Illumina MiSeq 16S rRNA Gene Library Preparation for Poultry Processing Microbiome Analyses.

The standardization of the microbiome sequencing of poultry rinsates is essential for generating comparable microbial composition data among poultry processing facilities if this technology is to be adopted by the industry. Samples must first be acquired, DNA must be extracted, and libraries must be constructed. In order to proceed to library sequencing, the samples should meet quality control standards. Finally, data must be analyzed using computer bioinformatics pipelines. This data can subsequently be incorporated into more advanced computer algorithms for risk assessment. Ultimately, *a uniform sequencing pipeline will enable both the government regulatory agencies and the poultry industry to identify potential weaknesses in food safety.This chapter presents the different steps for monitoring the population dynamics of the microbiome in poultry processing using 16S rDNA sequencing.

Rapid visual detection of Helicobacter pylori and vacA subtypes by Dual-Target RAA-LFD assay.

BACKGROUND: Helicobacter pylori (H. pylori) infects over 50% of the global population and is a significant risk factor for gastric cancer. The pathogenicity of H. pylori is primarily attributed to virulence factors such as vacA. Timely and accurate identification, along with genotyping of H. pylori virulence genes, are essential for effective clinical management and controlling its prevalence. METHODS: In this study, we developed a dual-target RAA-LFD assay for the rapid, visual detection of H. pylori genes (16s rRNA, ureA, vacA m1/m2), using recombinase aided amplification (RAA) combined with lateral flow dipstick (LFD) methods. Both 16s rRNA and ureA were selected as identification genes to ensure reliable detection accuracy. RESULTS: A RAA-LFD assay was developed to achieve dual-target amplification at a stable 37 °C within 20 min, followed by visualization using the lateral flow dipstick (LFD). The whole process, from amplification to results, took less than 30 min. The 95 % limit of detection (LOD) for 16 s rRNA and ureA, vacA m1, vacA m2 were determined as 3.8 × 10-2 ng/µL, 5.8 × 10-2 ng/µL and 1.4 × 10-2 ng/µL, respectively. No cross-reaction was observed in the detection of common pathogens including Escherichia coli, Klebsiella pneumoniae, Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus subtilis, showing the assay's high specificity. In the evaluation of the clinical performance of the RAA-LFD assay. A total of 44 gastric juice samples were analyzed, immunofluorescence staining (IFS) and quantitative polymerase chain reaction (qPCR) were used as reference methods. The RAA-LFD results for the 16s rRNA and ureA genes showed complete agreement with qPCR findings, accurately identifying H. pylori infection as confirmed by IFS in 10 out of the 44 patients. Furthermore, the assay successfully genotyped vacA m1/m2 among the positive samples, showing complete agreement with qPCR results and achieving a kappa (κ) value of 1.00. CONCLUSION: The dual-target RAA-LFD assay developed in this study provides a rapid and reliable method for detecting and genotyping H. pylori within 30 min, minimizing dependency on sophisticated laboratory equipment and specialized personnel. Clinical validation confirms its efficacy as a promising tool for effectively control of its prevalence and aiding in the precise treatment of H. pylori-associated diseases.

Aquibaculum sediminis sp. nov., a halotolerant bacteria isolated from salt lake sediment.

An aerobic, Gram-stain negative bacterium was isolated from sediment samples of Barkol salt lake in Hami City, Xinjiang Uygur Autonomous Region, China, with the number EGI_FJ10229T. The strain is ellipse-shaped, oxidase-negative, catalase-positive, and has white, round, smooth, opaque colonies on marine 2216 E agar plate. Growth occurs at 4.0-37.0 â„ƒ (optimal:30.0 â„ƒ), pH 7.0-9.0 (optimal: pH 8.0) and NaCl concentration of 0-8.0% (optimal: 3.0%). Phylogenetic analysis based on 16S rRNA gene and genome sequences indicated that the isolated strain should be assigned to the genus Aquibaculum and was most closely related to Aquibaculum arenosum CAU 1616 T. Average nucleotide identity (ANI) and Average amino-acid identity (AAI) values between the type species of the genus Aquibaculum and other related type species were lower than the threshold values recommended for bacterial species. The genomic DNA G + C content of EGI_FJ10229T was 65.41%. The major polar lipids were diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylcholine, phosphatidylethanolamine and unidentified phospholipid. The major fatty acids (> 5%) were C19:0 cyclo ω8c (42.0%) and C18:1 ω7c (33.78%). The respiratory quinone identified was Q-10. Differential phenotypic and genotypic characteristics of this strain and species of genus Aquibaculum showed that the strain should be classified as representing a new species belonging to this genus, for which the name Aquibaculum sediminis sp. nov. is proposed. The type strain of the proposed novel species is EGI_FJ10229T (= KCTC 8570 T = GDMCC 1.4598 T).

Stratification of human gut microbiomes by succinotype is associated with inflammatory bowel disease status.

BACKGROUND: The human gut microbiome produces and consumes a variety of compounds that interact with the host and impact health. Succinate is of particular interest as it intersects with both host and microbiome metabolism. However, which gut bacteria are most responsible for the consumption of intestinal succinate is poorly understood. RESULTS: We build upon an enrichment-based whole fecal sample culturing approach and identify two main bacterial taxa that are responsible for succinate consumption in the human intestinal microbiome, Phascolarctobacterium and Dialister. These two taxa have the hallmark of a functional guild and are strongly mutual exclusive across 21,459 fecal samples in 94 cohorts and can thus be used to assign a robust "succinotype" to an individual. We show that they differ with respect to their rate of succinate consumption in vitro and that this is associated with higher concentrations of fecal succinate. Finally, individuals suffering from inflammatory bowel disease (IBD) are more likely to have the Dialister succinotype compared to healthy subjects. CONCLUSIONS: We identified that only two bacterial genera are the key succinate consumers in human gut microbiome, despite the fact that many more intestinal bacteria encode for the succinate pathway. This highlights the importance of phenotypic assays in functionally profiling intestinal microbiota. A stratification based on "succinotype" is to our knowledge the first function-based classification of human intestinal microbiota. The association of succinotype with IBD thus builds a bridge between microbiome function and IBD pathophysiology related to succinate homeostasis. Video Abstract.

Gut microbiota and metabolomic profile changes play critical roles in tacrolimus-induced diabetes in rats.

Aims: Hyperglycemia is one of the adverse effects of tacrolimus (TAC), but the underlying mechanism is not fully identified. We used multi-omics analysis to evaluate the changes in the gut microbiota and metabolic profile of rats with TAC-induced diabetes. Methods: To establish a diabetic animal model, Sprague Dawley rats were divided randomly into two groups. Those in the TAC group received intraperitoneal injections of TAC (3 mg/kg) for 8 weeks, and those in the CON group served as the control. 16S rRNA sequencing was used to analyze fecal microbiota. The metabolites of the two groups were detected and analyzed by nontargeted and targeted metabolomics, including amino acids (AAs), bile acids (BAs), and short-chain fatty acids (SCFAs). Results: The rats treated with TAC exhibited hyperglycemia as well as changes in the gut microbiota and metabolites. Specifically, their gut microbiota had significantly higher abundances of Escherichia-Shigella, Enterococcus, and Allobaculum, and significantly lower abundances of Ruminococcus, Akkermansia, and Roseburia. In addition, they had significantly reduced serum levels of AAs including asparagine, aspartic acid, glutamic acid, and methionine. With respect to BAs, they had significantly higher serum levels of taurocholic acid (TCA), and glycochenodeoxycholic acid (GCDCA), but significantly lower levels of taurodeoxycholic acid (TDCA) and tauroursodeoxycholic acid (TUDCA). There were no differences in the levels of SCFAs between the two groups. Correlations existed among glucose metabolism indexes (fasting blood glucose and fasting insulin), gut microbiota (Ruminococcus and Akkermansia), and metabolites (glutamic acid, hydroxyproline, GCDCA, TDCA, and TUDCA). Conclusions: Both AAs and BAs may play crucial roles as signaling molecules in the regulation of TAC-induced diabetes.

Intratumoral and fecal microbiota reveals microbial markers associated with gastric carcinogenesis.

Background: The relationship between dysbiosis of the gastrointestinal microbiota and gastric cancer (GC) has been extensively studied. However, microbiota alterations in GC patients vary widely across studies, and reproducible diagnostic biomarkers for early GC are still lacking in multiple populations. Thus, this study aimed to characterize the gastrointestinal microbial communities involved in gastric carcinogenesis through a meta-analysis of multiple published and open datasets. Methods: We analyzed 16S rRNA sequencing data from 1,642 gastric biopsy samples and 394 stool samples across 11 independent studies. VSEARCH, QIIME and R packages such as vegan, phyloseq, cooccur, and random forest were used for data processing and analysis. PICRUSt software was employed to predict functions. Results: The α-diversity results indicated significant differences in the intratumoral microbiota of cancer patients compared to non-cancer patients, while no significant differences were observed in the fecal microbiota. Network analysis showed that the positive correlation with GC-enriched bacteria increased, and the positive correlation with GC-depleted bacteria decreased compared to healthy individuals. Functional analyses indicated that pathways related to carbohydrate metabolism were significantly enriched in GC, while biosynthesis of unsaturated fatty acids was diminished. Additionally, we investigated non-Helicobacter pylori (HP) commensals, which are crucial in both HP-negative and HP-positive GC. Random forest models, constructed using specific taxa associated with GC identified from the LEfSe analysis, revealed that the combination of Lactobacillus and Streptococcus included alone could effectively discriminate between GC patients and healthy individuals in fecal samples (area under the curve (AUC) = 0.7949). This finding was also validated in an independent cohort (AUC = 0.7712). Conclusions: This study examined the intratumoral and fecal microbiota of GC patients from a dual microecological perspective and identified Lactobacillus, Streptococcus, Roseburia, Faecalibacterium and Phascolarctobacterium as intratumoral and intestinal-specific co-differential bacteria. Furthermore, it confirmed the validity of the combination of Lactobacillus and Streptococcus as GC-specific microbial markers across multiple populations, which may aid in the early non-invasive diagnosis of GC.

Methanotrophic Communities and Cultivation of Methanotrophs from Rice Paddy Fields Fertilized with Pig-livestock Biogas Digestive Effluent and Synthetic Fertilizer in the Vietnamese Mekong Delta.

Biogas digestive effluent (BDE) has been applied to rice fields in the Vietnamese Mekong Delta (VMD). However, limited information is available on the community composition and isolation of methanotrophs in these fields. Therefore, the present study aimed (i) to clarify the responses of the methanotrophic community in paddy fields fertilized with BDE or synthetic fertilizer (SF) and (ii) to isolate methanotrophs from these fields. Methanotrophic communities were detected in rhizospheric soil at the rice ripening stage throughout 2 cropping seasons, winter-spring (dry) and summer-autumn (wet). Methanotrophs were isolated from dry-season soil samples. Although the continued application of BDE markedly reduced net methane oxidation potential and the copy number of pmoA genes, a dissimilarity ordination ana-lysis revealed no significant difference in the methanotrophic community between BDE and SF fields (P=0.167). Eleven methanotrophic genera were identified in the methanotrophic community, and Methylosinus and Methylomicrobium were the most abundant, accounting for 32.3-36.7 and 45.7-47.3%, respectively. Type-I methanotrophs (69.4-73.7%) were more abundant than type-II methanotrophs (26.3-30.6%). Six methanotrophic strains belonging to 3 genera were successfully isolated, which included type I (Methylococcus sp. strain BE1 and Methylococcus sp. strain SF3) and type II (Methylocystis sp. strain BE2, Methylosinus sp. strain SF1, Methylosinus sp. strain SF2, and Methylosinus sp. strain SF4). This is the first study to examine the methanotrophic community structure in and isolate several methanotrophic strains from BDE-fertilized fields in VMD.

Exploring the rhizosphere of perennial wheat: potential for plant growth promotion and biocontrol applications.

Perennial grains, which remain productive for multiple years, rather than growing for only one season before harvest, have deep, dense root systems that can support a richness of beneficial microorganisms, which are mostly underexplored. In this work we isolated forty-three bacterial strains associated with the rhizosphere of the OK72 perennial wheat line, developed from a cross between winter common wheat and Thinopyrum ponticum. Identified using 16S rDNA sequencing, these bacteria were assessed for plant growth-promoting traits such as indole-3-acetic acid, siderophores and ACC-deaminase acid production, biofilm formation, and the ability to solubilize phosphate and proteins. Twenty-five strains exhibiting in vitro significant plant growth promoting traits, belong to wheat keystone genera Pseudomonas, Microbacterium, Variovorax, Pedobacter, Dyadobacter, Plantibacter, and Flavobacterium. Seven strains, including Aeromicrobium and Okibacterium genera, were able to promote root growth in a commercial annual wheat cultivar while strains from Pseudomonas genus inhibited the growth of Aspergillus flavus and Fusarium species, using direct antagonism assays. The same strains produced a high amount of 1-undecanol a volatile organic compound, which may aid in suppressing fungal growth. The study highlights the potential of these bacteria to form new commercial consortia, enhancing the health and productivity of annual wheat crops within sustainable agricultural practices.

Enterobacter spp. isolates from an underground coal mine reveal ligninolytic activity.

Lignin, the second most abundant renewable carbon source on earth, holds significant potential for producing biobased specialty chemicals. However, its complex, highly branched structure, consisting of phenylpropanoic units and strong carbon-carbon and ether bonds, makes it highly resistant to depolymerisation. This recalcitrancy highlights the need to search for robust lignin-degrading microorganisms with potential for use as industrial strains. Bioprospecting for microorganisms from lignin-rich niches is an attractive approach among others. Here, we explored the ligninolytic potential of bacteria isolated from a lignin-rich underground coalmine, the Morupule Coal Mine, in Botswana. Using a culture-dependent approach, we screened for the presence of bacteria that could grow on 2.5% kraft lignin-supplemented media and identified them using 16 S rRNA sequencing. The potential ligninolytic isolates were evaluated for their ability to tolerate industry-associated stressors. We report the isolation of twelve isolates with ligninolytic abilities. Of these, 25% (3) isolates exhibited varying robust ligninolytic ability and tolerance to various industrial stressors. The molecular identification revealed that the isolates belonged to the Enterobacter genus. Two of three isolates had a 16 S rRNA sequence lower than the identity threshold indicating potentially novel species pending further taxonomic review. ATR-FTIR analysis revealed the ligninolytic properties of the isolates by demonstrating structural alterations in lignin, indicating potential KL degradation, while Py-GC/MS identified the resulting biochemicals. These isolates produced chemicals of diverse functional groups and monomers as revealed by both methods. The use of coalmine-associated ligninolytic bacteria in biorefineries has potential.

Strain-resolved de-novo metagenomic assembly of viral genomes and microbial 16S rRNAs.

BACKGROUND: Metagenomics is a powerful approach to study environmental and human-associated microbial communities and, in particular, the role of viruses in shaping them. Viral genomes are challenging to assemble from metagenomic samples due to their genomic diversity caused by high mutation rates. In the standard de Bruijn graph assemblers, this genomic diversity leads to complex k-mer assembly graphs with a plethora of loops and bulges that are challenging to resolve into strains or haplotypes because variants more than the k-mer size apart cannot be phased. In contrast, overlap assemblers can phase variants as long as they are covered by a single read. RESULTS: Here, we present PenguiN, a software for strain resolved assembly of viral DNA and RNA genomes and bacterial 16S rRNA from shotgun metagenomics. Its exhaustive detection of all read overlaps in linear time combined with a Bayesian model to select strain-resolved extensions allow it to assemble longer viral contigs, less fragmented genomes, and more strains than existing assembly tools, on both real and simulated datasets. We show a 3-40-fold increase in complete viral genomes and a 6-fold increase in bacterial 16S rRNA genes. CONCLUSION: PenguiN is the first overlap-based assembler for viral genome and 16S rRNA assembly from large and complex metagenomic datasets, which we hope will facilitate studying the key roles of viruses in microbial communities. Video Abstract.

The hepatopancreas microbiome of velvet crab, Necora puber.

Crustaceans are a valuable resource globally, both ecologically and economically, and investigations into their health are becoming increasingly important as exploitation rises. The microbiome plays a crucial role in crustacean immunity, and understanding its composition and structure can provide insights into the health of an organism and its interactions with various factors. In this study, we investigated the hepatopancreas microbiome of the velvet swimming crab, Necora puber, and compared its composition and structure with several study factors, including two different sampling points and infection with a paramyxid parasite, Paramarteilia canceri. To our knowledge, we provide the first description of a velvet crab microbiome, highlighting the dominance of a single microorganism, Candidatus hepatoplasma. We identified variations in microbiome composition between sampling points and discussed the possible processes affecting microbiome assembly. We also outline a core microbiome for the velvet crab hepatopancreas, consisting of 12 core phyla. Our study adds to the growing literature on crustacean microbiomes and provides a baseline for future investigations into the velvet crab microbiome and the health of this crustacean species.

Adlercreutzia wanghongyangiae sp. nov., and Adlercreutzia shanghongiae sp. nov., two new members of the genus Adlercreutzia isolated from plateau pika (Ochotona curzoniae).

Four anaerobic, Gram-stain-positive, non-motile, non-sporulating rod-shaped bacterial strains (R7T, R21, R22 and R25T) were isolated from the intestinal contents of plateau pika (Ochotona curzoniae) collected from the Qinghai-Tibet Plateau, PR China. The four isolates grew at between 25 and 42 °C (optimally at 35-37 °C), and with 0.3-3.3% NaCl (w/v) [optimum, 1.3% (w/v)]. Adding l-arginine to the medium could promote their growth. Strains R7T and R21 were most closely related to Adlercreutzia caecimuris B7T (97.48% 16S rRNA gene sequence similarity). Strains R25T and R22 were most closely related to Adlercreutzia equolifaciens DSM 19450T (98.25% 16S rRNA gene sequence similarity). The genome sequences of R7T and R25T were 2.89 and 2.90 Mb in size with 63.6 and 62.8 mol% DNA G+C contents, respectively. Phylogenetic analysis based on 16S rRNA gene sequences and core genes revealed that R7T and R21 were most closely related to A. caecimuris B7T and Adlercreutzia mucosicola DSM 19490T, whereas R25T and R22 were most closely related to A. equolifaciens DSM 19450T and Adlercreutzia rubneri ResAG-91T. R7T, R25T and the closely related species had average nucleotide identity (ANI) values of 81.9-83.2% as well as digital DNA-DNA hybridisation (dDDH) values between 27.3 and 27.9%, which clearly indicated that they represent two novel species within the genus Adlercreutzia. For R7T and R25T, meso-diaminopimelic acid was the diagnostic diamino acid in the cell-wall peptidoglycan, and the whole cell sugars included galactose, glucose and ribose. On the basis of these results, we propose that strains R7T and R25T represent two novel species of the genus Adlercreutzia, namely Adlercreutzia wanghongyangiae sp. nov. and Adlercreutzia shanghongiae sp. nov., respectively. The type strains are R7T (=GDMCC 1.4459T=KCTC 25860T) and R25T (=GDMCC 1.4458T=KCTC 25861T).

Pseudoalteromonas simplex sp. nov. Isolated from the Skin of Bandtail Puffer Fish (Sphoeroides spengleri).

A novel bacterial isolate A520T (A520T = CBAS 737T = CAIM 1944T) was obtained from the skin of bandtail puffer fish Sphoeroides spengleri (Tetraodontidae Family), collected in Arraial do Cabo (Rio de Janeiro, Brazil). A520T is Gram-stain-negative, flagellated and aerobic bacteria. Optimum growth occurs at 25-30 °C in the presence of 3% NaCl. The genome sequence of the novel isolate consisted of 4.5 Mb (4082 coding genes and G+C content of 41.1%). The closest phylogenetic neighbor was Pseudoalteromonas shioyasakiensis JCM 18891T (97.9% 16S rRNA sequence similarity, 94.8% Average Amino Acid Identity, 93% Average Nucleotide Identity and 51.8% similarity in Genome-to-Genome-Distance). Several in silico phenotypic features are useful to differentiate A520T from its closest phylogenetic neighbors, including trehalose, D-mannose, cellobiose, pyrrolidonyl-beta-naphthylamide, starch hydrolysis, D-xylose, lactose, tartrate utilization, sucrose, citrate, glycerol, mucate and acetate utilization, malonate, glucose oxidizer, gas from glucose, nitrite to gas, L-rhamnose, ornithine decarboxylase, lysine decarboxylase and yellow pigment. The genome of the novel species contains 3 gene clusters (~ 66.81 Kbp in total) coding for different types of bioactive compounds that could indicate ecological roles pertaining to the bandtail puffer fish host. Based on genome-based taxonomic approach, strain A520T (A520T = CBAS 737T = CAIM 1944T) is proposed as a new species, Pseudoalteromonas simplex sp. nov.

Chromobacterium indicum sp. nov., a Pigment-Producing Bacterium Isolated from Soil.

A purple colony, designated as TRC1.1.SA was isolated from a tea garden soil sample. It was a Gram-negative, rod-shaped, non-spore-forming and motile bacterium. The strain TRC1.1.SAT grew aerobically at temperatures 15-37 â„ƒ and pH levels 5.0-9.0. It showed both oxidase and catalase activity. The 16S rRNA gene sequence blast analysis revealed identity with the members of the genus Chromobacterium. The maximum identity was with the type strains of species Chromobacterium piscinae CCM 3329T (99.8%), C. vaccinii MWU205T (99.7%), and C. violaceum ATCC 12472T (98.7%). However, the average nucleotide identity (ANI) of the genome sequence showed less than 96% similarity with all species of the genus Chromobacterium. Further, digital DNA-DNA hybridization (dDDH) revealed the highest identity of 63.4% with its phylogenetic relative C. piscinae CCM 3329T. The G + C content of the strain was 63.9%. The major polar lipids identified were phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), and phosphoglyceraldehyde (PG). Fatty acid analysis showed C16:0, C16:1ω7c, C17:0 cyclo, and C18:1ω7c as the major fatty acids. RAST and antiSMASH analyses of the genome revealed the presence of a biosynthetic gene cluster (BGC) involved in the production of violacein pigment, as observed for type species C. violaceum ATCC 12472T. Considering the phenotypic differences and genomic identity, strain TRC1.1.SAT is assigned as a novel species of the genus Chromobacterium, for which the name Chromobacterium indicum is proposed. The type strain of prospective species is designated as TRC1.1.SAT (= MTCC 13391T; JCM 36723T; = KCTC 8324T).

Sex-specific post-inflammatory dysbiosis mediates chronic visceral pain in colitis.

BACKGROUND: Despite achieving endoscopic remission, over 20% of inflammatory bowel disease (IBD) patients experience chronic abdominal pain. Visceral pain and the microbiome exhibit sex-dependent interactions, while visceral pain in IBD shows a sex bias. Our aim was to evaluate whether post-inflammatory microbial perturbations contribute to visceral hypersensitivity in a sex-dependent manner. METHODS: Males, cycling females, ovariectomized, and sham-operated females were given dextran sodium sulfate to induce colitis and allowed to recover. Germ-free recipients received sex-appropriate and cross-sex fecal microbial transplants (FMT) from post-inflammatory donor mice. Visceral sensitivity was assessed by recording visceromotor responses to colorectal distention. The composition of the microbiota was evaluated via 16S rRNA gene V4 amplicon sequencing, while the metabolome was assessed using targeted (short chain fatty acids - SCFA) and semi-targeted mass spectrometry. RESULTS: Post-inflammatory cycling females developed visceral hyperalgesia when compared to males. This effect was reversed by ovariectomy. Both post-inflammatory males and females exhibited increased SCFA-producing species, but only males had elevated fecal SCFA content. FMT from post-inflammatory females transferred visceral hyperalgesia to both males and females, while FMT from post-inflammatory males could only transfer visceral hyperalgesia to males. CONCLUSIONS: Female sex, hormonal status as well as the gut microbiota play a role in pain modulation. Our data highlight the importance of considering biological sex in the evaluation of visceral pain.

Dynamic alterations and ecological implications of rice rhizosphere bacterial communities induced by an insect-transmitted reovirus across space and time.

BACKGROUND: Cereal diseases caused by insect-transmitted viruses are challenging to forecast and control because of their intermittent outbreak patterns, which are usually attributed to increased population densities of vector insects due to cereal crop rotations and indiscriminate use of pesticides, and lack of resistance in commercial varieties. Root microbiomes are known to significantly affect plant health, but there are significant knowledge gaps concerning epidemics of cereal virus diseases at the microbiome-wide scale under a variety of environmental and biological factors. RESULTS: Here, we characterize the diversity and composition of rice (Oryza sativa) root-associated bacterial communities after infection by an insect-transmitted reovirus, rice black-streaked dwarf virus (RBSDV, genus Fijivirus, family Spinareoviridae), by sequencing the bacterial 16S rRNA gene amplified fragments from 1240 samples collected at a consecutive 3-year field experiment. The disease incidences gradually decreased from 2017 to 2019 in both Langfang (LF) and Kaifeng (KF). BRSDV infection significantly impacted the bacterial community in the rice rhizosphere, but this effect was highly susceptible to both the rice-intrinsic and external conditions. A greater correlation between the bacterial community in the rice rhizosphere and those in the root endosphere was found after virus infection, implying a potential relationship between the rice-intrinsic conditions and the rhizosphere bacterial community. The discrepant metabolites in rhizosphere soil were strongly and significantly correlated with the variation of rhizosphere bacterial communities. Glycerophosphates, amino acids, steroid esters, and triterpenoids were the metabolites most closely associated with the bacterial communities, and they mainly linked to the taxa of Proteobacteria, especially Rhodocyclaceae, Burkholderiaceae, and Xanthomonadales. In addition, the greenhouse pot experiments demonstrated that bulk soil microbiota significantly influenced the rhizosphere and endosphere communities and also regulated the RBSDV-mediated variation of rhizosphere bacterial communities. CONCLUSIONS: Overall, this study reveals unprecedented spatiotemporal dynamics in rhizosphere bacterial communities triggered by RBSDV infection with potential implications for disease intermittent outbreaks. The finding has promising implications for future studies exploring virus-mediated plant-microbiome interactions. Video Abstract.

Molecular characteristics and antimicrobial susceptibility profiles of bovine mastitis agents in western Türkiye.

IMPORTANCE: Identifying bovine mastitis agents using molecular methods to reveal their phylogenetic relationships and antimicrobial resistance profiles is essential for developing up-to-date databases in mastitis cases that cause severe economic losses. OBJECTIVE: This study examined bacterial mastitis agents in cows with clinical and subclinical mastitis observed in various dairy cattle farms to reveal their phylogenetic relationships and antibiotic resistance properties. METHODS: Sixty-two clinical and subclinical bovine mastitis milk samples were collected from 15 dairy farms. The polymerase chain reaction (PCR) was used to amplify the 16S rRNA gene regions of the bacteria. The 16S rRNA gene sequences obtained from sequencing include the V4-V6 regions. The strains were compared using a similarity analysis method that produced phylogenetic trees using the Molecular Evolutionary Genetics Analysis 11 program. Antibiotic susceptibilities were determined using the Kirby-Bauer disk diffusion method. RESULTS: Sixty-three bacteria were isolated and identified in this study. The most isolated bacteria from all mastitis cases were Staphylococcus spp. (30.2%), Escherichia coli (25.4%), Streptococcus spp. (14.3%), and Aerococcus spp. (7.9%), respectively. The phylogenetic trees were drawn from the 16S rRNA sequences. Some of these bacteria showed resistance to different types of antibiotics at varying rates. CONCLUSIONS AND RELEVANCE: The bacteria isolated in this study originated from environmental sources. Regular cleaning of barns and proper hygiene practices are essential. Regular screenings for mastitis should be conducted in herds instead of the random or empirical use of antibiotics.

High-quality genome of a novel Thermosynechococcaceae species from Namibia and characterization of its protein expression patterns at elevated temperatures.

Thermophilic cyanobacteria thrive in extreme environments, making their thermoresistant enzymes valuable for industrial applications. Common habitats include hot springs, which act as evolutionary accelerators for speciation due to geographical isolation. The family Thermosynechococcaceae comprises thermophilic cyanobacteria known for their ability to thrive in high-temperature environments. These bacteria are notable for their photosynthetic capabilities, significantly contributing to primary production in extreme habitats. Members of Thermosynechococcaceae exhibit unique adaptations that allow them to perform photosynthesis efficiently at elevated temperatures, making them subjects of interest for studies on microbial ecology, evolution, and potential biotechnological applications. In this study, the genome of a thermophilic cyanobacterium, isolated from a hot spring near Okahandja in Namibia, was sequenced using a PacBio Sequel IIe long-read platform. Cultivations were performed at elevated temperatures of 40, 50, and 55°C, followed by proteome analyses based on the annotated genome. Phylogenetic investigations, informed by the 16S rRNA gene and aligned nucleotide identity (ANI), suggest that the novel cyanobacterium is a member of the family Thermosynechococcaceae. Furthermore, the new species was assigned to a separate branch, potentially representing a novel genus. Whole-genome alignments supported this finding, revealing few conserved regions and multiple genetic rearrangement events. Additionally, 129 proteins were identified as differentially expressed in a temperature-dependent manner. The results of this study broaden our understanding of cyanobacterial adaptation to extreme environments, providing a novel high-quality genome of Thermosynechococcaceae cyanobacterium sp. Okahandja and several promising candidate proteins for expression and characterization studies.

The phyllosphere of Nigerian medicinal plants, Euphorbia lateriflora and Ficus thonningii is inhabited by a specific microbiota.

The microbiota of medicinal plants is known to be highly specific and can contribute to medicinal activity. However, the majority of plant species have not yet been studied. Here, we investigated the phyllosphere composition of two common Nigerian medicinal plants, Euphorbia lateriflora and Ficus thonningii, by a polyphasic approach combining analyses of metagenomic DNA and isolates. Microbial abundance estimated via qPCR using specific marker gene primers showed that all leaf samples were densely colonized, with up to 108 per gram of leaf, with higher bacterial and fungal abundance than Archaea. While no statistically significant differences between both plant species were found for abundance, amplicon sequencing of 16S rRNA and ITS genes revealed distinct microbiota compositions. Only seven of the 27 genera isolated were represented on both plants, e.g. dominant Sphingomonas spp., and numerous members of Xanthomonadaceae and Enterobacteriaceae. The most dominant fungal families on both plants were Cladosporiaceae, Mycosphaerellaceae and Trichosphaeriaceae. In addition, 225 plant-specific isolates were identified, with Pseudomonadota and Enterobacteriaceae being dominant. Interestingly, 29 isolates are likely species previously unknown, and 14 of these belong to Burkholderiales. However, a high proportion, 56% and 40% of the isolates from E. lateriflora and F. thonningii, respectively, were characterized as various Escherichia coli. The growth of most of the bacterial isolates was not influenced by extractable secondary metabolites of plants. Our results suggest that a specific and diverse microbial community inhabits the leaves of both E. lateriflora and F. thonningii, including potentially new species and producers of antimicrobials.