Characterization of the Gut Microbial Community of Obese Patients Following a Weight-Loss Intervention Using Whole Metagenome Shotgun Sequencing

<div><p>Background/Objectives</p><p>Cross-sectional studies suggested that obesity is promoted by the gut microbiota. However, longitudinal data on taxonomic and functional changes in the gut microbiota of obese patients are scarce. The aim of this work is to study microbiota changes in the course of weight loss therapy and the following year in obese individuals with or without co-morbidities, and to asses a possible predictive value of the gut microbiota with regard to weight loss maintenance.</p><p>Subjects/Methods</p><p>Sixteen adult patients, who followed a 52-week weight-loss program comprising low calorie diet, exercise and behavioral therapy, were selected according to their weight-loss course. Over two years, anthropometric and metabolic parameters were assessed and microbiota from stool samples was functionally and taxonomically analyzed using DNA shotgun sequencing.</p><p>Results</p><p>Overall the microbiota responded to the dietetic and lifestyle intervention but tended to return to the initial situation both at the taxonomical and functional level at the end of the intervention after one year, except for an increase in <i>Akkermansia</i> abundance which remained stable over two years (12.7x10³ counts, 95%CI: 322–25100 at month 0; 141x10³ counts, 95%CI: 49-233x10³ at month 24; p = 0.005). The <i>Firmicutes/Bacteroidetes</i> ratio was higher in obese subjects with metabolic syndrome (0.64, 95%CI: 0.34–0.95) than in the “healthy obese” (0.27, 95%CI: 0.08–0.45, p = 0.04). Participants, who succeeded in losing their weight consistently over the two years, had at baseline a microbiota enriched in <i>Alistipes</i>, <i>Pseudoflavonifractor</i> and enzymes of the oxidative phosphorylation pathway compared to patients who were less successful in weight reduction.</p><p>Conclusions</p><p>Successful weight reduction in the obese is accompanied with increased <i>Akkermansia</i> numbers in feces. Metabolic co-morbidities are associated with a higher <i>Firmicutes/Bacteroidetes</i> ratio. Most interestingly, microbiota differences might allow discrimination between successful and unsuccessful weight loss prior to intervention.</p></div

Clinical parameters along the study period.

<p>Clinical parameters along the study period.</p

Clinical parameters of the study population.

<p>A. Relative weight loss during the observation period of two years consisting of one year intervention program with very low calorie diet (VLCD) during the first 3 months, reintroduction of normal food (reintrod.) during month 3–6, and weight maintenance therapy under normal diet during month 7–12, followed by a one-year-observation without intervention. Each line represents a patient (n = 16). Patients were grouped into those with persistent success (PS group, >10% RWL at T24, black lines and symbols) or no persistent success (NS group, <10% RWL at T24, grey lines and symbols). B. Change of insulin resistance during time. Insulin resistance was assessed using the HOMA-IR as described in Subjects and Methods. C. Change of liver steatosis assessed by sonography (circles) and fatty liver index (squares). Data in B and C are indicated as means +/- 95% confidence intervals (n = 16), **P<0.01 and ***P<0.001 (as compared to baseline, Wilcoxon’s test).</p

Abundance change of genera and metabolic pathways during the study.

<p>A. Relative abundance of five genera, which were the most abundant among those that changed during time. B. Akkermansia abundance. C. Relative abundance of all KEGG pathways that changed during whole study. D. Metabolic pathways that changed from baseline to T3. Abbreviations: bios, biosynthesis; met, metabolism. Statistics: Relative abundances are expressed in percent (abundance at T0 is 100%). Each dot is the mean at a given time point. Relative abundances at different time points were compared using the Friedman test (A, C: over the six time points), or the Wilcoxon test (B, D, *p < 0.05: between baseline and T3 or T24).</p

Bacterial species changes during weight loss intervention.

<p>Cladogram (based on 16S sequences) displaying the most abundant species from genera influenced by the intervention. Significant changes between T0 and T3 (left column of squares), between T3 and T6 (middle column), and between T6 and T24 (right column) are indicated by a star(p < 0.05). Blue squares indicate a decrease, red an increase in abundance. Species are colored according to the phyla they belong to (blue: Spirochaetae, pink: Bacteroidetes, green: Firmicutes, light pink: Proteobacteria, orange: Actinobacteria, brown: Verrucomicrobia, red: Synergistetes). This tree was created using the free software EvolView [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149564#pone.0149564.ref027" target="_blank">27</a>].</p

Correlations between bacterial genera or pathways and parameters related to the metabolic syndrome.

<p>Correlations between bacterial genera or pathways and parameters related to the metabolic syndrome.</p

Differences in gut microbiota between patients with different co-morbidities or different outcomes.

<p>We compared patients with or without metabolic syndrome (A, B), with or without non-alcoholic fatty liver disease (NAFLD, panels C, D), and with or without persistent success in weight loss (E, F). In patients with metabolic syndrome, the <i>Firmicutes/Bacteroidetes</i> (F/B) ratio (A) and the flagellin gene (KEGG K02406) abundance (B) were increased at T0. In patients with NAFLD, the abundance of <i>Lactococcus</i> (C) and “naphthalene degradation” pathway (D) were decreased at T24. In patients with persistent success in weight loss, the abundance of the “oxidative phosphorylation” pathway was increased (E), whereas the “PAH degradation” pathway was decreased (F) at T0. Statistics: *p<0.05; **p<0.01, ***p<0.001 (Mann-Whitney’s test).</p

<i>Lactobacillus rhamnosus GG</i> Protects against Non-Alcoholic Fatty Liver Disease in Mice

<div><p>Objective</p><p>Experimental evidence revealed that obesity-associated non-alcoholic fatty liver disease (NAFLD) is linked to changes in intestinal permeability and translocation of bacterial products to the liver. Hitherto, no reliable therapy is available except for weight reduction. Within this study, we examined the possible effect of the probiotic bacterial strain <i>Lactobacillus rhamnosus GG</i> (LGG) as protective agent against experimental NAFLD in a mouse model.</p><p>Methods</p><p>Experimental NAFLD was induced by a high-fructose diet over eight weeks in C57BL/J6 mice. Fructose was administered via the drinking water containing 30% fructose with or without LGG at a concentration resulting in approximately 5×10⁷ colony forming units/g body weight. Mice were examined for changes in small intestinal microbiota, gut barrier function, lipopolysaccharide (LPS) concentrations in the portal vein, liver inflammation and fat accumulation in the liver.</p><p>Results</p><p>LGG increased beneficial bacteria in the distal small intestine. Moreover, LGG reduced duodenal IκB protein levels and restored the duodenal tight junction protein concentration. Portal LPS (P≤0.05) was reduced and tended to attenuate TNF-α, IL-8R and IL-1β mRNA expression in the liver feeding a high-fructose diet supplemented with LGG. Furthermore liver fat accumulation and portal alanine-aminotransferase concentrations (P≤0.05) were attenuated in mice fed the high-fructose diet and LGG.</p><p>Conclusions</p><p>We show for the first time that LGG protects mice from NAFLD induced by a high-fructose diet. The underlying mechanisms of protection likely involve an increase of beneficial bacteria, restoration of gut barrier function and subsequent attenuation of liver inflammation and steatosis.</p></div

<i>Lactobacillus rhamnosus GG</i> elevates bacterial numbers in the distal small intestine.

<p>Total bacterial numbers (A), LGG numbers (B), the phyla Firmicutes (C), and Bacteriodetes (D) were measured via qPCR. Data are shown as means ± SEM (**P<0.01, ***P<0.001; <i>n</i> = 5–6). Abbreviations: see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080169#pone-0080169-g001" target="_blank">Figure 1</a>.</p

<i>Lactobacillus rhamnosus GG</i> improves diet-induced NAFLD.

<p>LGG ameliorates high-fructose diet-induced NAFLD via modulation of the intestinal microbiota. LGG products e.g. lactate may increase butyrate producing Firmicutes leading to an improved intestinal barrier and reduced portal plasma LPS concentrations as well as a decreased inflammation and fatty acid accumulation in the liver. Abbreviations: NAFLD, non-alcoholic fatty liver disease; LPS, lipopolysaccharides.</p