International Microbiology最新文献

{"title":"Alleviation of heavy metals chromium, cadmium and lead and plant growth promotion in Vigna radiata L. plant using isolated Pseudomonas geniculata.","authors":"Barkha Madhogaria, Sangeeta Banerjee, Sohini Chakraborty, Prasanta Dhak, Atreyee Kundu","doi":"10.1007/s10123-024-00546-2","DOIUrl":"10.1007/s10123-024-00546-2","url":null,"abstract":"<p><p>Plants exposed to heavy metals (HMs) stress negatively affect their development and production capacity. Chromium (Cr), Cadmium (Cd), and Lead (Pb) are the most common hazardous trace metals in agriculture. The physiological, biochemical, and molecular characteristics of crops are being affected. Phytoremediation is a method to alleviate heavy metals from the contaminated soil. The study aims to evaluate the phytoremediation ability of Vigna radiata L. (mung bean) in the absence and the presence of multi-metal tolerant and plant growth promoting Pseudomonas geniculata strain TIU16A3 isolated from soil of tannery industrial estate, Kolkata, West Bengal, India. The strain was further assessed with increasing concentrations of Cr, Cd, and Pb (10, 20, 40, and 80 µg/mL) when the mung bean plant was a test crop. The strain significantly increased plant growth, chlorophyll content, increased level of antioxidant enzymes such as superoxide dismutase, peroxidase, and catalase, and decreased oxidative stress indicators like H₂O₂ and electrolyte leakage in the presence of Cr, Cd, and Pb as compared to plants grown in the absence of Pseudomonas geniculata strain. Shoot length responsive gene (Aux/IAA) in the presence of heavy metal alone and Pseudomonas geniculata treated Cd and Cr showed higher relative expression of (Aux/IAA) compared to Pb. Due to these intrinsic abilities, Pseudomonas geniculata strain TIU16A3 can be a plant growth promoter and thus can help in the remediation of heavy metal (Cr, Cd, and Pb) contaminated soil.</p>","PeriodicalId":14318,"journal":{"name":"International Microbiology","volume":" ","pages":"133-149"},"PeriodicalIF":2.3,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141446125","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":"Traditional marketed meats as a reservoir of multidrug-resistant Escherichia coli.","authors":"Fernando Guibert, Kathya Espinoza, Clara Taboada-Blanco, Carla A Alonso, Rosario Oporto, Angie K Castillo, Beatriz Rojo-Bezares, María López, Yolanda Sáenz, Maria J Pons, Joaquim Ruiz","doi":"10.1007/s10123-023-00445-y","DOIUrl":"10.1007/s10123-023-00445-y","url":null,"abstract":"<p><p>This study aimed to analyze Escherichia coli from marketed meat samples in Peru. Sixty-six E. coli isolates were recovered from 21 meat samples (14 chicken, 7 beef), and antimicrobial resistance levels and the presence of mechanisms of antibiotic resistance, as well as clonal relationships and phylogeny of colistin-resistant isolates, were established. High levels of antimicrobial resistance were detected, with 93.9% of isolates being multi-drug resistant (MDR) and 76.2% of samples possessing colistin-resistant E. coli; of these, 6 samples from 6 chicken samples presenting mcr-1-producer E. coli. Colistin-resistant isolates were classified into 22 clonal groups, while phylogroup A (15 isolates) was the most common. Extended-spectrum β-lactamase- and pAmpC-producing E. coli were found in 18 and 8 samples respectively, with bla_CTX-M-55 (28 isolates; 16 samples) and bla_CIT (8 isolates; 7 samples) being the most common of each type. Additionally, bla_CTX-M-15, bla_CTX-M-65, bla_SHV-27, bla_{OXA-5/10-like}, bla_DHA, bla_EBC and narrow-spectrum bla_TEM were detected. In addition, 5 bla_CTX-M remained unidentified, and no sought ESBL-encoding gene was detected in other 6 ESBL-producer isolates. The tetA, tetE and tetX genes were found in tigecycline-resistant isolates. This study highlights the presence of MDR E. coli in Peruvian food-chain. The high relevance of CTX-M-55, the dissemination through the food-chain of pAmpC, as well as the high frequency of unrelated colistin-resistant isolates is reported.</p>","PeriodicalId":14318,"journal":{"name":"International Microbiology","volume":" ","pages":"27-43"},"PeriodicalIF":2.3,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138295147","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":"Construction of L-type lectin displaying Saccharomyces cerevisiae for Vibrio parahaemolyticus agglutination.","authors":"Phuong-Thao Thi Nguyen, Thuan-Thien Dinh, Hieu Tran-Van","doi":"10.1007/s10123-023-00440-3","DOIUrl":"10.1007/s10123-023-00440-3","url":null,"abstract":"<p><p>The utilization of Aga1P anchor protein in the display system for expressing heterologous proteins on the surface of Saccharomyces cerevisiae has been shown to be an ideal approach. This system has the ability to improve the expression of target proteins beyond the cell surface, resulting in increased activity and stability of the expression system. Recent studies have demonstrated that a new L-type lectin from Litopenaeus vannamei (LvLTLC1) has been found to possess the capability of agglutinating Vibrio parahaemolyticus, a pathogen responsible for causing acute hepatopancreatic necrosis disease (AHPND) in shrimp. In this study, LvLTLC1 protein was designed to be expressed on the surface of S. cerevisiae via Aga1P anchor. The expression of LvLTLC1 protein on the surface of S. cerevisiae::pYIP-LvLTLC1-Aga1P was confirmed through the use of analytical techniques including SDS-PAGE, dot blot, and fluorescent immunoassay with LvLTC1-specific antibody. Subsequently, the newly generated yeast strain was evaluated for its ability to agglutinate V. parahaemolyticus and A. hydrophila. The obtained results indicated that S. cerevisiae expressing LvLTLC1 protein on its surface had the ability to agglutinate both AHPND-causing V. parahaemolyticus and A. hydrophila. This newly generated yeast strain could be served as a feed supplement for controlling bacteria in general and AHPND in particular.</p>","PeriodicalId":14318,"journal":{"name":"International Microbiology","volume":" ","pages":"1-10"},"PeriodicalIF":2.3,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"54229072","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":"Composition of the rumen archaeal community of growing camels fed different concentrate levels.","authors":"Alaa Emara Rabee, Ahmed R Askar","doi":"10.1007/s10123-023-00459-6","DOIUrl":"10.1007/s10123-023-00459-6","url":null,"abstract":"<p><p>Understanding the rumen fermentation and methanogenic community in camels fed intensively is critical for optimizing rumen fermentation, improving feed efficiency, and lowering methane emissions. Using Illumina MiSeq sequencing, quantitative real-time PCR, and high-performance liquid chromatography, this study evaluates the influence of different concentrate supplement levels in the diet on rumen fermentation as well as the diversity and structure of the rumen methanogenic community for growing dromedary camels. Twelve growing camels were divided into three groups and given three levels of concentrate supplement, 0.7% (C1), 1% (C2), and 1.3% (C3) based on their body weight. All animals were fed alfalfa hay ad libitum. The levels of total volatile fatty acid, rumen ammonia, and methanogen copy number were unaffected by the supplementation level. Increasing the concentrate supplement level increased the proportion of propionic acid while decreasing the proportion of acetic acid. Increasing the level of concentrate in the diet had no effect on alpha diversity metrics or beta diversity of rumen methanogens. Methanobrevibacter and Methanosphaera predominated the methanogenic community and were declined as concentrate supplement level increased. This study sheds new light on the effect of concentrate supplement level in growing camels' diet on rumen fermentation and methanogenic community, which could help in the development of a strategy that aimed to reduce methane emissions and enhance feed efficiency.</p>","PeriodicalId":14318,"journal":{"name":"International Microbiology","volume":" ","pages":"53-62"},"PeriodicalIF":2.3,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12078364/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138498397","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":"Virulence genes of Pasteurella multocida cap B and its potential cross protection in mice.","authors":"Adam Bashir Tawor, Osman Erganiş, Aslı Balevi","doi":"10.1007/s10123-025-00658-3","DOIUrl":"https://doi.org/10.1007/s10123-025-00658-3","url":null,"abstract":"<p><p>Pasteurella multocida is a Gram-negative coccobacillus from the Pasteurellaceae family, commonly residing as a commensal organism in the respiratory tracts of healthy animals. However, it possesses multiple virulence factors and can cause severe respiratory diseases. This study aimed to characterize P. multocida and its virulence genes and evaluate the immunogenicity of an inactivated vaccine of serogroup B using different administration routes. A total of 250 samples were collected from animals showing respiratory symptoms. Using 5% blood agar, 27 P. multocida isolates were obtained, and 21 (8.4%) were confirmed via PCR targeting the kmt1 gene. Nineteen virulence-associated genes were screened, categorized into outer membrane, fimbrial, somatic antigen, and iron-binding genes. The plpB, tadD, gatG, and hgbA genes were detected in both serogroup B and E isolates, whereas ompA, toxA, pcgD, latB, nctB, ppgB, natG, hgbB, and exbB were absent in all isolates. The immunogenicity of an inactivated P. multocida vaccine was evaluated in mice using subcutaneous and intramuscular routes. Subcutaneous vaccination produced a significantly higher antibody titer at 3 and 5 weeks post-vaccination with a 0.5 mL dose; in contrast, intramuscular immunization resulted in a rapid increase after booster doses. Analysis of variance (ANOVA) and Duncan's multiple range test (p < 0.05) revealed statistically significant differences between treatments. The comparison between subcutaneous and intramuscular routes also showed a significant difference (p < 0.001). This study concludes that although P. multocida serogroup B harbors fewer virulence factors, it effectively induces an immune response in mice but fails to provide cross-protection against the local serogroup E strain.</p>","PeriodicalId":14318,"journal":{"name":"International Microbiology","volume":" ","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144010185","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":"Bio-control efficacy of selected indigenous nematophagous fungi against Meloidogyne enterolobii in vitro and on dry bean (Phaseolus vulgaris L.).","authors":"Ndivhuwo Ramatsitsi, Zakheleni Palane Dube, Khosi Ramachela, Tuelo Motloba","doi":"10.1007/s10123-024-00571-1","DOIUrl":"10.1007/s10123-024-00571-1","url":null,"abstract":"<p><p>Dry bean (Phaseolus vulgaris L.) is an important commercialized field crop in South Africa for aiding in food security as a cheap protein source. However, it is highly susceptible to root-knot nematodes (RKN), Meloidogyne species. Use of indigenous nematophagous fungi as bio-control agents (BCA) of Meloidogyne nematodes is a promising research focus area. This is because indigenous fungal species are naturally part of the ecosystem and therefore compatible with other biological processes unlike most synthetic chemicals. The objective of the study was to identify indigenous nematophagous fungal BCA and establish their potential efficacy in reducing M. enterolobii population densities on dry bean with and without incorporation of compost. Screened indigenous fungal species included Aspergillus terreus, Talaromyces minioluteus, T. sayulitensis, Trichoderma ghanense, and T. viride. There were observed significant parasitism differences (P ≤ 0.05) among the BCA, with T. ghanense showing the highest egg parasitism (86%), followed by T. minioluteus (72%) and T. sayulitensis (70%). On the other hand, the highest J2 parasitism was observed on T. minioluteus (95%), followed by A. terreus and T. viride (63%). A similar trend was observed under in vivo conditions, with higher efficacy with compost incorporation. This provides a highly encouraging alternative and ecologically complementary Meloidogyne management in dry bean production.</p>","PeriodicalId":14318,"journal":{"name":"International Microbiology","volume":" ","pages":"151-160"},"PeriodicalIF":2.3,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12078443/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141912753","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":"Determining the potential targets of silybin by molecular docking and its antibacterial functions on efflux pumps and porins in uropathogenic E. coli.","authors":"Shirin Fekri Kohan, Alireza Nouhi Kararoudi, Maryam Bazgosha, Somayeh Adelifar, Arman Hafezolghorani Esfahani, Fatemeh Ghaderi Barmi, Reyhaneh Kouchakinejad, Ebrahim Barzegari, Mahdi Shahriarinour, Najmeh Ranji","doi":"10.1007/s10123-024-00488-9","DOIUrl":"10.1007/s10123-024-00488-9","url":null,"abstract":"<p><strong>Background: </strong>One of the causes of antibiotic resistance is the reduced accumulation of antibiotics in bacterial cells through pumping out the drugs. Silybin, a key component of the Silybum marianum plant, exhibits various beneficial properties, including anti-bacterial, anti-inflammatory, antioxidant, and hepatoprotective effects.</p><p><strong>Methods and results: </strong>Clinical isolates of E. coli were procured from 17 Shahrivar Children's Hospital in Rasht, Guilan, located in northern Iran. Their susceptibility to six antibiotics was assessed using disc diffusion and broth dilution (MIC) methods. The antibacterial effects of silybin-loaded polymersome nanoparticles (SPNs) were investigated with broth dilution (MIC) and biofilm assays. Molecular docking was utilized to evaluate silybin's (the antibacterial component) binding affinity to efflux pumps, porins, and their regulatory elements. Additionally, qRT-PCR analysis explored the expression patterns of acrA, acrB, tolC, ompC, and ompF genes in both SPNs (sub-MIC) and ciprofloxacin (sub-MIC)-treated and untreated E. coli isolates. The combined use of SPNs and ciprofloxacin exhibited a notable reduction in bacterial growth and biofilm formation, in ciprofloxacin-resistant isolates. The study identified eight overlapping binding sites of the AcrABZ-TolC efflux pump in association with silybin, demonstrating a binding affinity ranging from -7.688 to -10.33 Kcal/mol. Furthermore, the qRT-PCR analysis showed that silybin upregulated AcrAB-TolC efflux pump genes and downregulated ompC and ompF porin genes in combination with ciprofloxacin in transcriptional level in uropathogenic E. coli.</p><p><strong>Conclusions: </strong>Silybin, a safe herbal compound, exhibits potential in inhibiting antibiotic resistance within bacterial isolates, potentially through the regulation of gene expression and plausible binding to target proteins.</p>","PeriodicalId":14318,"journal":{"name":"International Microbiology","volume":" ","pages":"63-74"},"PeriodicalIF":2.3,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139740986","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 of the inorganic phosphorus-solubilizing bacteria Lysinibacillus sphaericus and assessing its role in promoting rice growth.","authors":"Lin Pan, Qingshan Xu, Qianqian Wei, Yali Kong, Lianfeng Zhu, Wenhao Tian, Yulian Yan, Hangfeng Wang, Chunxin Chi, Junhua Zhang, Chunquan Zhu","doi":"10.1007/s10123-024-00538-2","DOIUrl":"10.1007/s10123-024-00538-2","url":null,"abstract":"<p><p>Soluble phosphorus scarcity severely limits plant growth and crop yield. In this study, a strain of inorganic phosphorus-solubilizing bacteria, Lysinibacillus sphaericus, was isolated from rice rhizosphere soil. The available phosphorus content in liquid inorganic phosphorus identification medium and in L. sphaericus-inoculated soil increased from 204.28 mg/L to 1124.68 mg/L and from 4.75 mg/kg to 7.04 mg/kg, respectively. The pH decreased significantly from 6.87 to 6.14. Incubation with L. sphaericus significantly increased malic and succinic acid content in the liquid inorganic phosphorus identification medium and increased acid phosphatase and alkaline phosphatase activity in the soil. Inoculation with L. sphaericus significantly increased rice growth, chlorophyll a/b content, and photosynthesis by increasing the soluble phosphorus content in the rice rhizosphere soil under phosphorus-deficient conditions. Further analysis revealed that L. sphaericus improved soil phosphorus release by decreasing soil pH and promoting acid phosphatase and alkaline phosphatase activity. This study supports the production of microbial fertilizers to improve rice yield in phosphorus-deficient conditions.</p>","PeriodicalId":14318,"journal":{"name":"International Microbiology","volume":" ","pages":"119-131"},"PeriodicalIF":2.3,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141157216","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":"Detection and evaluation of susceptibility to antibiotics in non-hydrogen sulfide-producing antibiotic-resistant soil microbe: Pseudomonas guariconensis.","authors":"Sahithya Selvakumar, Shubhi Singh, Priya Swaminathan","doi":"10.1007/s10123-024-00537-3","DOIUrl":"10.1007/s10123-024-00537-3","url":null,"abstract":"<p><p>Antimicrobial resistance in bacteria is a global threat that can make antibacterial treatments ineffective. One well-known method of antibiotic resistance and a common defensive mechanism in many harmful bacteria is the synthesis of endogenous hydrogen sulfide (H₂S) in bacteria. In this study, soil bacteria were screened using the lead acetate agar test and the triple sugar iron test to determine that they were non-endogenous H₂S producers. This was further validated by full genome analysis of the identified organism against the gene sequences of H₂S-producing genes. Antibacterial resistance of the bacteria was phenotypically analyzed using the Kirby-Bauer disk diffusion method. Then, the effect of exogenous H₂S on the antibiotic-resistant bacteria was checked in sodium sulfide, leading to antibiotic re-sensitization.</p>","PeriodicalId":14318,"journal":{"name":"International Microbiology","volume":" ","pages":"111-118"},"PeriodicalIF":2.3,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141064949","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":"Impact of kefir yeasts on Fusarium graminearum growth and production of deoxynivalenol.","authors":"Candela Moure, Diana Ramírez Albuquerque, Angela León Peláez, Virginia Fernández Pinto, Teresa Alconada","doi":"10.1007/s10123-025-00661-8","DOIUrl":"https://doi.org/10.1007/s10123-025-00661-8","url":null,"abstract":"<p><p>The kefir granules are a microbial consortium with numerous properties, among which their antifungal action has been reported. The aim of this research was to evaluate the antifungal capacity of yeasts isolated from kefir against Fusarium graminearum for its possible application as biocontrol agents. The inhibition of fungal growth was analyzed, according to dual tests, and the production of volatile organic compounds. The inhibition of the production of the mycotoxin deoxynivalenol was also analyzed. The six isolates studied belonged to the species Geotrichum candidum (2), Saccharomyces cerevisiae (1), Pichia kudriavzevii (2), and Pichia membranifaciens (1). They were isolated from kefir and identified in a previous work. The yeasts tested produced a reduction in the growth of F. graminearum in all assays. In particular, three of the isolates, 1 belonging to the species S. cerevisiae and 2 to the species P. kudriavzevii, generated volatile organic compounds that significantly inhibited the growth of the fungus in a 49.61, 51.46, and 49.61%, respectively. The yeasts produced an inhibition of the mycotoxin deoxynivalenol correlated with a reduction in the growth of the fungus. The isolates of Geotrichum candidum (Gc30) and Pichia kudriavzevii (Pk27, PK20) significantly reduced the deoxynivalenol content of F. graminearum by 86, 89, and 88%, and the growth inhibition in the assay was 34, 61, and 48%, respectively. The data obtained about yeasts of novel origin are promising for their possible use as fungal biocontrol agents in foods as an alternative to agrochemicals.</p>","PeriodicalId":14318,"journal":{"name":"International Microbiology","volume":" ","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-04-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144017117","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}