Background: Recent evidence suggests that there is a link between metabolic diseases and bacterial populations in the gut The aim of this study was to assess the differences between the composition of the intestinal microbiota in humans with type 2 diabetes and non-diabetic persons as control Methods and Findings: The study included 36 male adults with a broad range of age and body-mass indices (BMIs), among which 18 subjects were diagnosed with diabetes type 2 The fecal bacterial composition was investigated by real-time quantitative PCR (qPCR) and in a subgroup of subjects (N=20) by tag-encoded amplicon pyrosequencing of the V4 region of the 16S rRNA gene The proportions of phylum Firmicutes and class Clostridia were significantly reduced in the diabetic group compared to the control group (P=003) Furthermore, the ratios of Bacteroidetes to Firmicutes as well as the ratios of Bacteroides-Prevotella group to C coccoides-E rectale group correlated positively and significantly with plasma glucose concentration (P=004) but not with BMIs Similarly, class Betaproteobacteria was highly enriched in diabetic compared to non-diabetic persons (P=002) and positively correlated with plasma glucose (P=004) Conclusions: The results of this study indicate that type 2 diabetes in humans is associated with compositional changes in intestinal microbiota The level of glucose tolerance should be considered when linking microbiota with metabolic diseases such as obesity and developing strategies to control metabolic diseases by modifying the gut microbiota

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Gut Microbiota in Human Adults with Type 2 Diabetes Differs from Non-Diabetic Adults

OBJECTIVE.The aim of this study was to examine the contribution of a broad range of external influences to the gut microbiotic composition in early infancy. METHODS.Fecal samples from 1032 infants at 1 month of age, who were recruited from the KOALA Birth Cohort Study in the Netherlands, were subjected to quantitative real-time polymerase chain reaction assays for the enumeration of bifidobacteria, Escherichia coli, Clostridium difficile, Bacteroides fragilis group, lactobacilli, and total bacterial counts. Information on potential determinants of the gut microbiotic composition was collected with repeated questionnaires. The associations between these factors and the selected gut bacteria were analyzed with univariate and multivariate analyses. RESULTS.Infants born through cesarean section had lower numbers of bifidobacteria and Bacteroides, whereas they were more often colonized with C difficile, compared with vaginally born infants. Exclusively formula-fed infants were more often colonized with E coli, C difficile, Bacteroides, and lactobacilli, compared with breastfed infants. Hospitalization and prematurity were associated with higher prevalence and counts of C difficile. Antibiotic use by the infant was associated with decreased numbers of bifidobacteria and Bacteroides. Infants with older siblings had slightly higher numbers of bifidobacteria, compared with infants without siblings. CONCLUSIONS.The most important determinants of the gut microbiotic composition in infants were the mode of delivery, type of infant feeding, gestational age, infant hospitalization, and antibiotic use by the infant. Term infants who were born vaginally at home and were breastfed exclusively seemed to have the most “beneficial” gut microbiota (highest numbers of bifidobacteria and lowest numbers of C difficile and E coli).

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Factors influencing the composition of the intestinal microbiota in early infancy

Background and aims: The intestinal bacterial microflora plays an important role in the aetiology of inflammatory bowel disease (IBD). As most of the colonic bacteria cannot be identified by culture techniques, genomic technology can be used for analysis of the composition of the microflora. Patients and methods: The mucosa associated colonic microflora of 57 patients with active inflammatory bowel disease and 46 controls was investigated using 16S rDNA based single strand conformation polymorphism (SSCP) fingerprint, cloning experiments, and real time polymerase chain reaction (PCR). Results: Full length sequencing of 1019 clones from 16S rDNA libraries (n = 3) revealed an overall bacterial diversity of 83 non-redundant sequences—among them, only 49 known bacterial species. Molecular epidemiology of the composition of the colonic microflora was investigated by SSCP. Diversity of the microflora in Crohn’s disease was reduced to 50% compared with controls (21.7 v 50.4; p v 50.4; p Bacteroides species, Eubacterium species, and Lactobacillus species, as revealed by direct sequencing of variable bands and confirmed by real time PCR. Bacterial diversity in the Crohn’s group showed no association with CARD15/NOD2 status. Conclusions: Mucosal inflammation in inflammatory bowel disease is associated with loss of normal anaerobic bacteria. This effect is independent of NOD2/CARD15 status of patients.

https://gut.bmj.com/content/gutjnl/53/5/685.full.pdf

Reduction in diversity of the colonic mucosa associated bacterial microflora in patients with active inflammatory bowel disease

In vertebrates, including humans, individuals harbor gut microbial communities whose species composition and relative proportions of dominant microbial groups are tremendously varied. Although external and stochastic factors clearly contribute to the individuality of the microbiota, the fundamental principles dictating how environmental factors and host genetic factors combine to shape this complex ecosystem are largely unknown and require systematic study. Here we examined factors that affect microbiota composition in a large (n = 645) mouse advanced intercross line originating from a cross between C57BL/6J and an ICR-derived outbred line (HR). Quantitative pyrosequencing of the microbiota defined a core measurable microbiota (CMM) of 64 conserved taxonomic groups that varied quantitatively across most animals in the population. Although some of this variation can be explained by litter and cohort effects, individual host genotype had a measurable contribution. Testing of the CMM abundances for cosegregation with 530 fully informative SNP markers identified 18 host quantitative trait loci (QTL) that show significant or suggestive genome-wide linkage with relative abundances of specific microbial taxa. These QTL affect microbiota composition in three ways; some loci control individual microbial species, some control groups of related taxa, and some have putative pleiotropic effects on groups of distantly related organisms. These data provide clear evidence for the importance of host genetic control in shaping individual microbiome diversity in mammals, a key step toward understanding the factors that govern the assemblages of gut microbiota associated with complex diseases.

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Individuality in gut microbiota composition is a complex polygenic trait shaped by multiple environmental and host genetic factors

Aims: The microbiota of the human intestinal tract constitutes a complex ecosystem. We report the design and optimization of an extensive set of 16S rDNA-targeted species- and group-specific primers for more accurate quantification of bacteria from faecal samples with real-time PCR.

Methods and Results:  A linear range of quantification between 0·1–10 pg and 10 ng of specific target genome was obtained, which corresponds to detection of ca 30–4500 to 1·9 × 106–6·0 × 106 target bacterial genomes. Functionality of the assays was confirmed by quantification of target bacterial DNA from faecal DNA preparations of healthy volunteers and irritable bowel syndrome (IBS) patients. Additionally, spiking of faecal preparations with Helicobacter pylori, Clostridium difficile or Campylobacter jejuni was used to confirm the accurate and sensitive quantification.

Conclusions:  Real-time PCR is a very sensitive and precise technique for an extensive quantitative evaluation of gut microbiota and is feasible for detection of human pathogens from faecal samples.

Significance and Impact of the Study:  To design and optimize an extensive set of real-time PCR assays targeting a large group of predominant and pathogenic GI microbial species for further use in updating the current knowledge of the putative role of gut microbiota in health and disease.

https://sfamjournals.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2672.2004.02409.x

Development of an extensive set of 16S rDNA‐targeted primers for quantification of pathogenic and indigenous bacteria in faecal samples by real‐time PCR

Denaturing gradient gel electrophoresis (DGGE) of DNA fragments generated by PCR with 16S ribosomal DNA-targeted group-specific primers was used to detect lactic acid bacteria (LAB) of the genera Lactobacillus, Pediococcus, Leuconostoc, and Weissella in human feces. Analysis of fecal samples of four subjects revealed individual profiles of DNA fragments originating not only from species that have been described as intestinal inhabitants but also from characteristically food-associated bacteria such as Lactobacillus sakei, Lactobacillus curvatus, Leuconostoc mesenteroides, and Pediococcus pentosaceus. Comparison of PCR-DGGE results with those of bacteriological culture showed that the food-associated species could not be cultured from the fecal samples by plating on Rogosa agar. On the other hand, all of the LAB species cultured from feces were detected in the DGGE profile. We also detected changes in the types of LAB present in human feces during consumption of a milk product containing the probiotic strain Lactobacillus rhamnosus DR20. The analysis of fecal samples from two subjects taken before, during, and after administration of the probiotic revealed that L. rhamnosus was detectable by PCR-DGGE during the test period in the feces of both subjects, whereas it was detectable by culture in only one of the subjects.

Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella Species in Human Feces by Using Group-Specific PCR Primers and Denaturing Gradient Gel Electrophoresis

In this study, we characterized the genetic background of various nleA variants in 106 Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic Escherichia coli (EPEC) strains. The flanking regions of eight nleA variants were analyzed by DNA sequencing and compared with the corresponding regions of five previously described NleA-encoding prophages. The analyzed nleA variants were all located downstream of the DNA region responsible for phage morphogenesis. In particular, the type III effector genes avrA, ospB, nleH, and nleG and IS elements were detected in the neighborhood of nleA. The structure of the eight analyzed regions flanking nleA primarily resembled the corresponding region of the NleA4795-encoding prophage BP-4795. Using PCR, the gene order flanking 13 nleA variants in strains of different serogroups was compared to the respective regions in reference strains. The analyses showed that strains which harbor prophages with conserved flanking regions of a particular nleA variant predominantly occurred, and IS elements were additionally detected in these regions. We were able to mobilize nleA by transduction in 20% of strains determined, which comprised in particular EPEC strains harboring an nleA variant, the gene encoding the protein known as “EspI-like.” Plaque hybridization was used to identify phages that harbor the genes stx and nleA. However, only two strains harbored variant nleA4795 in the genome of an Stx1 prophage.

Claudia M. Lis

Papers

Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella Species in Human Feces by Using Group-Specific PCR Primers and Denaturing Gradient Gel Electrophoresis

Genetic background and mobility of variants of the gene nleA in attaching and effacing Escherichia coli.