MicrobiologyOpen最新文献

{"title":"Monitoring the Persistence of Pseudomonas sivasensis Strain CF10PS3 in Cereal Fields","authors":"Mathieu Delitte,&nbsp;Benjamin Dubois,&nbsp;Jacques Mahillon,&nbsp;Frédéric Debode,&nbsp;Claude Bragard","doi":"10.1002/mbo3.70005","DOIUrl":"10.1002/mbo3.70005","url":null,"abstract":"<p>The persistence and efficacy of biocontrol agents in agricultural fields are crucial for sustainable crop production. In this study, we investigated the persistence of the introduced bacterial strain <i>Pseudomonas sivasensis</i> CF10PS3 in the wheat phyllosphere using a novel qPCR probe protocol. The CF10PS3 strain, known for its in vitro biocontrol properties against wheat pathogens, was applied through foliar spray, and its persistence was monitored over 7 weeks. Our qPCR assays, designed to specifically detect CF10PS3, distinguished it from naturally occurring <i>P. sivasensis</i> strains, providing precise insights into its dynamics in the field. The experimental results indicated that CF10PS3 was already present on the wheat leaves before its application, suggesting its natural adaptation to the foliar environment. Following initial application, a significant increase in CF10PS3 was observed, though subsequent environmental factors such as rain and wind might have caused notable fluctuations in its population. Despite these variations, the introduced strain showed considerable persistence, with population levels significantly higher than those in untreated plots by the end of the study period. This research underscores the importance of understanding bacteria dynamics in the field, highlighting the influence of environmental conditions on their efficacy. The use of specific qPCR probes proved effective in monitoring introduced strains, offering valuable insights for optimizing biocontrol agent application strategies. Our findings contribute to the development of robust biocontrol methods, promoting sustainable agricultural practices and enhancing crop protection.</p>","PeriodicalId":18573,"journal":{"name":"MicrobiologyOpen","volume":"13 6","pages":""},"PeriodicalIF":3.9,"publicationDate":"2024-11-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/mbo3.70005","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142648702","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Bacillus xiamenensis Inhibits the Growth of Moraxella osloensis by Producing Indole-3-Carboxaldehyde","authors":"Masahiro Watanabe,&nbsp;Yuika Sekino,&nbsp;Kouji Kuramochi,&nbsp;Yuuki Furuyama","doi":"10.1002/mbo3.70009","DOIUrl":"10.1002/mbo3.70009","url":null,"abstract":"<p><i>Moraxella osloensis</i>, a gram-negative rod-shaped bacterium found on human skin, produces 4-methyl-3-hexenoic acid, contributing to clothing and body malodor. <i>M. osloensis</i> is resistant to UV light, drying, and antimicrobials, making its eradication challenging. As the skin is low in nutrients, commensal bacteria compete for resources and use diverse strategies to inhibit their competitors. Therefore, skin-derived bacteria that exhibited growth-inhibitory activity against <i>M. osloensis</i> were searched. Screening skin-derived bacteria using a coculture halo assay revealed that <i>Bacillus xiamenensis</i> formed an inhibition zone with <i>M. osloensis</i>. Coculture plates were extracted with ethyl acetate and fractionated using a silica gel column and preparative thin-layer chromatography to isolate the active compound from the <i>B. xiamenensis</i> metabolites. Nuclear magnetic resonance spectroscopy identified the active compound as indole-3-carboxaldehyde, which has low toxicity in humans. At soluble concentrations, indole-3-carboxaldehyde does not inhibit the growth of other bacteria, such as <i>Staphylococcus aureus</i>, <i>Escherichia coli</i>, and <i>Bacillus subtilis</i>, suggesting <i>M. osloensis</i> is highly sensitive to indole-3-carboxaldehyde. These findings highlight <i>B. xiamenensis</i> as a promising candidate for the development of a skin probiotic to promote skin health and combat malodor-causing bacteria.</p>","PeriodicalId":18573,"journal":{"name":"MicrobiologyOpen","volume":"13 6","pages":""},"PeriodicalIF":3.9,"publicationDate":"2024-11-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11558204/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142623836","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The Effects of Carbonate on Candida albicans Filamentation, Biofilm Formation, and Antifungal Resistance","authors":"Trenton P. Miedema,&nbsp;Kayla E. Grooters,&nbsp;Ian A. Cleary","doi":"10.1002/mbo3.70008","DOIUrl":"10.1002/mbo3.70008","url":null,"abstract":"<p><i>Candida albicans</i>, a member of the normal microbial population of healthy humans, is an opportunistic pathogen that can cause serious disease in immunocompromised patients. An important virulence factor of <i>C. albicans</i> is the formation of biofilms. These organized communities of cells are efficient at attaching to host cells and implanted medical devices. Carbonate has been studied as an agricultural antifungal agent, and here we demonstrate that carbonate can affect filamentation, biofilm formation, and antifungal drug resistance in <i>C. albicans</i>.</p>","PeriodicalId":18573,"journal":{"name":"MicrobiologyOpen","volume":"13 6","pages":""},"PeriodicalIF":3.9,"publicationDate":"2024-11-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11558181/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142623743","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evaluation of DNA Extraction Methods for Microbial Community Profiling in Deadwood Decomposition","authors":"Yanmei Zhang,&nbsp;Zewei Song,&nbsp;Jonathan S. Schilling","doi":"10.1002/mbo3.70007","DOIUrl":"10.1002/mbo3.70007","url":null,"abstract":"<p>As technologies advance alongside metabarcoding and metagenomic resources, particularly for larger fungal genomes, DNA extraction methods must be optimized to meet higher thresholds, especially from complex environmental substrates. This study focused on extracting fungal genomic compounds from woody substrates, a challenge due to the embedment of endophytic and saprotrophic fungi within wood cells, the physical recalcitrance of wood, the adsorption of nucleic acids to wood polymers, and the release of downstream inhibitors. Hypothesizing that cetyltrimethylammonium bromide would be the best option, we compared prominent methods by extracting and sequencing microbial DNA from sound and decayed birch (<i>Betula papyrifera</i>) and pine (<i>Pinus resinosa</i>). DNA quantities varied significantly depending on extraction methods and decay stage. The quality of DNA, in terms of purity and integrity, significantly impacted whether the samples could be amplified and sequenced. However, amplicon sequencing of bacterial and fungal communities revealed no significant extraction bias. This, along with the sequencing effectiveness and cost/time efficiency, indicates that Qiagen is the gold standard for woody substrates. This study increases confidence in published amplicon data sets regardless of the extraction methods, provides a cost-benefit table for making protocol decisions, and offers guidance on fungal DNA extractions from complex organic substrates (sound and decayed wood) that would best suit future metagenomic efforts.</p>","PeriodicalId":18573,"journal":{"name":"MicrobiologyOpen","volume":"13 6","pages":""},"PeriodicalIF":3.9,"publicationDate":"2024-11-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11561137/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142623838","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Physiological Effects of TolC-Dependent Multidrug Efflux Pumps in Escherichia coli: Impact on Motility and Growth Under Stress Conditions","authors":"Amanda M. Di Maso,&nbsp;Cristian Ruiz","doi":"10.1002/mbo3.70006","DOIUrl":"10.1002/mbo3.70006","url":null,"abstract":"<p>Enterobacteriaceae possess eight TolC-dependent multidrug efflux pumps: AcrAB-TolC, AcrAD-TolC, AcrEF-TolC, MdtEF-TolC, MdtABC-TolC, EmrAB-TolC, EmrYK-TolC, and MacAB-TolC, which efflux bile salts, antibiotics, metabolites, or other compounds. However, our understanding of their physiological roles remains limited, especially for less-studied pumps like EmrYK-TolC. In this study, we tested the effects on swimming motility and growth under stress conditions of <i>Escherichia coli</i> mutants individually deleted for each inner-membrane transporter component of all eight TolC-dependent pumps, a mutant deleted for the AcrB-accessory protein AcrZ, and a mutant simultaneously deleted for all eight pumps (Δ<i>tolC</i>). We found that all mutants tested, except the Δ<i>emrY</i> and Δ<i>acrZ</i> mutants, displayed increased swimming motility. Additionally, the loss of each individual TolC-dependent pump or AcrZ did not reduce growth and sometimes even enhanced it compared to the parental strain under various growth conditions: temperature (LB at 25, 30, 37, and 42°C), pH (LB at pH 6.0, 7.4, and 9.0; and LB buffered to pH 6.0, 7.4, and 8.25), LB with limited air exchange, and nutritional stress (M9-glucose or M9-glycerol). In contrast, the Δ<i>tolC</i> mutant grew significantly slower than the parental strain under all conditions tested except in LB-TRIS pH 7.4 and LB with limited air exchange. Overall, these findings indicate that while individual TolC-dependent pumps are generally dispensable for growth under many stress conditions in the absence of antimicrobials, possibly due to their partially overlapping substrate profiles, TolC-dependent efflux is required for maximal growth under most conditions.</p>","PeriodicalId":18573,"journal":{"name":"MicrobiologyOpen","volume":"13 6","pages":""},"PeriodicalIF":3.9,"publicationDate":"2024-11-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11554990/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142623741","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Listeria monocytogenes in the seafood industry: Exploring contamination sources, outbreaks, antibiotic susceptibility and genetic diversity","authors":"Karlene Lambrechts,&nbsp;Diane Rip","doi":"10.1002/mbo3.70003","DOIUrl":"https://doi.org/10.1002/mbo3.70003","url":null,"abstract":"<p>Fish and seafood are rich sources of protein, vitamins, and minerals, significantly contributing to individual health. A global increase in consumption has been observed. <i>Listeria monocytogenes</i> is a known problem in food processing environments and is found in various seafood forms, including raw, smoked, salted, and ready-to-eat. Without heat treatment and given <i>L. monocytogenes</i>' ability to multiply under refrigerated conditions, consuming seafood poses a substantial health hazard, particularly to immunocompromised individuals. Numerous global outbreaks of listeriosis have been linked to various fish products, underscoring the importance of studying <i>L. monocytogenes</i>. Different strains exhibit varying disease-causing abilities, making it crucial to understand and monitor the organism's virulence and resistance aspects for food safety. This paper aims to highlight the genetic diversity of <i>L. monocytogenes</i> found in fish products globally and to enhance understanding of contamination routes from raw fish to the final product.</p>","PeriodicalId":18573,"journal":{"name":"MicrobiologyOpen","volume":"13 5","pages":""},"PeriodicalIF":3.9,"publicationDate":"2024-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/mbo3.70003","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142449052","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Influence of microbiota on the growth and gene expression of Clostridioides difficile in an in vitro coculture model","authors":"Elisa Martinez,&nbsp;Noémie Berg,&nbsp;Cristina Rodriguez,&nbsp;Georges Daube,&nbsp;Bernard Taminiau","doi":"10.1002/mbo3.70001","DOIUrl":"https://doi.org/10.1002/mbo3.70001","url":null,"abstract":"<p><i>Clostridioides difficile</i> is an anaerobic, spore-forming, Gram-positive pathogenic bacterium. This study aimed to analyze the effect of two samples of healthy fecal microbiota on <i>C. difficile</i> gene expression and growth using an in vitro coculture model. The inner compartment was cocultured with spores of the <i>C. difficile</i> polymerase chain reaction (PCR)-ribotype 078, while the outer compartment contained fecal samples from donors to mimic the microbiota (FD1 and FD2). A fecal-free plate served as a control (CT). RNA-Seq and quantitative PCR confirmation were performed on the inner compartment sample. Similarities in gene expression were observed in the presence of the microbiota. After 12 h, the expression of genes associated with germination, sporulation, toxin production, and growth was downregulated in the presence of the microbiota. At 24 h, in an iron-deficient environment, <i>C. difficile</i> activated several genes to counteract iron deficiency. The expression of genes associated with germination and sporulation was upregulated at 24 h compared with 12 h in the presence of microbiota from donor 1 (FD1). This study confirmed previous findings that <i>C. difficile</i> can use ethanolamine as a primary nutrient source. To further investigate this interaction, future studies will use a simplified coculture model with an artificial bacterial consortium instead of fecal samples.</p>","PeriodicalId":18573,"journal":{"name":"MicrobiologyOpen","volume":"13 5","pages":""},"PeriodicalIF":3.9,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/mbo3.70001","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142438999","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The metagenomic landscape of a high-altitude geothermal spring in Tajikistan reveals a novel Desulfurococcaceae member, Zestomicrobium tamdykulense gen. nov., sp. nov","authors":"Munavvara Dzhuraeva,&nbsp;Khursheda Bobodzhanova,&nbsp;Nils-Kåre Birkeland","doi":"10.1002/mbo3.70004","DOIUrl":"10.1002/mbo3.70004","url":null,"abstract":"<p>Metagenomic analysis was conducted to assess the microbial community in the high-altitude Tamdykul geothermal spring in Tajikistan. This analysis yielded six high-quality bins from the members of Thermaceae, Aquificaceae, and Halothiobacillaceae, with a 41.2%, 19.7%, and 18.1% share in the total metagenome, respectively. Minor components included <i>Schleiferia thermophila</i> (1.6%) and members of the archaeal taxa <i>Pyrobaculum</i> (1.2%) and <i>Desulfurococcaceae</i> (0.7%). Further analysis of the metagenome-assembled genome (MAG) from the <i>Desulfurococcaceae</i> family (MAG002) revealed novel taxonomy with only 80.95% closest placement average nucleotide identity value to its most closely related member of the <i>Desulfurococcaceae</i> family, which is part of the <i>Thermoproteota</i> phylum comprising hyperthermophilic members widespread in geothermal environments. MAG002 consisted of 1.3 Mbp, distributed into 48 contigs with 1504 predicted coding sequences, had an average GC content of 41.3%, a completeness and contamination rate of 98.7% and 2.6%, respectively, and branched phylogenetically between the <i>Ignisphaera</i> and <i>Zestosphaera</i> lineages. Digital DNA-DNA hybridization values compared with <i>Ignisphaera aggregans</i> and <i>Zestosphaera tikiterensis</i> were 33.7% and 19.4%, respectively, suggesting that this MAG represented a novel species and genus. Its 16S rRNA gene contained a large 421 bp intron. It encodes a complete gluconeogenesis pathway involving a bifunctional fructose-1,6-bisphosphate phosphatase/aldolase; however, the glycolysis pathway is incomplete. The ribulose monophosphate pathway enzymes could be used for pentose synthesis. MAG002 encodes several hydrogen-evolving hydrogenases, with possible roles as hydrogen sinks during fermentation. We propose the name <i>Zestomicrobium tamdykulense</i> gen. nov. sp. nov. for this organism; it is the first thermophilic genome reported from Tajikistan.</p>","PeriodicalId":18573,"journal":{"name":"MicrobiologyOpen","volume":"13 5","pages":""},"PeriodicalIF":3.9,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/mbo3.70004","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142400712","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"High-quality genome of a novel Thermosynechococcaceae species from Namibia and characterization of its protein expression patterns at elevated temperatures","authors":"Nathanael D. Arnold,&nbsp;Michael Paper,&nbsp;Tobias Fuchs,&nbsp;Nadim Ahmad,&nbsp;Patrick Jung,&nbsp;Michael Lakatos,&nbsp;Katia Rodewald,&nbsp;Bernhard Rieger,&nbsp;Farah Qoura,&nbsp;Martha Kandawa-Schulz,&nbsp;Norbert Mehlmer,&nbsp;Thomas B. Brück","doi":"10.1002/mbo3.70000","DOIUrl":"10.1002/mbo3.70000","url":null,"abstract":"<p>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.</p>","PeriodicalId":18573,"journal":{"name":"MicrobiologyOpen","volume":"13 5","pages":""},"PeriodicalIF":3.9,"publicationDate":"2024-10-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11450739/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142372310","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The effects of stress on gut virome: Implications on infectious disease and systemic disorders","authors":"Francesca Talarico,&nbsp;Bruno Tilocca,&nbsp;Rocco Spagnuolo,&nbsp;Ludovico Abenavoli,&nbsp;Francesco Luzza,&nbsp;Paola Roncada","doi":"10.1002/mbo3.1434","DOIUrl":"10.1002/mbo3.1434","url":null,"abstract":"<p>The role of gut microbiota in health and disease is being thoroughly examined in various contexts, with a specific focus on the bacterial fraction due to its significant abundance. However, despite their lower abundance, viruses within the gut microbiota are gaining recognition for their crucial role in shaping the structure and function of the intestinal microbiota, with significant effects on the host as a whole, particularly the immune system. Similarly, environmental factors such as stress are key in modulating the host immune system, which in turn influences the composition of the gut virome and neurological functions through the bidirectional communication of the gut–brain axis. In this context, alterations in the host immune system due to stress and/or dysbiosis of the gut virome are critical factors in the development of both infectious and noninfectious diseases. The molecular mechanisms and correlation patterns between microbial species are not yet fully understood. This literature review seeks to explore the interconnected relationship between stress and the gut virome, with a focus on how this interaction is influenced by the host's immune system. We also discuss how disturbances in this finely balanced system can lead to the onset and/or progression of diseases.</p>","PeriodicalId":18573,"journal":{"name":"MicrobiologyOpen","volume":"13 5","pages":""},"PeriodicalIF":3.9,"publicationDate":"2024-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/mbo3.1434","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291197","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}