Despite a long-suspected role in the development of human colorectal cancer (CRC), the composition of gut microbiota in CRC patients has not been adequately described. In this study, fecal bacterial diversity in CRC patients (n=46) and healthy volunteers (n=56) were profiled by 454 pyrosequencing of the V3 region of the 16S ribosomal RNA gene. Both principal component analysis and UniFrac analysis showed structural segregation between the two populations. Forty-eight operational taxonomic units (OTUs) were identified by redundancy analysis as key variables significantly associated with the structural difference. One OTU closely related to Bacteroides fragilis was enriched in the gut microbiota of CRC patients, whereas three OTUs related to Bacteroides vulgatus and Bacteroides uniformis were enriched in that of healthy volunteers. A total of 11 OTUs belonging to the genera Enterococcus, Escherichia/Shigella, Klebsiella, Streptococcus and Peptostreptococcus were significantly more abundant in the gut microbiota of CRC patients, and 5 OTUs belonging to the genus Roseburia and other butyrate-producing bacteria of the family Lachnospiraceae were less abundant. Real-time quantitative PCR further validated the significant reduction of butyrate-producing bacteria in the gut microbiota of CRC patients by measuring the copy numbers of butyryl-coenzyme A CoA transferase genes (Mann-Whitney test, P<0.01). Reduction of butyrate producers and increase of opportunistic pathogens may constitute a major structural imbalance of gut microbiota in CRC patients.

Lipopolysaccharide endotoxin is the only known bacterial product which, when subcutaneously infused into mice in its purified form, can induce obesity and insulin resistance via an inflammation-mediated pathway. Here we show that one endotoxin-producing bacterium isolated from a morbidly obese human's gut induced obesity and insulin resistance in germfree mice. The endotoxin-producing Enterobacter decreased in relative abundance from 35% of the volunteer's gut bacteria to non-detectable, during which time the volunteer lost 51.4 kg of 174.8 kg initial weight and recovered from hyperglycemia and hypertension after 23 weeks on a diet of whole grains, traditional Chinese medicinal foods and prebiotics. A decreased abundance of endotoxin biosynthetic genes in the gut of the volunteer was correlated with a decreased circulating endotoxin load and alleviated inflammation. Mono-association of germfree C57BL/6J mice with strain Enterobacter cloacae B29 isolated from the volunteer's gut induced fully developed obesity and insulin resistance on a high-fat diet but not on normal chow diet, whereas the germfree control mice on a high-fat diet did not exhibit the same disease phenotypes. The Enterobacter-induced obese mice showed increased serum endotoxin load and aggravated inflammatory conditions. The obesity-inducing capacity of this human-derived endotoxin producer in gnotobiotic mice suggests that it may causatively contribute to the development of obesity in its human host.

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.

Gut microbiota-derived endotoxin has been linked to human nonalcoholic fatty liver disease (NAFLD), but the specific causative agents and their molecular mechanisms remain elusive. In this study, we investigated whether bacterial strains of endotoxin-producing pathogenic species overgrowing in obese human gut can work as causative agents for NAFLD. We further assessed the role of lipopolysaccharide (LPS)-Toll-like receptor 4 (TLR4) cross talk in this pathogenicity. Nonvirulent strains of Gram-negative pathobionts were isolated from obese human gut and monoassociated with C57BL/6J germfree (GF) mice fed a high-fat diet (HFD). Deletion of waaG in the bacterial endotoxin synthetic pathway and knockout of TLR4 in GF mice were used to further study the underlying mechanism for a causal relationship between these strains and the development of NAFLD. Three endotoxin-producing strains, Enterobacter cloacae B29, Escherichia coli PY102, and Klebsiella pneumoniae A7, overgrowing in the gut of morbidly obese volunteers with severe fatty liver, induced NAFLD when monoassociated with GF mice on HFD, while HFD alone did not induce the disease in GF mice. The commensal Bacteroides thetaiotaomicron (ATCC 29148), whose endotoxin activity was markedly lower than that of Enterobacteriaceae strains, did not induce NAFLD in GF mice. B29 lost its proinflammatory properties and NAFLD-inducing capacity upon deletion of the waaG gene. Moreover, E. cloacae B29 did not induce NAFLD in TLR4-deficient GF mice. These nonvirulent endotoxin-producing strains in pathobiont species overgrowing in human gut may work as causative agents, with LPS-TLR4 cross talk as the most upstream and essential molecular event for NAFLD. IMPORTANCE Recent studies have reported a link between gut microbiota and nonalcoholic fatty liver disease (NAFLD), showing that germfree (GF) mice do not develop metabolic syndromes, including NAFLD. However, the specific bacterial species causing NAFLD, as well as their molecular cross talk with the host for driving liver disease, remain elusive. Here, we found that nonvirulent endotoxin-producing strains of pathogenic species overgrowing in obese human gut can act as causative agents for induction of NAFLD and related metabolic disorders. The cross talk between endotoxin from these specific producers and the host’s TLR4 receptor is the most upstream and essential molecular event for inducing all phenotypes in NAFLD and related metabolic disorders. These nonvirulent endotoxin-producing strains of gut pathogenic species overgrowing in human gut may collectively become a predictive biomarker or serve as a novel therapeutic target for NAFLD and related metabolic disorders.