A gut microbiota-targeted dietary intervention for amelioration of chronic inflammation underlying metabolic syndrome.

Chronic inflammation induced by endotoxin from a dysbiotic gut microbiota contributes to the development of obesity-related metabolic disorders. Modification of gut microbiota by a diet to balance its composition becomes a promising strategy to help manage obesity. A dietary scheme based on whole grains, traditional Chinese medicinal foods, and prebiotics (WTP diet) was designed to meet human nutritional needs as well as balance the gut microbiota. Ninety-three of 123 central obese volunteers (BMI ≥ 28 kg m(-2) ) completed a self-controlled clinical trial consisting of 9-week intervention on WTP diet followed by a 14-week maintenance period. The average weight loss reached 5.79 ± 4.64 kg (6.62 ± 4.94%), in addition to improvement in insulin sensitivity, lipid profiles, and blood pressure. Pyrosequencing of fecal samples showed that phylotypes related to endotoxin-producing opportunistic pathogens of Enterobacteriaceae and Desulfovibrionaceae were reduced significantly, while those related to gut barrier-protecting bacteria of Bifidobacteriaceae increased. Gut permeability, measured as lactulose/mannitol ratio, was decreased compared with the baseline. Plasma endotoxin load as lipopolysaccharide-binding protein was also significantly reduced, with concomitant decrease in tumor necrosis factor-α, interleukin-6, and an increase in adiponectin. These results suggest that modulation of the gut microbiota via dietary intervention may enhance the intestinal barrier integrity, reduce circulating antigen load, and ultimately ameliorate the inflammation and metabolic phenotypes.

Assessment of the modulating effects of fructo-oligosaccharides on fecal microbiota using human flora-associated piglets.

We first used human flora-associated (HFA) piglets, a significantly improved model for research on human gut microbiota, to study the effects of short-chain fructo-oligosaccharides (scFOS) on the gut bacterial populations. Ten neonatal HFA piglets were assigned to receive basal diets alone or supplemented with scFOS (0.5 g/kg body weight/day) from 1 to 37 days after birth (DAB). The impact of scFOS on the fecal bacterial populations of the piglets before (12 DAB), during (17 DAB), and after (25 and 37 DAB) weaning were monitored by PCR-denaturing gradient gel electrophoresis and real-time quantitative PCR. The Bifidobacterium genus was stimulated consistently, except during weaning, confirming the bifidogenic property of scFOS. At 12 DAB, the Clostridium leptum subgroup was decreased and two unknown Bacteroides-related species were increased; at 25 DAB, the C. leptum subgroup and Subdoligranulum variabile-like species were elevated, whereas one unknown Faecalibacterium-related species was suppressed; and at 37 DAB, the Bacteroides genus was decreased. The results showed that effects of scFOS on non-bifidobacteria varied at different developmental stages of the animals, warranting further investigation into the host-development-related effects of prebiotics on the gut microbiota and the host physiology using the HFA piglets as a model for humans.

Fatal infection in human flora-associated piglets caused by the opportunistic pathogen Klebsiella pneumoniae from an apparently healthy human donor.

Seventeen out of 24 human flora-associated (HFA) piglets died after oral administration of whole fecal flora from an apparently healthy human donor. The bacteria isolated from the organs of the infected piglets were identified as Klebsiella pneumoniae by bacteriological and biochemical tests and 16S rRNA gene sequence analysis. The identical K. pneumoniae strain was also isolated from the donor's fecal flora. All three neonatal piglets inoculated with K. pneumoniae from the donor's fecal flora developed severe diarrhea, with 2 eventually dying. This strongly suggests that the opportunistic pathogen K. pneumoniae from the human donor caused the fatal infection in the HFA piglets. The results underscore the importance of safety evaluation of the human donor's fecal flora for HFA piglet development.

[Specific-PCR and denaturing gradient gel electrophoresis assistant isolation of Thauera spp. from a coking wastewater treatment plant].

OBJECTIVE: We used specific-PCR and denaturing gradient gel electrophoresis (DGGE) to isolate Thauera spp. from a coking wastewater treatment plant. METHODS AND RESULTS: To isolate Thauera from the denitrifying bioreactor of a coking wastewater treatment, biofilm was inoculated to six different media and cultured them under both aerobic and anaerobic conditions. We then compared the composition of Thauera spp. using Thauera-specific PCR-DGGE method. The media 1/10 NB and MMQ which grew higher diverse Thauera spp. and fewer colonies, were used to isolate Thauera sp. under aerobic condition. The colonies were then screened by Thauera-specific PCR. The purity of the colonies that shown Thauera-specific PCR positive signal was then checked by DGGE. The colonies with multiple species were further streaked on different media. DGGE analysis showed that Thauera in colony Q20 was enriched in medium MMP. The colony was finally purified by streaking on MMP medium for several rounds. The composition of the colonies were tracked by Thauera-specific PCR and DGGE at each step. Finally, three strains were purified, which were identified as Thauera sp. according to their 16S rRNA gene sequences. CONCLUSION: Guiding with specific biomarker, the efficiency and sensitivity of bacteria isolation can be largely improved.

Molecular profiling of the Clostridium leptum subgroup in human fecal microflora by PCR-denaturing gradient gel electrophoresis and clone library analysis.

A group-specific PCR-based denaturing gradient gel electrophoresis (DGGE) method was developed and combined with group-specific clone library analysis to investigate the diversity of the Clostridium leptum subgroup in human feces. PCR products (length, 239 bp) were amplified using C. leptum cluster-specific primers and were well separated by DGGE. The DGGE patterns of fecal amplicons from 11 human individuals revealed host-specific profiles; the patterns for fecal samples collected from a child for 3 years demonstrated the structural succession of the population in the first 2 years and its stability in the third year. A clone library was constructed with 100 clones consisting of 1,143-bp inserts of 16S rRNA gene fragments that were amplified from one adult fecal DNA with one forward universal bacterial primer and one reverse group-specific primer. Eighty-six of the clones produced the 239-bp C. leptum cluster-specific amplicons, and the remaining 14 clones did not produce these amplicons but still phylogenetically belong to the subgroup. Sixty-four percent of the clones were related to Faecalibacterium prausnitzii (similarity, 97 to 99%), 6% were related to Subdoligranulum variabile (similarity, approximately 99%), 2% were related to butyrate-producing bacterium A2-207 (similarity, 99%), and 28% were not identified at the species level. The identities of most bands in the DGGE profiles for the same adult were determined by comigration analysis with the 86 clones that harbored the 239-bp group-specific fragments. Our results suggest that DGGE combined with clone library analysis is an effective technique for monitoring and analyzing the composition of this important population in the human gut flora.

[Analysis of Bifidobacteria spp. composition in human gut via temperature gradient gel electrophoresis (TGGE)].

Temperature Gradient Gel Electrophoresis (TGGE) method combined with 16S rDNA clone library profiling was used to analyze Bifidobacteria spp. composition in human gut in this study. Bifidobacteria group-specific TGGE patterns of 10 healthy human individuals showed that the Bifidobacteria population in humans was host-specific. The genomic diversity and species composition of Bifidobacteria in different individual was dissimilar. Through sequencing and TGGE comigration analysis of the Bifidobacteria group-specific amplicons for one healthy boy, it was revealed that the TGGE bands of this individual represented species Bifidobacterium bifidum, B. infantis, B. longum, B. adolescentis, B. pseudocatenulatum and one potentially new species, respectively. B. pseudocatenulatum was the most common species of tested samples. While as control, only two species- B. pseudocatenulatum and B. longum were isolated using traditional culture method. Bifidobacteria group-specific PCR based TGGE method combined with 16S rDNA clone library profiling is a sensitive and effective approach to resolve the population structure of Bifidobacteria in microflora of human intestinal tract.