Gut Microbiota Alteration is Characterized by a Proteobacteria and Fusobacteria Bloom in Kwashiorkor and a Bacteroidetes Paucity in Marasmus.

Kwashiorkor and marasmus are considered to be two different clinical diseases resulting from severe malnutrition, but this distinction has been questioned. In a previous study comparing children with kwashiorkor and healthy children from Niger and Senegal, we found a dramatic gut microbiota alteration with a predominant depletion of anaerobes and enrichment in Proteobacteria and Fusobacteria in kwashiorkor. However, it remained unknown whether this association was related to malnutrition or was a specific feature of kwashiorkor. In this continuation study, we added 7 new marasmus subjects and 71,162 new colonies from the same countries. Our results showed that, compared to marasmus, the kwashiorkor gut microbiota was characterized by an increased proportion of Proteobacteria (culturomics, Marasmus 5.0%, Kwashiorkor 16.7%, p < 0.0001; metagenomics, Marasmus 14.7%, Kwashiorkor 22.0%, p = 0.001), but there was a decreased proportion of Bacteroidetes in marasmus (culturomics, Marasmus 0.8%, Kwashiorkor 6.5%, p = 0.001; metagenomics, Marasmus 5.4%, Kwashiorkor 7.0%, p = 0.03). Fusobacterium was more frequently cultured from kwashiorkor. All detected potential pathogenic species were enriched in the kwashiorkor gut microbiota. These results provide a biological basis to support the usage of an antibiotic therapy more effective in suppressing the overgrowth of bacterial communities resistant to penicillin, combined with antioxidants and probiotics for nutritional recovery therapies, particularly for kwashiorkor.

Anaerococcus rubiinfantis sp. nov., isolated from the gut microbiota of a Senegalese infant with severe acute malnutrition.

Anaerococcus rubiinfantis sp. nov. strain mt16(T) is a new species within the genus Anaerococcus, which was isolated by the culturomics approach from the gut microbiota of an infant suffering from kwashiorkor. A phenotypic, biochemical and proteomic description of this strain is hereby presented alongside a complete annotation of its genome. This strictly anaerobic species forms Gram-positive non-sporeforming cocci. The major fatty acid was hexadecanoic acid. The phylogenetic analysis of strain mt16(T) showed a 97.9% similarity level with Anaerococcus vaginalis, the closest validly published species. Its genome is 1,929,161 bp long with 29.5% G + C content and contains 1808 protein-coding genes and 56 RNA genes, among which are six rRNA genes. Genomic analysis identified 41/1864 coding genes as ORFans (2.2%) and at least 620/1808 (34.9%) orthologous proteins which are not shared with the closest phylogenetic species. We believe that the extension of the human anaerobic gut compendium by culturomics is one of the first steps that will improve the understanding of the links between the microbiome and health or disease.

Glycans affect DNA extraction and induce substantial differences in gut metagenomic studies.

Exopolysaccharides produced by bacterial species and present in feces are extremely inhibitory to DNA restriction and can cause discrepancies in metagenomic studies. We determined the effects of different DNA extraction methods on the apparent composition of the gut microbiota using Illumina MiSeq deep sequencing technology. DNA was extracted from the stool from an obese female using 10 different methods and the choice of DNA extraction method affected the proportional abundance at the phylum level, species richness (Chao index, 227 to 2,714) and diversity (non parametric Shannon, 1.37 to 4.4). Moreover DNA was extracted from stools obtained from 83 different individuals by the fastest extraction assay and by an extraction assay that degradated exopolysaccharides. The fastest extraction method was able to detect 68% to 100% genera and 42% to 95% species whereas the glycan degradation extraction method was able to detect 56% to 93% genera and 25% to 87% species. To allow a good liberation of DNA from exopolysaccharides commonly presented in stools, we recommend the mechanical lysis of stools plus glycan degradation, used here for the first time. Caution must be taken in the interpretation of current metagenomic studies, as the efficiency of DNA extraction varies widely among stool samples.

Possible role of the microbiome in the development of acute malnutrition and implications for food-based strategies to prevent and treat acute malnutrition.

A pattern of changes in the microbiome composition have been observed in the normal maturation of the human gut. Perturbations from this pattern have been described in malnourished humans and reproduced in animal models of severe malnutrition. Treatment and prevention of malnutrition in the future may be more effective if the interventions not only restore body composition, but the composition of the microbiome as well.

Functional characterization of IgA-targeted bacterial taxa from undernourished Malawian children that produce diet-dependent enteropathy.

To gain insights into the interrelationships among childhood undernutrition, the gut microbiota, and gut mucosal immune/barrier function, we purified bacterial strains targeted by immunoglobulin A (IgA) from the fecal microbiota of two cohorts of Malawian infants and children. IgA responses to several bacterial taxa, including Enterobacteriaceae, correlated with anthropometric measurements of nutritional status in longitudinal studies. The relationship between IgA responses and growth was further explained by enteropathogen burden. Gnotobiotic mouse recipients of an IgA(+) bacterial consortium purified from the gut microbiota of undernourished children exhibited a diet-dependent enteropathy characterized by rapid disruption of the small intestinal and colonic epithelial barrier, weight loss, and sepsis that could be prevented by administering two IgA-targeted bacterial species from a healthy microbiota. Dissection of a culture collection of 11 IgA-targeted strains from an undernourished donor, sufficient to transmit these phenotypes, disclosed that Enterobacteriaceae interacted with other consortium members to produce enteropathy. These findings indicate that bacterial targets of IgA responses have etiologic, diagnostic, and therapeutic implications for childhood undernutrition.

Gut microbiomes of Malawian twin pairs discordant for kwashiorkor.

Kwashiorkor, an enigmatic form of severe acute malnutrition, is the consequence of inadequate nutrient intake plus additional environmental insults. To investigate the role of the gut microbiome, we studied 317 Malawian twin pairs during the first 3 years of life. During this time, half of the twin pairs remained well nourished, whereas 43% became discordant, and 7% manifested concordance for acute malnutrition. Both children in twin pairs discordant for kwashiorkor were treated with a peanut-based, ready-to-use therapeutic food (RUTF). Time-series metagenomic studies revealed that RUTF produced a transient maturation of metabolic functions in kwashiorkor gut microbiomes that regressed when administration of RUTF was stopped. Previously frozen fecal communities from several discordant pairs were each transplanted into gnotobiotic mice. The combination of Malawian diet and kwashiorkor microbiome produced marked weight loss in recipient mice, accompanied by perturbations in amino acid, carbohydrate, and intermediary metabolism that were only transiently ameliorated with RUTF. These findings implicate the gut microbiome as a causal factor in kwashiorkor.

Antibodies to Herpesvirus hominis types 1 and 2 in malnourished Nigerian children.

Antibodies to Herpesvirus hominis (HVH) types 1 and 2 were determined by a micro-neutralisation method in 37 children with kwashiorkor, 16 with marasmus, and in 64 well-nourished control children. All the children were aged between 1 and 4 years. The prevalence of antibodies was similar in the two sexes and at different ages. HVH-1 antibodies were present in 51% of children with kwashiorkor, in 44% with marasmus, and in 26% of well-nourished children, reflecting the very poor socioeconomic conditions of malnourished children. HVH-2 antibodies too were present in about 19% of children with kwashiorkor, and in 2% of well-nourished controls; they were absent in marasmic children. It is suggested that HVH-2 infection in malnourished children is facilitated by the communal use of fomites--such as bedclothes and underwear.

Septicaemia in kwashiorkor.

In blood and stool cultures from 90 Black children with kwashiorkor, the commonest organisms isolated were Gram-negative aerobic rods. The mortality rate was highest in the group who developed septicaemia. In patients with negative blood cultures, the outcome was much more favourable, although the majority of them showed evidence of respiratory and/or bowel infections. It is felt that routine antibiotic therapy is advisable in the management of kwashiorkor.

Isolation of Campylobacter fetus from recent cases of human vibriosis.

Campylobacter fetus was isolated from five recent cases of human vibriosis, of which two were adults and three were children. One adult presented with pericarditis and the other with recurrent pyrexia. Campylobacter fetus subsp. intestinalis which resembled cattle strains serologically, was isolated under CO2 or anaerobic conditions from blood cultures of these patients. Two of the three children had kwashiorkor and the third was only 8 days old. Isolates identified as Campylobacter fetus subsp. jejuni were cultured from blood of these patients, two of whom had diarrhoea. Three patients succumbed, despite adequate antibiotic therapy. The epidemiology of the disease is discussed and it is suggested that infection may have been from the patients' own flora.

Pathogenic mechanisms of a non-agglutinable Vibrio cholerae strain: demonstration of invasive and enterotoxigenic properties.

A non-agglutinable Vibrio cholerae strain isolated from the blood of a child with kwashiorkor and fever was shown to have the potential to invade as well as to produce a toxin resembling cholera toxin.

Jejunal microflora in malnourished Gambian children.

Growth of bacteria greater than 10-5 organisms/ml was found in 22 children, of whom 17 gave a histroy of chronic diarrhoea. The other 8 children had either no diarrhoea or where having an acute attack lasting for a few days. In those with chronic diarrhoea, Esch. coli, bacteroides, and enterococci tended to occur more frequently, whereas streptococci occurred more frequently in those with acute diarrhoea. Bacilli, staphylococci, micrococci, klebsiellas, pseudomonads, and candidas often occurred in both groups and in large numbers in those with chronic diarrhoea. This confirms previous reports in other parts of the world that some children with malnutrition have considerable bacterial contamination of the jejunum, and that this may be of aetiological significance as a cause of much of the diarrhoea seen in malnourished children. It is possible too that this may be important in the pathogenesis of malnutrition. The presence of intestinal parasites in these malnourished children is also noted. A double-blind trial in the use of antibiotics in this condition is advocated to determine whether it is possible to break the diarrhoea-malabsorption-malnutrition cycle. At the same time the effect of simply removing the child to a more sanitary environment, together with an estimate of the natural clearance of bacteria from the upper intestine, should be evaluated.