rDNA Sequences Research Articles

Tropical fruit-derived Lactiplantibacillus as potential probiotic and antifungal agents against Fusarium oxysporum

Abstract Fifty-five lactic acid bacteria (LAB) were isolated from seven selected tropical fruits, with Solanum nigrum exhibiting the highest LAB prevalence and Couroupita guianenis and Musa fruits showing the lowest counts. Two strains isolated from Ficus racemosa demonstrated significant antifungal activity against Fusarium oxysporum. 16S rDNA sequencing identified these strains as Lactiplantibacillus plantarum MYSVCF3 and Lpb. argentoratensis MYSVCF5. The isolates displayed adaptability to a broad range of environmental conditions, including temperatures of 10–45 °C, pH 2–6, and salt up to 7%. The strains tolerated simulated gastrointestinal conditions of acid (pH-2), phenol (0.6%), and bile (0.3%) suggesting potential probiotic attributes. Lpb. argentoratensis MYSVCF5 inhibited F. oxysporum, two ESKAPE group bacteria (P. aeruginosa, S. aureus) plus S. paratyphi and E. coli. The cell-free supernatant (CFS) of Lpb. argentoratensis MYSVCF5 reduced the growth of fungal biomass by 94% and completely inhibited conidial germination, retaining activity even after extended cold storage. LC–MS/MS analysis identified organic acids in the CFS, with citric acid as the most abundant at 34.9 (± 0.3) µg/mL, followed by lactic (8.3 µg/mL) and malic acids (5.2 µg/mL). This study isolated a novel LAB, a potential candidate having probiotics and antifungal properties for application in food and agriculture.

Morphology and phylogenetic position of two novel Dysteria species (Ciliophora: Cyrtophoria) from deep-sea hydrothermal vents on the Central Indian Ridge, Indian Ocean

This study reports the discovery of two novel ciliate species – Dysteria brevicauda sp. nov. and Dysteria parasemilunaris sp. nov. – inhabiting unique environments in recently explored hydrothermal vent fields at the Onnuri and Onbada vents (1978 m and 2523 m depths, respectively) along the Central Indian Ridge in the Indian Ocean. The species were identified using a combination of morphological and molecular approaches. The morphology of D. brevicauda sp. nov. (65–110 × 20–30 μm) is characterized by a protruding dorso-anterior part, a short tail-like structure, converging outer two right kineties at the posterior end, and two frontoventral kineties with a few gaps in the anterior part. D. parasemilunaris sp. nov. (50–70 × 25–35 μm) can be recognized by its elongated oval shape with a dorsoapical collar-like protrusion, a terminally positioned stout podite, and five right kineties, including two frontoventral kineties. D. parasemilunaris sp. nov., which inhabits both the Onnuri and Onbada vents, is morphologically almost identical to Dysteria sp. collected from another hydrothermal vent, Clam Acres, in 1985. Thus, these are considered conspecific species. Phylogenetic analysis based on SSU rDNA sequences places the two new species within Dysteria Group III and V clades, respectively. D. brevicauda sp. nov. is positioned between Spirodysteria kahli (97.3% similarity) and D. subtropica (97.1% similarity). Meanwhile, D. parasemilunaris sp. nov. forms a cluster with D. semilunaris (95.3% similarity) and branches off from D. lanceolata (94.3% similarity). These findings contribute to the understanding of ciliate biodiversity in deep-sea hydrothermal vents. http:zoobank.org/urn:lsid:zoobank.org:pub:FE45985A-C0C1-4BAC-9471-3CC5D6180FEC.

Exploring the Effects of Gracilaria lemaneiformis Polysaccharides on the Fecal Microbiota and Fecal Metabolites of Fattening Pigs Based on 16S rDNA and Metabolome Sequencing

Exploring the Effects of Gracilaria lemaneiformis Polysaccharides on the Fecal Microbiota and Fecal Metabolites of Fattening Pigs Based on 16S rDNA and Metabolome Sequencing

Therapeutic Potential of Cajanus cajan (L.) Millsp. Leaf Extract in Modulating Gut Microbiota and Immune Response for the Treatment of Inflammatory Bowel Disease

Background/Objectives: Inflammatory bowel disease (IBD) is a persistent inflammatory condition affecting the gastrointestinal tract, distinguished by the impairment of the intestinal epithelial barrier, dysregulation of the gut microbiota, and abnormal immune responses. Cajanus cajan (L.) Millsp., traditionally used in Chinese herbal medicine for gastrointestinal issues such as bleeding and dysentery, has garnered attention for its potential therapeutic benefits. However, its effects on IBD remain largely unexplored. Methods: In this study, the major compounds from Cajanus cajan leaf extract (CCLE) were initially characterized by LCMS-IT-TOF. The IBD model was developed in C57BL/6 mice by administering continuous 4% (w/v) dextran sodium sulfate (DSS) aqueous solution over a period of seven days. The body weight, colon length, disease activity index (DAI), and histopathological examination using hematoxylin and eosin (H&E) staining were performed in the IBD model. The levels of the main inflammatory factors, specifically TNF-α, IL-1β, IL-6, and myeloperoxidase (MPO), were quantified by employing enzyme-linked immunosorbent assay (ELISA) kits. Additionally, the levels of tight junction proteins (ZO-1, Occludin) and oxidative stress enzymes (iNOS, SOD1, CAT) were investigated by qPCR. Subsequently, flow cytometry was employed to analyze the populations of various immune cells within the spleen, thereby assessing the impact of the CCLE on the systemic immune homeostasis of IBD mice. Finally, 16S rDNA sequencing was conducted to examine the composition and relative abundance of gut microbiota across different experimental groups. In addition, molecular docking analysis was performed to assess the interaction between the principal components of CCLE and the aryl hydrocarbon receptor (AHR). Results: We identified seven bioactive compounds in CCLE: catechin, cajachalcone, 2-hydroxy-4-methoxy-6-(2-phenylcinyl)-benzoic acid, longistylin A, longistylin C, pinostrobin, amorfrutin A, and cajaninstilbene acid. Our results demonstrated that oral administration of CCLE significantly alleviates gastrointestinal symptoms in DSS-induced IBD mice by modulating the balance of gut-derived pro- and anti-inflammatory cytokines. This modulation is associated with a functional correction in M1/M2 macrophage polarization and the Th17/Treg cell balance in splenic immune cells, as well as shifts in the populations of harmful bacteria (Erysipelatoclostridium and Staphylococcus) and beneficial bacteria (Odoribacter, unidentified Oscillospiraceae, Lachnoclostridium, and Oscillibacter) in the gut. Furthermore, cajaninstilbene acid, longistylin A, and longistylin C were identified as potential AhR agonists. Conclusions: The present results suggested that CCLE, comprising stilbenes like cajaninstilbene acid, longistylin A, and longistylin C, protects the epithelial barrier’s structure and function against DSS-induced acute IBD by restoring gut microbiota balance and systemic immune response as AhR agonists. Overall, CCLE represents a promising natural product-based therapeutic strategy for treating IBD by restoring gut microbiota balance and modulating systemic immune responses.

Effect of intermittent water flow on biodegradation of organic micropollutants in the hyporheic zone.

Water scarcity in the Mediterranean area has increased the number of intermittent rivers. Recently, hyporheic zones (HZ) of intermittent rivers have gained attention since a substantial part of the stream's natural purification capacity is located within these zones. Thus, understanding the flow dynamics in HZs is crucial for gaining insights into the degradation of organic micropollutants. A lab-scale study using column experiments was conducted in an attempt to elucidate the environmental processes accounting for the biodegradation capacity of the HZ under flow intermittency. A mixture of six compounds including pesticides (chloranthraniliprole, fluopyram and trifloxystrobin) and pharmaceuticals (venlafaxine, amisulpride and paroxetine) spiked at 1μg/L level was used for degradation kinetic studies and at 1mg/L for transformation products identification using suspect/non-target liquid chromatography high-resolution mass spectrometry approaches. The experiments lasted 60 days, divided into two 14-day phases: one before and one after a 5-week desiccation period. Bacterial community was charaterized by high-throughput DNA sequencing. The results suggested that intermittent flows stimulated the biodegradation of three compounds namely fluopyram, trifloxystrobin and venlafaxine, showing a large range of biodegradation profiles in batch water/sediment testing system according to OECD 308 tests. Biodegradation rate enhancement was ascribed to the occurrence of additional transformation routes after the desiccation period of river sediment, with the formation of new transformation products reported for the first time in the present work. 16S rDNA sequencing revealed that the desiccation period favored the growth of nitrifying and denitrifying bacteria which could partially explain the emergence of the new transformation pathways and most specifically those leading to N-oxide derivatives. Identification of transformation products also revealed that reductive transformation routes were relevant for this study, being dehydrogenation, dehalogenation, ether bond cleavage and sulfone reduction into sulphide important reactions. These results suggest that the intermittent flow conditions can influence the HZ biodegradation capacity.

Molecular Phylogenetic Relationships and Cultural Conditions of Calocybe indica

Calocybe indica known as a milky white, indigenous and medicinally important edible mushroom. The cultural conditions, molecular identification, and phylogenetic relationships of selected strains of Calocybe indica were examined. The optimal temperature for mycelial development was found to be 30°C, whereas the lowest growth occurred at 15°C. The most favorable growth occurs at a pH of 5. Based on the growth characteristics of various culture media, potato dextrose agar proved to be the most advantageous, while Hennerberg and Czapek Dox media were less suitable. Molecular identification and the phylogenetic relationships of the chosen IUM strains of C. indica were examined through their ITS and RAPD analyses. The selected strains of C. indica had total sequence lengths of ITS1, ITS2, and 5.8S rDNA ranging from 570 to 620 bp. The sizes of the ITS1 and ITS2 regions vary among strains, with ITS2 showing greater variety than ITS1. All strains contained identical 5.8S rDNA sequences, according to sequencing analysis. Eleven out of the twenty arbitrary 10-base oligonucleotide primers utilized to amplify genomic DNA segments for the chosen C. indica strains were effective. RAPD-PCR resulted in distinct multiple products, demonstrating significant variability among the strains assessed. The quantity of amplified bands differed based on the primers utilized or the strains evaluated. The DNA polymorphisms displayed consistent traits in the replicated trials. Thus, it can be concluded that ITS sequencing and RAPD methods were effective in identifying the genetic diversity of the chosen strains of Calocybe indica. Plant Tissue Cult. & Biotech. 34(2): 205-216, 2024 (December)

Gut microbial composition associated with risk of premature aging in women with Yin-deficiency constitution

BackgroundYin-deficiency constitution (YinDC) refers to a traditional Chinese medicine concept, characterized by an imbalance state that includes an imbalance in the gut microbiota, resulting from a relative deficiency of Yin fluids within the body. In recent years, it has become apparent that the composition and structure of the gut microbiota play a significant role in the aging process. The imbalance of gut microbiota in YinDC may accelerate the aging process. However, the specific gut microbiota compositions involved in the YinDC premature aging process remain unknown.MethodsIn this study, we conducted a cohort study including 60 women with YinDC and BC to analyze their gut microbiota composition. We integrated 16S rDNA sequencing with machine learning methods to reveal the association between gut microbiota and premature aging in YinDC women.ResultsWe found a significant difference in the composition of gut microbiota between the YinDC and the BC group. At the phylum level, Cyanobacteria and Synerobacteria only emerged in the YinDC group. At the genus level, Bacteroides, Bifidobacterium, Haemophilus, Alistipes, and Dialister showed higher abundance in the YinDC group. Bilophila, Eubacterium, and Aeromonas were the most significant indicators influencing the YinDC premature aging. The YinDC group had the most functional gene pathways associated with the metabolism.ConclusionOur study demonstrated that the gut microbiota was associated with premature aging in women with YinDC, potentially providing preliminary evidence and guidance for personalized anti-aging strategies.

Two new myxozoan parasites, Myxobolus mayarum n. sp. and Kudoa mayarum n. sp., infecting the neotropical fish Mayan Cichlid, Mayaheros urophthalmus (Günther, 1862) in the Yucatán Peninsula, Mexico.

The biodiversity of freshwater fishes is extensive in Mexico; however, knowledge of their associated myxozoan parasites is limited. This study aimed to recognize myxozoan parasites in the endemic fish Mayaheros urophthalmus. Two new species, Myxobolusmayarum n. sp. and Kudoamayarum n. sp. were described from M. urophthalmus collected in the Celestún Coastal Lagoon and Baldiosera Freshwater Spring from the Yucatán Peninsula. Myxobolusmayarum n. sp. was found in 100% of the examined fish, infecting the gills, kidney, pectoral fins, and spleen. The spores were round, 11.34 ± 0.67 μm length, 10.03 ± 0.44 μm width, and 7.22 ± 0.45 μm thickness. The polar capsules were pyriform and equal in size, 3.85 ± 0.29 μm length and 2.27 ± 0.20 μm width. The polar tubule 30.71 ± 2.10 μm long and coiled 5-6 times. Kudoamayarum n. sp. was found in 30% of fish, infecting the heart, oesophagus, and stomach. The spores were subspherical, 5.40 ± 0.40 μm length and 5.76 ± 0.37 μm width. The polar capsules were ellipsoidal and equal in size, 1.90 ± 0.27 μm length and 1.46 ± 0.20 μm width. The phylogenetic analyses based on the small and large subunits ribosomal DNA (18S rDNA and 28S rDNA) sequences demonstrated that these two species are distinct from other published myxozoans, providing evidence to support the description of two new species. This study constitutes the first record of myxozoans from the Yucatán Peninsula, contributing to increase the knowledge about the biodiversity of myxozoans in neotropical regions.

The role of the microbiota and metabolites in the treatment of pulmonary fibrosis with UC-MSCs: Integrating fecal metabolomics and 16S rDNA analysis.

Pulmonary fibrosis (PF) is a chronic and irreversible interstitial lung disease characterized by a lack of effective therapies. Mesenchymal stem cells (MSCs) have garnered significant interest in the realm of lung regeneration due to their abundant availability, ease of isolation, and capacity for expansion. The objective of our study was to investigate the potential therapeutic role of umbilical cord-derived MSCs (UC-MSCs) in the management of PF, with a focus on the alterations in the gut microbiota and its metabolites during the use of UC-MSCs for the treatment of pulmonary fibrosis, as well as the possible mechanisms involved. Bleomycin injection was utilized to establish a mouse model of lung fibrosis, followed by the application of 16S rDNA sequencing and LC-MS/MS metabolomics to explore the underlying mechanism of UC-MSC treatment for lung fibrosis. Seventy-five mice were allocated into five groups, namely Control, Model, and low/medium/high dose of UC-MSCs groups, and survival metrics, lung morphology, and the levels of the inflammatory cytokines TNF-α, IL-1β, IL-6, and TGF-β1 were subsequently evaluated. Fecal samples from six mice in each of the Control group, Model group, and UC-MSCs-M groups were collected randomly for 16S rDNA sequencing to analyze the gut microbiota and nontargeted metabolomics. In comparison to IPF model mice, the three treatment groups exhibited increased survival rates, restored alveolar morphology, and reduced levels of the inflammatory cytokines TNF-α, IL-1β, IL-6, and TGF-β1, confirming the anti-inflammatory properties of UC-MSCs in IPF treatment. The findings from the 16S rDNA assay indicate that UC-MSCs treatment effectively lower α-diversity induced such as Chao 1 and ACE, as well as β-diversity, leading to a decrease in microbiota abundance. The findings from the metabolomics analysis revealed that the metabolites exhibiting notable variances were primarily composed of Lipids and lipid-like molecules, Organoheterocyclic compounds, Organic acids and derivatives, and Benzenoids, indicating the potential of UC-MSCs to exert antifibrotic effects via these metabolic pathways. Umbilical cord-derived mesenchymal stem cells (UC-MSCs) ameliorate bleomycin-induced pulmonary fibrosis symptoms in mice by exerting anti-inflammatory effects and mitigating pulmonary fibrosis through the modulation of gut microbiota disorders and their metabolism. These findings offer novel insights into the potential mechanisms and clinical utility of stem cell therapy for pulmonary fibrosis.

Lachnoclostridium intestinal flora is associated with immunotherapy efficacy in nasopharyngeal carcinoma.

Effective biomarkers for assessing anti-PD-1/PD-L1 therapy efficacy in patients with nasopharyngeal carcinoma (NPC) are still lacking. The human gut microbiota has been shown to influence clinical response to anti-PD-1/PD-L1 therapy in many cancers. However, the relationship between the gut microbiota and the efficacy of immunotherapy in patients with nasopharyngeal carcinoma has not been determined. We conducted a prospective study in which fecal and blood samples from patients with NPC were subjected to 16S rDNA sequencing and survival analysis. To investigate potential differences in the gut microbiome between these groups and to identify potential biomarkers indicative of immunotherapy efficacy, patients were categorized into two groups according to their clinical response to immunotherapy, the responder group (R group) and the non-responder group (NR group). Progression-free survival (PFS) between these subgroups was analyzed using Kaplan-Meier survival analysis with the log-rank test. Additionally, we performed univariate and multivariate analyses to evaluate prognostic factors. Finally, we carried out non-targeted metabolomics to examine the metabolic effects associated with the identified microbiome. Our 16S rDNA sequencing results showed that the abundance of Lachnoclostridium was higher in the NR group than in the R group (p = 0.003), and alpha diversity analysis showed that the abundance of microbiota in the NR group was higher than that in the R group (p = 0.050). Patients with a lower abundance of Lachnoclostridium had better PFS (p = 0.048). Univariate (p = 0.017) and multivariate analysis (p = 0.040) showed that Lachnoclostridium was a predictor of PFS. Non-targeted metabolomics analysis revealed that Lachnoclostridium affects the efficacy of immunotherapy through the usnic acid. High abundance of Lachnoclostridium predicts poor prognosis in patients with NPC receiving immunotherapy.

Oral-Delivery Lactococcus lactis Expressing mCherry Fusion Lactoferrin Peptides against Infection of Avian Pathogenic Escherichia coli in Chickens

Avian pathogenic Escherichia coli (APEC) infections result in significant economic losses and reduced animal welfare. Historically, antibiotics and vaccinations currently control APEC infections in poultry, however, antibiotic-resistant strains and heterologous serotypes limit their effectiveness. Meanwhile, antibiotic-resistant strains can be transmitted to humans via contact with animals, food or their environment. Probiotics and antimicrobial peptides (AMPs) are potential alternatives to antibiotics and represent promising strategies to combat APEC. Bovine lactoferricin and lactoferrampin possess anti-bacterial, anti-inflammatory, and anti-oxidant properties. Lactococcus lactis (L. lactis) is an excellent vector for delivering recombinant proteins. In this research, we generated a recombinant L. lactis strain MG1363 expressing lactoferrin peptides, which was labeled with a fluorescent marker mCherry and lacked an antibiotic resistance gene (LL-EFLmC). Our investigation focused on the impact of LL-EFLmC strain on the gut microbiota composition and avian pathogenic E. coli O78 challenge. Our findings indicate that LL-EFLmC exhibits inhibitory effects against APEC-O78 and Staphylococcus aureus CVCC26003 (S. aureus CVCC26003) in vitro. Furthermore, the inclusion of LL-EFLmC in chicken feed significantly improved the average daily intake and gain to feed ratio. Additionally, LL-EFLmC treatment resulted in a significant increase in serum IgG and intestinal mucus SIgA levels. Administration of LL-EFLmC was found to effectively suppress APEC-O78 infection and mitigate the expression of pro-inflammatory cytokines, including IL-1β, IL-12, IFN-γ, and TNF-α. Additionally, 16S rDNA sequencing data revealed that LL-EFLmC was able to restore the intestinal flora that had been disrupted by APEC-O78. These findings suggest that LL-EFLmC may serve as a promising feed additive and antibiotic alternative in chicken production, due to its potential to enhance immune regulation, promote growth, and confer resistance against APEC-O78 infection.

Progesterone Regulates Gut Microbiota Mediating Bone Marrow MSCs Injury in ITP Patients during Pregnancy.

Immune thrombocytopenia during pregnancy (PITP) is the most common cause of platelet reduction in early and mid-pregnancy. However, the pathogenesis of PITP is still unclear. To determine the characteristics of bone marrow mesenchymal stem cells (BM-MSCs) in PITP patients and to explore the associations between metabolites, the gut microbiota, and BM-MSCs in PITP. The characteristics of BM-MSCs were detected through in vitro and in vivo experiments. Non-targeted metabolomics was used to screen metabolites. The features of the gut microbiota were analyzed by 16S rDNA sequencing. PITP and a fecal microbiota transplantation (FMT) mouse model were established to explore the associations between metabolites, the gut microbiota, and BM-MSCs. BM-MSCs from PITP patients had significant senescence and apoptosis, as well as impaired immunoregulatory function. Metabolomic analysis indicated that progesterone was the most significant specific metabolite in PITP patients. In vivo studies showed that progesterone mediated the MSCs injury. Further analysis of the gut microbiota and FMT experiments revealed that progesterone mediated BM-MSCs injury by regulating the the composition of the gut microbiota in the PITP. RNA-seq analysis of BM-MSCs from FMT mice revealed abnormal expression of genes related to cell aging and the NOD-like receptor signaling pathway. In conclusion, BM-MSCs in the PITP were significantly impaired, which was associated with increased progesterone and changes in the gut microbiota regulated by progesterone. Intervening with the gut microbiota may become a new treatment for PITP.

Gongronella fusoacuminata (Cunninghamellaceae, Mucorales), a new species from Taiwan

Rhizosphere fungi play crucial roles in plant health and soil ecology. We describe and illustrate Gongronella fusoacuminata, a new species isolated from the rhizosphere soil of pine and mallow in Taiwan. Phylogenetic analyses based on both ITS and LSU rDNA sequences, in conjunction with unique morphological characteristics, confirmed its distinct taxonomic classification within the genus. The phylogenetic analyses revealed that G. fusoacuminata possesses distinctive sequences, clearly differentiating it from other species within the genus. In addition, G. fusoacuminata is distinguished by its fusiform sporangiospores with acuminate apices and their relatively large size, clearly discerning it from other documented species.

Taxonomic and molecular characterization of Pseudosteringophorusprofundis sp. nov. (Digenea, Fellodistomidae), a parasite of Macrourusholotrachys Günther, 1878 (Gadiformes, Macrouridae) from the deep sea southeastern Pacific Ocean.

Pseudosteringophorusprofundis sp. nov. a new species of deep-sea digenean, parasitizing the gallbladder of the "Bigeye grenadier" (Macrourusholotrachys Günther, 1878) in the deep waters of the southeastern Pacific Ocean is described on the basis of morphological and molecular (28S rRNA) data. The new species is distinguishable from Pseudosteringophorushoplognathi Yamaguti, 1940, the only other member of the genus, by its subterminal oral sucker, the position of the ovary and testes, the larger anterior seminal vesicle compared to the posterior one, and its larger eggs. In addition, the new species is a parasite of a deep-sea fish, whereas P.hoplognathi is a parasite of shallow-water fish. A phylogenetic tree, based on 28S rDNA sequences, indicates that this species is included in a clade of deep-sea fellodistomid species (Steringophorus spp.). We provide the first molecular data on the genus Pseudosteringophorus Yamaguti, 1940 and expand the molecular database for the family Fellodistomidae. Further studies, including sequences from other fellodistomid taxa, are needed to more precisely infer relationships within this family.

Elucidation of intragenomic variation of ribosomal DNA sequences in the enigmatic fungal genus Ceraceosorus, including a newly described species Ceraceosorus americanus

AbstractMulticopy nuclear ribosomal DNA (rDNA) genes have been used as markers for fungal identification for three decades. The rDNA sequences in a genome are thought to be homogeneous due to concerted evolution. However, intragenomic variation of rDNA sequences has recently been observed in many fungi, which may make fungal identification and species abundance estimation based on these loci problematic. Ceraceosorus is an enigmatic genus in the smut lineage Ustilaginomycotina for which very limited distribution data exist. Our previous research demonstrated intragenomic variation in the internal transcribed spacer (ITS1-5.8S-ITS2) region of two Ceraceosorus species. In this study, we described the fourth known species of Ceraceosorus, C. americanus, isolated from an asymptomatic rosemary leaf collected in Louisiana, USA. This is the first report of this genus in the Americas. We then selected all four known Ceraceosorus species, plus exemplar smut fungi representing all major lineages of subphylum Ustilaginomycotina, to examine sequence heterogeneity in three regions of the rDNA repeat (partial 18S, ITS, and partial 28S regions). Three methods were used: PCR-cloning-Sanger sequencing, targeted amplicon high-throughput sequencing, and whole-genome shotgun high-throughput sequencing. Our results show that Ceraceosorus is the only sampled fungal genus in Ustilaginomycotina with significant intragenomic variation at the ITS, with up to 25 nucleotide variant sites in the ITS1–5.8S–ITS2 region and 2.6% divergence among analyzed ITS haplotypes. We found many conflicting patterns across the three detection methods, with up to 27 conflicting variant sites recorded from a single individual. At least 40% of the conflicting patterns are possibly due to PCR-cloning-sequencing errors, as the corresponding variant sites were not observed in the other detection methods. Based on our data and the literature, we evaluated the characteristics and advantages/disadvantages of each detection method. Finally, a model for how intragenomic variation in the rDNA copies within a genome may arise is presented.

Exploring the Link Between Obligate Anaerobe-Related Dysbiosis and Prostate Cancer Development: A Pilot Study.

Several independent studies have associated prostate cancer (PCa) with specific groups of bacteria, most of them reporting the presence of anaerobic or microaerophilic species such as Cutibacterium acnes (C. acnes). Such findings suggest a prostate cancer-related bacterial dysbiosis, in a manner similar to the association between Helicobacter pylori infection and gastric cancer. In an earlier exploratory study looking for such dysbiosis events, using a culturomics approach, we discovered that the presence of obligate anaerobes (OAs) along with C. acnes was associated with increased prostate-specific antigen (PSA) levels in 39 participants. Building on this, in this study, we analyzed 89 post-rectal examination urine samples, from men with prostate cancer attending the PROVENT trial, using 16S rDNA sequencing. Our investigation focused on the impact of six previously identified OA genera (Finegoldia, Fusobacterium, Prevotella, Peptoniphilus_A, Peptostreptococcus, and Veillonella_A) on PSA levels. However, an additional data-driven approach was followed to uncover more taxa linked to increased PSA. Our analysis revealed a statistically significant association between Peptostreptococcus and elevated PSA levels. Additionally, there were potential interactions between Prevotella and Fusobacterium. Interestingly, we also found that an aerobe, Ochrobactrum_A,was significantly linked to higher PSA levels. These findings suggest that OA-related dysbiosis may contribute to elevated PSA levels through prostate cell damage even before prostate cancer develops, possibly playing a role in chronic inflammation and the hypervascular changes seen in precancerous lesions. Future clinical trials with larger cohorts are needed to further evaluate the role of OA in prostate cancer development and progression.

Green synthesis of silver nanoparticles from endophytic fungus Colletotrichum sp. with special emphasis on antibacterial, antioxidant and plant growth promoting potential

The creation of "microbial nanotechnology" for quick and large-scale manufacturing of nanoparticles would be possible with an effective biosynthesis method. Plumbago zeylanica L. is a medicinal plant from which the endophytic fungus Colletotrichum sp. was isolated and identified by 18S rDNA sequencing and microscopic studies. Cell filtrate of Colletotrichum sp. was employed to produce biological silver nanoparticles which acted as a reducing and stabilising agent during this process. The brown colour proved that silver nanoparticles (AgNPs) are being produced. In characterization of AgNPs, ultraviolet-visible spectroscopy revealed maximum absorption at 425 nm. From transmission electron microscopy and field emission scanning electron microscopic study the shape of the silver nanoparticle was found spherical with 10–30 nm diameters. The existence of elemental silver (peak) was verified by the silver signal in the energy-dispersive X?ray spectroscopy. Fourier-transform infrared analysis showed some major and minor shifts in the peaks of some chemical bonding. Synthesised AgNP had strong antibacterial action against pathogenic bacterial strains in 200 g/mL concentration and against Pseudomonas aeruginosa MTCC 741, it showed the highest inhibition. The AgNP concentration of 150 g/mL demonstrated the highest competency for antioxidant capabilities while ascorbic acid was used as the reference. Also, AgNP exhibited plant growth-promoting ability as in 40 mg/L concentration; it significantly enhanced the shoot and root growth, total soluble protein, sugar, and indole acetic acid content of P. zeylanica L. This AgNP can be useful in pharmaceutical industries and agriculture as nano fertilizers for all these purposes.

First Report of 'Candidatus Phytoplasma trifolii'-Related Strain Associated with Vaccinium Reddish Witches'-Broom Disease of European Blueberry in Lithuania.

European blueberries (Vaccinium myrtillus L.) can be found across the Northern Hemisphere, particularly in cool, temperate forests. These shrubs produce dark blue berries that are rich in vitamins, antioxidants, and anthocyanins making them valuable for both human consumption and food supplements. Blueberries play a crucial ecological role as a food source for various animals (Pires et al., 2020). In Oct 2023, symptomatic plants were observed in the Aukštagiris Geomorphological Reserve (54°44'N.; 25°21'E.), Lithuania. These plants exhibited symptoms characteristic of phytoplasma infection, including reduced leaf size with a reddish color, witches'-broom structures, and stunted growth. Samples were collected from 5 asymptomatic and 20 symptomatic plants. The disease was designated as Vaccinium reddish witches'-broom (VacRWB). Total DNA was extracted from all samples and used as a template for nested PCR using the universal primer pairs P1/P7 and R16F2n/R16R2 to amplify a 1.2 kb fragment of the phytoplasma 16S rRNA gene (Lee et al., 1998)., Primers Tuf340/Tuf890 and Tuf400/Tuf835 were used to amplify a 0.5 kb fragment of the phytoplasma elongation factor Tu (tuf) gene (Makarova et al., 2012). PCR amplicons were obtained from 18 symptomatic plants. Enzymatic RFLP analysis of the 1.2 kb 16S rDNA revealed that the phytoplasma is a member of 16SrVI-A and a 'Candidatus Phytoplasma trifolii'-related strain. Three R16F2n/R16R2 and three Tuf400/Tuf835 amplicons were cloned, sequenced and were respectively identical. BLAST analysis of obtained sequences against the NCBI nt database showed 99.44% to 100% identities for 16S rRNA gene and 96.42% to 99.49% identities for tuf gene sequences compared to previously identified 'Ca. P. trifolii'strains (first hits: PP237769 (matched 1250/1250 bp) and JQ824231 (389/391 bp), respectively). iPhyClassifier (Zhao et al., 2009) was used to generate in silico RFLP profiles of the 16S rDNA sequences, revealing an identical pattern (similarity coefficient = 1.00) to the reference pattern of subgruop 16SrVI-A (acc.: AY390261). Furthermore, the enzymatic and virtual RFLP profiles (using 14 endonucleases) matched each other. Phylogenetic analysis of the obtained VacRWB gene sequences confirmed the classification. Selected sequences were deposited in GenBank (NCBI) under Acc. nos PQ585819 (16S rRNA gene) and PQ594182 (tuf gene). Previously, a phytoplasma strain of group 16SrVI was reported in association with V. myrtillus plants in Austria (Borroto Fernandez et al., 2007). In Lithuania and other parts of Europe, infections by phytoplasmas of the 16SrIII-F subgroup are commonly associated with wild blueberries (Valiunas et al. 2004), often exhibiting slightly different symptoms (e.g., without reddish coloration and more severe stunting). To our knowledge, this is the first report of a 'Ca. P. trifolii'-related strain infecting European blueberry in Northern Europe, including Lithuania. Recently, a 'Ca. P. trifolii'-related strain (LingbSY) has been identified in lingonberries in Lithuania (Dėlkus et al., 2024). The susceptibility of both wild blueberries and lingonberries to related phytoplasma strains suggests the potential involvement of a shared insect vector, which warrants further investigations. These diseases pose a significant threat to forest ecosystems and could potentially spread to cultivated blueberries and lingonberries emphasizing the need for a comprehensive control strategy.

Exploring the Pharmacokinetics and Gut Microbiota Modulation of Hesperidin and Nobiletin from Mandarin Orange Peel in Experimental Dogs: A Pilot Study

Background/Objectives: Mandarin orange peel (MOP) is recognized for its traditional medicinal properties due to its high flavonoid content. This study aimed to analyze MOP harvested in Japan for specific bioactive compounds and to explore its health applications in dogs, including effects on gut microbiota and cognitive symptoms. Methods: Flavonoid content (particularly hesperidin and nobiletin) of MOP was measured. High-flavonoid MOP was then incorporated into gelatin cubes. In the time–course blood concentration study, experimental beagle dogs received a single oral dose (4 g). For intestinal microbiota analysis and blood biochemical tests, beagle dogs were administered 1 g twice daily. For the Demonstration Test, older dogs (a 14-year-old female Shiba Inu, a 14-year-old female Miniature Dachshund, and a 19-year-old male Miniature Dachshund) were administered 1 g twice daily. Analysis included microbiota profiling via 16S rDNA sequencing and observational assessment of cognitive indicators in a pilot study involving senior dogs with Alzheimer’s disease. Results: The MOP powder contained 9.3% hesperidin in early-ripening varieties (October harvest) and 6.9% in ripe varieties (December harvest). Nobiletin content was 41 mg/100 g (0.041%) and 35 mg/100 g (0.035%) for the early and late harvests, respectively. Administration of MOP-enriched gelatin cubes reduced Fusobacteriaceae and increased Eggerthellaceae. Cognitive symptoms like howling and counterclockwise turning showed improvement in senior dogs (n = 3). Conclusions: This study provides preliminary support for the potential health benefits of MOP in canine dietary applications, particularly for gut health and cognitive function. Improvement in cognitive symptoms may be due to the anxiolytic effects of mandarin peel.

Urbanization enhances consumer protist-driven ARGs dissemination in riverine ecosystems.

Despite the emergence of antibiotic resistance genes (ARGs) and antibiotic resistant bacteria (ARBs), how biological inter-trophic interactions, modulated by watershed urbanization, shape the resistome remains unexplored. We collected water samples from the highly urbanized (western: 65% built land, sewage-affected) and lesser-urbanized (northern: 25% built land, drinking water source) downstream tributaries of the Jiulong River in southeast China over dry and wet seasons. We utilized metagenomic and amplicon (16S and 18S rDNA) sequencing to investigate the relationships among microeukaryotic algae, consumer protists, bacterial communities, and the resistome. Metagenomic results showed that ARG-MGE-carrying contigs (mobile ARGs), rather than ARG-carrying contigs (non-mobile ARGs), exhibited more pronounced discrepancies between tributaries. A higher total abundance of ARGs and a greater number of co-shared ARGs between pathogen and non-pathogen bacteria were observed in the more urbanized western tributary. Structural equation modeling revealed that consumer protist-bacteria and algae-bacteria cohesions predominantly influenced the resistome in the western and northern tributaries, respectively. Additionally, consumer protists had more significant associations (511 out of 634) with bacteria carrying mobile ARGs in western tributary, while algae had more significant associations (73 out of 105) in northern tributary. These results highlight the distinct inter-trophic driving factors of the resistome modulated by watershed urbanization.