The protective effects of walnut green husk polysaccharide on liver injury, vascular endothelial dysfunction and disorder of gut microbiota in high fructose-induced mice.

This study aimed to investigate the protective effects of walnut green husk polysaccharide (WGHP) on liver injury, vascular endothelial dysfunction and disorder of gut microbiota in mice induced by high fructose (HF) diet. The chemical analysis results show that the walnut green husk polysaccharide is a low molecular weight acidic heteropolysaccharide, composed mainly of glucuronic acid, arabinose and galactose. Biochemical analysis showed that WGHP significantly improved glucose metabolism and lipid metabolism and decreased oxidative stress in HF-diet induced obesity mice. Histopathological observation of liver and cardiovascular aorta confirmed the protective effects of WGHP on hepatic steatosis and vascular endothelial dysfunction. Furthermore, 16S rRNA sequencing results demonstrated that WGHP reversed the disorders of gut microbiota caused by HF, decreased the relative abundance of Verrucomicrobia and increased the relative abundance of Deferribacteres at the phylum level, decreased the relative abundance of Akkermansia, Lachnoclostridium and norank_f__Muribaculaceae and increased the relative abundance of Prevotellaceae_UCG-001, Helicobacter, Alloprevotella and Allobaculum at the genus levels. Our results indicate that WGHP may act as a functional polysaccharide for protecting liver and cardiovascular in HF-fed mice.

Lactobacillus rhamnosus Granules Dose-Dependently Balance Intestinal Microbiome Disorders and Ameliorate Chronic Alcohol-Induced Liver Injury.

As the functions of Lactobacilli become better understood, there are increasing numbers of applications for Lactobacillus products. Previously, we have demonstrated that Lactobacillus rhamnosus GG (LGG) can prevent alcoholic liver injury. LGG granules were produced by fluid bed granulation with a media composed of starch, skimmed milk powder, whey powder, microcrystalline cellulose and maltose, and LGG fermented liquid that comprised 30-50% of the total weight. We found LGG granules dose-dependently protected against chronic alcoholic liver disease. When alcohol was consumed for 8 weeks with LGG treatment during the last 2 weeks, we demonstrated that the dose dependence of LGG granules can improve alcohol-induced liver injury through decreasing the levels of lipopolysaccharide and tumor necrosis factor-α in serum and prevent liver steatosis by suppressing triglyceride, free fatty acid, and malondialdehyde production in liver. Alcohol feeding caused a decline in the number of both Lactobacillus and Bifidobacterium, with a proportional increase in the number of Clostridium perfringens in ileum, and expansion of the Gram-negative bacteria Proteobacteria, Campylobacterales, and Helicobacter in cecum. However, LGG granule treatment restored the content of these microorganisms. In conclusion, LGG granule supplementation can improve the intestinal microbiota, reduce the number of gram-negative bacteria, and ameliorate alcoholic liver injury.

Comparison of media and growth conditions for culturing enterohepatic Helicobacter species.

This research aims to compare the culturing conditions for enterohepatic Helicobacter, evaluating culture media, incubation atmosphere and susceptibility to antimicrobials used to generate selective conditions. Four common media for the closely related genus Campylobacter (Columbia, Bolton, Brucella and CCDA agar), as well as the need for hydrogen in the microaerobic incubation atmosphere, were evaluated. Serial dilutions of 13 strains belonging to six species (H. apodemus, H. bilis, H. canicola, H. canis, H. equorum and Helicobacter sp.) were inoculated in each media and incubated at 37°C for 48 to 96 h using CampyGen (OXOID) and gaseous exchange (including hydrogen) in parallel. Columbia or Brucella agars were the most appropriate for culturing EHH (P < 0·05). However, there was no significant difference between the atmospheres evaluated (P = 0·13). In addition, minimal inhibitory concentration for six antibiotics showed that all isolates were resistant to trimethoprim, whereas for the rest of the antibiotics (cephalothin, cefoperazone, cefsulodin, teicoplanin and vancomycin) the inhibition range was between 8 and 64 µg ml- 1 . Our findings suggest that Columbia or Brucella media, regardless of the use of hydrogen, can be used for the EHH isolation. In addition, the concentration of antibiotics included in commercial campylobacteria supplements is suitable for EHH species recovery. SIGNIFICANCE AND IMPACT OF THE STUDY: Enterohepatic Helicobacter (EHH) infections have been associated with several diseases in humans such as acute gastroenteritis, inflammatory bowel disease and hepatobiliary diseases. Although they are frequently detected in clinical samples by molecular methods, only occasionally they are isolated using culture conditions described for the taxonomic related pathogen Campylobacter sp. This is because the optimal conditions for the isolation of EHH have not yet been described, which results in an underestimation of the prevalence and clinical importance of these emerging pathogens. Therefore, this study provides insight for culturing EHH species.

Comparison of the clinical and microbiological characteristics of Campylobacter and Helicobacter bacteremia: the importance of time to blood culture positivity using the BACTEC blood culture systems.

OBJECTIVE: Campylobacter spp. and Helicobacter spp. are rare but important causes of bacteremia in humans. Distinguishing these bacteria is complicated because of their similar phenotypic profiles. We conducted clinical and microbiological investigations of Campylobacter spp. or Helicobacter spp. bacteremia. Patients diagnosed with bacteremia from 2008 to 2014 were included. The clinical and microbiological characteristics of Campylobacter spp. and Helicobacter spp. bacteremia were compared. The BACTEC system was used in blood cultures. A receiver operating characteristic curve was plotted based on the time to blood culture positivity. RESULTS: Sixteen cases of Helicobacter spp. bacteremia (patient age: 61 ± 18 years) and 14 cases of Campylobacter spp. bacteremia (patient age: 49 ± 21 years) were identified. Median time to blood culture positivity was longer for the Helicobacter spp. cases than the Campylobacter spp. cases (91.4 h vs 55.3 h, p < 0.01). A time to blood culture positivity > 75 h predicted Helicobacter spp. bacteremia with a sensitivity of 0.88 and a specificity of 0.93 (area under the receiver operating characteristic curve of 0.90). In conclusion, a time to blood culture positivity was useful in distinguishing Helicobacter spp. bacteremia from Campylobacter spp. bacteremia.

Extraintestinal infection of Helicobacter cinaedi induced by oral administration to Balb/c mice.

Although Helicobacter cinaedi was initially considered an opportunistic pathogen in immunocompromised patients, it was later shown to also infect immunocompetent and healthy individuals. Sporadic bacteremia due to H. cinaedi has frequently been reported; however, whether the bacterium can be translocated after passage through the intestinal mucosa remains unclear. In the present study, a preclinical small animal model that faithfully reproduces H. cinaedi infection in humans was developed. Balb/c male mice were orally inoculated with a single dose of 6.8 × 107 CFU of a human clinical H. cinaedi strain. The organism persistently colonized the intestinal tract of the mice, particularly the cecum and colon, for at least 56 days, and the bacteria were excreted in the feces. Although inoculated bacteria were recovered from the spleen, liver, kidney, lung, bladder and mesenteric lymph nodes during the first 2 weeks of bacteremia, the organism was not isolated from these organs after 4 weeks, suggesting that complement- and antibody-mediated serum sensitivity account for the relatively low frequency of systemic infection. However, H. cinaedi was isolated from the biceps femoris, triceps branchii, latissimus dorsi, and trapezius muscles beyond 2 weeks after infection and after production of specific anti-H. cinaedi IgM and IgG antibodies. The present findings suggest that experimental infection of Balb/c mice with H. cinaedi may be a useful model for further studies of H. cinaedi pathogenesis, prophylaxis or therapeutic interventions in vivo.

Chronic Opisthorchis viverrini Infection Changes the Liver Microbiome and Promotes Helicobacter Growth.

Adults of Opisthorchis viverrini reside in the biliary system, inducing inflammation of bile ducts and cholangitis, leading to hepatobiliary disease (HBD) including cholangiocarcinoma. O. viverrini infection also has major implications for the bacterial community in bile ducts and liver. To investigate this in chronic O. viverrini infection (≥ 8 months p.i.), bacterial genomic DNA from livers of hamsters and from worms was investigated using culture techniques, PCR for Helicobacter spp. and high-throughput next-generation sequencing targeting the V3-V4 hypervariable regions of prokaryotic 16S rRNA gene. Of a total of 855,046 DNA sequence reads, 417,953 were useable after filtering. Metagenomic analyses assigned these to 93 operational taxonomic units (OTUs) consisting of 80 OTUs of bacteria, including 6 phyla and 42 genera. In the chronic O. viverrini-infected group, bacterial community composition and diversity were significantly increased compared to controls. Sequences of Fusobacterium spp. were the most common (13.81%), followed by Streptococcus luteciae (10.76%), Escherichia coli (10.18%), and Bifidobacterium spp. (0.58%). In addition, Helicobacter pylori (0.17% of sequences) was also identified in the liver of chronic O. viverrini infections, but not in normal liver. The presence of H. pylori was confirmed by PCR and by use of an antibody against bacterial antigen, supporting the metagenomics data. The identities of bacteria cultured for enrichment suggested that chronic O. viverrini infection changes the liver microbiome and promotes Helicobacter spp. growth. There may be synergy between O. viverrini and the liver microbiome in enhancing immune response-mediated hepatobiliary diseases.

Epithelial Coculture and l-Lactate Promote Growth of Helicobacter cinaedi under H2-Free Aerobic Conditions.

Helicobacter cinaedi is an emerging opportunistic pathogen associated with infections of diverse anatomic sites. Nevertheless, the species demonstrates fastidious axenic growth; it has been described as requiring a microaerobic atmosphere, along with a strong preference for supplemental H2 gas. In this context, we examined the hypothesis that in vitro growth of H. cinaedi could be enhanced by coculture with human epithelial cells. When inoculated (in Ham's F12 medium) over Caco-2 monolayers, the type strain (ATCC BAA-847) gained the ability to proliferate under H2-free aerobic conditions. Identical results were observed during coculture with several other monolayer types (LS-174T, AGS, and HeLa). Under chemically defined conditions, 40 amino acids and carboxylates were screened for their effect on the organism's atmospheric requirements. Several molecules promoted H2-free aerobic proliferation, although it occurred most prominently with millimolar concentrations of l-lactate. The growth response of H. cinaedi to Caco-2 cells and l-lactate was confirmed with a collection of 12 human-derived clinical strains. mRNA sequencing was next performed on the type strain under various growth conditions. In addition to providing a whole-transcriptome profile of H. cinaedi, this analysis demonstrated strong constitutive expression of the l-lactate utilization locus, as well as differential transcription of terminal respiratory proteins as a function of Caco-2 coculture and l-lactate supplementation. Overall, these findings challenge traditional views of H. cinaedi as an obligate microaerophile. IMPORTANCE: H. cinaedi is an increasingly recognized pathogen in people with compromised immune systems. Atypical among other members of its bacterial class, H. cinaedi has been associated with infections of diverse anatomic sites. Growing H. cineadi in the laboratory is quite difficult, due in large part to the need for a specialized atmosphere. The suboptimal growth of H. cinaedi is an obstacle to clinical diagnosis, and it also limits investigation into the organism's biology. The current work shows that H. cinaedi has more flexible atmospheric requirements in the presence of host cells and a common host-derived molecule. This nutritional interplay raises new questions about how the organism behaves during human infections and provides insights for how to optimize its laboratory cultivation.

Effects of taurine on gut microbiota and metabolism in mice.

As being a necessary amino acid, taurine plays an important role in the regulation of neuroendocrine functions and nutrition. In this study, effects of taurine on mice gut microbes and metabolism were investigated. BALB/C mice were randomly divided into three experimental groups: The first group was administered saline (CK), the second was administered 165 mg/kg natural taurine (NE) and the third one administered 165 mg/kg synthetic taurine (CS). Gut microbiota composition in mice feces was analyzed by metagenomics technology, and the content of short-chain fatty acids (SCFA) in mice feces was detected by gas chromatography (GC), while the concentrations of lipopolysaccharide (LPS) and superoxide dismutase (SOD) were detected by a LPS ELISA kit and a SOD assay kit, respectively. The results showed that the effect of taurine on gut microbiota could reduce the abundance of Proteobacteria, especially Helicobacter. Moreover, we found that the SCFA content was increased in feces of the NE group while LPS content was decreased in serum of the NE group; the SOD activity in serum and livers of the NE and CS groups were not changed significantly compare to that of the CK group. In conclusion, taurine could regulate the gut micro-ecology, which might be of benefit to health by inhibiting the growth of harmful bacteria, accelerating the production of SCFA and reducing LPS concentration.

Oral glutathione supplementation drastically reduces Helicobacter-induced gastric pathologies.

Helicobacter (H.) suis causes gastric pathologies in both pigs and humans. Very little is known on the metabolism of this bacterium and its impact on the host. In this study, we have revealed the importance of the glutamate-generating metabolism, as shown by a complete depletion of glutamine (Gln) in the medium during H. suis culture. Besides Gln, H. suis can also convert glutathione (GSH) to glutamate, and both reactions are catalyzed by the H. suis γ-glutamyltranspeptidase (GGT). Both for H. pylori and H. suis, it has been hypothesized that the degradation of Gln and GSH may lead to a deficiency for the host, possibly initiating or promoting several pathologies. Therefore the in vivo effect of oral supplementation with Gln and GSH was assessed. Oral supplementation with Gln was shown to temper H. suis induced gastritis and epithelial (hyper)proliferation in Mongolian gerbils. Astonishingly, supplementation of the feed with GSH, another GGT substrate, resulted in inflammation and epithelial proliferation levels returning to baseline levels of uninfected controls. This indicates that Gln and GSH supplementation may help reducing tissue damage caused by Helicobacter infection in both humans and pigs, highlighting their potential as a supportive therapy during and after Helicobacter eradication therapy.

Helicobacter cinaedi induced typhlocolitis in Rag-2-deficient mice.

BACKGROUND: Helicobacter cinaedi, an enterohepatic helicobacter species (EHS), is an important human pathogen and is associated with a wide range of diseases, especially in immunocompromised patients. It has been convincingly demonstrated that innate immune response to certain pathogenic enteric bacteria is sufficient to initiate colitis and colon carcinogenesis in recombinase-activating gene (Rag)-2-deficient mice model. To better understand the mechanisms of human IBD and its association with development of colon cancer, we investigated whether H. cinaedi could induce pathological changes noted with murine enterohepatic helicobacter infections in the Rag2(-/-) mouse model. MATERIALS AND METHODS: Sixty 129SvEv Rag2(-/-) mice mouse were experimentally or sham infected orally with H. cinaedi strain CCUG 18818. Gastrointestinal pathology and immune responses in infected and control mice were analyzed at 3, 6 and 9 months postinfection (MPI). H. cinaedi colonized the cecum, colon, and stomach in infected mice. RESULTS: H. cinaedi induced typhlocolitis in Rag2(-/-) mice by 3 MPI and intestinal lesions became more severe by 9 MPI. H. cinaedi was also associated with the elevation of proinflammatory cytokines, interferon-γ, tumor-necrosis factor-α, IL-1ß, IL-10; iNOS mRNA levels were also upregulated in the cecum of infected mice. However, changes in IL-4, IL-6, Cox-2, and c-myc mRNA expressions were not detected. CONCLUSIONS: Our results indicated that the Rag2(-/-) mouse model will be useful to continue investigating the pathogenicity of H. cinaedi, and to study the association of host immune responses in IBD caused by EHS.