Fems Microbiology Letters最新文献

{"title":"Contributions of Hemolytic Proteins in Virulent Aeromonas hydrophila to Motile Aeromonas Septicemia Disease of Channel Catfish (Ictalurus punctatus).","authors":"Dunhua Zhang, Jun Feng, Yi Wang, Craig A Shoemaker, Allison A Wise, Benjamin H Beck","doi":"10.1093/femsle/fnae108","DOIUrl":"https://doi.org/10.1093/femsle/fnae108","url":null,"abstract":"<p><p>Hemolytic proteins are a major group of virulence factors in pathogenic Aeromonas hydrophila. Six genes encoding presumable hemolytic proteins were revealed from the genome of virulent A. hydrophila (vAh) that caused severe disease in channel catfish. The aim of this study was to assess the contribution of these hemolytic proteins to the virulence of this bacterium. Genes coding for following six proteins were investigated: aerolysin (Arl), 21-kDa hemolysin (Hly1), thermostable hemolysin (Hly2), phospholipase/lecithinase-related hemolysin (Hly3), membrane-associated hemolysin III (Hly4), and cytolysin-associated hemolysin (Hly5). Individual genes were deleted from the bacterium using CRISPR-Cas9 mediated methods. Assessment showed that deletion of Arl gene (Δarl) completely abolished hemolytic activity of this mutant while Δhly1-Δhly5 mutants had the same activity as the wild vAh. Extracellular proteins (ECP) of the Δarl mutant caused significantly (p < 0.01) less cell death in vitro with viability increased by approximately 20%, compared to the wild vAh. ECPs of mutants Δhly1-Δhly5 remained the same cell toxicity as the wild vAh. A second deletion of hly5 from the Δarl mutant further lowered the cell toxicity of the ECP of the mutant (Δarl+Δhly5). Assays in vivo showed that both Δarl and Δhly5 mutants caused less fish mortality with reduction of 57% and 16%, respectively, compared to the wild vAh; the Δarl+Δhly5 mutant caused the least mortality with approximately 87% of reduction; and other mutants had the same virulence as the wild vAh. Analyses of SDS-PAGE and Western blotting evidently indicate that both Arl and Hly5 proteins formed hexamer-like stable structures post secretion from the bacterium. Arl and Hly5 apparently had synergistic action in cytotoxicity and causing disease and were the major virulence factors among the six hemolytic proteins analyzed in this study.</p>","PeriodicalId":12214,"journal":{"name":"Fems Microbiology Letters","volume":" ","pages":""},"PeriodicalIF":2.2,"publicationDate":"2024-12-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142806554","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Excitation of filamentous growth in dekkera spp. By quorum sensing aromatic alcohols 2-phenylethanol and tryptophol.","authors":"Scott J Britton, Thijs Dingemans, Lisa Rogers, Jane S White, Dawn L Maskell","doi":"10.1093/femsle/fnae105","DOIUrl":"https://doi.org/10.1093/femsle/fnae105","url":null,"abstract":"<p><p>Fungi from the genus Dekkera, also known as Brettanomyces, are significant contaminants in commercial beer and wine production, and when present unintentionally, these non-domesticated yeasts result in the development of undesirable sensorial characteristics, in part due to the production of volatile phenols and acetate esters. The persistence of Dekkera spp. in industrial manufacturing environments can be attributed to its strong bioadhesive properties, allowing it to attach to various surfaces and form biofilms, which often contribute to recurrent contaminations. In other fungi, the yeast-to-filamentous transition is pivotal in enhancing bioadhesive properties, a process tightly regulated by density-dependent quorum-sensing mechanisms. However, there is no documented evidence regarding the influence of fungal quorum-sensing compounds on the yeast-to-filamentous transition in Dekkera, nor is there any evidence of existing quorum-sensing circuits in this genus. In this investigation, two Dekkera spp. were cultivated on a modified nitrogen-limiting SLAD medium supplemented with exogenous concentrations of quorum-sensing molecules 2-phenylethanol and tryptophol. Following cultivation, whole colonies were imaged and analyzed with a whole colony filamentation algorithm to quantify their filamentation. Our results demonstrate that the quorum-sensing compounds 2-phenylethanol and tryptophol significantly promote the yeast-to-filamentous transition in Dekkera spp., underscoring the broader presence of quorum-regulated social behaviors within this genus.</p>","PeriodicalId":12214,"journal":{"name":"Fems Microbiology Letters","volume":" ","pages":""},"PeriodicalIF":2.2,"publicationDate":"2024-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142827957","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Isolation and characterization of a multidrug-resistant Staphylococcus aureus infecting phage and its therapeutic use in mice","authors":"Zhen Xiao, Hongyi Xu, Juan Wang, Xueyuan Hu, Xiumei Huang, Shiping Song, Qingqing Zhang, Yanxin Liu, Yaopeng Liu, Na Liu, Junhui Liu, Ge Zhao, Xiyue Zhang, Yuehua Li, Jianmei Zhao, Junwei Wang, Huanqi Liu, Lin Wang, Zhina Qu","doi":"10.1093/femsle/fnae072","DOIUrl":"https://doi.org/10.1093/femsle/fnae072","url":null,"abstract":"In recent years, the emergence of multidrug-resistant bacteria has limited the selection of drugs for treating bacterial infections, reduced clinical efficacy, and increased treatment costs and mortality. It is urgent to find alternative antibiotics. In order to explore a new method for controlling methicillin resistant Staphylococcus aureus (S. aureus) , this study isolated and purified a multi drug resistant S. aureus broad-spectrum phage JPL-50 from wastewater. JPL-50 belongs to the Siphoviridae family after morphological observation, biological characterization, and transmission electron microscopy (TEM) fragmentation spectrum analysis. It can cleave 84% of tested S. aureus (168/200) , in which 100% of tested mastitis-associated strains (48/48) and 72.04% of MRSA strains (67/93) were lysed. In addition, it has an optimal growth temperature of about 30°C, a high activity within a wide pH range (pH 3–10) , and an optimal multiplicity of infection of 0.01. The one-step growth curve shows a latent time of 20 minutes, an explosive time of 80 minutes. JPL-50 was 16, 927 bp in length and was encoded by double-stranded DNA, with no genes associated with bacterial resistance or virulence factors detected. In a therapeutic study, injection of the phage JPL-50 once and for 7 times in 7 days protected 40% and 60% of the mice from fatal S.aureus infection, respectively. More importantly, JPL-50-doxycycline combination could effectively inhibit host S.aureus in vitro and reduce the use of doxycycline within 8 hours. In conclusion, the bacteriophage JPL-50 has a wide lysis spectrum, high lysis rate, high tolerance to extreme environments, and moderate in vivo activity, providing ideas for developing multidrug-resistant S. aureus infections.","PeriodicalId":12214,"journal":{"name":"Fems Microbiology Letters","volume":"65 1","pages":""},"PeriodicalIF":2.1,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142252124","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Organization, Conservation, and Diversity of Biosynthetic Gene Clusters in Bacillus sp. BH32 and Its Closest Relatives in the Bacillus cereus Group","authors":"Hadj Ahmed Belaouni, Amine Yekkour, Abdelghani Zitouni, Atika Meklat","doi":"10.1093/femsle/fnae071","DOIUrl":"https://doi.org/10.1093/femsle/fnae071","url":null,"abstract":"This study explores the organization, conservation, and diversity of biosynthetic gene clusters (BGCs) among Bacillus sp. strain BH32, a plant-beneficial bacterial endophyte, and its closest non-type Bacillus cereus group strains. BGC profiles were predicted for each of the 17 selected strains using antiSMASH, resulting in the detection of a total of 198 BGCs. We quantitatively compared the BGCs and analyzed their conservation, distribution, and evolutionary relationships. The study identified both conserved and singleton BGCs across the studied Bacillus strains, with minimal variation, and discovered two major BGC synteny blocks composed of homologous BGCs conserved within the B. cereus group. The identified BGC synteny blocks provide insight into the evolutionary relationships and diversity of BGCs within this complex group.","PeriodicalId":12214,"journal":{"name":"Fems Microbiology Letters","volume":"23 1","pages":""},"PeriodicalIF":2.1,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142223408","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Dynamic responses of Salmonella Typhimurium to re-exposure to sublethal ciprofloxacin","authors":"Jiseok Yi, Junhwan Kim, Juhee Ahn","doi":"10.1093/femsle/fnae050","DOIUrl":"https://doi.org/10.1093/femsle/fnae050","url":null,"abstract":"This study was designed to evaluate the history-dependent behaviors of Salmonella Typhimurium re-exposed to sublethal levels of ciprofloxacin. The S. Typhimurium cells were pre-exposed to 0 (CON), 1/16 (LOW), 1/8 (MED), and 1/4 (HIGH) MICs of ciprofloxacin, followed by re-exposure to the same concentrations. The bacterial growth, post-antibiotic effect (PAE), relative fitness, and swimming motility of treatments were evaluated in the absence of ciprofloxacin. The lag phase duration (LPD) was estimate to assess bacterial recovery under ciprofloxacin exposure. A disk diffusion assay was used to determine the cross-resistance and collateral sensitivity of CON, LOW, MED, and HIGH treatments to ciprofloxacin (CIP), ceftriaxone (CEF), erythromycin (ERY), gentamicin (GEN), and polymyxin B (POL). The S. Typhimurium cells pre-exposed to ciprofloxacin were susceptible in antibiotic-free media, showing delayed growth. The highest PAE (&amp;gt; 1 h) and bacterial fluctuation (CV = 5%) were observed at the High treatment compared to the CON. The HIGH treatment had the lowest relative fitness levels (0.87) and swimming motility (55 mm). The LPD was significantly decreased at the LOW treatment (1.8 h) when re-exposed to 1/16× MIC of ciprofloxacin. The LOW, MED, and HIGH treatments showed the cross-resistance to POL and the collateral sensitivity to CEF, ERY, and GEN. The pre-exposure to ciprofloxacin could induce phenotypic diversity, corresponding to the history-dependent behaviors. These results provide important insights for the dynamic nature of bacterial populations when re-exposed to sublethal concentrations of antibiotics.","PeriodicalId":12214,"journal":{"name":"Fems Microbiology Letters","volume":"140 1","pages":""},"PeriodicalIF":2.1,"publicationDate":"2024-06-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141502758","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comparison of PetrifilmTM AC and Pour plate techniques used for the heterotrophic aerobic bacterial count in water","authors":"Faith Mkhwanazi, Tshilidzi Mazibuko, Olivia Mosoma, Malefaso Rathebe, Mrudula Patel","doi":"10.1093/femsle/fnae029","DOIUrl":"https://doi.org/10.1093/femsle/fnae029","url":null,"abstract":"Heterotrophic bacteria (HPC) are commonly found in water samples. While these bacterial counts do not necessarily indicate a health hazard, high counts provide a good indication of the efficiency of water disinfection and integrity of distribution systems. The aim of this study was to compare the PetrifimTM AC method to the Pour Plate technique for the testing of HPC in water samples. Artificially contaminated (192 samples) and natural water samples (25) were processed using two methods. Both methods accurately detected high, medium and low counts of HPC, producing average Z scores between -2 and + 2. Paired-wise student t-test and correlation coefficient showed nonsignificant differences between the results of two methods. Acceptable repeatability and reproducibility was obtained using both the methods. Uncertainty of measurement for PetrifilmTM AC and Pour Plate method was found to be 2.9% and 5.4% respectively. PetrifilmTM AC proved to be robust at 33 °C and 37 °C. In conclusion, PetrifimTM AC, which is easy to process, read and less time consuming, proved to be comparable to the conventional Pour Plate method in establishing HPC in water. In addition, PetrifimTM AC requires less space for the processing and incubation, generate small volume of waste for disposal and requires no equipment, except for the incubator.","PeriodicalId":12214,"journal":{"name":"Fems Microbiology Letters","volume":"56 1","pages":""},"PeriodicalIF":2.1,"publicationDate":"2024-04-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140841262","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Identification and characterization of a novel type of ketohexokinase from the haloarchaeon Haloferax volcanii","authors":"Marius Ortjohann, Peter Schönheit","doi":"10.1093/femsle/fnae026","DOIUrl":"https://doi.org/10.1093/femsle/fnae026","url":null,"abstract":"Ketohexokinase (KHK) catalyzes the ATP-dependent phosphorylation of fructose, forming fructose-1-phosphate and ADP. The enzyme is well studied in Eukarya, in particular in human and other vertebrates, but homologs have not been identified in Bacteria and Archaea. Here we report the identification of a novel type of KHK from the haloarchaeon Haloferax volcanii (HvKHK). The encoding gene khk was identified as HVO_1812. The gene was expressed as 90 kDa homodimeric protein, catalyzing the phosphorylation of fructose with a Vmax value of 59 U/mg and apparent KM values for ATP and fructose of 0.47 mM and 1.29 mM, respectively. Homologs of HvKHK were only identified in few haloarchaea and halophilic Bacteria. The protein showed low sequence identity to characterized KHKs from eukaryotes and phylogenetic analyses indicate that haloarchaeal KHKs are largely separated from eukaryal KHKs. This is the first report of the identification of KHKs in prokaryotes that form a novel cluster of sugar kinases within the ribokinase/pfkB superfamily.","PeriodicalId":12214,"journal":{"name":"Fems Microbiology Letters","volume":"84 1","pages":""},"PeriodicalIF":2.1,"publicationDate":"2024-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140583638","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Analysis of CRISPR-Cas loci distribution in Xanthomonas citri and its possible control by the quorum sensing system","authors":"Paula Maria Moreira Martins, Laís Moreira Granato, Túlio Morgan, Julia Lopes Nalin, Marco Aurélio Takita, Poliane Alfenas-Zerbini, Alessandra Alves de Souza","doi":"10.1093/femsle/fnae005","DOIUrl":"https://doi.org/10.1093/femsle/fnae005","url":null,"abstract":"Xanthomonas is an important genus of plant-associated bacteria that causes significant yield losses of economically important crops worldwide. Different approaches have assessed genetic diversity and evolutionary interrelationships among the Xanthomonas species. However, information from clustered regularly interspaced short palindromic repeats (CRISPR) has yet to be explored. In this work, we analyzed the architecture of CRISPR-Cas loci and presented a sequence similarity-based clustering of conserved Cas proteins in different species of Xanthomonas. Although absent in many investigated genomes, Xanthomonas harbors subtype I-C and I-F CRISPR-Cas systems. The most represented species, Xanthomonas citri, presents a great diversity of genome sequences with an uneven distribution of CRISPR-Cas systems among the subspecies/pathovars. Only Xanthomonas citri subsp. citri and Xanthomonas citri pv. punicae have these systems, exclusively of subtype I-C system. Moreover, the most likely targets of the X. citri CRISPR spacers are viruses (phages). At the same time, few are plasmids, indicating that CRISPR/Cas system is possibly a mechanism to control the invasion of foreign DNA. We also showed in X. citri susbp. citri that the cas genes are regulated by the diffusible signal factor (DSF), the quorum sensing (QS) signal molecule, according to cell density increases, and under environmental stress like starvation. These results suggest that the regulation of CRISPR-Cas by QS occurs to activate the gene expression only during phage infection or due to environmental stresses, avoiding a possible reduction in fitness. Although more studies are needed, CRISPR-Cas systems may have been selected in the Xanthomonas genus throughout evolution, according to the cost-benefit of protecting against biological threats and fitness maintenance in challenging conditions.","PeriodicalId":12214,"journal":{"name":"Fems Microbiology Letters","volume":"78 1","pages":""},"PeriodicalIF":2.1,"publicationDate":"2024-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139510140","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A comprehensive diversity analysis on the gut microbiomes of ASD patients: from alpha, beta to gamma diversities.","authors":"Hongju Daisy Chen, Lianwei Li, Fubing Yu, Zhanshan Sam Ma","doi":"10.1093/femsle/fnae014","DOIUrl":"10.1093/femsle/fnae014","url":null,"abstract":"<p><p>Autism spectrum disorder (ASD) is estimated to influence as many as 1% children worldwide, but its etiology is still unclear. It has been suggested that gut microbiomes play an important role in regulating abnormal behaviors associated with ASD. A de facto standard analysis on the microbiome-associated diseases has been diversity analysis, and nevertheless, existing studies on ASD-microbiome relationship have not produced a consensus. Here, we perform a comprehensive analysis of the diversity changes associated with ASD involving alpha-, beta-, and gamma-diversity metrics, based on 8 published data sets consisting of 898 ASD samples and 467 healthy controls (HC) from 16S-rRNA sequencing. Our findings include: (i) In terms of alpha-diversity, in approximately 1/3 of the studies cases, ASD patients exhibited significantly higher alpha-diversity than the HC, which seems to be consistent with the \"1/3 conjecture\" of diversity-disease relationship (DDR). (ii) In terms of beta-diversity, the AKP (Anna Karenina principle) that predict all healthy microbiomes should be similar, and every diseased microbiome should be dissimilar in its own way seems to be true in approximately 1/2 to 3/4 studies cases. (iii) In terms of gamma-diversity, the DAR (diversity-area relationship) modeling suggests that ASD patients seem to have large diversity-area scaling parameter than the HC, which is consistent with the AKP results. However, the MAD (maximum accrual diversity) and RIP (ratio of individual to population diversity) parameters did not suggest significant differences between ASD patients and HC. Throughout the study, we adopted Hill numbers to measure diversity, which stratified the diversity measures in terms of the rarity-commonness-dominance spectrum. It appears that the differences between ASD patients and HC are more propounding on rare-species side than on dominant-species side. Finally, we discuss the apparent inconsistent diversity-ASD relationships among different case studies and postulate that the relationships are not monotonic.</p>","PeriodicalId":12214,"journal":{"name":"Fems Microbiology Letters","volume":" ","pages":""},"PeriodicalIF":2.1,"publicationDate":"2024-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139989705","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Release of intracellular enzymes increases the total extracellular activities of carbohydrate-processing enzymes in marine environment.","authors":"Ke-Xuan Huang, Yu-Xuan Jiang, Yan-Ru Dang, Qi-Long Qin","doi":"10.1093/femsle/fnae077","DOIUrl":"10.1093/femsle/fnae077","url":null,"abstract":"<p><p>Microbial extracellular enzymatic activities (EEAs) produced by microbes to degrade biopolymers are the 'gatekeeper' of carbon cycle in the marine ecosystem. It is usually assumed that these extracellular enzymes are actively secreted by microbes. However, biopolymer-degrading enzymes also exist in the intracellular space. Cell lysis will passively release these enzymes into the environments and contribute to the total EEAs. However, to what extent the cell lysis can contribute to the total EEAs are still unclear. Here, using extreme cell lysis method, we evaluated the maximum contribution of cell lysis to total EEAs in culturable marine bacteria and coastal seawater. For carbohydrate-processing enzymes (β-glucosidase, alginate lyase, and chitinase), the release of intracellular enzymes could contribute positively (up to 56.1% increase for β-glucosidase in seawater) to the total EEAs. For protease and leucine aminopeptidase, the cell lysis did not increase and even decreased the total EEAs. For alkaline phosphatase, the intracellular enzymes generally had no contribution to the total EEAs. These results showed that passively released intracellular enzymes could substantially increase the total extracellular activities of carbohydrate-processing enzymes, which should be considered in building the link between the EEAs and organic carbon cycle in the ocean.</p>","PeriodicalId":12214,"journal":{"name":"Fems Microbiology Letters","volume":" ","pages":""},"PeriodicalIF":2.2,"publicationDate":"2024-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142344383","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}