Gut Microbial Dysbiosis is Correlated with Stroke Severity Markers in Aged Rats Following Stroke

Background: An imbalanced gut microbial community, or dysbiosis, has been shown to occur following stroke. It is possible that this dysbiosis negatively impacts stroke recovery and rehabilitation. Species level resolution measurements of the gut microbiome following stroke are needed to develop and test precision interventions such as probiotic or fecal microbiota transplant therapies that target the gut microbiome following stroke. Previous studies have used 16S rRNA amplicon sequencing in young male mice to obtain broad profiling of the gut microbiome at the genus level following stroke, but further investigations will be needed with whole genome shotgun sequencing in aged rats of both sexes to obtain species level resolution in a model which will better translate to the demographics of human stroke patients.Results: 39 aged male and female rats underwent middle cerebral artery occlusion. Fecal samples were collected before stroke and three days post stroke to measure gut microbiome. Machine learning was used to identify the top ranked bacteria which were changed following stroke. MRI imaging was used to obtain infarct and edema size and cerebral blood flow (CBF). ELISA was used to obtain inflammatory markers. Dysbiosis was demonstrated by an increase in pathogenic bacteria such as Butyricimonas virosa (15.52 fold change, p<0.0001), Bacteroides vulgatus (7.36 fold change, p<0.0001), and Escherichia coli (47.67 fold change, p<0.0001). These bacteria were positively associated with infarct and edema size and with the inflammatory markers Ccl19, Ccl24, IL17a, IL3, and complement C5; they were negatively correlated with CBF. Conversely, beneficial bacteria such as Ruminococcus flavefaciens (0.14 fold change, p<0.0001), Akkermansia muciniphila (0.78 fold change, p<0.0001), and Lactobacillus murinus (0.40 fold change, p<0.0001) were decreased following stroke and associated with all the previous parameters in the opposite direction of the pathogenic species. There were not significant microbiome differences between the sexes.Conclusion: The species level resolution measurements found here can be used as a foundation to develop and test precision interventions targeting the gut microbiome following stroke. Probiotics that include Ruminococcus flavefaciens, Akkermansia muciniphila, and Lactobacillus murinus should be developed to target the deficit following stroke to measure the impact on stroke severity.

Investigating the Reciprocal Interrelationships among the Ruminal Microbiota, Metabolome, and Mastitis in Early Lactating Holstein Dairy Cows

Mastitis in dairy cow significantly affects animal performance, ultimately reducing profitability. The reciprocal interrelationships among ruminal microbiota, metabolome, and mastitis combining early inflammatory factors (serum proinflammatory cytokines) in lactating dairy cows has not been explored, thus, this study evaluated these reciprocal interrelationships in early lactating Holstein dairy cows to identify potential microbial biomarkers and their relationship with ruminal metabolites. The ruminal fluid was sampled from 8 healthy and 8 mastitis cows for the microbiota and metabolite analyses. The critical ruminal microbial biomarkers and metabolites related to somatic cell counts (SCC) and serum proinflammatory cytokines were identified by the linear discriminant analysis effect size (LEfSe) algorithm and Spearman’s correlation analysis, respectively. The SCC level and proinflammatory cytokines positively correlated with Sharpea and negatively correlated with Ruminococcaceae UCG-014, Ruminococcus flavefaciens, and Treponema saccharophilum. Furthermore, the metabolites xanthurenic acid, and 1-(1H-benzo[d]imidazol-2-yl) ethan-1-ol positively correlated with microbial biomarkers of healthy cows, whereas, xanthine, pantothenic acid, and anacardic acid were negatively correlated with the microbial biomarkers of mastitis cows. In conclusion, Ruminococcus flavefaciens and Treponema saccharophilum are potential strains for improving the health of dairy cows. The current study provides a novel perspective to assist in targeting the ruminal microbiota with preventive/therapeutic strategies against inflammatory diseases in the future.

A trimodular family 16 glycoside hydrolase from the cellulosome of Ruminococcus flavefaciens displays highly specific licheninase (EC 3.2.1.73) activity

Cellulosomes are highly complex cell-bound multi-enzymatic nanomachines used by anaerobes to break down plant carbohydrates. The genome sequence of Ruminococcus flavefaciens revealed a remarkably diverse cellulosome composed of more than 200 cellulosomal enzymes. Here we provide a detailed biochemical characterization of a highly elaborate R. flavefaciens cellulosomal enzyme containing an N-terminal dockerin module, which anchors the enzyme into the multi-enzyme complex through binding of cohesins located in non-catalytic cell-bound scaffoldins, and three tandemly repeated family 16 glycoside hydrolase (GH16) catalytic domains. The DNA sequence encoding the three homologous catalytic domains was cloned and hyper-expressed in Escherichia coli BL21 (DE3) cells. SDS-PAGE analysis of purified His6 tag containing RfGH16_21 showed a single soluble protein of molecular size ~89 kDa, which was in agreement with the theoretical size, 89.3 kDa. The enzyme RfGH16_21 exhibited activity over a wide pH range (pH 5.0–8.0) and a broad temperature range (50–70 °C), displaying maximum activity at an optimum pH of 7.0 and optimum temperature of 55 °C. Substrate specificity analysis of RfGH16_21 revealed maximum activity against barley β-d-glucan (257 U mg−1) followed by lichenan (247 U mg−1), but did not show significant activity towards other tested polysaccharides, suggesting that it is specifically a β-1,3-1,4-endoglucanase. TLC analysis revealed that RfGH16_21 hydrolyses barley β-d-glucan to cellotriose, cellotetraose and a higher degree of polymerization of gluco-oligosaccharides indicating an endo-acting catalytic mechanism. This study revealed a fairly high, active and thermostable bacterial endo-glucanase which may find considerable biotechnological potentials.

Atividade antimicrobiana in vitro de uma combinação de óleos vegetais de caju e mamona e de óleos essenciais de cravo, eugenol, timol e vanilina contra bactérias Gram-negativas e Gram-positivas no rúmen de bovinos

A preocupação pública com o uso rotineiro de antibióticos e ionóforos na dieta de ruminantes aumentou devido ao surgimento de bactérias resistentes aos antibióticos que podem representar riscos à saúde humana. Assim, esforços têm sido empregados para o desenvolvimento de compostos alternativos para substituir antibióticos e ionóforos na dieta de ruminantes. Este estudo foi realizado para avaliar os efeitos in vitro de uma mistura contendo óleos vegetais de caju e mamona e óleos essenciais de cravo-da-índia, eugenol, timol e vanilina sobre a atividade das bactérias Gram-negativas e Gram-positivas presentes no rúmen. O experimento foi elaborado de forma que cada bactéria fosse exposta às doses de 1,5, 3, 5 e 6,0 mg/mL da mistura, com seis repetições. As bactérias foram cultivadas em meio M2 de Hobson em tubos Hungate. A atividade antimicrobiana foi avaliada em espectrofotômetro a 600 nm. As leituras foram realizadas 0, 8, 12 e 24 horas após a inoculação a 39º C. As quatro concentrações (1,5, 3,0, 4,5 e 6,0 mg/mL) da mistura de óleos vegetais e essenciais inibiram o crescimento da Prevotella albensis, Prevotella bryantii, Prevotella ruminicola e Anaerovibrio lipolyticus 8 e 12 horas após a incubação. Para Ruminococcus albus e Ruminococcus flavefaciens a adição da mistura de óleos essenciais nas concentrações de 3,0 e 4,5 mg/mL resultou em maior impacto na dinâmica de crescimento, com redução na densidade óptica após 12 h de incubação. Em conclusão, os resultados mostram que a ação combinada dos óleos vegetais e essenciais influencia o crescimento de bactérias gram-negativas e gram-positivas e pode ser usado como um modulador ruminal. Deste modo, o estudo contribui com novas informações sobre a ação combinada dos óleos vegetais e essenciais como agentes antimicrobianos na produção de ruminantes.

Characteristics of gut microbiota in 100 children with mycoplasma pneumonia in China

Exploring the "lung-intestine"axis of gut microbiota is a hot spot in recent years. There have been few studies on the correlation between gut microbiota and asthma and allergies, of which gut microbiota-mediated molecular regulation of the pathogenesis of infectious pathogens is a blind area. Therefore, we investigated gut microecological changes in MPP patients and the gut microecological characteristics of wheezing MPP pediatric patients.30 healthy children, 50 nonwheezing MPP pediatric patients, and 50 wheezing MPP pediatric patients were collected. 16srDNA gene sequencing results showed that there are differences in gut microbiota abundances between MPP pediatric patients and healthy children. Quantitative real-time polymerase chain reaction (QRT-PCR) validation showed that the abundances of Ruminococcus flavefaciens, Clostridium butyricum, Lactobacillus, and Bifidobacterium in the guts of MPP children decreased compared with healthy children. Compared with nonwheezing MPP children, the abundances of Ruminococcus flavefaciens and Clostridium butyricum were significantly decreased in the guts of wheezing MPP children. This indicates that Ruminococcus flavefaciens and Clostridium butyricum are significantly decreased in nonwheezing MPP children and could be potential risk factors for wheezing in MPP children.

Gut Microbiota Assessment in Obese Children and Adolescents by Machine Learning Algorithms

Gut microbiota has been recently established to play a contributory role in the development and progression of obesity, a multifactorial disease predisposing to several comorbidities. Our aim was to evaluate the gut microbiota composition by machine learning algorithms in 33 Italian obese children and adolescents. Patients were divided in 3 groups: simple obesity (n=10, mean age 11.6 +3.0, median 10.8), metabolic syndrome (n=16, mean age 13.3+3.0, median 13.5) or Prader Willi syndrome (n=7, mean age 8.3+5.3, median 8.7). Inclusion criteria were living in Northern Italy, born singleton birth, personal history negative for acute or chronic gastrointestinal diseases and/or antibiotic or probiotics administration in the previous month. As controls 17 healthy control (mean age 12.0+2.4 median 10.6) were analyzed using the same approach. Statistical analysis for sparse high-throughput sequencing data algorithm (metagenomeSeq) using cumulative sum scaling for data normalization was performed. False discovery rate adjusted p-value using zero-inflated Gaussian fit statistical model (indicated with q). Over all analyses Parasutterella resulted with a q=0.014424, the comparison between obese patients and controls was q=0.021194. In the overall analysis Acidaminococcus intestini showed q=0.039528 while the comparison in pairs of two between metabolic syndrome and controls was q=0.03979. Using the EdgeR algorithm Clostridium bartlettii abundance between Prader Willi patients and controls resulted in q=0.02189. In overall analysis Ruminococcus flavefaciens resulted q=6.1528E-17 (using the DESeq2 algorithm); in pairs analysis Ruminococcus flavefaciens showed significant difference in Prader Willi patients as compared to obese (q=0.013755) and metabolic syndrome ones (q=0.021898). In overall analysis Veillonellaceae showed a FDR q=0.029303. while its richness resulted more than 150 times higher in metabolic syndrome patients than in controls (q=0.039793 evaluated with DESeq2 algorithm). Among Veillonellaceae descendants, the Veillonella rogosae showed, on the contrary, the lowest abundance in metabolic syndrome patients as compared to other groups. In detail, Veillonella rogosae abundances were 13 (FDR q=0.014566), around 20 times (FDR q=0.010646) and &gt;20 (FDR q=0.0025008) less abundant in metabolic syndrome patients than obese, Prader Willi patients and controls, respectively. Significant differences in gut microbiota composition was found among patients with different degrees of obesity and controls. Further, Prader Willi patients showed a peculiar microbiota assessment.

In Vivo Competitions between Fibrobacter succinogenes, Ruminococcus flavefaciens, and Ruminoccus albus in a Gnotobiotic Sheep Model Revealed by Multi-Omic Analyses

ABSTRACT Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens are the three predominant cellulolytic bacterial species found in the rumen. In vitro studies have shown that these species compete for adherence to, and growth upon, cellulosic biomass. Yet their molecular interactions in vivo have not heretofore been examined. Gnotobiotically raised lambs harboring a 17-h-old immature microbiota devoid of culturable cellulolytic bacteria and methanogens were inoculated first with F. succinogenes S85 and Methanobrevibacter sp. strain 87.7, and 5 months later, the lambs were inoculated with R. albus 8 and R. flavefaciens FD-1. Longitudinal samples were collected and profiled for population dynamics, gene expression, fibrolytic enzyme activity, in sacco fibrolysis, and metabolite profiling. Quantitative PCR, metagenome and metatranscriptome data show that F. succinogenes establishes at high levels initially but is gradually outcompeted following the introduction of the ruminococci. This shift resulted in an increase in carboxymethyl cellulase (CMCase) and xylanase activities but not in greater fibrolysis, suggesting that F. succinogenes and ruminococci deploy different but equally effective means to degrade plant cell walls. Expression profiles showed that F. succinogenes relied upon outer membrane vesicles and a diverse repertoire of CAZymes, while R. albus and R. flavefaciens preferred type IV pili and either CBM37-harboring or cellulosomal carbohydrate-active enzymes (CAZymes), respectively. The changes in cellulolytics also affected the rumen metabolome, including an increase in acetate and butyrate at the expense of propionate. In conclusion, this study provides the first demonstration of in vivo competition between the three predominant cellulolytic bacteria and provides insight on the influence of these ecological interactions on rumen fibrolytic function and metabolomic response. IMPORTANCE Ruminant animals, including cattle and sheep, depend on their rumen microbiota to digest plant biomass and convert it into absorbable energy. Considering that the extent of meat and milk production depends on the efficiency of the microbiota to deconstruct plant cell walls, the functionality of predominant rumen cellulolytic bacteria, Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens, has been extensively studied in vitro to obtain a better knowledge of how they operate to hydrolyze polysaccharides and ultimately find ways to enhance animal production. This study provides the first evidence of in vivo competitions between F. succinogenes and the two Ruminococcus species. It shows that a simple disequilibrium within the cellulolytic community has repercussions on the rumen metabolome and fermentation end products. This finding will have to be considered in the future when determining strategies aiming at directing rumen fermentations for animal production.