Robin Mesnage, Marta Calatayud, Cindy Duysburgh, Massimo Marzorati, Michael N Antoniou
{"title":"Alterations in infant gut microbiome composition and metabolism after exposure to glyphosate and Roundup and/or a spore-based formulation using the SHIME technology.","authors":"Robin Mesnage, Marta Calatayud, Cindy Duysburgh, Massimo Marzorati, Michael N Antoniou","doi":"10.1017/gmb.2022.5","DOIUrl":"https://doi.org/10.1017/gmb.2022.5","url":null,"abstract":"<p><p>Despite extensive research into the toxicology of the herbicide glyphosate, there are still major unknowns regarding its effects on the human gut microbiome. We describe the effects of glyphosate and a Roundup glyphosate-based herbicide on infant gut microbiota using SHIME technology. SHIME microbiota culture was undertaken in the presence of a concentration of 100-mg/L glyphosate and the same glyphosate equivalent concentration of Roundup. Roundup and to a lesser extent glyphosate caused an increase in fermentation activity, resulting in acidification of the microbial environment. This was also reflected by an increase in lactate and acetate production concomitant to a decrease in the levels of propionate, valerate, caproate and butyrate. Ammonium production reflecting proteolytic activities was increased by Roundup exposure. Global metabolomics revealed large-scale disturbances, including an increased abundance of long-chain polyunsaturated fatty acids. Changes in bacterial composition measured by qPCR and 16S rRNA suggested that lactobacilli had their growth stimulated as a result of microenvironment acidification. Co-treatment with the spore-based probiotic formulation MegaSporeBiotic reverted some of the changes in short-chain fatty acid levels. Altogether, our results suggest that glyphosate can exert effects on human gut microbiota.</p>","PeriodicalId":73187,"journal":{"name":"Gut microbiome (Cambridge, England)","volume":"3 ","pages":"e6"},"PeriodicalIF":0.0,"publicationDate":"2022-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11406414/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142302555","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Petra Louis, Sylvia Helen Duncan, Paul Owen Sheridan, Alan William Walker, Harry James Flint
{"title":"Microbial lactate utilisation and the stability of the gut microbiome.","authors":"Petra Louis, Sylvia Helen Duncan, Paul Owen Sheridan, Alan William Walker, Harry James Flint","doi":"10.1017/gmb.2022.3","DOIUrl":"10.1017/gmb.2022.3","url":null,"abstract":"<p><p>The human large intestinal microbiota thrives on dietary carbohydrates that are converted to a range of fermentation products. Short-chain fatty acids (acetate, propionate and butyrate) are the dominant fermentation acids that accumulate to high concentrations in the colon and they have health-promoting effects on the host. Although many gut microbes can also produce lactate, it usually does not accumulate in the healthy gut lumen. This appears largely to be due to the presence of a relatively small number of gut microbes that can utilise lactate and convert it to propionate, butyrate or acetate. There is increasing evidence that these microbes play important roles in maintaining a healthy gut environment. In this review, we will provide an overview of the different microbes involved in lactate metabolism within the gut microbiota, including biochemical pathways utilised and their underlying energetics, as well as regulation of the corresponding genes. We will further discuss the potential consequences of perturbation of the microbiota leading to lactate accumulation in the gut and associated disease states and how lactate-utilising bacteria may be employed to treat such diseases.</p>","PeriodicalId":73187,"journal":{"name":"Gut microbiome (Cambridge, England)","volume":" ","pages":"e3"},"PeriodicalIF":0.0,"publicationDate":"2022-05-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11406415/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47263021","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Marie-Luise Puhlmann, Roosa Jokela, Katja Catharina Wilhelmina van Dongen, Thi Phuong Nam Bui, Roland Willem Jan van Hangelbroek, Hauke Smidt, Willem Meindert de Vos, Edith Johanna Maria Feskens
{"title":"Dried chicory root improves bowel function, benefits intestinal microbial trophic chains and increases faecal and circulating short chain fatty acids in subjects at risk for type 2 diabetes.","authors":"Marie-Luise Puhlmann, Roosa Jokela, Katja Catharina Wilhelmina van Dongen, Thi Phuong Nam Bui, Roland Willem Jan van Hangelbroek, Hauke Smidt, Willem Meindert de Vos, Edith Johanna Maria Feskens","doi":"10.1017/gmb.2022.4","DOIUrl":"10.1017/gmb.2022.4","url":null,"abstract":"<p><p>We investigated the impact of dried chicory root in a randomised, placebo-controlled trial with 55 subjects at risk for type 2 diabetes on bowel function, gut microbiota and its products, and glucose homeostasis. The treatment increased stool softness (+1.1 ± 0.3 units; <i>p =</i> 0.034) and frequency (+0.6 ± 0.2 defecations/day; <i>p</i> < 0.001), strongly modulated gut microbiota composition (7 % variation; <i>p =</i> 0.001), and dramatically increased relative levels (3-4-fold) of <i>Anaerostipes</i> and <i>Bifidobacterium</i> spp., in a dose-dependent, reversible manner. A synthetic community, including selected members of these genera and a <i>Bacteroides</i> strain, generated a butyrogenic trophic chain from the product. Faecal acetate, propionate and butyrate increased by 25.8 % (+13.0 ± 6.3 mmol/kg; <i>p</i> = 0.023) as did their fasting circulating levels by 15.7 % (+7.7 ± 3.9 μM; <i>p =</i> 0.057). In the treatment group the glycaemic coefficient of variation decreased from 21.3 ± 0.94 to 18.3 ± 0.84 % (<i>p =</i> 0.004), whereas fasting glucose and HOMA-ir decreased in subjects with low baseline <i>Blautia</i> levels (-0.3 ± 0.1 mmol/L fasting glucose; <i>p =</i> 0.0187; -0.14 ± 0.1 HOMA-ir; <i>p =</i> 0.045). Dried chicory root intake rapidly and reversibly affects bowel function, benefits butyrogenic trophic chains, and promotes glycaemic control.</p>","PeriodicalId":73187,"journal":{"name":"Gut microbiome (Cambridge, England)","volume":"3 1","pages":"e4"},"PeriodicalIF":0.0,"publicationDate":"2022-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11407914/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45022875","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Iñaki Milton-Laskibar, Laura Judith Marcos-Zambrano, Saioa Gómez-Zorita, Enrique Carrillo de Santa Pau, Alfredo Fernández-Quintela, Jose Alfredo Martínez, María Puy Portillo
{"title":"Involvement of microbiota and short-chain fatty acids on non-alcoholic steatohepatitis when induced by feeding a hypercaloric diet rich in saturated fat and fructose.","authors":"Iñaki Milton-Laskibar, Laura Judith Marcos-Zambrano, Saioa Gómez-Zorita, Enrique Carrillo de Santa Pau, Alfredo Fernández-Quintela, Jose Alfredo Martínez, María Puy Portillo","doi":"10.1017/gmb.2022.2","DOIUrl":"10.1017/gmb.2022.2","url":null,"abstract":"<p><p>Consumption of high-energy-yielding diets, rich in fructose and lipids, is a factor contributing to the current increase in non-alcoholic fatty liver disease prevalence. Gut microbiota composition and short-chain fatty acids (SCFAs) production alterations derived from unhealthy diets are considered putative underlying mechanisms. This study aimed to determine relationships between changes in gut microbiota composition and SCFA levels by comparing rats featuring diet-induced steatohepatitis with control counterparts fed a standard diet. A high-fat high-fructose (HFHF) feeding induced higher body, liver and mesenteric adipose tissue weights, increased liver triglyceride content and serum transaminase, glucose, non-HDL-c and MCP-1 levels. Greater liver malondialdehyde levels and glutathione peroxidase activity were also observed after feeding the hypercaloric diet. Regarding gut microbiota composition, a lowered diversity and increased abundances of bacteria from the <i>Clostridium</i> sensu stricto 1, <i>Blautia</i>, <i>Eubacterium coprostanoligenes</i> group, <i>Flavonifractor</i>, and UBA1819 genera were found in rats featuring diet-induced steatohepatitis, as well as higher isobutyric, valeric and isovaleric acids concentrations. These results suggest that hepatic alterations produced by a hypercaloric HFHF diet may be related to changes in overall gut microbiota composition and abundance of specific bacteria. The shift in SCFA levels produced by this unbalanced diet cannot be discarded as potential mediators of the reported hepatic and metabolic alterations.</p>","PeriodicalId":73187,"journal":{"name":"Gut microbiome (Cambridge, England)","volume":" ","pages":"e5"},"PeriodicalIF":0.0,"publicationDate":"2022-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11406367/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"48271976","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Maria LaBouyer, Grietje Holtrop, Graham Horgan, Silvia W Gratz, Alvaro Belenguer, Nicola Smith, Alan W Walker, Sylvia H Duncan, Alexandra M Johnstone, Petra Louis, Harry J Flint, Karen P Scott
{"title":"Higher total faecal short-chain fatty acid concentrations correlate with increasing proportions of butyrate and decreasing proportions of branched-chain fatty acids across multiple human studies.","authors":"Maria LaBouyer, Grietje Holtrop, Graham Horgan, Silvia W Gratz, Alvaro Belenguer, Nicola Smith, Alan W Walker, Sylvia H Duncan, Alexandra M Johnstone, Petra Louis, Harry J Flint, Karen P Scott","doi":"10.1017/gmb.2022.1","DOIUrl":"10.1017/gmb.2022.1","url":null,"abstract":"<p><p>Metabolites produced by microbial fermentation in the human intestine, especially short-chain fatty acids (SCFAs), are known to play important roles in colonic and systemic health. Our aim here was to advance our understanding of how and why their concentrations and proportions vary between individuals. We have analysed faecal concentrations of microbial fermentation acids from 10 human volunteer studies, involving 163 subjects, conducted at the Rowett Institute, Aberdeen, UK over a 7-year period. In baseline samples, the % butyrate was significantly higher, whilst % iso-butyrate and % iso-valerate were significantly lower, with increasing total SCFA concentration. The decreasing proportions of iso-butyrate and iso-valerate, derived from amino acid fermentation, suggest that fibre intake was mainly responsible for increased SCFA concentrations. We propose that the increase in % butyrate among faecal SCFA is largely driven by a decrease in colonic pH resulting from higher SCFA concentrations. Consistent with this, both total SCFA and % butyrate increased significantly with decreasing pH across five studies for which faecal pH measurements were available. Colonic pH influences butyrate production through altering the stoichiometry of butyrate formation by butyrate-producing species, resulting in increased acetate uptake and butyrate formation, and facilitating increased relative abundance of butyrate-producing species (notably <i>Roseburia</i> and <i>Eubacterium rectale</i>).</p>","PeriodicalId":73187,"journal":{"name":"Gut microbiome (Cambridge, England)","volume":" ","pages":"e2"},"PeriodicalIF":0.0,"publicationDate":"2022-03-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11406374/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45872434","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Alaa Bawaneh, Carol A Shively, Janet Austin Tooze, Katherine Loree Cook
{"title":"Impact of gut permeability on the breast microbiome using a non-human primate model.","authors":"Alaa Bawaneh, Carol A Shively, Janet Austin Tooze, Katherine Loree Cook","doi":"10.1017/gmb.2022.9","DOIUrl":"https://doi.org/10.1017/gmb.2022.9","url":null,"abstract":"<p><p>We previously demonstrated in non-human primates (NHP) that Mediterranean diet consumption shifted the proportional abundance of <i>Lactobacillus</i> in the breast and gut. This data highlights a potential link about gut-breast microbiome interconnectivity. To address this question, we compared bacterial populations identified in matched breast and faecal samples from our NHP study. Dietary pattern concurrently shifted two species in both regions; <i>Streptococcus lutetiensis and Ruminococcus torques</i>. While we observe similar trends in <i>Lactobacillus</i> abundances in the breast and gut, the species identified in each region vary; Mediterranean diet increased <i>Lactobacillus_unspecified species</i> in breast but regulated <i>L. animalis</i> and <i>L. reuteri</i> in the gut.We also investigated the impact of gut permeability on the breast microbiome. Regardless of dietary pattern, subjects that displayed increased physiological measures of gut permeability (elevated plasma lipopolysaccharide, decreased villi length, and decreased goblet cells) displayed a significantly different breast microbiome. Gut barrier dysfunction was associated with increased α-diversity and significant different β-diversity in the breast tissue. Taken together our data supports the presence of a breast microbiome influenced by diet that largely varies from the gut microbiome population but is, however, sensitive to gut permeability.</p>","PeriodicalId":73187,"journal":{"name":"Gut microbiome (Cambridge, England)","volume":"3 ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9990890/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9090006","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Débora Campos, Ricardo Goméz-García, Diana Oliveira, Ana Raquel Madureira
{"title":"Intake of nanoparticles and impact on gut microbiota: <i>in vitro</i> and animal models available for testing.","authors":"Débora Campos, Ricardo Goméz-García, Diana Oliveira, Ana Raquel Madureira","doi":"10.1017/gmb.2021.5","DOIUrl":"10.1017/gmb.2021.5","url":null,"abstract":"<p><p>The oral delivery of compounds associated with diet or medication have an impact on the gut microbiota balance, which in turn, influences the physiologic process. Several reports have shown significant advances in clarifying the impact, interactions and outcomes of oral intake of nanoparticles and the human gut. These interactions may affect the bioavailability of the delivered compounds. In addition, there is a considerable breakthrough in the development of antimicrobial nanoparticles for intestinal pathogenic bacteria. Several <i>in vitro</i> fermentation and <i>in vivo</i> models have been developed throughout the years and were used to test these systems. The methodologies and studies carried out so far on the modulation of human and animal gut microbiome by oral delivery nanosized materials were reviewed. Overall, the available <i>in vitro</i> studies mimic the real physiological events enabling to select the best production conditions of nanoparticulate systems in a preliminary stage of research. On the other hand, animal studies can be used to access the dosage effect, safety and correlation between haematological, biochemical and symptoms, with gut microbiota groups and metabolites.</p>","PeriodicalId":73187,"journal":{"name":"Gut microbiome (Cambridge, England)","volume":"3 1","pages":"e1"},"PeriodicalIF":0.0,"publicationDate":"2021-12-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11406378/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47034389","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Conference report: the importance of the gut microbiome and nutrition on health.","authors":"Derek Ball, Spiridoula Athanasiadou","doi":"10.1017/gmb.2021.4","DOIUrl":"10.1017/gmb.2021.4","url":null,"abstract":"<p><p>The Nutrition Society Spring Conference (28-29 March 2021) focussed on the gut microbiome and health that was divided across three separate but inter-related areas from the impact of nutrition on the gut microbiome, the cause and effect of nutrition and health on the gut microbiome to the interaction between pathogens and gut microbiota. The program was supported by two plenary lectures, the first discussed the computational methods commonly employed to examine gut microbiota and the concluding lecture presented the interaction between gut microbiome, nutrition and health in older populations.</p>","PeriodicalId":73187,"journal":{"name":"Gut microbiome (Cambridge, England)","volume":" ","pages":"e4"},"PeriodicalIF":0.0,"publicationDate":"2021-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11406383/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"48356903","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Dietary fibre and the gut-brain axis: microbiota-dependent and independent mechanisms of action.","authors":"Danique La Torre, Kristin Verbeke, Boushra Dalile","doi":"10.1017/gmb.2021.3","DOIUrl":"10.1017/gmb.2021.3","url":null,"abstract":"<p><p>Dietary fibre is an umbrella term comprising various types of carbohydrate polymers that cannot be digested nor absorbed by the human small intestine. Consumption of dietary fibre is linked to beneficial effects on cognitive and affective processes, although not all fibres produce the same effects. Fibres that increase short-chain fatty acid (SCFA) production following modulation of the gut microbiota are thought to be the most potent fibres to induce effects on cognitive and affective processes. SCFAs can exert their effects by improving central, peripheral and systemic immunity, lowering hypertension and enhancing intestinal barrier integrity. Here, we propose additional mechanisms by which dietary fibres may contribute to improvements in affective and cognitive processes. Fibre-induced modulation of the gut microbiota may influence affective processes and cognition by increasing brain-derived neurotrophic factor levels. Depending on the physicochemical properties of dietary fibre, additional effects on affect and cognition may occur via non-microbiota-related routes, such as enhancement of the immune system and lowering cholesterol levels and subsequently lowering blood pressure. Mechanistic randomised placebo-controlled trials are needed to establish the effects of dietary fibre consumption and the magnitude of explained variance in affect and cognition when incorporating measurements of microbiota-dependent and microbiota-independent mechanisms in humans.</p>","PeriodicalId":73187,"journal":{"name":"Gut microbiome (Cambridge, England)","volume":"2 1","pages":"e3"},"PeriodicalIF":0.0,"publicationDate":"2021-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11406392/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45714894","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Jessica A Davis, Fiona Collier, Mohammadreza Mohebbi, Julie A Pasco, Nitin Shivappa, James R Hébert, Felice N Jacka, Amy Loughman
{"title":"The associations of butyrate-producing bacteria of the gut microbiome with diet quality and muscle health.","authors":"Jessica A Davis, Fiona Collier, Mohammadreza Mohebbi, Julie A Pasco, Nitin Shivappa, James R Hébert, Felice N Jacka, Amy Loughman","doi":"10.1017/gmb.2021.2","DOIUrl":"10.1017/gmb.2021.2","url":null,"abstract":"<p><p>This study aimed to investigate the relationships between diet quality, the relative abundance of butyrate-producing bacteria of the gut microbiome and muscle mass, strength and function. In this cross-sectional study, <i>n</i> = 490 men (64.4 ± 13.5 years) from the Geelong Osteoporosis Study provided food frequency questionnaire data, from which the Australian Recommended Food Score (ARFS) and Dietary Inflammatory Index (DII) score were calculated. Muscle mass (skeletal muscle index from DXA-derived lean mass), muscle strength (handgrip strength) and muscle function (Timed Up-and-Go test) were measured. Participants provided stool samples for 16S rRNA gene sequencing. There was no evidence of associations between alpha or beta diversity and muscle health measures. A healthier ARFS score was positively associated with the relative abundance of butyrate-producing bacteria (<i>β</i> 0.09, 95%CI 0.03, 0.15) and a higher (pro-inflammatory) DII score was associated with lower relative abundance of butyrate-producing bacteria (<i>β</i> -0.60, 95%CI -1.06, -0.15). The relative abundance of butyrate-producing bacteria was positively associated with healthier muscle mass, strength and function; however, these relationships were attenuated in multivariable models. These findings support the role of diet quality in achieving a healthier gut microbiome, however, further evidence is required for a gut-muscle axis in humans.</p>","PeriodicalId":73187,"journal":{"name":"Gut microbiome (Cambridge, England)","volume":" ","pages":"e2"},"PeriodicalIF":0.0,"publicationDate":"2021-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11406371/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46008115","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}