The influences of low protein diet on the intestinal microbiota of mice.

Recent research suggests that protein deficiency symptoms are influenced by the intestinal microbiota. We investigated the influence of low protein diet on composition of the intestinal microbiota through animal experiments. Specific pathogen-free (SPF) mice were fed one of four diets (3, 6, 9, or 12% protein) for 4 weeks (n = 5 per diet). Mice fed the 3% protein diet showed protein deficiency symptoms such as weight loss and low level of blood urea nitrogen concentration in their serum. The intestinal microbiota of mice in the 3% and 12% protein diet groups at day 0, 7, 14, 21 and 28 were investigated by 16S rRNA gene sequencing, which revealed differences in the microbiota. In the 3% protein diet group, a greater abundance of urease producing bacterial species was detected across the duration of the study. In the 12% diet protein group, increases of abundance of Streptococcaceae and Clostridiales families was detected. These results suggest that protein deficiency may be associated with shifts in intestinal microbiota.

Gut microbial features can predict host phenotype response to protein deficiency.

Malnutrition remains a major health problem in low- and middle-income countries. During low protein intake, <0.67 g/kg/day, there is a loss of nitrogen (N2 ) balance, due to the unavailability of amino acid for metabolism and unbalanced protein catabolism results. However, there are individuals, who consume the same low protein intake, and preserve N2 balance for unknown reasons. A novel factor, the gut microbiota, may account for these N2 balance differences. To investigate this, we correlated gut microbial profiles with the growth of four murine strains (C57Bl6/J, CD-1, FVB, and NIH-Swiss) on protein deficient (PD) diet. Results show that a PD diet exerts a strain-dependent impact on growth and N2 balance as determined through analysis of urinary urea, ammonia and creatinine excretion. Bacterial alpha diversity was significantly (P < 0.05, FDR) lower across all strains on a PD diet compared to normal chow (NC). Multi-group analyses of the composition of microbiomes (ANCOM) revealed significantly differential microbial signatures between the four strains independent of diet. However, mice on a PD diet demonstrated differential enrichment of bacterial genera including, Allobaculum (C57Bl6/J), Parabacteroides (CD-1), Turicibacter (FVB), and Mucispirillum (NIH-Swiss) relative to NC. For instance, selective comparison of the CD-1 (gained weight) and C57Bl6/J (did not gain weight) strains on PD diet also demonstrated significant pathway enrichment of dihydroorodate dehydrogenase, rRNA methyltransferases, and RNA splicing ligase in the CD-1 strains compared to C57Bl6/J strains; which might account in their ability to retain growth despite a protein deficient diet. Taken together, these results suggest a potential relationship between the specific gut microbiota, N2 balance and animal response to malnutrition.

Protein- and zinc-deficient diets modulate the murine microbiome and metabolic phenotype.

BACKGROUND: Environmental enteropathy, which is linked to undernutrition and chronic infections, affects the physical and mental growth of children in developing areas worldwide. Key to understanding how these factors combine to shape developmental outcomes is to first understand the effects of nutritional deficiencies on the mammalian system including the effect on the gut microbiota. OBJECTIVE: We dissected the nutritional components of environmental enteropathy by analyzing the specific metabolic and gut-microbiota changes that occur in weaned-mouse models of zinc or protein deficiency compared with well-nourished controls. DESIGN: With the use of a 1H nuclear magnetic resonance spectroscopy-based metabolic profiling approach with matching 16S microbiota analyses, the metabolic consequences and specific effects on the fecal microbiota of protein and zinc deficiency were probed independently in a murine model. RESULTS: We showed considerable shifts within the intestinal microbiota 14-24 d postweaning in mice that were maintained on a normal diet (including increases in Proteobacteria and striking decreases in Bacterioidetes). Although the zinc-deficient microbiota were comparable to the age-matched, well-nourished profile, the protein-restricted microbiota remained closer in composition to the weaned enterotype with retention of Bacteroidetes. Striking increases in Verrucomicrobia (predominantly Akkermansia muciniphila) were observed in both well-nourished and protein-deficient mice 14 d postweaning. We showed that protein malnutrition impaired growth and had major metabolic consequences (much more than with zinc deficiency) that included altered energy, polyamine, and purine and pyrimidine metabolism. Consistent with major changes in the gut microbiota, reductions in microbial proteolysis and increases in microbial dietary choline processing were observed. CONCLUSIONS: These findings are consistent with metabolic alterations that we previously observed in malnourished children. The results show that we can model the metabolic consequences of malnutrition in the mouse to help dissect relevant pathways involved in the effects of undernutrition and their contribution to environmental enteric dysfunction.

Defined Nutrient Diets Alter Susceptibility to Clostridium difficile Associated Disease in a Murine Model.

BACKGROUND: Clostridium difficile is a major identifiable and treatable cause of antibiotic-associated diarrhea. Poor nutritional status contributes to mortality through weakened host defenses against various pathogens. The primary goal of this study was to assess the contribution of a reduced protein diet to the outcomes of C. difficile infection in a murine model. METHODS: C57BL/6 mice were fed a traditional house chow or a defined diet with either 20% protein or 2% protein and infected with C. difficile strain VPI10463. Animals were monitored for disease severity, clostridial shedding and fecal toxin levels. Select intestinal microbiota were measured in stool and C. difficile growth and toxin production were quantified ex vivo in intestinal contents from untreated or antibiotic-treated mice fed with the different diets. RESULTS: C. difficile infected mice fed with defined diets, particularly (and unexpectedly) with protein deficient diet, had increased survival, decreased weight loss, and decreased overall disease severity. C. difficile shedding and toxin in the stool of the traditional diet group was increased compared with either defined diet 1 day post infection. Mice fed with traditional diet had an increased intestinal Firmicutes to Bacteroidetes ratio following antibiotic exposure compared with either a 2% or 20% protein defined nutrient diet. Ex vivo inoculation of cecal contents from antibiotic-treated mice showed decreased toxin production and C. difficile growth in both defined diets compared with a traditional diet. CONCLUSIONS: Low protein diets, and defined nutrient diets in general, were found to be protective against CDI in mice. Associated diet-induced alterations in intestinal microbiota may influence colonization resistance and clostridial toxin production in a defined nutrient diet compared to a traditional diet, leading to increased survival. However, mechanisms which led to survival differences between 2% and 20% protein defined nutrient diets need to be further elucidated.

Coagulation activation in an experimental pneumonia model in malnourished mice.

Malnutrition induces a decrease in immunity that affects the ability of the organism to deal with an infectious challenge. The clotting system is considered a branch of immunity and its activation is important in the pathogenesis of an infectious disease. This work was conducted to determine coagulation modifications in malnourished hosts before and during infection. Weaned mice were malnourished via a protein-free diet. Well-nourished control mice (WNC) consumed a balanced conventional diet. Malnourished mice (MN) and WNC were challenged intranasally with Streptococcus pneumoniae. Blood, bronchoalveolar lavages (BAL), and lung samples were taken at different times post infection. The results were that MN showed altered hemostatic tests and fibrin(ogen) deposits in the lung. Thus, an increase in thrombin-antithrombin complexes (TATc) in plasma and BAL was observed. In the MN group, infection induced a rise in TATc in plasma and BAL and increased plasma fibrinogen and fibrin(ogen) deposits in the lung. A decrease in activated protein C and antithrombin in BAL and an early decrease followed by an increase in plasma Factor VIII were also observed. Thus, malnourishment induced a procoagulant state increased by infection. This is the first work that presents results of an exhaustive study of coagulation in malnourished hosts before and during an infection.

A dietary nucleoside-nucleotide mixture inhibits endotoxin-induced bacterial translocation in mice fed protein-free diet.

Nucleosides and nucleotides are important substrates utilized by the intestinal mucosa. To determine the relative effect of dietary nucleosides and nucleotides on the gut, we investigated the effects of these compounds on endotoxin-induced bacterial translocation, cecal bacterial populations and ileal histology in protein-malnourished mice. There was an inhibition of gram-negative enteric bacteria in the mesenteric lymph node and spleen of the surviving mice fed the protein-free diet supplemented with a nucleoside-nucleotide mixture compared with the nonsupplemented group. Histologically, the damage to the gut mucosal barrier was more pronounced in the nonsupplemented group than in the nucleoside-nucleotide supplemented group. However, the cecal bacterial populations in the groups were not different. The villous height, crypt depth and total wall thickness were more developed in the supplemented group compared with the nonsupplemented group, indicating that the nucleoside-nucleotide mixture blocked bacterial translocation by preventing endotoxin-induced mucosal or epithelial damage. These results suggest that the nucleoside-nucleotide mixture could be used to inhibit or reduce the incidence of bacterial translocation, decrease intestinal injury and improve survival in a lethal model of protein deficiency and endotoxemia.

Intraperitoneal administration of nucleoside-nucleotide mixture inhibits endotoxin-induced bacterial translocation in protein-deficient mice.

Nucleosides and nucleotides as a precursor for nucleic acid synthesis may be essential for rapidly growing cells, since intestinal epithelial cells have limited capacity for the de novo purine and pyrimidine synthesis. The present study was undertaken to determine the effect of intraperitoneal administration of nucleoside-nucleotide mixture (NNM) or saline on endotoxin-induced bacterial translocation, ileal histology, and cecal population levels in protein-deficient mice. Intraperitoneal administration of NNM for 14 days was associated with reduced translocation of gram-negative enterics to the mesenteric lymph node and spleen in comparison to saline. Histologically, the extent of the damage to the gut mucosa was greater in the saline group. This was confirmed by the profound diminution of the villous height, crypt depth, and the intestinal wall in the saline treated group as compared to the NNM treated group, suggestive of the efficacy of NNM in improving the gut and epithelial mucosal cells. However, the cecal population levels in both groups were not different. Additionally, the mice in the saline group were more susceptible to the lethal effects of endotoxin as compared to the NNM group suggesting that NNM may be essential for the enhancement of the host defense system. These results suggest that NNM may be used to an advantage to inhibit or reduce the incidence of endotoxin-induced bacterial translocation and improved survival in protein-deficient mice.

Effects of protein malnutrition and endotoxin on the intestinal mucosal barrier to the translocation of indigenous flora in mice.

Since protein malnourished or endotoxemic patients are at increased risk of developing nosocomial infections with enteric organisms, we investigated the effects of these risk factors alone and in combination on the intestinal mucosal barrier to bacteria. Protein malnutrition resulted in severe ileal atrophy that was directly related to the length of time the mice were protein malnourished. Although protein malnutrition did not promote bacterial translocation from the gut to systemic organs, the protein-malnourished mice were more susceptible to endotoxin-induced bacterial translocation than normally nourished mice (p less than 0.01). Since the gross epithelial damage documented after endotoxin administration in normally nourished mice was diminished after protein malnutrition, there was no correlation between the gross appearance of the epithelial mucosal barrier and the extent of endotoxin-induced bacterial translocation. These results suggest that the synergistic effect of endotoxin plus protein malnutrition on bacterial translocation is not primarily related to failure of the gut mucosal barrier. Nonetheless, it appears that protein-malnourished mice are less able to clear translocating bacteria than normally nourished mice.

Effects of dietary composition on growth of M. leprae in mouse footpads.

The number of bacteria per mouse footpad were measured at intervals beginning with the third month in male, weanling BALB/c mice infected with M. leprae and fed for a period of 6 months to test the effects of diet on multiplication of bacteria. The mean bacteria count per footpad in mice remaining at 6 months in the two high fat diets was higher (p = 0.014) than the mean of the two low fat diets. Likewise, the pooled mean bacterial count of mice fed the two diets of animal origin had a tendency to a higher mean bacterial count compared to mice fed the two diets of plant origin. Low level of dietary protein in early life also seemed to predispose to M. leprae multiplication. Our data in mice suggest that the association of diet with human leprosy should be investigated.

Impact of protein malnutrition on exogenous reinfection with Mycobacterium tuberculosis.

Malnutrition may be a predisposing host factor in the development of exogenous-reinfection tuberculosis. Outbred Hartley guinea pigs were given isocaloric diets containing either 30% ovalbumin (control animals) or 10% ovalbumin (low-protein-fed [LP] animals). Equal numbers of control and LP animals were assigned to one of three infection groups: (i) primary pulmonary infection with a low-virulence, streptomycin-resistant (LVsr) isolate of Mycobacterium tuberculosis and then reinfection 6 weeks later by the same route with a high-virulence (HV) isolate; (ii) only the primary infection (LVsr isolate); and (iii) only the secondary infection (HV isolate). Each infection resulted in the development of 4 to 12 pulmonary tubercles. Guinea pigs were skin tested with purified protein derivative and killed 6 weeks after the second infection. Protein deprivation suppressed the dermal responses to purified protein derivative in all infection groups. Primary infection of well-nourished animals with the LVsr isolate induced significant protection against infection with the HV isolate in the reinfected group, based upon the numbers of viable mycobacteria in the lung and spleen. Protein malnutrition did not exacerbate disease in the reinfected group beyond that observed in malnourished animals infected with the HV isolate only, but neither did the infection with the LVsr isolate protect the LP animals against reinfection with the HV isolate. We conclude that malnutrition interferes with the protection normally afforded by primary infection but does not result in more severe disease in reinfected individuals than would be observed in singly infected subjects.

The effect of oral pancreatic extract on jejunal bactericidal activity in protein-deficient vervet monkeys challenged with Vibrio cholerae.

Eleven vervet monkeys (Cercopithecus aethiops) were fed with an "O" protein diet. After the serum albumin level fell below 2.5 g/100 ml the animals and 4 controls, which received regular monkey chow, were orally infected with a monkey-adapted strain of Vibrio cholerae. The total bactericidal activity of the jejunal fluid decreased during feeding with "O" protein diet, but increased after challenge with V. cholerae in all groups. The non-immunoglobulin-bound bactericidal activity, which also decreased during protein depletion, remained less in those animals receiving placebo instead of pancreatic extract after challenge.

Ulcerative dermatitis in rats with over fifteen generations of protein malnutrition.

1. Male and female rats with histories of up to twenty generations of protein malnutrition were found to be at a higher risk for the development of ulcerative dermatitis than rats maintained on a low-protein diet for one generation or in controls on an adequate-protein intake. 2. In all groups, female rats were more likely to have dermatitis than male rats. 3. Bacteriologic examination was performed in the intergenerationally malnourished and control animals; Staphylococcus aureus was isolated from the skins of animals in both groups, whether or not any lesion was present. In these two groups of animals, experimental inoculation with S. aureus produced dermatitis only in the malnourished animals.