Microbiological research最新文献

{"title":"Leuconostoc mesenteroides strain MS4-derived bacteriocins: A potent antimicrobial arsenal for controlling Xylella fastidiosa infection.","authors":"Miloud Sabri, Kaoutar El Handi, Cosima Damiana Calvano, Mariachiara Bianco, Angelo De Stradis, Franco Valentini, Toufic Elbeaino","doi":"10.1016/j.micres.2025.128071","DOIUrl":"https://doi.org/10.1016/j.micres.2025.128071","url":null,"abstract":"<p><p>Xylella fastidiosa subsp. pauca (Xfp) currently presents a serious threat to agriculture in Europe and in the Mediterranean, following its discovery in several countries. Addressing this bacterial plant disease with traditional agricultural practices and management strategies has proven inadequate, highlighting the urgent need for effective and environmentally safe antibacterial solutions. In this study, we explored the antibacterial activity of the lactic acid bacterium Leuconostoc mesenteroides strain MS4-derived bacteriocins against Xfp, utilizing a combination of in vitro and in planta experiments. In particular, the cell-free precipitate (CFP) derived from strain MS4 culture in MRS broth, suppressed Xfp growth on BCYE agar plate, whereas protease K-treated CFP was inactive, highlighting the presence of antimicrobial compounds of proteinaceous nature. Additionally, fluorescence and transmission electron microscopy analyses showed that the CFP exhibits a bactericidal effect on Xfp cells, characterized by membrane disruption and subsequent cellular damage. The whole-genome sequencing and bioinformatic analysis revealed that MS4 genome consists of a circular chromosome of 1860,891 bp and a circular plasmid of 37,317 bp and most importantly to encompass six bacteriocin-encoding genes, with a peptide size ranging from 45 to 59 amino acids. MALDI-TOF/TOF MS and RPLC-ESI-MS assays performed on cell-free supernatant (CFS) confirmed the secretion of four (out of 6) bacteriocins (denoted MK-45, MR-53, MW-56, and MG-58) by MS4 in MRS broth. In spot assays, these bacteriocins displayed significant lethality against Xfp, with a minimum lethal concentration between 0.2 and 0.4 mg/mL. The application of CFP on Xfp-infected Nicotiana benthamiana plants, implemented both as preventive and curative approach, successfully controlled the infection, resulting in no visible symptoms 40 days post-inoculation. The finding of MS4 as a natural source of various potent bacteriocins against Xfp, coupled with a significant production under low-cost and uncomplicated laboratory conditions, make of MS4 a cost-effective and realistic option for sustainable management of Xf-related diseases.</p>","PeriodicalId":18564,"journal":{"name":"Microbiological research","volume":"293 ","pages":"128071"},"PeriodicalIF":6.1,"publicationDate":"2025-01-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008304","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"De novo evolution of antibiotic resistance to Oct-TriA₁.","authors":"Farhan R Chowdhury, Laura Domínguez Mercado, Katya Kharitonov, Brandon L Findlay","doi":"10.1016/j.micres.2025.128056","DOIUrl":"https://doi.org/10.1016/j.micres.2025.128056","url":null,"abstract":"<p><p>The rise of antimicrobial resistance as a global health concern has led to a strong interest in compounds able to inhibit the growth of bacteria without detectable levels of resistance evolution. A number of these compounds have been reported in recent years, including the tridecaptins, a small family of lipopeptides typified by the synthetic analogue octyl-tridecaptin A₁. Hypothesizing that prior reports of negligible resistance evolution have been due in part to limitations in the laboratory evolution systems used, we have attempted to select for resistant mutants using a soft agar gradient evolution (SAGE) system developed by our lab. Following optimization of the media conditions by incorporation of the anti-synaeresis agent xanthan gum into the agar matrix, we successfully evolved high-level resistance to both octyl-tridecaptin A₁ as well as the challenging lipopeptide antibiotic polymyxin B. Decreased tridecaptin susceptibility was linked to mutations in outer membrane proteins ompC, lptD and mlaA, with the effect of these genes confirmed through a mix of allelic replacement and knockout studies. Overall, this work demonstrates the robust evolutionary potential of bacteria, even in the face of challenging antimicrobial agents.</p>","PeriodicalId":18564,"journal":{"name":"Microbiological research","volume":"293 ","pages":"128056"},"PeriodicalIF":6.1,"publicationDate":"2025-01-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008303","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Transcriptomic analysis of sub-MIC Eugenol exposition on antibiotic resistance profile in Multidrug Resistant Enterococcus faecalis E9.8.","authors":"Natacha Caballero Gómez, Julia Manetsberger, Sonia Castillo-Gutiérrez, Charles W Knapp, Nabil Benomar, Hikmate Abriouel","doi":"10.1016/j.micres.2025.128057","DOIUrl":"https://doi.org/10.1016/j.micres.2025.128057","url":null,"abstract":"<p><p>The spread of multidrug-resistant (MDR) bacteria and their resistance genes along the food chain and the environment has become a global threat aggravated by incorrect disinfection strategies. This study analysed the effect of induction by sub-inhibitory concentrations of eugenol - a major ingredient in clove essential oil commonly used in disinfectant agents - on the phenotypic and genotypic response of MDR Enterococcus faecalis E9.8 strain, selected based on the phenotypic response of other enterococci. Eugenol treatment irreversibly reduced several antibiotics' minimum inhibitory concentration (MIC), confirmed by kinetic studies for kanamycin, erythromycin, and tetracycline. Furthermore, transcriptomic analysis indicated the reversion of antibiotic resistance through direct and indirect measures, such as down-regulation of genes coding for proteins involved in antibiotic resistance, toxin resistance and virulence factors. Regarding antibiotic resistance genes (ARGs), ten differentially expressed genes (five down-regulated and five up-regulated genes) were related to the main transporter families, which present key targets in antibiotic resistance reversion. Our study thus highlights the importance of considering indirectly related genes as targets for antibiotic resistance reversion besides ARGs sensu stricto. These results allow us to propose using eugenol as an antibiotic resistance reversing agent to be included in disinfectant solutions as an excellent alternative to limit the spread of MDR bacteria and their ARGs in the food chain and the environment.</p>","PeriodicalId":18564,"journal":{"name":"Microbiological research","volume":"293 ","pages":"128057"},"PeriodicalIF":6.1,"publicationDate":"2025-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008308","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Pseudomonas aeruginosa T6SS secretes an oxygen-binding hemerythrin to facilitate competitive growth under microaerobic conditions.","authors":"Chunhui Luo, Huawei Gu, Damin Pan, Yixin Zhao, Anqi Zheng, Hai Zhu, Chen Zhang, Chen Li, Jing Zhang, Can Chen, Lei Xu, Junfeng Pan, Xihui Shen, Yao Wang","doi":"10.1016/j.micres.2025.128052","DOIUrl":"https://doi.org/10.1016/j.micres.2025.128052","url":null,"abstract":"<p><p>Pseudomonas aeruginosa is a prominent respiratory pathogen in cystic fibrosis (CF) patients, thriving in the hypoxic airway mucus. Previous studies have established the role of the oxygen-binding hemerythrin, Mhr, in enhancing P. aeruginosa's fitness under microaerobic conditions. However, the specific mechanisms by which Mhr operates remain unclear. This study uniquely identifies Mhr as an effector of the H2-Type VI Secretion System (H2-T6SS) and elucidates its role in the transport and interaction mechanisms that confer a growth advantage under microaerobic conditions. Our findings demonstrate that mhr expression is directly regulated by Anr and Dnr. Western blot analysis confirms that Mhr is secreted extracellularly via the H2-T6SS. The oxygen-binding Mhr re-enters P. aeruginosa through the OprG porin. Then, Mhr interacts with cbb3-type cytochrome c oxidase (cbb3-CcO) subunits CcoP1/CcoP2, significantly impacting intracellular NADH/NAD⁺ levels. These insights suggest that the T6SS-mediated secretion and transport of Mhr represent a novel mechanism by which P. aeruginosa acquires and delivers oxygen, potentially enhancing microaerobic respiration, energy production, and growth under microaerobic conditions.</p>","PeriodicalId":18564,"journal":{"name":"Microbiological research","volume":"293 ","pages":"128052"},"PeriodicalIF":6.1,"publicationDate":"2025-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008306","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"GlSIRT1 deacetylates and activates pyruvate kinase to improve pyruvate content and enhance heat stress resistance in Ganoderma lucidum.","authors":"Jing Han, Xin Tang, Lingshuai Wang, Huhui Chen, Rui Liu, Mingwen Zhao","doi":"10.1016/j.micres.2025.128055","DOIUrl":"https://doi.org/10.1016/j.micres.2025.128055","url":null,"abstract":"<p><p>Heat stress is a prevalent environmental stressor. Previous studies have shown that heat stress drives many cellular changes in Ganoderma lucidum. Interestingly, glycolysis is activated during heat stress, which could contribute to increased heat resistance. However, the molecular mechanisms underlying the enhanced heat resistance of G. lucidum following heat exposure are not yet fully understood. In this study, we explored the possibility that acetylation modification plays a significant role in responses to abiotic stress. After heat treatment, an enhanced interaction between the deacetylase GlSIRT1 and pyruvate kinase (PK) was observed, and the acetylation level of PK was decreased. Further studies revealed that GlSIRT1 increases PK activity through deacetylation, thereby increasing pyruvate content. Consistent with these findings, both PK activity and pyruvate content were reduced in GlSIRT1 knockdown strains, which exhibited greater sensitivity to heat stress compared to the wild-type (WT) strain. Collectively, our results reveal a novel molecular mechanism by which heat treatment increases pyruvate content.</p>","PeriodicalId":18564,"journal":{"name":"Microbiological research","volume":"293 ","pages":"128055"},"PeriodicalIF":6.1,"publicationDate":"2025-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142984186","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Androgen deficiency-induced loss of Lactobacillus salivarius extracellular vesicles is associated with the pathogenesis of osteoporosis.","authors":"Wenjun Zhang, Jian Xie, Zhuoya Wang, Yuchun Zhong, Li Liu, Jun Liu, Wenming Zhang, Yimin Pi, Furui Tang, Zehong Liu, Yinjin Shao, Tian Liu, Cihua Zheng, Jun Luo","doi":"10.1016/j.micres.2025.128047","DOIUrl":"https://doi.org/10.1016/j.micres.2025.128047","url":null,"abstract":"<p><p>Male osteoporosis is primarily caused by a decrease in testicular testosterone production. Male osteoporosis remains a disease with insufficient diagnosis and treatment, and its consequences are severe, especially in older patients. The gut microbiota plays a crucial role in its occurrence and development. Our study found that the relative abundance of Lactobacillus salivarius in the fecal microbiota of male patients with osteoporosis was significantly lower than that in healthy volunteers. Animal experiments have shown that orchiectomy (ORX) can induce osteoporosis and disrupt the intestinal mucosal barrier, and intestinal microbiota. In addition, we discovered a potential etiological connection between the decreased abundance of the intestinal bacterium L. salivarius and the occurrence of ORX-induced osteoporosis. Cohousing or direct colonization of the intestinal microbiota from healthy rats or direct oral administration of the bacteria alleviated ORX-induced osteoporosis and repaired the intestinal mucosal barrier. Finally, we demonstrated that the extracellular vesicles (EVs) of L. salivarius could be transported to the bones and mitigate ORX-induced osteoporosis in rats. Our results indicate that the gut microbiota participates in protecting bones by secreting and delivering bacterial EVs, and that the reduction of L. salivarius and its EVs is closely related to the development of androgen deficiency-related osteoporosis.</p>","PeriodicalId":18564,"journal":{"name":"Microbiological research","volume":"293 ","pages":"128047"},"PeriodicalIF":6.1,"publicationDate":"2025-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008302","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Microbial diversity and interactions: Synergistic effects and potential applications of Pseudomonas and Bacillus consortia.","authors":"Lixue Wang, Xinyi Zhang, Jiahui Lu, Lingxia Huang","doi":"10.1016/j.micres.2025.128054","DOIUrl":"https://doi.org/10.1016/j.micres.2025.128054","url":null,"abstract":"<p><p>Microbial diversity and interactions in the rhizosphere play a crucial role in plant health and ecosystem functioning. Among the myriads of rhizosphere microbes, Pseudomonas and Bacillus are prominent players known for their multifaceted functionalities and beneficial effects on plant growth. The molecular mechanism of interspecies interactions between natural isolates of Bacillus and Pseudomonas in medium conditions is well understood, but the interaction between the two in vivo remains unclear. This paper focuses on the possible synergies between Pseudomonas and Bacillus associated in practical applications (such as recruiting beneficial microbes, cross-feeding and niche complementarity), and looks forward to the application prospects of the consortium in agriculture, human health and bioremediation. Through in-depth understanding of the interactions between Pseudomonas and Bacillus as well as their application prospects in various fields, this study is expected to provide a new theoretical basis and practical guidance for promoting the research and application of rhizosphere microbes.</p>","PeriodicalId":18564,"journal":{"name":"Microbiological research","volume":"293 ","pages":"128054"},"PeriodicalIF":6.1,"publicationDate":"2025-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142971595","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Akkermansia muciniphila inhibits jejunal lipid absorption and regulates jejunal core bacteria.","authors":"Qiming Ma, Xincheng Zhou, Weikang Su, Qingyu Wang, Guoxing Yu, Weihua Tao, Zhiyong Dong, Cunchuan Wang, Chi-Ming Wong, Tiemin Liu, Shiqi Jia","doi":"10.1016/j.micres.2025.128053","DOIUrl":"https://doi.org/10.1016/j.micres.2025.128053","url":null,"abstract":"<p><p>Insufficiency of Akkermansia muciniphila (Akk) has been implicated in the pathogenesis of metabolic diseases, and administration or restoration of Akk has ameliorated these disorders. Recently, Pasteurized Akk (PA-Akk) has been approved as a functional food. However, the impact of Akk on lipid absorption in the proximal intestine, which is directly exposed to orally administered Akk, remains largely unexplored. In this study, we orally administered Akk and PA-Akk to mice and investigated the subsequent lipid absorption. Long-term administration of Akk resulted in reduced lipid deposits in the liver and adipocytes, along with improved glucose metabolism. This was primarily attributed to a reduction in lipid absorption by epithelial cells in the proximal jejunum. Mechanistically, Akk activated AMP-activated protein kinase (AMPK) and directly inhibit lipids absorption in both mouse and human jejunal epithelial cells. Furthermore, we demonstrated that Akk treatment, but not PA-Akk treatment, promotes the abundance of genera that are highly abundant in the normal jejunum and belong to the phylum Firmicutes. Thus, our study concludes that oral administration of Akk provides beneficial effects on metabolism, partially through inhibiting jejunal lipid absorption and promoting the abundance of core jejunal microbes.</p>","PeriodicalId":18564,"journal":{"name":"Microbiological research","volume":"293 ","pages":"128053"},"PeriodicalIF":6.1,"publicationDate":"2025-01-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142971560","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Microbiome succession on the pomegranate phylloplane during bacterial blight dysbiosis: Functional implications for blight suppression.","authors":"Vinod Chouhan, Sunil Thalor, K Charishma, Mohammed Javed, Shanu Kumar, Jyotsana Sharma, Vibuthi Munjal, Aundy Kumar","doi":"10.1016/j.micres.2025.128050","DOIUrl":"https://doi.org/10.1016/j.micres.2025.128050","url":null,"abstract":"<p><p>Bacterial blight of pomegranate caused by Xanthomonas axonopodis pv. punicae poses significant challenges to sustainable cultivation, necessitating eco-friendly management strategies, and this study explores the role of the phylloplane microbiome in disease suppression through metabarcoding, traditional microbiology, and antibacterial screening of microbial candidates. Here, we mapped the phylloplane microbiome of pomegranate cultivar 'Bhagwa' during bacterial blight development using metabarcoding sequencing (2443,834 reads), traditional microbiological methods (nutrient-rich and minimal media), and scanning electron microscopy. We observed shifts in microbial diversity, with Xanthomonas typically released through stomata as the blight progressed from water-soaked early lesion to advanced necrotic lesion. The Shannon diversity index peaked at 2.6 in early necrotic stages but dropped to 2.1 in advanced blight. Proteobacteria and Firmicutes were the dominant phyla, with significant compositional changes between disease stages. Bacillus species were prevalent throughout, peaking in both early and severe lesions. Pantoea and Curtobacterium increased during severe blight, while Exiguobacterium thrived on the abaxial surface. A core microbiome, including Pantoea, Enterobacter, and Pseudomonas, remained consistent across stages. Antibacterial screening of 116 bacterial candidates, dominated by Pantoea (32), Bacillus (18), and Pseudomonas (11), revealed multipronged activities against X. axonopodis pv. punicae. Bacillus amyloliquefaciens P2-1 and Pantoea dispersa Pg-Slp-6 suppressed the pathogen through secreted metabolites, while Pantoea dispersa Pg-Slp-6, Pseudomonas oryzihabitans Pg-Slp-82, and Pantoea dispersa Pg-slp-117 exhibited volatile-mediated suppression. Among these, Bacillus amyloliquefaciens P2-1 and Pantoea dispersa Pg-slp-6 showed 55 % and 42 % blight suppression, respectively, highlighting their potential as biocontrol agents.</p>","PeriodicalId":18564,"journal":{"name":"Microbiological research","volume":"293 ","pages":"128050"},"PeriodicalIF":6.1,"publicationDate":"2025-01-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008305","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Strain diversity and host specificity of the gut symbiont Gilliamella in Apis mellifera, Apis cerana and Bombus terrestris.","authors":"Nihong Zhou, Qiulan Zheng, Yao Liu, Zhichu Huang, Ye Feng, Yanping Chen, Fuliang Hu, Huoqing Zheng","doi":"10.1016/j.micres.2025.128048","DOIUrl":"https://doi.org/10.1016/j.micres.2025.128048","url":null,"abstract":"<p><p>Social bees, with their specialized gut microbiota and societal transmission between individuals, provide an ideal model for studying host-gut microbiota interactions. While the functional disparities arising from strain-level diversity of gut symbionts and their effects on host health have been studied in Apis mellifera and bumblebees, studies focusing on host-specific investigations of individual strains across different honeybee hosts remain relatively unexplored. In this study, the complete genomic sequences of 17 strains of Gilliamella from A. mellifera, Apis cerana and Bombus terrestris were analyzed. The analysis revealed that the strains of A. mellifera display a more expansive genomic and functional content compared to the strains of A. cerana and B. terrestris. Phylogenetic analysis showed a deep divergence among the Gilliamella strains from different hosts. Additionally, biochemistry tests and antibiotic susceptibility tests revealed that gut strains from A. mellifera exhibited a more extensive pathway for carbohydrate metabolism and a greater resistance to antibiotics than gut strains from A. cerana and B. terrestris. Strains from A. mellifera and A. cerana showed higher colonization efficiency and competitive ability whithin their respective host species, indicating a higher degree of host-specific adaptation of local gut microbiota. In addition, colonization by A. mellifera-derived strain triggers a stronger transcriptional response in the host than A. cerana-derived strain. The variation in the number of differentially expressed genes and the involvement of distinct signaling pathways across these two host species suggest species-specific adaptations to Gilliamella strains. These findings suggest that despite occupying similar niches in the bee gut, strain-level variations can influence microbial functions, and their impact on host physiological functions may vary across different strains.</p>","PeriodicalId":18564,"journal":{"name":"Microbiological research","volume":"293 ","pages":"128048"},"PeriodicalIF":6.1,"publicationDate":"2025-01-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008307","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}