing for B. velezensis VJH504 and obtained a single circular chromosome with a size of 3.79 Mb. A phylogenetic tree was constructed based on the 16S rRNA gene sequences of B. velezensis VJH504 and 13 other Bacillus species, and Average Nucleotide Identity (ANI) analysis was performed using their whole-genome sequences, confirming isolateVJH504 as B. velezensis. Following this, based on the complete genome sequence od B. velezensis VJH504, specific functional analysis, Carbohydrate-Active Enzymes (CAZymes) analysis, and secondary metabolite analysis were carried out, predicting organism's abilities for biofilm formation, production of antifungal CAZymes, and synthesis of antagonistic secondary metabolites against pathogens. Afterwards, a comparative genomic analysis was performed between B. velezensis VJH504 and three other B. velezensis strains, revealing subtle differences in their genomic sequences and suggesting the potential for the discovery of novel antimicrobial substances in B. velezensis VJH504. In conclusion, the mechanism of B. velezensis VJH504 in controlling cucumber fusarium wilt was predicted to appear that B. velezensis VJH504is a promising biocontrol agent, showcasing excellent application potential in agricultural production.

Unexpected Diversity of Pseudomonads Associated with Bacterial Leaf Spot of Cucurbits in the Southeastern United States.

Bacterial leaf spot of cucurbits (BLS) is an emerging disease in the southeastern United States that is capable of causing widespread outbreaks under conducive conditions. Historically attributed solely to the bacterium Pseudomonas syringae pv. lachrymans, recent studies have identified additional P. syringae pathovars as causal agents of the disease. To further investigate the identity and diversity of P. syringae strains associated with BLS in the southeastern United States, 47 bacterial isolates were recovered from symptomatic cucurbits from Florida, Alabama, and Georgia. Strains were characterized using the LOPAT testing scheme, fluorescence, and pathogenicity to watermelon and squash seedlings. Thirty-eight fluorescent isolates underwent whole-genome sequencing and were further characterized with 16S rRNA, four gene multilocus sequence analysis (MLSA) phylogeny, and average nucleotide identity analysis. Thirty-four isolates were identified as members of the P. syringae species complex, including P. syringae sensu stricto (12), P. alliivorans (12), P. capsici (nine), and P. viridiflava (one). An additional four isolates were found to belong to the Pseudomonas genus outside of the syringae species complex, though they did not share 95% or greater average nucleotide identity to any validly published species and are believed to belong to three novel Pseudomonas species. These results reveal an unpredicted level of diversity of Pseudomonas strains associated with BLS in the region and show the benefits of whole-genome sequencing for strain identification. Identification of P. capsici, which is capable of causing disease at higher temperatures than P. syringae, as a causal agent of BLS may also affect management strategies in the future.

Taxonomic considerations on Aerococcus urinae with proposal of subdivision into Aerococcus urinae, Aerococcus tenax sp. nov., Aerococcus mictus sp. nov., and Aerococcus loyolae sp. nov.

Average nucleotide identity analysis, based on whole genome sequences of 115 strains previously identified as Aerococcus urinae, an emerging uropathogen, discriminates at least six unique genomic taxa. The whole genome analysis affords clearer species boundaries over 16S rRNA gene sequencing and traditional phenotypic approaches for the identification and phylogenetic organization of Aerococcus species. The newly described species can be differentiated by matrix-assisted laser desorption ionization time-of-flight analysis of protein signatures. We propose the emendation of the description of A. urinae (type strain ATCC 51268T = CCUG 34223T=NCFB 2893) and the names of Aerococcus tenax sp. nov. (ATCC TSD-302T = DSM 115700T = CCUG 76531T=NR-58630T), Aerococcus mictus sp. nov. (ATCC TSD-301T = DSM 115699T = CCUG 76532T=NR-58629T), and Aerococcus loyolae sp. nov. (ATCC TSD-300T = DSM 115698T = CCUG 76533T=NR-58628T) for three of the newly identified genomic taxa.

Streptomyces fuscus sp. nov., a brown-black pigment producing actinomycete isolated from dry mudflat sand.

A novel Gram-stain-positive, aerobic actinobacterial strain, designated GXMU-J15T, was isolated from dry mudflat sand. A polyphasic approach was employed for its taxonomic characterization. The strain developed extensively branched yellowish white to light yellow substrate mycelia and white aerial mycelia, and produced smooth cylindrical spores in a loose straight spore chain on International Streptomyces Project 2-7 agar media. Strain GXMU-J15T grew at 20-50 °C (optimum, 35 °C), at pH 5.0-8.0 (optimum, pH 7.0) and in the presence of 0-8 % (w/v) NaCl. Analysis of 16S rRNA gene sequences indicated that strain GXMU-J15T represents a member of the genus Streptomyces. Strain GXMU-J15T showed the highest 16S rRNA gene sequence similarity to Streptomyces lusitanus CGMCC 4.1745T (99.1 %) and Streptomyces thermocarboxydus CGMCC 4.1883T (98.8 %). Phylogenetic tree analysis based on multilocus sequence analysis (MLSA) and whole genome sequence construction revealed that strain GXMU-J15T was most closely related to Streptomyces cupreus PSKA01T, Streptomyces cinnabarinus DSM 40467T and Streptomyces davaonensis JCM 4913T. The MLSA and genome-to-genome distances between strain GXMU-J15T and its relatives were 0.0418, 0.0443 and 0.0485 and 0.1237, 0.1188 and 0.1179, respectively. The results of orthologous average nucleotide identity and digital DNA-DNA hybridization analysis corroborated the results of the MLSA and whole genome sequence evolution analysis, indicating that the novel isolate represents a distinct species of the genus Streptomyces. The whole-cell sugars of strain GXMU-J15T were xylose, glucose and galactose. The characteristic diamino acid in the cell-wall hydrolysate was ll-diaminopimelic acid. The lipids contained diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, phosphatidylglycerides, phosphatidylcholine, two phospholipids of an unknown structure containing glucosamine, one unknown phospholipid and two unknown lipids. The major cellular fatty acid components were iso-C15 : 0, anteiso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0. The main respiratory quinone types were MK-9(H6) and MK-9(H8). The whole genome size of strain GXMU-J15T was 8.68 Mbp, with 71.23 mol% G+C content. Genomic analysis indicated that strain GXMU-J15T has the potential to synthesize polyketides, terpenes and a series of important antibiotics besides the gene cluster for melanin synthesis. Based on these genotypic and phenotypic data, strain GXMU-J15T is proposed to represent a new species of the genus Streptomyces named Streptomyces fuscus sp. nov. The type strain is GXMU-J15T (=MCCC 1K08211T=JCM 35917T).

Large-scale characterization of hospital wastewater system microbiomes and clinical isolates from infected patients: profiling of multi-drug-resistant microbial species

Hospital-acquired infection (HAI) and infectious agents exhibiting antimicrobial resistance (AMR) are challenges globally. Environmental patient-facing wastewater apparatus including handwashing sinks, showers and toilets are increasingly identified as sources of infectious agents and AMR genes. We performed large-scale metagenomic analysis of the wastewater system in a large teaching hospital in the Republic of Ireland experiencing multidrug-resistant HAI outbreaks. Immediately prior to refurbishment of a medical ward where HAI has been endemic, wastewater pipe sections (n=20) were removed. These comprised toilet u-bends, sink and shower drains and, following DNA extraction, each underwent metagenomic analysis. Diverse taxonomic and resistome profiles were observed, with members of phyla Proteobacteria and Actinobacteria dominating (38.23 ± 5.68% and 15.78 ± 3.53%, respectively). Genomes of five clinical isolates were analysed. These antimicrobial-resistant bacterial isolates were from patients >48 hours post-admission to the ward. Genomic analysis determined that the isolates bore a high number of antimicrobial resistance genes (ARGs). Comparison of resistome profiles of these isolates and wastewater pipe metagenomes revealed a high degree of similarity, with many identical ARGs shared between clinical isolates and the wastewater environment, suggesting probable acquisition post-admission. The highest numbers of ARGs observed were those encoding resistance to significant clinically- and commonly-used antibiotic classes. Average nucleotide identity analysis confirmed presence of highly similar or identical genomes in the clinical isolates and wastewater pipes. These unique large-scale analyses reinforce the need for regular cleaning and decontamination of patient-facing hospital wastewater pipes and effective infection control policies to prevent the transmission of nosocomial infection and emergence of AMR within potential wastewater reservoirs.

Whole genome sequencing and functional analysis of a novel biofilm-eradicating strain Nocardiopsis lucentensis EMB25.

The process of biofilm formation is intricate and multifaceted, requiring the individual cells to secrete extracellular polymeric substances (EPS) that subsequently aggregate and adhere to various surfaces. The issue of biofilms is a significant concern for public health due to the increased resistance of microorganisms associated with biofilms to antimicrobial agents. The current study describes the whole genome and corresponding functions of a biofilm inhibiting and eradicating actinobacteria isolate identified as Nocardiopsis lucentensis EMB25. The N. lucentensis EMB25 has 6.5 Mbp genome with 71.62% GC content. The genome analysis by BLAST Ring Image Generator (BRIG) revealed it to be closely related to Nocardiopsis dassonvillei NOCA502F. Interestingly, based on orthologous functional groups reflected by average nucleotide identity (ANI) analysis, it was 81.48% similar to N. arvandica DSM4527. Also, it produces lanthipeptides and linear azole(in)e-containing peptides (LAPs) akin to N. arvandica. The secondary metabolite search revealed the presence of major gene clusters involved in terpene, ectoine, siderophores, Lanthipeptides, RiPP-like, and T1PKS biosynthesis. After 24h of treatment, the cell-free extract effectively eradicates the pre-existing biofilm of P. aeruginosa PseA. Also, the isolated bacteria exhibited antibacterial activity against MRSA, Staphylococcus aureus and Bacillus subtilis bacteria. Overall, this finding offers valuable insights into the identification of BGCs, which contain enzymes that play a role in the biosynthesis of natural products. Specifically, it sheds light on the functional aspects of these BGCs in relation to N. lucentensis.

Occurrence, Characteristics, and qPCR-Based Identification of Pectobacterium versatile Causing Soft Rot of Chinese Cabbage in China.

Pectobacterium is one of the most important genera of phytopathogenic bacteria. It can cause soft-rot diseases on a wide range of plant species across the world. In this study, three Pectobacterium strains (KC01, KC02, and KC03) were isolated from soft-rotted Chinese cabbage in Beijing, China. These three strains were identified as Pectobacterium versatile based on phylogenetic analysis of Pectobacterium 16S ribosomal RNA, pmrA, and 504 Pectobacterium core genes, as well as a genomic average nucleotide identity analysis. Their biochemical characteristics were found to be similar to the P. versatile type strain ICMP9168T but differed in response to citric acid, stachyose, D-glucuronic acid, dextrin, and N-acetyl-β-D-mannosamine. All of the tested P. versatile strains showed different carbohydrate utilization abilities compared with P. carotovorum and P. odoriferum, particularly in their ability to utilize D-arabitol, L-rhamnose, and L-serine. Under laboratory conditions, the maceration ability of P. versatile on Chinese cabbage was the highest at 28°C, compared with those at 13, 28, 23, and 33°C. Additionally, P. versatile could infect all of the 17 known Pectobacterium host plants, except for Welsh onion (Allium fistulosum). A SYBR Green quantitative PCR (qPCR) detection system was developed to distinguish P. versatile from other soft-rot bacteria based on the combined performance of melting curve (with a single melting peak at around 85°C) and fluorescence curve (with cycle threshold <30) when the bacterial genomic DNA concentration was in the range of 10 pg/µl to 10 ng/µl. This study is the first to report the presence of P. versatile on Chinese cabbage in China, as well as a specific and sensitive qPCR assay that can be used to quickly identify P. versatile. The work contributes to a better understanding of P. versatile and will facilitate the effective diagnosis of soft-rot disease, ultimately benefitting commercial crop production.

Exploring the microbial diversity of novel misos with metagenomics

Interest in fermented foods, especially plant-based ones, has increased considerably in the last decade. Miso—a Japanese paste traditionally fermented with soybeans, salt, and kōji (Aspergillus oryzae grown on grains or beans)—has gained attention among chefs for its rich flavour and versatility. Some chefs have even been experimenting with making novel misos with untraditional substrates to create new flavours. Such novel fermented foods also offer new scientific opportunities. To explore the microbial diversity of these new traditional foods, we sampled six misos made by the team at a leading restaurant called Noma in Copenhagen (Denmark), using yellow peas (including a nixtamalised treatment), lupin seeds, Swedish Vreta peas, grey peas, and Gotland lentils as substrates. All misos were made with the same recipe and fermented for 3 months at 28 °C. Samples were collected at the end of fermentation for subsequent shotgun metagenomic sequencing and a genome-resolved metagenomic analysis. The taxonomic profile of the samples revealed the presence of kōji mould (A. oryzae) and Bacillus amyloliquefaciens in all misos. Various species of the genera Latilactobacillus, Lactiplantibacillus, Pediococcus and Staphylococcus were also detected. The Metagenome-Assembled Genomes (MAGs) revealed genomic sequences belonging to 12 different species and functional analyses of these MAGs were performed. Notably, we detected the presence of Exiguobacterium—the first reported instance of the genus in miso—and Average Nucleotide Identity (ANI) analyses suggest a potentially new species. We hope these results will improve the scientific literature on misos and contribute to developing novel fermented plant-based foods.

Characterization of drought stress-mitigating Rhizobium from faba bean (Vicia faba L.) in the Chinese Qinghai-Tibet Plateau.

Rhizobium-driven symbiotic nitrogen-fixation in legumes not only benefits the growth but also enhances the stress tolerance of plants. Isolating and characterizing efficient, drought-tolerant rhizobia is a central goal for improving crop yields in arid regions. Here, we phylogenetically and biochemically characterized a novel strain of Rhizobium ('QHCD11') sampled from the root nodules of faba beans growing in an arid agricultural area in Qinghai-Tibet. We further tested the drought tolerance of the strain as well as of 'Qingcan 14' faba bean seedlings inoculated with it. Biochemical characterization involved bromothymol blue (BTB) tests, carbon metabolic profiling (Biolog GENIII), DNA-DNA hybridization (dDDH) assays, average nucleotide identity (ANI) analyses, and 16S rRNA sequencing. The result indicated that strain 'QHCD11' likely belongs to the Rhizobium indicum species. Drought stress tolerance was assessed by exposure to polyethylene glycol (PEG-6000) at concentrations of 0, 10, 15, and 20%. Increasing concentrations of PEG-6000 tended to result in decreased growth of 'QHCD11', although the strain performed better at 20% PEG 6000 than at 15%. Inoculation of drought-stressed faba bean seedlings with strain 'QHCD11' improved root vitality, chlorophyll content, antioxidant enzyme activities, and plant height. We suggest that inoculation of faba beans with 'QHCD11' is an environmentally sound strategy for mitigating crop drought stress in arid and semi-arid regions. In addition, the results presents here will benefit future studies into faba bean-rhizobia symbioses under drought stress.

Mesomycoplasma ovipneumoniae from goats with respiratory infection: pathogenic characteristics, population structure, and genomic features

BackgroundMycoplasma ovipneumoniae is a critical pathogen that causes respiratory diseases that threaten Caprini health and cause economic damage. A genome-wide study of M. ovipneumoniae will help understand the pathogenic characteristics of this microorganism.ResultsToxicological pathology and whole-genome sequencing of nine M. ovipneumoniae strains isolated from goats were performed using an epidemiological survey. These strains exhibited anterior ventral lung consolidation, typical of bronchopneumonia in goats. Average nucleotide identity and phylogenetic analysis based on whole-genome sequences showed that all M. ovipneumoniae strains clustered into two clades, largely in accordance with their geographical origins. The pan-genome of the 23 M. ovipneumoniae strains contained 5,596 genes, including 385 core, 210 soft core, and 5,001 accessory genes. Among these genes, two protein-coding genes were annotated as cilium adhesion and eight as paralog surface adhesins when annotated to VFDB, and no antibiotic resistance-related genes were predicted. Additionally, 23 strains carried glucosidase-related genes (ycjT and group_1595) and glucosidase-related genes (atpD_2), indicating that M. ovipneumoniae possesses a wide range of glycoside hydrolase activities.ConclusionsThe population structure and genomic features identified in this study will facilitate further investigations into the pathogenesis of M. ovipneumoniae and lay the foundation for the development of preventive and therapeutic methods.

Brachybacterium atlanticum sp. nov., a novel marine bacterium isolated from the Atlantic Ocean.

A bacterial strain, PhyBa_CO2_2T, was isolated from the North Atlantic Gyre, offshore Terceira Island in the Azores. Initially, the NCBI nucleotide blast analysis based on 16S rRNA gene sequences revealed that the strain belongs to the genus Brachybacterium, with a 100 % identity with Brachybacterium paraconglomeratum LMG 19861T. However, further genomic characterization through average nucleotide identity (ANI) and digital DNA-DNA hybridization analyses showed values of 96.06 and 64.80 %, respectively. Comparative genomics also highlighted differences in gene content. The genome size of PhyBa_CO2_2T is 3.6 Mbp and the DNA G+C content is 72.1 mol%. Chemotaxonomic analysis demonstrated that the composition of the fatty acids was mainly composed of anteiso-C15 : 0 (46.04 %), iso-C16 : 0 (13.70 %) and anteiso-C17 : 0 (9.48 %), and the polar lipids were mainly diphosphatidylglycerol, phosphatidylglycerol and two unidentified glycolipids. Furthermore, the diagnostic amino acid of the cell wall was meso-diaminopimelic acid and the predominant menaquinone was MK7. Finally, phenotypic analysis revealed differences in biochemical profiles between PhyBa_CO2_2T and its closely related strains in terms of indole production, urease and β-glucuronidase activity. Therefore, based on the genomic, chemotaxonomic and phenotypic data obtained, we concluded that strain PhyBa_CO2_2T represents a new species, for which the name Brachybacterium atlanticum sp. nov. is proposed in reference to its isolation site. The type strain is PhyBa_CO2_2T (=DSM 114113T= CECT 30695T).

Identification of two distinct phylogenomic lineages and model strains for the understudied cystic fibrosis lung pathogen Burkholderia multivorans.

Burkholderia multivorans is the dominant Burkholderia pathogen recovered from lung infection in people with cystic fibrosis. However, as an understudied pathogen there are knowledge gaps in relation to its population biology, phenotypic traits and useful model strains. A phylogenomic study of B. multivorans was undertaken using a total of 283 genomes, of which 73 were sequenced and 49 phenotypically characterized as part of this study. Average nucleotide identity analysis (ANI) and phylogenetic alignment of core genes demonstrated that the B. multivorans population separated into two distinct evolutionary clades, defined as lineage 1 (n=58 genomes) and lineage 2 (n=221 genomes). To examine the population biology of B. multivorans, a representative subgroup of 77 B. multivorans genomes (28 from the reference databases and the 49 novel short-read genome sequences) were selected based on multilocus sequence typing (MLST), isolation source and phylogenetic placement criteria. Comparative genomics was used to identify B. multivorans lineage-specific genes - ghrB_1 in lineage 1 and glnM_2 in lineage 2 - and diagnostic PCRs targeting them were successfully developed. Phenotypic analysis of 49 representative B. multivorans strains showed considerable inter-strain variance, but the majority of the isolates tested were motile and capable of biofilm formation. A striking absence of B. multivorans protease activity in vitro was observed, but no lineage-specific phenotypic differences were demonstrated. Using phylogenomic and phenotypic criteria, three model B. multivorans CF strains were identified, BCC0084 (lineage 1), BCC1272 (lineage 2a) and BCC0033 lineage 2b, and their complete genome sequences determined. B. multivorans CF strains BCC0033 and BCC0084, and the environmental reference strain, ATCC 17616, were all capable of short-term survival within a murine lung infection model. By mapping the population biology, identifying lineage-specific PCRs and model strains, we provide much needed baseline resources for future studies of B. multivorans.

Description of Streptococcus thalassemiae sp. nov., a Bacterium Isolated from Human Blood.

Blood is a precious biological liquid that is normally sterile. Therefore, bacteria in the bloodstream are shown a priori anomaly. A blood culture is systematically performed to diagnose the cause of the bacteremia. Indeed, a patient received in our service had a thalassemia major and underwent a genoidentical transplant. Then, a blood test was performed to diagnose a four-day fever. In this context, we have isolated strain Marseille-Q2617 from the blood sample. It revealed a new bacterial strain that belongs to the genus Streptococcus. It is a Gram-positive coccus, nonmotile, and nonspore forming. The major fatty acid found is hexadecanoic acid, with 49.5%. A taxonomic method was used to characterize the strain by studying their phenotypic, phylogenetic, and genomic characteristics. In addition, sequence analysis of the 16S rRNA gene shows that the strain Marseille-Q2617 has 99.94% sequence similarity to Streptococcus mitis. Average nucleotide identity (ANI) analysis for strain Marseille-Q2617T showed the highest similarity of 92.9% with S. mitis. The DNA-DNA hybridization value obtained (50.2%) between strain Marseille-Q2607 and S. mitis, its closest related species, was below the recommended threshold (<70%). Strain Marseille-Q2617T has a genome size of 2.02 Mbp with 40.5 mol% of G + C content. Based on these results, we propose a new species of the genus Streptococcus, for which the name Streptococcus thalassemiae sp. nov., Marseille-Q2617T (=CSUR Q2617 = CECT 30109) was proposed.

Detection of NDM-1 and OXA-10 Co-Producing Providencia rettgeri Clinical Isolate.

The coexistence of blaNDM-1 with other resistance determinants is rarely reported for Providencia rettgeri. Therefore, this study investigates the phenotypic and genetic characteristics of a multidrug-resistant P. rettgeri strain YQ150713. P. rettgeri YQ150713 was identified as carrying blaNDM-1. S1-pulsed-field gel electrophoresis (S1-PFGE), Southern blotting, and conjugation experiments were used to determine plasmid characteristics. An antimicrobial susceptibility test was conducted. The complete genomic sequence of YQ150713 was obtained using Illumina NovaSeq 6000 and Oxford nanopore platforms.To further characterize the phylogenetic structure of P. rettgeri YQ150713, average nucleotide identity (ANI) and phylogenetic analyses were conducted. The S1-PFGE, Southern blot, and conjugation assays have confirmed that the isolate P. rettgeri YQ150713 contains the blaNDM-1 gene on a conjugative plasmid pYQ150713-NDM-1. Antimicrobial susceptibility testing has indicated that strain YQ150713 was resistant to various common antibiotics, except aztreonam and fosfomycin. Bioinformatics analysis has further shown that pYQ150713-NDM-1 was a novel plasmid with a size of 265,883 bp, and blaNDM-1 and blaOXA-10 were co-located on it. Phylogenetic analysis suggesting P. rettgeri has spread widely throughout the world. In this study, blaNDM-1 and blaOXA-10 were co-localized on a novel plasmid pYQ150713-NDM-1 with a horizontal transfer function. To reduce the risk of the dissemination of such P. rettgeri isolates in clinical settings, more surveillance will be required in the future.