A global phylogenomic and metabolic reconstruction of the large intestine bacterial community of domesticated cattle.

BACKGROUND: The large intestine is a colonization site of beneficial microbes complementing the nutrition of cattle but also of zoonotic and animal pathogens. Here, we present the first global gene catalog of cattle fecal microbiomes, a proxy of the large intestine microbiomes, from 436 metagenomes from six countries. RESULTS: Phylogenomics suggested that the reconstructed genomes and their close relatives form distinct branches and produced clustering patterns that were reminiscent of the metagenomics sample origin. Bacterial taxa had distinct metabolic profiles, and complete metabolic pathways were mainly linked to carbohydrates and amino acids metabolism. Dietary changes affected the community composition, diversity, and potential virulence. However, predicted enzymes, which were part of complete metabolic pathways, remained present, albeit encoded by different microbes. CONCLUSIONS: Our findings provide a global insight into the phylogenetic relationships and the metabolic potential of a rich yet understudied bacterial community and suggest that it provides valuable services to the host. However, we tentatively infer that members of that community are not irreplaceable, because similar to previous findings, symbionts of complex bacterial communities of mammals are expendable if there are substitutes that can perform the same task. Video Abstract.

Sporulation of Clostridium cellulolyticum while grown in cellulose-batch and cellulose-fed continuous cultures on a mineral-salt based medium.

Clostridium cellulolyticum sporulation was investigated during growth on cellulose fibers in a mineral-salt based medium which corresponds to conditions linked to its natural ecological niche. At steady state of the continuous cultures under limitation and with an excess of cellulose and/or ammonium, bacterial cells mainly sporulated at low dilution rates (D), at least 10% sporulation being observed at the lowest D tested. Increasing the cellulose concentration in the feed-medium reservoir increased the percentage of spores in the bioreactor. It appeared that the remaining undigested cellulose could serve as an exogenous carbon source supply at a continuous but limited rate throughout the sporulation process. In addition to the proportion of carbon and nitrogen, the influence of the environmental pH on spore formation was studied. In cellulose-fed continuous cultures at a constant D and a pH decreasing from 7.2 to 6.4, the percentage of spores increased to 14% at the lowest pH tested. When C. cellulolyticum was grown in batch culture, the level of sporulation was dramatically higher in unregulated-pH fermentation compared to pH-controlled growth conditions at pH 7.2 since in the former it reached 45% within 5 days of cultivation. It then appeared that a low specific growth rate and a low environmental pH in the presence of an insoluble carbon substrate were the major factors inducing sporulation in C. cellulolyticum. Furthermore, since the spores adhere to the carbon substrate (the cellulose) the bacteria gain advantages when the environment allows germination thanks to the recovery of suitable growth conditions. By allowing the maintenance and the integrity of the bacteria in the microbiota, spore formation could then explain the successful survival of C. cellulolyticum in cellulosic anaerobic habitats where low environmental pH conditions are often found.

Kinetics and metabolism of cellulose degradation at high substrate concentrations in steady-state continuous cultures of Clostridium cellulolyticum on a chemically defined medium.

The hydrolysis and fermentation of insoluble cellulose were investigated using continuous cultures of Clostridium cellulolyticum with increasing amounts of carbon substrate. At a dilution rate (D) of 0.048 h(-1), biomass formation increased proportionately to the cellulose concentration provided by the feed reservoir, but at and above 7.6 g of cellulose x liter(-1) the cell density at steady state leveled off. The percentage of cellulose degradation declined from 32.3 to 8.3 with 1.9 and 27.0 g of cellulose x liter(-1), respectively, while cellodextrin accumulation rose and represented up to 4.0% of the original carbon consumed. The shift from cellulose-limited to cellulose-sufficient conditions was accompanied by an increase of both the acetate/ethanol ratio and lactate biosynthesis. A kinetics study of C. cellulolyticum metabolism in cellulose saturation was performed by varying D with 18.1 g of cellulose x liter(-1). Compared to cellulose limitation (M. Desvaux, E. Guedon, and H. Petitdemange, J. Bacteriol. 183:119-130, 2001), in cellulose-sufficient continuous culture (i) the ATP/ADP, NADH/NAD+, and q(NADH produced)/q(NADH used) ratios were higher and were related to a more active catabolism, (ii) the acetate/ethanol ratio increased while the lactate production decreased as D rose, and (iii) the maximum growth yield (Y(max)X/S) (40.6 g of biomass per mol of hexose equivalent) and the maximum energetic yield (Y(max)ATP) (19.4 g of biomass per mol of ATP) were lowered. C. cellulolyticum was then able to regulate and optimize carbon metabolism under cellulose-saturated conditions. However, the facts that some catabolized hexose and hence ATP were no longer associated with biomass production with a cellulose excess and that concomitantly lactate production and pyruvate leakage rose suggest the accumulation of an intracellular inhibitory compound(s), which could further explain the establishment of steady-state continuous cultures under conditions of excesses of all nutrients. The following differences were found between growth on cellulose in this study and growth under cellobiose-sufficient conditions (E. Guedon, S. Payot, M. Desvaux, and H. Petitdemange, Biotechnol. Bioeng. 67:327-335, 2000): (i) while with cellobiose, a carbon flow into the cell of as high as 5.14 mmol of hexose equivalent g of cells(-1) x h(-1) could be reached, the maximum entering carbon flow obtained here on cellulose was 2.91 mmol of hexose equivalent g of cells(-1) x h(-1); (ii) while the NADH/NAD+ ratio could reach 1.51 on cellobiose, it was always lower than 1 on cellulose; and (iii) while a high proportion of cellobiose was directed towards exopolysaccharide, extracellular protein, and free amino acid excretions, these overflows were more limited under cellulose-excess conditions. Such differences were related to the carbon consumption rate, which was higher on cellobiose than on cellulose.

Flux analysis of the metabolism of Clostridium cellulolyticum grown in cellulose-fed continuous culture on a chemically defined medium under ammonium-limited conditions.

An investigation of cellulose degradation by the nonruminal, cellulolytic, mesophilic bacterium Clostridium cellulolyticum was performed in cellulose-fed chemostat cultures with ammonium as the growth-limiting nutrient. At any dilution rate (D), acetate was always the main product of the catabolism, with a yield of product from substrate ranging between 37.7 and 51.5 g per mol of hexose equivalent fermented and an acetate/ethanol ratio always higher than 1. As D rose, the acetyl coenzyme A was rerouted in favor of ethanol pathways, and ethanol production could represent up to 17.7% of the carbon consumed. Lactate was significantly produced, but with increasing D, the specific lactate production rate declined, as did the specific rate of production of extracellular pyruvate. The proportion of the original carbon directed towards phosphoglucomutase remained constant, and the carbon surplus was balanced mainly by exopolysaccharide and glycogen biosyntheses at high D values, while cellodextrin excretion occurred mainly at lower ones. With increasing D, the specific rate of carbon flowing down catabolites increased as well, but when expressed as a percentage of carbon it declined, while the percentage of carbon directed through biosynthesis pathways was enhanced. The maximum growth and energetic yields were lower than those obtained in cellulose-limited chemostats and were related to an uncoupling between catabolism and anabolism leading to an excess of energy. Compared to growth on cellobiose in ammonium-limited chemostats (E. Guedon, M. Desvaux, and H. Petitdemange, J. Bacteriol. 182:2010-2017, 2000), (i) a specific consumption rate of carbon of as high as 26.72 mmol of hexose equivalent g of cells(-1) x h(-1) could not be reached and (ii) the proportions of carbon directed towards cellodextrin, glycogen, and exopolysaccharide pathways were not as high as first determined on cellobiose. While the use of cellobiose allows highlighting of metabolic limitation and regulation of C. cellulolyticum under ammonium-limited conditions, some of these events should then rather be interpreted as distortions of the metabolism. Growth of cellulolytic bacteria on easily available carbon and nitrogen sources represents conditions far different from those of the natural lignocellulosic compounds.

Carbon flux distribution and kinetics of cellulose fermentation in steady-state continuous cultures of Clostridium cellulolyticum on a chemically defined medium.

The metabolic characteristics of Clostridium cellulolyticum, a mesophilic cellulolytic nonruminal bacterium, were investigated and characterized kinetically for the fermentation of cellulose by using chemostat culture analysis. Since with C. cellulolyticum (i) the ATP/ADP ratio is lower than 1, (ii) the production of lactate at low specific growth rate (mu) is low, and (iii) there is a decrease of the NADH/NAD(+) ratio and q(NADH produced)/ q(NADH used) ratio as the dilution rate (D) increases in carbon-limited conditions, the chemostats used were cellulose-limited continuously fed cultures. Under all conditions, ethanol and acetate were the main end products of catabolism. There was no shift from an acetate-ethanol fermentation to a lactate-ethanol fermentation as previously observed on cellobiose as mu increased (E. Guedon, S. Payot, M. Desvaux, and H. Petitdemange, J. Bacteriol. 181:3262-3269, 1999). The acetate/ethanol ratio was always higher than 1 but decreased with D. On cellulose, glucose 6-phosphate and glucose 1-phosphate are important branch points since the longer the soluble beta-glucan uptake is, the more glucose 1-phosphate will be generated. The proportion of carbon flowing toward phosphoglucomutase remained constant (around 59.0%), while the carbon surplus was dissipated through exopolysaccharide and glycogen synthesis. The percentage of carbon metabolized via pyruvate-ferredoxin oxidoreductase decreased with D. Acetyl coenzyme A was mainly directed toward the acetate formation pathway, which represented a minimum of 27.1% of the carbon substrate. Yet the proportion of carbon directed through biosynthesis (i.e., biomass, extracellular proteins, and free amino acids) and ethanol increased with D, reaching 27.3 and 16.8%, respectively, at 0.083 h(-1). Lactate and extracellular pyruvate remained low, representing up to 1.5 and 0.2%, respectively, of the original carbon uptake. The true growth yield obtained on cellulose was higher, [50.5 g of cells (mol of hexose eq)(-1)] than on cellobiose, a soluble cellodextrin [36.2 g of cells (mol of hexose eq)(-1)]. The rate of cellulose utilization depended on the solid retention time and was first order, with a rate constant of 0.05 h(-1). Compared to cellobiose, substrate hydrolysis by cellulosome when bacteria are grown on cellulose fibers introduces an extra means for regulation of the entering carbon flow. This led to a lower mu, and so metabolism was not as distorted as previously observed with a soluble substrate. From these results, C. cellulolyticum appeared well adapted and even restricted to a cellulolytic lifestyle.

Cellulose catabolism by Clostridium cellulolyticum growing in batch culture on defined medium.

A reinvestigation of cellulose degradation by Clostridium cellulolyticum in a bioreactor with pH control of the batch culture and using a defined medium was performed. Depending on cellulose concentration, the carbon flow distribution was affected, showing the high flexibility of the metabolism. With less than 6.7 g of cellulose liter(-1), acetate, ethanol, H(2), and CO(2) were the main end products of the fermentation and cellulose degradation reached more than 85% in 5 days. The electron flow from the glycolysis was balanced by the production of H(2) and ethanol, the latter increasing with increasing initial cellulose concentration. From 6.7 to 29.1 g of cellulose liter(-1), the percentage of cellulose degradation declined; most of the cellulase activity remained on the cellulose fibers, the maximum cell density leveled off, and the carbon flow was reoriented from ethanol to acetate. In addition to that of previously indicated end products, lactate production rose, and, surprisingly enough, pyruvate overflow occurred. Concomitantly the molar growth yield and the energetic yield of the biomass decreased. Growth arrest may be linked to sufficiently high carbon flow, leading to the accumulation of an intracellular inhibitory compound(s), as observed on cellobiose (E. Guedon, M. Desvaux, S. Payot, and H. Petitdemange, Microbiology 145:1831-1838, 1999). These results indicated that bacterial metabolism exhibited on cellobiose was distorted compared to that exhibited on a substrate more closely related to the natural ecosystem of C. cellulolyticum. To overcome growth arrest and to improve degradation at high cellulose concentrations (29.1 g liter(-1)), a reinoculation mode was evaluated. This procedure resulted in an increase in the maximum dry weight of cells (2,175 mg liter(-1)), cellulose solubilization (95%), and end product concentrations compared to a classical batch fermentation with a final dry weight of cells of 580 mg liter(-1) and 45% cellulose degradation within 18 days.

Kinetic analysis of Clostridium cellulolyticum carbohydrate metabolism: importance of glucose 1-phosphate and glucose 6-phosphate branch points for distribution of carbon fluxes inside and outside cells as revealed by steady-state continuous culture.

During the growth of Clostridium cellulolyticum in chemostat cultures with ammonia as the growth-limiting nutrient, as much as 30% of the original cellobiose consumed by C. cellulolyticum was converted to cellotriose, glycogen, and polysaccharides regardless of the specific growth rates. Whereas the specific consumption rate of cellobiose and of the carbon flux through glycolysis increased, the carbon flux through the phosphoglucomutase slowed. The limitation of the path through the phosphoglucomutase had a great effect on the accumulation of glucose 1-phosphate (G1P), the precursor of cellotriose, exopolysaccharides, and glycogen. The specific rates of biosynthesis of these compounds are important since as much as 16.7, 16.0, and 21.4% of the specific rate of cellobiose consumed by the cells could be converted to cellotriose, exopolysaccharides, and glycogen, respectively. With the increase of the carbon flux through glycolysis, the glucose 6-phosphate (G6P) pool decreased, whereas the G1P pool increased. Continuous culture experiments showed that glycogen biosynthesis was associated with rapid growth. The same result was obtained in batch culture, where glycogen biosynthesis reached a maximum during the exponential growth phase. Glycogen synthesis in C. cellulolyticum was also not subject to stimulation by nutrient limitation. Flux analyses demonstrate that G1P and G6P, connected by the phosphoglucomutase reaction, constitute important branch points for the distribution of carbon fluxes inside and outside cells. From this study it appears that the properties of the G1P-G6P branch points have been selected to control excretion of carbon surplus and to dissipate excess energy, whereas the pyruvate-acetyl coenzyme A branch points chiefly regulate the redox balance of the carbon catabolism as was shown previously (E. Guedon et al., J. Bacteriol. 181:3262-3269, 1999).

Relationships between cellobiose catabolism, enzyme levels, and metabolic intermediates in Clostridium cellulolyticum grown in a synthetic medium.

Continuous cultures, under cellobiose sufficient concentrations (14. 62 mM) using a chemically defined medium, were examined to determine the carbon regulation selected by Clostridium cellulolyticum. Using a synthetic medium, a q(cellobiose) of 2.57 mmol g cells(-1) h(-1) was attained whereas the highest value obtained on complex media was 0.68 mmol g cells(-1) h(-1) (Payot et al. 1998. Microbiology 144:375-384). On a synthetic medium at D = 0.035 h(-1) under cellobiose excess, lactate and ethanol biosynthesis were able to use the reducing equivalents supplied by acetic acid formation and the H(2)/CO(2) ratio was found equal to 1. At a higher dilution rate (D = 0.115 h(-1)), there was no lactate production and the pathways toward ethanol and NADH-ferredoxin-hydrogenase contributed to balance the reducing equivalents; in this case a H(2)/CO(2) ratio of 1.54 was found. With increasing D, there was a progressive increase (i) in the steady-state concentration of NADH and NAD(+) pools from 11.8 to 22.1 micromol (g cells) (-1), (ii) in the intracellular NADH/NAD(+) ratios from 0.43 to 1.51. On synthetic media, under cellobiose excess the carbon flow was also equilibrated by three overflows: exopolysaccharide, extracellular protein, and amino acid excretions. At D = 0.115 h(-1), 34% of the cellobiose consumed was converted into exopolysaccharides; this deviation of the carbon flow and the increase of the phosphoroclastic activity decreased dramatically the pyruvate excretion and explained the break in lactate production. Whatever the dilution rate, C. cellulolyticum, using ammonium and cellobiose excess, always spilled usual amino acids accompanied by other amino compounds. In vitro, GAPDH, phosphoroclastic reaction, alcohol dehydrogenase, and acetate kinase activities were high under conditions giving high in vivo specific production rates. There were also correlations between the in vitro lactate dehydrogenase activity and in vivo lactate production, but in contrast with the preceding activities, these two parameters decreased with D. All the results demonstrate that C. cellulolyticum was able to optimize carbon catabolism from cellulosic substrates in a synthetic medium.

Effect of dilution rate, cellobiose and ammonium availabilities on Clostridium cellulolyticum sporulation.

The nutritional and physiological factors affecting sporulation of Clostridium cellulolyticum were studied using steady-state continuous cultures grown in both complex and synthetic media. Under cellobiose limitation, the probability that cells will sporulate appears to be directly related to the growth rate. In complex medium, the highest percentage of sporulation was 20% at a dilution rate of 0.015 h-1 whereas in synthetic medium it was 10% at 0.035 h-1. In both media, when the dilution rate was either higher or lower the percentage of sporulation decreased by between 2% and 4%. At low dilution rates, endospore formation was repressed under cellobiose-sufficient concentrations, suggesting catabolite repression by cellobiose. Furthermore, the concentration of ammonium was important in determining the percentage of sporulation, as ammonium limitation induced extensive sporulation at low growth rates even in an excess of cellobiose. The sporulation process is not triggered when cells are cellobiose-exhausted both in complex and synthetic media. These data suggest that, in C. cellulolyticum, an exogenous supply of carbon is required throughout the sporulation process. In the experimental conditions used in this work, no relationship between glycogen accumulation or glycogen mobilization and endospore formation was detected in C. cellulolyticum.

Growth inhibition of Clostridium cellulolyticum by an inefficiently regulated carbon flow.

Carbon flow in Clostridium cellulolyticum was investigated either in batch or continuous culture using a synthetic medium with cellobiose as the sole source of carbon and energy. Previous experiments carried out using a complex growth medium led to the conclusion that the carbon flow was stopped by intracellular NADH. In this study, results showed that cells cultured in a synthetic medium were better able to control electron flow since the NADH/NAD+ ratios were in the range 0.3-0.7, whereas a ratio as high as 57 was previously found in cells cultured on a complex medium. Furthermore, a specific rate of cellobiose consumption of 2.13 mmol (g cells)-1 h-1 was observed on synthetic medium whereas the highest value obtained on complex medium was 0.68 mmol (g cells)-1 h-1. When C. cellulolyticum was grown in continuous culture and cellobiose in the feed medium was increased from 5.84 to 17.57 mM in stepwise fashion, there was an increase in cellobiose utilization without growth inhibition. In contrast, when the reactor was fed directly with 14.62 mM cellobiose, residual cellobiose was observed (4.24 mM) and growth was limited. These data indicate that C. cellulolyticum is not able to optimize its growth and carbon flow in response to a sudden increase in the concentration of growth substrate cellobiose. This interpretation was confirmed (i) by the study of cellobiose batch fermentation where it was demonstrated that growth inhibition was not due to nutritional limitation or inhibition by fermentation products but was associated with carbon excess and (ii) by the growth of C. cellulolyticum in dialysis culture where no growth inhibition was observed due to the limitation of carbon flow by the low rate of cellobiose diffusion through the dialysis tubing.

Carbon and electron flow in Clostridium cellulolyticum grown in chemostat culture on synthetic medium.

Previous results indicated poor sugar consumption and early inhibition of metabolism and growth when Clostridium cellulolyticum was cultured on medium containing cellobiose and yeast extract. Changing from complex medium to a synthetic medium had a strong effect on (i) the specific cellobiose consumption, which was increased threefold; and (ii) the electron flow, since the NADH/NAD+ ratios ranged from 0.29 to 2.08 on synthetic medium whereas ratios as high as 42 to 57 on complex medium were observed. These data indicate a better control of the carbon flow on mineral salts medium than on complex medium. By continuous culture, it was shown that the electron flow from glycolysis was balanced by the production of hydrogen gas, ethanol, and lactate. At low levels of carbon flow, pyruvate was preferentially cleaved to acetate and ethanol, enabling the bacteria to maximize ATP formation. A high catabolic rate led to pyruvate overflow and to increased ethanol and lactate production. In vitro, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and ethanol dehydrogenase levels were higher under conditions giving higher in vivo specific production rates. Redox balance is essentially maintained by NADH-ferredoxin reductase-hydrogenase at low levels of carbon flow and by ethanol dehydrogenase and lactate dehydrogenase at high levels of carbon flow. The same maximum growth rate (0.150 h-1) was found in both mineral salts and complex media, proving that the uptake of nutrients or the generation of biosynthetic precursors occurred faster than their utilization. On synthetic medium, cellobiose carbon was converted into cell mass and catabolized to produce ATP, while on complex medium, it served mainly as an energy supply and, if present in excess, led to an accumulation of intracellular metabolites as demonstrated for NADH. Cells grown on synthetic medium and at high levels of carbon flow were able to induce regulatory responses such as the production of ethanol and lactate dehydrogenase.

In vivo determination of the caloric value of gum arabic in the male rat--biological data.

Sprague-Dawley male rats were given normal diets (UAR A 03) containing various amounts of gum arabic, sucrose and lignin for eight consecutive weeks. The caloric values of the diets were derived by utilizing the indirect method comparing the slopes from food consumption and weight gain curves. While the data obtained from sucrose diets showed that these diets caused a clear incremental effect on energy production with respect to both test animals and controls, and data from lignin diets were found to produce a negative effect on energy yields, gum arabic diets produced results indicating that at certain levels of intake, gum arabic may contribute energy values slightly superior to those of lignin (0 cal-g), possibly between zero and 1 cal/g. However, it was not found possible to attribute a clear-cut quantitative caloric value to this additive from data obtained in this study.

[Anesthesia and resuscitation in emergency maxillofacial surgery]. / Anesthésie et réanimation en chirurgie maxillo-faciale d'urgence

A series of 290 cases of maxillo facial trauma permitted the authors to draw up the indications for resuscitation and surgery. Restoration of the permeability of the airways is most often obtained by oral intubation under non-depressor pharmcological agents. The difficulties encountered during intubation were usually linked to pain, but sometimes had a mainly mechanical origin, particularly during zygomatic and malar bone fractures. Tracheotomy was exceptional. Hemorrhage was often the major obstacle and, in certain cases, could only be controlled by immobilisation of the fragments. The anaesthetic technique is very similar to that used in neurosurgery.