Microbiology最新文献

{"title":"Phylogenetic Analysis of phn Transporters of Achromobacter insolitus LCu2","authors":"Ye. V. Kryuchkova, G. L. Burygin","doi":"10.1134/s0026261723605122","DOIUrl":"https://doi.org/10.1134/s0026261723605122","url":null,"abstract":"<h3 data-test=\"abstract-sub-heading\">Abstract</h3><p>Phosphonates are alternative phosphorus sources for bacteria. The genome of <i>Achromobacter insolitus</i> strain LCu2 contains three predicted <i>phn</i> clusters of ABC-type phosphonate transporters into the cell. To understand the functional, evolutionary, and ecological role of the <i>phn</i> clusters, phylogenetic analysis of substrate-binding PhnD proteins from strain LCu2 with their homologs in other <i>Achromobacter</i> species and in closely related genera of the family <i>Alcaligenaceae</i> was carried out. The PhnD transporters formed three separate clusters, which indicated the differences in their structural composition. PhnD₁ and PhnD₂ were present in the genomes of all <i>Achromobacter</i> species and grouped separately from those of other members of the family <i>Alcaligenaceae</i>, which indicated vertical inheritance of the <i>phn</i>D₁ and <i>phn</i>D₂ genes and their involvement in the life-supporting processes. <i>Phn</i>D₃ was found in the genomes of seven <i>Achromobacter</i> species. The <i>phn</i>D₃ gene was probably acquired via horizontal transfer or duplication and is induced during adaptation to changing environmental conditions. Maintenance of three structurally different <i>phn</i> clusters of transporters is probably ecologically advantageous to <i>A. insolitus</i> LCu2, providing for phosphorus retrieval from synthetic and natural organophosphonates as well as other sources.</p>","PeriodicalId":18514,"journal":{"name":"Microbiology","volume":"61 1","pages":""},"PeriodicalIF":1.5,"publicationDate":"2024-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141770044","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":"The Genome of a New Halorubrum distributum Strain ICIS4 Isolated from the Culture of a Microalga Dunaliella salina","authors":"E. A. Selivanova, A. S. Balkin, Yu. A. Khlopko, V. Ya. Kataev, A. O. Plotnikov","doi":"10.1134/s0026261723604426","DOIUrl":"https://doi.org/10.1134/s0026261723604426","url":null,"abstract":"<h3 data-test=\"abstract-sub-heading\">\u0000<b>Abstract</b>\u0000</h3><p>The complete genome sequence of a new strain of the haloarchaeaon <i>Halorubrum distributum</i> ICIS4 was revealed and analyzed. The strain was isolated from a culture of a carotenogenic microalga <i>Dunaliella salina</i> maintained in laboratory conditions for a long period of time. The genome (3.32 Mb) contained 3236 protein-coding genes. Of the 2817 groups of homologous genes, 11 were unique to this strain. In the genome, the genes were revealed, which were responsible for utilization of glycerol and starch and for synthesis of vitamins, pigments, and siderophores. These genes may be involved in formation and maintenance of the association with microalgae. A region similar to the HRPV9 virus and another circular contig similar to a phage of the haloarchaeon <i>Haloquadratum walsbyi</i> were revealed in the genome assembly.</p>","PeriodicalId":18514,"journal":{"name":"Microbiology","volume":"49 1","pages":""},"PeriodicalIF":1.5,"publicationDate":"2024-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141770046","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":"Westiellopsis tiwarii sp. nov., a New Cyanobacterium from North-East India Soils","authors":"M. Parmar, T. P. Jaiswal, S. Bhattacharjee, S. S. Singh, A. K. Mishra","doi":"10.1134/s0026261723602154","DOIUrl":"https://doi.org/10.1134/s0026261723602154","url":null,"abstract":"<h3 data-test=\"abstract-sub-heading\">Abstract</h3><p>In modern times, cyanobacterial identification, characterization, and classification have adopted a pragmatic polyphasic approach assigning equal weight to the ecological, morphological, physiological, and molecular parameters. The paucity of data on cyanobacterial diversity across unexplored regions resulted in poor taxonomic characterization of cyanobacteria. Hence, the poorly explored North-Eastern lower Himalayan region of India was targeted and a true-branched soil inhabiting cyanobacterial strain KFS2 was isolated. Along with habitat analysis, morphological and 16S-rRNA gene-based molecular characterization of the isolated strain have been performed. The complex cellular morphology, uni-seriate to multi-seriate main filaments and branches, along with the 16S rRNA gene sequence-based phylogeny indicated affiliation of this strain with the genus <i>Westiellopsis.</i> Furthermore, the strain KFS2 showed unique morphological features such as the multi-seriate base of lateral branches, the presence of both intercalary and terminal heterocytes in the main filaments along with the intercalary position of heterocytes in the lateral branches, and was found to form a separate node with the <i>Westiellopsis</i> clade having strong bootstrap support in the 16S-rRNA gene-based phylogeny. Moreover, comparative analysis of folded secondary structures of D1-D1' and box-B in the 16S-23S ITS region of the strain also exhibited striking differences with other members of the genus. Considering all the unique morphological and molecular characteristics of the strain KFS2, it was suggested to represent a new species of the genus <i>Westiellopsis</i>, with the name <i>Westiellopsis tiwarii</i> being proposed in accordance with the International Code of Nomenclature of algae, fungi, and plants.</p>","PeriodicalId":18514,"journal":{"name":"Microbiology","volume":"61 1","pages":""},"PeriodicalIF":1.5,"publicationDate":"2024-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141770134","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":"Bioinformatic and Functional Analysis of the pSID Siderophore Biosynthesis Plasmid of Rhodococcus pyridinivorans 5Ap","authors":"M. I. Mandryk, A. A. Vysotskaya, Yu. V. Yahorava, D. U. Surzhyk, A. Yu. Larchenka, S. L. Vasylenko","doi":"10.1134/s0026261724604603","DOIUrl":"https://doi.org/10.1134/s0026261724604603","url":null,"abstract":"<h3 data-test=\"abstract-sub-heading\">\u0000<b>Abstract</b>—</h3><p>Complete genome sequencing of <i>R. pyridinivorans</i> strain 5Ap revealed the pSID plasmid (CP063453.1) 250 428 bp in size. The gene responsible for replication of this plasmid is, most probably, <i>dnaB</i>. The genes which may be involved in the replication (<i>dnaB, ssb</i>) and plasmid separation after replication (<i>parA</i>) showed the highest similarity to the determinants located on large (224‒343 kb) plasmids of rhodococci: unnamed1 of <i>R. pyridinivorans</i> YF3, unnamed1 of <i>R. rhodochrous</i> LH-B3, pRJH1 of <i>R. pyridinivorans</i> YC-JH2, pRDE01 of <i>Rhodococcus</i> sp. RDE2, and pRho-VOC14-C342 of <i>R. opacus</i> VOC-14. The pSID plasmid was found to contain two loci responsible for the synthesis of secondary metabolites, one of them determining the synthesis of a polyketide compound (similar sequences have been revealed on plasmids of other rhodococci) and the other one probably determines the synthesis of a siderophore: the genes for biosynthesis of this compound (<i>sid1–5</i>) exhibited the highest similarity (not exceeding 75%) with the sequences from <i>Streptomyces vilmorinianum</i> YP1 (CP040244.1), <i>S. ficellus</i> NRRL 8067 (CP034279.1), <i>Streptomyces</i> sp. NBC00162 (CP102509.1), and some other streptomycetes, while showing no similarity to the known siderophore biosynthesis genes of rhodococci. The locus of the pSID plasmid responsible for the siderophore synthesis had a unique organization, since transcription of the <i>sid5</i> (<i>iucC</i>) gene occurs in the opposite direction, while in other bacteria it belongs to an operon and is located at one of its termini. Inactivation of the <i>sid1</i> gene was found to result in decreased antagonistic activity of <i>R. pyridinivorans</i> 5Ap against plant-pathogenic bacteria <i>P. carotovorum</i> 2.18, lower resistance to iron and cadmium ions and arsenate, as well as in emergence of phytotoxic properties against radish, while wild-type bacteria exhibit plant growth-promoting activity.</p>","PeriodicalId":18514,"journal":{"name":"Microbiology","volume":"30 1","pages":""},"PeriodicalIF":1.5,"publicationDate":"2024-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141770040","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 New Bacteriophage Pseudomonas Phage Ka1 from a Tributary Stream of Lake Baikal","authors":"M. S. Fedorova, A. E. Gatina, V. N. Ilyina, L. L. Yadykova, V. V. Drukker, A. S. Gorshkova, A. R. Kayumov, E. Y. Trizna","doi":"10.1134/s0026261723604906","DOIUrl":"https://doi.org/10.1134/s0026261723604906","url":null,"abstract":"<h3 data-test=\"abstract-sub-heading\">Abstract</h3><p>The bacteriophage <i>Pseudomonas</i> phage Ka1 was isolated from the water of a tributary stream of Lake Baikal, in the area of discharge of treated wastewater of the city of Slyudyanka. The bacteriophage genome contains 46 092 bp and has a 50% G+C pairs (AN OP455935.1). It exhibits 91% identity with the <i>Pseudomonas</i> phage PSA37 genome, belonging to <i>Bruynoghevirus</i> from the class <i>Caudoviricetes</i> suggesting that Ka1 is a new representative of Luz24-like phages (<i>Bruynoghevirus</i>). The bacteriophage lyzes 62% of <i>P. aeruginosa</i> clinical isolates and increases the efficiency of gentamicin, ciprofloxacin, imipenem and meropenem by 4–8-fold against these bacteria. Integrases, transposases and recombinases have not been identified in the genome of the bacteriophage <i>Pseudomonas</i> phage Ka1; which allows using it in the complex therapy of infections caused by <i>P. aeruginosa.</i></p>","PeriodicalId":18514,"journal":{"name":"Microbiology","volume":"2 1","pages":""},"PeriodicalIF":1.5,"publicationDate":"2024-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141770051","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":"Microalgae from Eroded Soils in the Northern Fergana Valley, Uzbekistan","authors":"Yu. A. Tukhtaboeva, E. S. Krivina, V. V. Red’kina, A. D. Temraleeva","doi":"10.1134/s0026261724605013","DOIUrl":"https://doi.org/10.1134/s0026261724605013","url":null,"abstract":"<h3 data-test=\"abstract-sub-heading\">\u0000<b>Abstract</b>\u0000</h3><p>For the first time, the cultivated diversity of microalgae in eroded soils in the northern part of the Fergana Valley in Uzbekistan has been studied based on both morphological and molecular genetic analysis. Ten strains of green microalgae (<i>Chlorophyta</i>) and one charophyte strain (<i>Charophyta</i>) were revealed. Only seven strains could be identified at the species level: <i>Chlorella vulgaris, Chromochloris zofingiensis, Deuterostichococcus epilithicus, Pseudomuriella schumacherensis</i>, and <i>Pseudostichococcus monallantoides</i>. Another four strains were identified only at the genus level and require further study: <i>Bracteacoccus</i> sp., <i>Chlorosarcinopsis</i> sp., <i>Klebsormidium</i> sp., and <i>Tetratostichococcus</i> sp. The low species diversity in the microalgae is likely due to both the low fertility of the eroded soils on the slopes, and the limitations of the culture-based approach that only reveals a fraction of the overall microbial diversity. Microalgal colonization of eroded soils in the arid foothill zone can be facilitated by various adaptations, such as small cell size and the production of extracellular polysaccharides, mycosporine-like aminoacids, and secondary carotenoids. The present work may contribute to the further development of highly functional microalgal consortia, which can lead to improvements and sustainable development of low-productivity, arid, and degraded terrestrial ecosystems.</p>","PeriodicalId":18514,"journal":{"name":"Microbiology","volume":"73 1","pages":""},"PeriodicalIF":1.5,"publicationDate":"2024-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141770039","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":"Antimicrobial Activities and Characterization of Bacteriocin-Like Substances Isolated from Lactic Acid Bacteria of Local Fermented Foods","authors":"R. C. Inovejas, C. C. Divina, J. K. Jacob, J. B. Abucay Jr., E. L. C. Inovejas","doi":"10.1134/s0026261723601999","DOIUrl":"https://doi.org/10.1134/s0026261723601999","url":null,"abstract":"<h3 data-test=\"abstract-sub-heading\">Abstract</h3><p>Bacteriocins produced by a number of lactic acid bacteria (LAB) comprise a variety of antimicrobial proteins that may be used as antibiotic alternative in food preservation. The present study aimed to isolate and characterize LAB-derived bacteriocin-like substances (BLS) from local fermented foods. A total of twenty-one bacterial strains were isolated and subjected to an antibacterial assay. Five isolates designated as AB1, AB2, AK1, AK2, AnK1 showed inhibitory activity against selected indicator organisms and were subjected to further characterization. Upon BLAST analysis of the 16S rRNA gene sequences, the isolates showed close relationship to <i>Lactobacillus plantarum</i> with similarity ranging from 91 to 97%. Moreover, the antibacterial metabolite production of the isolates was observed to peak on the 5th to 7th day of incubation. Since AnK1 showed the highest zone of inhibition among 5 isolates, it was selected for characterization of the BLS isolated by ammonium sulfate precipitation and anion exchange chromatography. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the AnK1-BLS had a single band and a molecular weight around 15 kDa, and a total protein concentration of 7.06 µg/mL. Interestingly, AnK1-BLS had a broad antimicrobial spectrum on both gram-negative and gram-positive food-borne pathogens, including <i>Escherichia coli</i>, <i>Salmonella enterica</i>, <i>Pseudomonas aeruginosa</i>, and <i>Bacillus subtilis</i>. Moreover, the purified AnK1-BLS exhibited good thermal stability when heated at 80‒121°C. Likewise, its inhibitory activity was not affected by pH in the range of 3.0 to 6.5, with maximum activity recorded at pH 3.0. The results suggest that AnK1-BLS may have application prospects in the food industry. Further studies on the complete protein characterization of AnK1-BLS is recommended to fully harness its capabilities.</p>","PeriodicalId":18514,"journal":{"name":"Microbiology","volume":"66 1","pages":""},"PeriodicalIF":1.5,"publicationDate":"2024-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141770043","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":"Assessment of Bioactive Potential and Characterization of an Anticancer Compound from the Endophytic Fungi of Ocimum sanctum","authors":"A. Verma, K. Kumar, U. Talukdar, G. Pal, D. Kumar, P. Shukla, S. Patel, A. Kumar, A. Kumar, R. N. Kharwar, S. K. Verma","doi":"10.1134/s002626172360252x","DOIUrl":"https://doi.org/10.1134/s002626172360252x","url":null,"abstract":"<h3 data-test=\"abstract-sub-heading\">Abstract</h3><p>The goal of this study was to isolate endophytic fungi from <i>Ocimum sanctum</i> L. and to evaluate their biological potential, including antibacterial, antioxidant, and enzymatic activities and further characterization of the bioactive compounds. Nine endophytic fungi were isolated from the leaves and stem tissues of <i>O. sanctum</i> collected from the botanical garden of Banaras Hindu University, Varanasi, India. All isolates were identified based on their microscopic structures and molecular sequencing of the ITS rDNA. <i>Aspergillus clavatonanicus</i> (SS7) and <i>Cochliobolus hawaiiensis</i> (SL3) showed the highest colonization frequencies in the stem and leaves (16 and 14%, respectively). All fungal isolates were tested for extracellular enzymatic activities of amylase, cellulase, and pectinase. Of the nine fungal isolates, 60% tested positive for amylase and cellulase, whereas 50% showed pectinase activity. Using a disc diffusion assay, the extracted secondary metabolites were checked for antibacterial activity against three human pathogenic bacteria. Two isolates, SL2 and SS7, exhibited the highest antibacterial activity against all pathogens, including <i>Enterococcus faecalis, Klebsiella pneumoniae</i>, and methicillin-resistant <i>Staphylococcus aureus</i> (MRSA). Crude extracts of the six fungal isolates showed positive antioxidant activity. The crude extract of <i>Aspergillus allahabadii</i> (isolate SL2) showed strong antibacterial and antioxidant activities and crystallized during purification. X-ray crystallography confirmed the identity of the crystal as citrinin, which also exhibited strong anticancer activity against Dalton’s lymphoma cells. The results of this study suggest that endophytic fungi isolated from the leaf and stem tissues of <i>Ocimum sanctum</i> are potential sources of antibacterial, antioxidant, and anticancer compounds.</p>","PeriodicalId":18514,"journal":{"name":"Microbiology","volume":"24 1","pages":""},"PeriodicalIF":1.5,"publicationDate":"2024-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141770042","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":"Effect of Electrical Stimulation of the External Circuit of Membraneless Bioelectrochemical Systems on Imidacloprid Degradation and Representation of the mtrB and DyP-Type Peroxidases Genes","authors":"A. A. Samkov, S. M. Samkova, M. N. Kruglova","doi":"10.1134/s0026261723604396","DOIUrl":"https://doi.org/10.1134/s0026261723604396","url":null,"abstract":"<h3 data-test=\"abstract-sub-heading\">\u0000<b>Abstract</b>\u0000</h3><p>In all variants where carbon felt was present, imidacloprid degradation by the microflora of bottom sediments was many times higher than in the control, reaching 84.0 ± 1.7% under the polar connection of an external voltage source (1.2 V). When <i>Shewanella oneidensis</i> MR-1 was introduced, almost complete degradation of the pollutant was observed, while in the control without electrodes, it was 29.7 ± 6.0%. The relative representation of the genes of the MtrB transmembrane protein of the respiratory chain, which is associated with exoelectrogenesis, depended on the external chain and had a maximum value when the voltage source was connected polarly, correlating with the pesticide degradation by the autochthonous microflora, similar to the DyP-type peroxidase genes. The introduction of <i>S. oneidensis</i> MR-1 resulted in an almost tenfold increase in the relative representation of DyP-type peroxidase genes. In all experimental variants, the values of the DyP relative representation were significantly higher than in the control without carbon felt, as well as the degree of imidacloprid degradation under these experimental conditions.</p>","PeriodicalId":18514,"journal":{"name":"Microbiology","volume":"347 1","pages":""},"PeriodicalIF":1.5,"publicationDate":"2024-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141770047","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":"The Effect of Probiotic Lactobacilli on the Morphological and Physiological Parameters and Intestinal Microbiota of Quails","authors":"E. A. Gavrilova, O. S. Karaseva, Y. N. Monir, A. M. Ezhkova, V. O. Ezhkov, R. A. Volkov, A. M. Senina, D. R. Khusnutdinova, E. V. Nikitina, D. R. Yarullina, A. R. Kayumov","doi":"10.1134/s0026261723604669","DOIUrl":"https://doi.org/10.1134/s0026261723604669","url":null,"abstract":"<h3 data-test=\"abstract-sub-heading\">Abstract</h3><p>The paper shows the effect of introducing <i>Lactiplantibacillus plantarum</i> AG10 biomass into quail feed. Quails that received a probiotic feed additive for seven weeks demonstrated a significant improvement in such economic indicators as the dynamics of live weight, the mass of gutted carcasses and feed consumption per kg of gain. Using the analysis of 16S rRNA gene sequence libraries sequenced on the Illumina MiSeq platform, shifts in the microbiota of the cecum have been detected, which may contribute to improving the morphological and physiological parameters of quails.</p>","PeriodicalId":18514,"journal":{"name":"Microbiology","volume":"66 1","pages":""},"PeriodicalIF":1.5,"publicationDate":"2024-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141770092","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}