International Microbiology最新文献

{"title":"Calonectria eucalyptorum sp. nov., a new leaf blight pathogen of Eucalyptus from India.","authors":"Nitika Negi, Ramkrishna, Rajendra K Meena, Maneesh S Bhandari, Shailesh Pandey","doi":"10.1007/s10123-024-00602-x","DOIUrl":"https://doi.org/10.1007/s10123-024-00602-x","url":null,"abstract":"<p><p>In this study, Calonectria eucalyptorum sp. nov. is described from the blighted leaves of Eucalyptus in India using morphological and multi-locus phylogenetic analyses. The new species belongs to the Calonectria cylindrospora species complex, and its unique microscopic features and DNA sequence information enable clear separation from the 12 currently accepted species in this complex. Conidia of the new taxon are slightly longer than those of its phylogenetic neighbors. Additionally, this species produces central as well as lateral stipe extensions, which is a feature not known for the other members of the C. cylindrospora species complex. Analyses of the combined partial calmodulin, histone, translation elongation factor-1α, and β-tubulin gene regions revealed a distinct phylogenetic position for C. eucalyptorum. Recombination analysis provided additional support for the new species hypothesis. Koch's postulates for the new taxon as a foliar pathogen of Eucalyptus were fulfilled. The discovery of novel and pathogenic Calonectria species is important because it sheds light on species diversity, potential threats, and disease control.</p>","PeriodicalId":14318,"journal":{"name":"International Microbiology","volume":" ","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-10-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142380846","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":"Carlos Asensio and the dawn of molecular microbial ecology.","authors":"Víctor de Lorenzo, Fernando Baquero, Alfredo Aguilar","doi":"10.1007/s10123-024-00596-6","DOIUrl":"https://doi.org/10.1007/s10123-024-00596-6","url":null,"abstract":"<p><p>At near 50 years of the discovery of microcins, this article highlights the pivotal-but under-recognised-influence of Spanish biochemist Carlos Asensio (1925-1982) in contemporary microbiology, featuring the epistemological, sociological, and cultural impact of his scientific achievements. At a time when the intestinal microbiome is central to current biomedical research, it is due to emphasise his role in the establishment of new scientific fields that are now considered fundamental. Despite his premature death at the peak of his conceptual and experimental creativity, many of his ideas about microbial communication in complex communities inspired a generation of researchers and opened new topics reach to this day. Asensio was also a trailblazer in Spain, advocating for fundamental research within the socio-economic context of his time. He foresaw the shift towards what is now termed the knowledge-based bioeconomy, recognised the need for multidisciplinary research teams, and advocated integration science into societal and political agendas. These facets became evident during his research on microcins, low molecular weight bioactive compounds produced by enterobacteria. These molecules were hypothesised as mediators of microbial interactions in the human gut and were considered potential new antibiotics and even antitumoral agents. His research mobilised young talent and attracted unprecedented resources in Spain during the late 1970s-early 1980s. It underscored the medical value of microbial ecology and exemplified the benefits of collaboration between academia and industry. Asensio played a pivotal role in the emergence of molecular microbial ecology as a research discipline and its foundational and applied significance in biotechnology.</p>","PeriodicalId":14318,"journal":{"name":"International Microbiology","volume":" ","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142365208","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":"Investigating the antifungal potential of genetically modified hybrid chitinase enzymes derived from Bacillus subtilis and Serratia marcescens.","authors":"Nahla Alsayd Bouqellah, Lina Jamil Mohamed Abdel-Hafez, Islam Yousif Mostafa, Ahmed Hassan Ibrahim Faraag","doi":"10.1007/s10123-024-00591-x","DOIUrl":"https://doi.org/10.1007/s10123-024-00591-x","url":null,"abstract":"<p><p>Chitinases are glycosyl hydrolase enzymes that break down chitin, an integral component of fungal cell walls. Bacteria such as Bacillus subtilis and Serratia marcescens produce chitinases with antifungal properties. In this study, we aimed to generate hybrid chitinase enzymes with enhanced antifungal activity by combining functional domains from native chitinases produced by B. subtilis and S. marcescens. Chitinase genes were cloned from both bacteria and fused together using overlap extension PCR. The hybrid constructs were expressed in E. coli and the recombinant enzymes purified. Gel electrophoresis and computational analysis confirmed the molecular weights and isoelectric points of the hybrid chitinases were intermediate between the parental enzymes. Antifungal assays demonstrated that the hybrid chitinases inhibited growth of the fungus Fusarium oxysporum significantly more than the native enzymes and also showed fungicidal activity against Candida albicans, Alternaria solani, and Rhizoctonia solani. The results indicate that hybrid bacterial chitinases are a promising approach to engineer novel antifungal proteins. This study provides insight into structure-function relationships of chitinases and strategies for generating biotherapeutics with enhanced bioactive properties. These hybrid chitinases result in a more potent and versatile antifungal agent.</p>","PeriodicalId":14318,"journal":{"name":"International Microbiology","volume":" ","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142361486","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":"Integrated processes for olive mill wastewater treatment and its revalorization for microalgae culture.","authors":"Rihab Hachicha, Jihen Elleuch, Pascal Dubessay, Ridha Hachicha, Slim Abdelkafi, Philippe Michaud, Imen Fendri","doi":"10.1007/s10123-024-00600-z","DOIUrl":"https://doi.org/10.1007/s10123-024-00600-z","url":null,"abstract":"<p><p>The olive oil industry generates 30 million cubic meters of olive mill wastewaters (OMWWs) annually. OMWWs are a major environmental concern in the Mediterranean region due to their high organic matter content, suspended solids, unpleasant odor, and dark color. The application of primary treatments such as coagulation-flocculation, adsorption, and hybrid systems combining coagulation-flocculation with adsorption has enabled to remove part of the organic matter, color, turbidity, and growth-inhibiting compounds from OMWWs. Among these methods, the hybrid system combining activated carbon and chitosan has proven to be the best removal efficiency. Subsequently, secondary treatment involving the cultivation of Chlorella sp. on OMWWs pretreated with chitosan achieved the highest maximal specific growth rate (0.513 ± 0.022 day⁻¹) and biomass productivity (0.621 ± 0.021 g/L/day). Notably, the fatty acids (FA) profile produced by Chlorella sp. cells grown under these conditions differed, underscoring the potential of OMWWs as a microalgal growth medium. This innovative approach not only addresses environmental issues but also opens new avenues for sustainable bioproducts.</p>","PeriodicalId":14318,"journal":{"name":"International Microbiology","volume":" ","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142365209","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":"Theophylline as a quorum sensing and biofilm inhibitor in Pseudomonas aeruginosa and Chromobacterium violaceum.","authors":"Aparna Mohan, Pooja P Rajan, Praveen Kumar, Devi Jayakumar, Minsa Mini, Sneha Asha, Parvathi Vaikkathillam","doi":"10.1007/s10123-024-00487-w","DOIUrl":"10.1007/s10123-024-00487-w","url":null,"abstract":"<p><p>Quorum sensing (QS) is pivotal in coordinating virulence factors and biofilm formation in various pathogenic bacteria, making it a prime target for disrupting bacterial communication. Pseudomonas aeruginosa is a member of the \"ESKAPE\" group of bacterial pathogens known for their association with antimicrobial resistance and biofilm formation. The current antibiotic arsenal falls short of addressing biofilm-related infections effectively, highlighting the urgent need for novel therapeutic agents. In this study, we explored the anti-QS and anti-biofilm properties of theophylline against two significant pathogens, Chromobacterium violaceum and P. aeruginosa. The production of violacein, pyocyanin, rhamnolipid, and protease was carried out, along with the evaluation of biofilm formation through methods including crystal violet staining, triphenyl tetrazolium chloride assay, and fluorescence microscopy. Furthermore, computational analyses were conducted to predict the targets of theophylline in the QS pathways of P. aeruginosa and C. violaceum. Our study demonstrated that theophylline effectively inhibits QS activity and biofilm formation in C. violaceum and P. aeruginosa. In P. aeruginosa, theophylline inhibited the production of key virulence factors, including pyocyanin, rhamnolipid, protease, and biofilm formation. The computational analyses suggest that theophylline exhibits robust binding affinity to CviR in C. violaceum and RhlR in P. aeruginosa, key participants in the QS-mediated biofilm pathways. Furthermore, theophylline also displays promising interactions with LasR and QscR in P. aeruginosa. Our study highlights theophylline as a versatile anti-QS agent and offers a promising avenue for future research to develop novel therapeutic strategies against biofilm-associated infections.</p>","PeriodicalId":14318,"journal":{"name":"International Microbiology","volume":" ","pages":"1457-1471"},"PeriodicalIF":2.3,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139717536","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":"Functional characterization of biodegradable films obtained from whole Paecilomyces variotii biomass.","authors":"Ezequiel A Martinez, Andrés G Salvay, Macarena R Sanchez-Díaz, Vanesa Ludemann, Mercedes A Peltzer","doi":"10.1007/s10123-024-00501-1","DOIUrl":"10.1007/s10123-024-00501-1","url":null,"abstract":"<p><p>The indiscriminate use of petroleum-based polymers and plastics for single-use food packaging has led to serious environmental problems due the non-biodegradable characteristics. Thus, much attention has been focused on the research of new biobased and biodegradable materials. Yeast and fungal biomass are low-cost and abundant sources of biopolymers with highly promising properties for the development of biodegradable materials. This study aimed to select a preparation method to develop new biodegradable films using the whole biomass of Paecilomyces variotii subjected to successive physical treatments including ultrasonic homogenization (US) and heat treatment. Sterilization process had an important impact on the final filmogenic dispersion and mechanical properties of the films. Longer US treatments produced a reduction in the particle size and the application of an intermediate UT treatment contributed favorably to the breaking of agglomerates allowing the second US treatment to be more effective, achieving an ordered network with a more uniform distribution. Samples that were not filtrated after the sterilization process presented mechanical properties similar to plasticized materials. On the other hand, the filtration process after sterilization eliminated soluble and hydratable compounds, which produced a reduction in the hydration of the films.</p>","PeriodicalId":14318,"journal":{"name":"International Microbiology","volume":" ","pages":"1573-1585"},"PeriodicalIF":2.3,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140131393","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":"Genome sequencing and analysis of penicillin V producing Penicillium rubens strain BIONCL P45 isolated from India.","authors":"Amol M Sawant, Vishwambar D Navale, Koteswara Rao Vamkudoth","doi":"10.1007/s10123-024-00491-0","DOIUrl":"10.1007/s10123-024-00491-0","url":null,"abstract":"<p><strong>Background: </strong>A filamentous fungus Penicillium rubens is widely recognized for producing industrially important antibiotic, penicillin at industrial scale.</p><p><strong>Objective: </strong>To better comprehend, the genetic blueprint of the wild-type P. rubens was isolated from India to identify the genetic/biosynthetic pathways for phenoxymethylpenicillin (penicillin V, PenV) and other secondary metabolites.</p><p><strong>Method: </strong>Genomic DNA (gDNA) was isolated, and library was prepared as per Illumina platform. Whole genome sequencing (WGS) was performed according to Illumina NovoSeq platform. Further, SOAPdenovo was used to assemble the short reads validated by Bowtie-2 and SAMtools packages. Glimmer and GeneMark were used to dig out total genes in genome. Functional annotation of predicted proteins was performed by NCBI non-redundant (NR), UniProt, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Ontology (GO) databases. Moreover, secretome analysis was performed by SignalP 4.1 and TargetP v1.1 and carbohydrate-active enzymes (CAZymes) and protease families by CAZy database. Comparative genome analysis was performed by Mauve 2.4.0. software to find genomic correlation between P. rubens BIONCL P45 and Penicillium chrysogenum Wisconsin 54-1255; also phylogeny was prepared with known penicillin producing strains by ParSNP tool.</p><p><strong>Results: </strong>Penicillium rubens BIONCL P45 strain was isolated from India and is producing excess PenV. The 31.09 Mb genome was assembled with 95.6% coverage of the reference genome P. chrysogenum Wis 54-1255 with 10687 protein coding genes, 3502 genes had homologs in NR, UniProt, KEGG, and GO databases. Additionally, 358 CAZymes and 911 transporter coding genes were found in genome. Genome contains complete pathways for penicillin, homogentisate pathway of phenyl acetic acid (PAA) catabolism, Andrastin A, Sorbicillin, Roquefortine C, and Meleagrin. Comparative genome analysis of BIONCL P45 and Wis 54-1255 revealed 99.89% coverage with 2952 common KEGG orthologous protein-coding genes. Phylogenetic analysis revealed that BIONCL P45 was clustered with Fleming's original isolate P. rubens IMI 15378.</p><p><strong>Conclusion: </strong>This genome can be a helpful resource for further research in developing fermentation processes and strain engineering approaches for high titer penicillin production.</p>","PeriodicalId":14318,"journal":{"name":"International Microbiology","volume":" ","pages":"1473-1484"},"PeriodicalIF":2.3,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139931076","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":"Unveiling the bioactive potential of Actinomycetota from the Tagus River estuary.","authors":"José Diogo Neves Dos Santos, Eugénia Pinto, Jesús Martín, Francisca Vicente, Fernando Reyes, Olga Maria Lage","doi":"10.1007/s10123-024-00483-0","DOIUrl":"10.1007/s10123-024-00483-0","url":null,"abstract":"<p><p>The increase in global travel and the incorrect and excessive use of antibiotics has led to an unprecedented rise in antibiotic resistance in bacterial and fungal populations. To overcome these problems, novel bioactive natural products must be discovered, which may be found in underexplored environments, such as estuarine habitats. In the present work, estuarine actinomycetotal strains were isolated with conventional and iChip techniques from the Tagus estuary in Alcochete, Portugal, and analysed for different antimicrobial bioactivities. Extracts were produced from the isolated cultures and tested for bioactivity against Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922, Aspergillus fumigatus ATCC 240305, Candida albicans ATCC 10231 and Trichophyton rubrum FF5. Furthermore, bioactive extracts were subjected to dereplication by high-performance liquid chromatography (HPLC) and high-resolution mass spectrometry (HRMS) to putatively identify their chemical components. In total, 105 isolates belonging to 3 genera were obtained. One which was isolated, MTZ3.1^T, represents a described novel taxon for which the name Streptomyces meridianus was proposed. Regarding the bioactivity testing, extracts from 12 strains proved to be active against S. aureus, 2 against E. coli, 4 against A. fumigatus, 3 against C. albicans and 10 against T. rubrum. Dereplication of bioactive extracts showed the presence of 28 known bioactive molecules, 35 hits have one or more possible matches in the DNP and 18 undescribed ones. These results showed that the isolated bacteria might be the source of new bioactive natural products.</p>","PeriodicalId":14318,"journal":{"name":"International Microbiology","volume":" ","pages":"1357-1372"},"PeriodicalIF":2.3,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11452475/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139485537","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Endophytic bacilli from Cyamopsis tetragonoloba (L.) Taub. induces plant growth and drought tolerance.","authors":"Vaishali Umrao, Sushma Yadav, Pradeep Semwal, Sankalp Misra, Shashank Kumar Mishra, Puneet Singh Chauhan, Pramod Arvind Shirke","doi":"10.1007/s10123-024-00499-6","DOIUrl":"10.1007/s10123-024-00499-6","url":null,"abstract":"<p><p>Cyamopsis tetragonoloba (L.) Taub. (guar) is a commercially important crop known for its galactomannan content in seeds. Drought stress is a significant global concern that compromises the productivity of major legumes including guar. The endophytic microbes associated with plants play a significant role in enhancing plant growth and modulating the impact of abiotic stress(s). The present study involved the isolation of 73 endophytic bacteria from the guar seeds of drought-tolerant (RGC-1002 and RGC-1066) and sensitive (Sarada and Varsha) varieties. Based on multiple PGP attributes and drought tolerance, at 50% PEG6000 w/v, 11 efficient isolates were selected and identified through 16S rRNA gene sequencing. Isolates belonging to ten different species of bacilli including Cytobacillus oceanisediminis, Mesobacillus fermenti, Peribacillus simplex from sensitive and Bacillus zanthoxyli, B. safensis, B. velezensis, B. altitudinis, B. licheniformis, B. tequilensis, and B. paralicheniformis isolated from tolerant varieties. A greenhouse experiment with a drought-sensitive guar variety demonstrated that inoculation of selected isolates showed comparatively better plant growth, higher relative water content (RWC), decreased carbon isotope discrimination ratio (Δ¹³C), increased chlorophyll, carotenoids, anthocyanin, and proline content, decreased malondialdehyde (MDA) and modulated defense enzymes as compared to their uninoculated controls. Tolerant variety isolates B. tequilensis NBRI14G and B. safensis NBRI10R showed the most promising results in improving plant growth and also drought stress tolerance in guar plants. This study represents for the first time that seed endophytic bacterial strains from guar can be utilized to develop the formulation for improving the productivity of guar under drought-stress conditions.</p>","PeriodicalId":14318,"journal":{"name":"International Microbiology","volume":" ","pages":"1541-1556"},"PeriodicalIF":2.3,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140110232","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":"Influence of coloured lights on growth and enzyme production of beneficial endophytic fungi.","authors":"Adeline Su Yien Ting, Peck Ting Gan","doi":"10.1007/s10123-024-00486-x","DOIUrl":"10.1007/s10123-024-00486-x","url":null,"abstract":"<p><p>The influence of light regulation on fungal growth and enzyme production was tested on endophytic isolates of Fusarium proliferatum (CCH), Colletotrichum boninense (PL1, PL9, OL2), Colletotrichum gloeosporiodes (OL3) and Colletotrichum siamense (PL3). The isolates were treated with blue, red, green, and yellow light, while white fluorescent light (12 h light/12 h dark photoperiod) and 24 h dark conditions were applied as control. Results revealed that coloured light treatments induced formation of circadian rings, while exposure to white light and dark conditions showed less pronounced circadian rings. Growth and sporulation of endophytes were not significantly influenced by light. By contrast, enzyme production was affected by coloured light treatments, notably with red (amylase), blue (cellulase) and yellow (cellulase, xylanase, L-asparaginase) light, resulting in lower enzyme levels for certain isolates. Under control conditions, enzyme production was relatively higher for amylase, cellulase, xylanase (for cultures incubated in the dark), and for L-asparaginase (for cultures incubated in white fluorescent light). Among the endophytic isolates, F. proliferatum (CCH) showed better response to coloured light treatment as higher sporulation and enzyme production was detected, although growth was significantly suppressed. On the contrary, C. gloeosporiodes (OL3) showed better growth but significantly lower enzyme production and sporulation when treated with the various coloured light. This study revealed that coloured light may have the potential to manipulate growth, sporulation and enzyme production in certain fungal species as strategies for fungal control or for harnessing of valuable enzymes.</p>","PeriodicalId":14318,"journal":{"name":"International Microbiology","volume":" ","pages":"1405-1416"},"PeriodicalIF":2.3,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139563617","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}