Long-Term Changes to the Microbiome, Blood Lipid Profiles and IL-6 in Female and Male Swedish Patients in Response to Bariatric Roux-en-Y Gastric Bypass.

Lipid metabolism dysregulation is a critical factor contributing to obesity. To counteract obesity-associated disorders, bariatric surgery is implemented as a very effective method. However, surgery such as Roux-en-Y gastric bypass (RYGB) is irreversible, resulting in life-long changes to the digestive tract. The aim of the present study was to elucidate changes in the fecal microbiota before and after RYGB in relation to blood lipid profiles and proinflammatory IL-6. Here, we studied the long-term effects, up to six years after the RYGB procedure, on 15 patients' gut microbiomes and their post-surgery well-being, emphasizing the biological sex of the patients. The results showed improved health among the patients after surgery, which coincided with weight loss and improved lipid metabolism. Health changes were associated with decreased inflammation and significant alterations in the gut microbiome after surgery that differed between females and males. The Actinobacteriota phylum decreased in females and increased in males. Overall increases in the genera Prevotella, Paraprevotella, Gemella, Streptococcus, and Veillonella_A, and decreases in Bacteroides_H, Anaerostipes, Lachnoclostridium_B, Hydrogeniiclostridium, Lawsonibacter, Paludicola, and Rothia were observed. In conclusion, our findings indicate that there were long-term changes in the gut microbiota after RYGB, and shifts in the microbial taxa appeared to differ depending on sex, which should be investigated further in a larger cohort.

Synergistic effect of haloduracin and chloramphenicol against clinically important Gram-positive bacteria.

The emergence of drug-resistant pathogens has triggered the search for more efficient antimicrobial agents and formulations for treatment of infections. In recent years, combination therapy has become one of the effective clinical practices in treating infections. The present study deals with the effect of haloduracin, a lantibiotic bateriocin and chloramphenicol against clinically important bacteria. The combined use of haloduracin and chloramphenicol resulted in remarkable synergy against a spectrum of microorganisms including strains of Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis and different groups of Streptococcus. The synergy allowed using these antimicrobial agents at substantially reduced concentrations without compromising their efficiency. Use of lower doses of chloramphenicol can avoid the severity of its side effects. In addition to minimizing undesirable side effects of some drugs, this approach brings the possibility of using antibiotics that are no longer effective due to drug resistance. Furthermore, the observed synergy between haloduracin and chloramphenicol opens a new window of using bacteriocins and antibiotics in combination therapy of infections.

A novel multi-strain probiotic and synbiotic supplement for prevention of Clostridium difficile infection in a murine model.

The protective effect of a multi-strain probiotic and synbiotic formulation was evaluated in C57BL/6 mice infected with Clostridium difficile (CD) NAP1/027. Antibiotic-treated mice were divided into the following four groups: Group 1, fed with a synbiotic formulation consisting of Lactobacillus plantarum F44, L. paracasei F8, Bifidobacterium breve 46, B. lactis 8:8, galacto-oligosaccharides, isomalto-oligosaccharides, and resistant starch; Group 2, fed with the same four probiotic strains as Group 1; Group 3, fed with the same prebiotic supplements as Group 1 for 7 days before CD infection; and Group 4 (control group) antibiotic treated and infected with NAP1/027 strain. Feces and cecal contents were collected for microbial cell viability, quantitative PCR (qPCR), toxin analyses and histopathology. Synbiotics- and probiotics-fed mice showed a significant increase in total bifidobacteria (P < 0.05). The total lactobacilli count was increased in Group 1. Tests for cecal toxins were negative in Group 2 mice, whereas one sample each from Group 1 and 3 was positive. qPCR of cecal contents showed significant reduction in NAP1/027 DNA copies in Groups 1 and 2 and significantly higher numbers of B. breve 46, L. plantarum F44, and L. paracasei F8 in Groups 1 and 2 (P < 0.05); these changes were much less pronounced in Groups 3 and 4. Our findings indicate that the newly developed synbiotic or multi-strain probiotic formulation confers protection against NAP1/027 infection in C57BL/6 mice. This holds promise for performing human studies.

Survival and synergistic growth of mixed cultures of bifidobacteria and lactobacilli combined with prebiotic oligosaccharides in a gastrointestinal tract simulator.

BACKGROUND: Probiotics, especially in combination with non-digestible oligosaccharides, may balance the gut microflora while multistrain preparations may express an improved functionality over single strain cultures. In vitro gastrointestinal models enable to test survival and growth dynamics of mixed strain probiotics in a controlled, replicable manner. METHODS: The robustness and compatibility of multistrain probiotics composed of bifidobacteria and lactobacilli combined with mixed prebiotics (galacto-, fructo- and xylo-oligosaccharides or galactooligosaccharides and soluble starch) were studied using a dynamic gastrointestinal tract simulator (GITS). The exposure to acid and bile of the upper gastrointestinal tract was followed by dilution with a continuous decrease of the dilution rate (de-celerostat) to simulate the descending nutrient availability of the large intestine. The bacterial numbers and metabolic products were analyzed and the growth parameters determined. RESULTS: The most acid- and bile-resistant strains were Lactobacillus plantarum F44 and L. paracasei F8. Bifidobacterium breve 46 had the highest specific growth rate and, although sensitive to bile exposure, recovered during the dilution phase in most experiments. B. breve 46, L. plantarum F44, and L. paracasei F8 were selected as the most promising strains for further studies. CONCLUSIONS: De-celerostat cultivation can be applied to study the mixed bacterial cultures under defined conditions of decreasing nutrient availability to select a compatible set of strains.

Bile enhances cell surface hydrophobicity and biofilm formation of bifidobacteria.

Twenty-four human bifidobacterial strains were analysed for cell surface hydrophobicity (CSH) using a salt aggregation test (SAT) and a Congo red binding (CRB) assay. Three strains were selected for a systematic study on the CSH and biofilm formation: Bifidobacterium breve 46, Bifidobacterium animalis ssp. lactis 8:8 and a reference strain B. animalis ssp. lactis JCM 10602. CRB of the B. breve 46 and B. animalis ssp. lactis JCM 10602 was significantly enhanced (P < 0.05) when grown in deMan-Rogosa-Sharpe cysteine (MRSC) broth supplemented with taurocholic acid (TA) or native porcine bile (PB). An enhanced CSH of the strains grown with PB and gastric mucin correlated with an increased mucin binding and an enhanced biofilm formation in prebiotic oligosaccharide-supplemented cultures. The three strains showed late bile-induced biofilm (72 h) under an anaerobic growth condition, and both B. animalis ssp. lactis strains showed a late bile-induced biofilm formation under aerobic conditions shown by crystal violet staining. These two strains were thus considered to be oxygen tolerant and more robust. Furthermore, enhanced biofilm formation of these robust bifidobacterial strains in the presence of prebiotics may allow for strong colonisation in the gastrointestinal tract when administered to in vivo models as a "synbiotic supplement".

Prebiotic-non-digestible oligosaccharides preference of probiotic bifidobacteria and antimicrobial activity against Clostridium difficile.

Bifidobacterium breve 46, Bifidobacterium lactis 8:8 and Bifidobacterium longum 6:18 and three reference strains B. breve CCUG 24611, B. lactis JCM 10602, and Bifidobacterium pseudocatenulatum JCM 1200 were examined for acid and bile tolerance, prebiotic utilization and antimicrobial activity against four Clostridium difficile (CD) strains including the hypervirulent strain, PCR ribotype NAP1/027. B. lactis 8:8 and B. lactis JCM 10602 exhibited a high tolerance in MRSC broth with pH 2.5 for 30 min. B. breve 46 and B. lactis 8:8 remained 100% viable in MRSC broth with 5% porcine bile after 4 h. All six strains showed a high prebiotic degrading ability (prebiotic score) with galactooligosaccharides (GOS), isomaltooligosaccharides (IMOS) and lactulose as carbon sources and moderate degradation of fructooligosaccharides (FOS). Xylooligosaccharides (XOS) was metabolized to a greater extent by B. lactis 8:8, B. lactis JCM 10602, B. pseudocatenulatum JCM 1200 and B. longum 6:18 (prebiotic score >50%). All strains exhibited extracellular antimicrobial activity (AMA) against four CD strains including the CD NAP1/027. AMA of B. breve 46, B. lactis 8:8 and B. lactis JCM 10602 strains was mainly ascribed to a combined action of organic acids and heat stable, protease sensitive antimicrobial peptides when cells were grown in MRSC broth with glucose and by acids when grown with five different prebiotic-non-digestible oligosaccharides (NDOs). None of C. difficile strains degraded five prebiotic-NDOs. Whole cells of B. breve 46 and B. lactis 8:8 and their supernatants inhibited the growth and toxin production of the CD NAP1/027 strain.

Bile stimulates cell surface hydrophobicity, Congo red binding and biofilm formation of Lactobacillus strains.

Seventeen Lactobacillus strains were tested for cell surface hydrophobicity (CSH) using the salt aggregation test (SAT) and Congo red binding (CRB) assay. CRB was dependent on pH and ionic strength and was protease-sensitive. In the presence of 100 µg mL(-1) cholesterol, the CRB was significantly reduced. Autoaggregating (AA) Lactobacillus crispatus strains showed 50% more CRB than the reference strain, the curli-producing Escherichia coli MC4 100. CRB of L. crispatus 12005, L. paracasei F8, L. plantarum F44 and L. paracasei F19 were enhanced when grown in Man Rogosa Sharpe (MRS) broth with 0.5% taurocholic acid (TA) or 5% porcine bile (PB) (P < 0.05). CSH was also enhanced for the non-AA strains L. plantarum F44, L. paracasei F19 and L. rhamnosus GG when grown in MRS broth with 0.5% TA, 5% PB or 0.25% mucin, with enhanced biofilm formation in MRS broth with bile (P < 0.05). Two AA strains, L. crispatus 12005 and L. paracasei F8, developed biofilm independent of bile or mucin. In summary, under bile-stressed growth conditions, early (24-h cultures) biofilm formation is associated with an increase in hydrophobic cell surface proteins and high CRB. Late mature (72-h culture) biofilm contained more carbohydrates, as shown by crystal violet staining.

Helicobacter species and gut bacterial DNA in Meckel's diverticulum and the appendix.

AIM: To analyse the possible association of various Helicobacter species and certain common gut bacteria in patients with Meckel's diverticulum and appendicitis. METHODS: A nested-polymerase chain reaction (PCR), specific to 16S rRNA of the Helicobacter genus, was performed on paraffin embedded samples, 50 with acute appendicitis, 50 normal appendixes, and 33 Meckel's diverticulum with gastric heterotopia and/or ulcer. Helicobacter genus positive samples were sequenced for species identification. All samples were also analysed for certain gut bacteria by PCR. RESULTS: Helicobacter pullorum DNA was found in one out of 33 cases and Enterobacteria in two cases of Meckel's diverticulum. Helicobacter pylori (H. pylori) was found in three, Enterobacter in 18, and Bacteroides in 19 out of 100 appendix samples by PCR. Enterococcus was not found in any MD or appendix samples. All H. pylori positive cases were from normal appendixes. CONCLUSION: Helicobacter is not an etiological agent in the pathogenesis of symptomatic Meckel's diverticulum or in acute appendicitis.

Effect of heparin, fucoidan and other polysaccharides on adhesion of enterohepatic helicobacter species to murine macrophages.

Helicobacter species have been isolated and cultured from both the gastric and enterohepatic niches of the gastrointestinal tract and are associated with a wide spectrum of diseases. Some members of the enterohepatic Helicobacter species (EHS), which include Helicobacter bilis, Helicobacter hepaticus and Helicobacter pullorum, are associated with chronic inflammatory and proliferative bowel inflammation, hepatitis and in experimental murine studies with hepatic cancer. The present study aimed to explore if polysulphated polysaccharides can prevent adhesion of EHS to the murine macrophage cell line J774A.1. A competitive binding assay showed that heparin and heparan sulphate at a concentration of 1.25 mg/ml reduced binding of H. hepaticus and H. pullorum to the host cells, but not H. bilis. Of the tested Helicobacter spp, the highest inhibition by heparin was demonstrated for H. pullorum (P < 0.01), the most hydrophilic strain. Partially or completely de-sulphated heparin derivatives lost the ability to inhibit adherence of EHS, indicating the importance of sulphated groups of heparin. The most efficient inhibitor of EHS binding to macrophages was fucoidan, which reduced bacterial adhesion of the three enterohepatic Helicobacter species to a greater extent than heparin, 60-90% inhibition vs 30-70% inhibition by heparin. Identification of receptors that EHS ligands bind to is important for understanding the development of infection and may provide a rational target to prevent infection and therapy.

Helicobacter species and common gut bacterial DNA in gallbladder with cholecystitis.

AIM: To analyze the association between Helicobacter spp. and some common gut bacteria in patients with cholecystitis. METHODS: A nested-polymerase chain reaction (PCR), specific to 16S rRNA of Helicobacter spp. was performed on paraffin-embedded gallbladder samples of 100 cholecystitis and 102 control cases. The samples were also analyzed for some common gut bacteria by PCR. Positive samples were sequenced for species identification. RESULTS: Helicobacter DNA was found in seven out of 100 cases of acute and chronic cholecystitis. Sequence analysis displayed Helicobacter pullorum (H. pullorum) in six cases and Helicobacter pylori in one; H. pullorum was only found in cases with metaplasia. Control samples were negative for Helicobacter spp. and some common gut bacteria. There was a significant difference (P = 0.007) between cholecystitis and control samples for Helicobacter DNA. CONCLUSION: A possible relationship was detected between Helicobacter DNA and cholecystitis. Further serological and immunohistochemical studies are needed to support these data.

Characterization of the properties of Pediococcus parvulus for probiotic or protective culture use.

Pediococcus parvulus 2.6 (previously Pediococcus damnosus 2.6, here confirmed as P. parvulus by 16S DNA sequencing) displayed antibacterial activity toward several bacterial species, including isolates found as contaminants in oats, herein genetically identified as Bacillus cereus. No inhibition of Listeria monocytogenes was found under the conditions used. Antibacterial activity was retrieved after ammonium sulfate or acetone precipitation showed it to be peptide mediated. P. parvulus 2.6 has previously shown good technological properties in oat-based products. This, together with the currently found inhibition of food spoilage microorganisms like B. cereus, makes it suitable as a food protective culture. Survival trials of P. parvulus 2.6 at conditions mimicking the gastrointestinal tract were prompted by previously found cholesterol-lowering effects in humans after consumption of oat products cofermented by using P. parvulus 2.6 and Bifidobacterium spp. Viability was measured with in vitro, gutlike simulations at 37 degrees C. High survival was shown under two of three conditions (gastric juice, bile, and small intestine juice), defined as main obstacles of the gastrointestinal tract. The critical step was bile exposure. At a concentration of 20%, viability was low, but 0.3% bile (mean concentration in the intestine) did not have a major influence on growth. Viability of P. parvulus 2.6 was significantly decreased in gastric juice at pH 1.5 (with pepsin), but it was not significantly affected at pH 2.5, and was also improved at a lower pH in 20% oat milk. Viability was judged sufficient for colonization at gutlike conditions, qualifying the strain for further probiotic studies.

Immunoblot analysis as an alternative method to diagnose enterohepatic Helicobacter infections.

INTRODUCTION: Enterohepatic Helicobacter species have been associated with chronic infections of the hepatobiliary tract and lower bowel in naturally and experimentally infected mice, Helicobacter-infected animals should thus not be used in studies of diseases associated with chronic inflammation. Helicobacter species induce inflammation and modulate host immune responses, thus emphasizing the need to diagnose these infections in laboratory animals. MATERIALS AND METHODS: An immunoblot assay was developed to analyze antibodies to enterohepatic Helicobacter species in naturally colonized laboratory mouse colonies. We evaluated the serum antibody responses to cell surface proteins of H. bilis, H. hepaticus, and H. ganmani in 188 mouse sera from four different university animal facilities. Lower bowel tissue specimens from 56 of these animals were available and analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and the results compared with matched immunoblot patterns. RESULTS: Specific antibody reactivity to H. bilis was detected in 8 of 186 (4.3%) sera, to H. hepaticus in 45 of 184 (24%) sera, and to H. ganmani in 51 of 188 (27%) of tested sera. These results were compared with PCR-DGGE analyses of tissue samples of corresponding animals, and concordance between the two diagnostic tests was found in 96% for H. bilis, in 91% for H. hepaticus, and in 82% for H. ganmani. The PCR-DGGE also detected DNA of H. typhlonius, H. sp. flexispira, and H. rodentium. CONCLUSIONS: Infection with enterohepatic species was common in the laboratory mouse colonies tested, independent of strain and stock. Immunoblot analysis seems to be a promising diagnostic tool to monitor enterohepatic Helicobacter species infections of laboratory rodents.

First attempt to produce experimental Campylobacter concisus infection in mice.

AIM: To infect mice with atypical Campylobacter concisus (C. concisus) for the first time. METHODS: Three separate experiments were conducted in order to screen the ability of five clinical C. concisus isolates of intestinal origin and the ATCC 33237 type strain of oral origin to colonize and produce infection in immunocompetent BALB/cA mice. The majority of the BALB/cA mice were treated with cyclophosphamide prior to C. concisus inoculation to suppress immune functions. Inoculation of C. concisus was performed by the gastric route. RESULTS: C. concisus was isolated from the liver, ileum and jejunum of cyclophosphamide-treated mice in the first experiment. No C. concisus strains were isolated in the two subsequent experiments. Mice infected with C. concisus showed a significant loss of body weight from day two through to day five of infection but this decreased at the end of the first week. Histopathological examination did not consistently find signs of inflammation in the gut, but occasionally microabscesses were found in the liver of infected animals. CONCLUSION: Transient colonization with C. concisus was observed in mice with loss of body weight. Future studies should concentrate on the first few days after inoculation and in other strains of mice.

Permanent atrial fibrillation in patients without structural heart disease is not associated with signs of infection by Chlamydia pneumoniae and Helicobacter pylori.

OBJECTIVE: The objective of this study was to explore the role of Chlamydia pneumoniae and Helicobacter pylori infections in patients with idiopathic permanent atrial fibrillation. METHODS AND RESULTS: Sera from 72 patients with permanent atrial fibrillation without structural heart disease (mean age 69.6 years, 23 women) were analysed for IgG antibodies against Chlamydia pneumoniae and Helicobacter pylori and compared in a I:I age- and sex-matched case:control manner with those pooled from a healthy reference population of 72 individuals from the same geographical area. After excluding patients with other possible or definite factors known either to cause atrial fibrillation or to affect the prevalence of seropositivity to these agents, the frequency of seropositivity due to one or both of the infectious agents was compared. Serum C-reactive protein (CRP) level was assessed using immunoturbidimetry technique. Both agents were equally common in men and women. Neither seropositivity to Chlamydia pneumoniae (76% vs. 83%, patients vs. control subjects, ns) nor to Helicobacter pylori (57% contra 55%, patients vs. controls, ns) alone reached significance in the comparisons between patients with atrial fibrillation and control subjects. Serum CRP was higher in patients with AF (5.3 mg/L vs. 2.8 mg/L, P < 0.001). CONCLUSIONS: Though presence of permanent AF is associated with elevated CRP levels, this elevation is not the result of earlier infections with Chlamydia pneumoniae or Helicobacter pylori or their combination.

Cross-reactivity between immune responses to Helicobacter bilis and Helicobacter pylori in a population in Thailand at high risk of developing cholangiocarcinoma.

Helicobacter bilis DNA has been detected in human tissue and is a candidate for etiologic investigations on the causes of hepatic and biliary tract diseases, but reliable serologic tests need to be developed in order to pursue such investigations. The scope of this study was to assess the specificity of two assays for H. bilis immune response allowing for H. pylori, and their cross-reactivity in a population in Thailand at high risk for cholangiocarcinoma. Plasma samples from 92 Thai volunteers were independently tested in two laboratories (Massachusetts Institute of Technology [MIT] and Lund). MIT performed three analyses of H. pylori and H. bilis based either on (i) outer membrane protein (OMP) with no preabsorption or on antigens derived from whole-cell sonicate before (ii) or after (iii) preabsorption with H. pylori sonicate protein. Lund used cell surface proteins from H. pylori and H. bilis as antigens. Testing for H. bilis was preabsorbed with a whole-cell lysate of H. pylori. More than 80% of the samples were positive for H. pylori in both laboratories. As tested by MIT, 58.7% (95% confidence interval, 47.9 to 68.9%) were positive for H. bilis by OMP and 44.5% (34.1 to 55.3%) were positive for H. bilis sonicate protein, but only 15.2% (8.6 to 24.2%) remained positive after preabsorption with H. pylori sonicate protein. Lund found 34.5% of the samples positive for H. bilis (22.0 to 41.0%), which was statistically compatible with all three MIT results. Serologic responses to OMPs of the two bacteria coincided in 66 and 45% of the samples in the MIT and Lund assays, respectively. We found high cross-reactivity between the immune responses to H. pylori and H. bilis antigens. More-specific H. bilis antigens need to be isolated to develop serologic tests suitable for epidemiological studies.

Abdominal aortic aneurysm and infection with CagA positive strains of Helicobacter pylori.

The aim of the present study was to investigate whether virulent CagA positive Helicobacter pylori strains are those preferentially related to abdominal aortic aneurysm (AAA) rupture. Several microorganisms have been linked to aneurysm development. Chronic Chlamydophila pneumoniae infection has been suggested as a possible contributing factor for the development and expansion of AAA. Previous studies have shown increased risk of carotid atherosclerosis and coronary heart disease in subjects harbouring CagA positive strains of H. pylori. The relevance of CagA positive H. pylori involved in the processes underlying aneurysmal development, expansion, and rupture is unknown. In a case-control study, 119 patients with AAA and 36 matched controls were prospectively investigated with H. pylori serology. Patients with ruptured AAA have similar levels of IgG antibodies against H. pylori to patients with electively operated AAA, small AAA, and controls. In conclusion, this study fails to demonstrate a connection between H. pylori CagA seropositivity and abdominal aortic aneurysm rupture.

Abdominal aortic aneurysm and cytomegalovirus infection.

Cytomegalovirus (CMV) has been implicated in the pathogenesis of atherosclerosis. Abdominal aortic aneurysm is regarded traditionally as a consequence of atherosclerosis. Several microorganisms have been suggested as possible contributing factors for the development of abdominal aortic aneurysm. The relevance of CMV in the processes underlying the development, expansion, and rupture of abdominal aortic aneurysm is unknown. The aim of the present study was to investigate whether CMV infection is related to abdominal aortic aneurysm rupture. One hundred nineteen patients with abdominal aortic aneurysm and 36 matched controls without abdominal aortic aneurysm were investigated prospectively by CMV serology. Patients with ruptured abdominal aortic aneurysm have similar levels of IgG antibodies against CMV as patients with nonruptured abdominal aortic aneurysm, small abdominal aortic aneurysm, and controls without abdominal aortic aneurysm. In conclusion, this study fails to demonstrate a connection between CMV infection and abdominal aortic aneurysm rupture.

Detection of Helicobacter species in chronic liver disease and chronic inflammatory bowel disease.

AIM: To study the association between helicobacters and chronic liver diseases and chronic inflammatory bowel diseases. PATIENTS AND METHODS: Thirty-two patients with various chronic liver diseases and 137 patients with inflammatory bowel disease were enrolled. Antibodies to H. pylori, H. hepaticus, H. bilis, and H. pullorum were measured by enzyme immunoassay (EIA), and sera positive in a non-pylori helicobacter EIA were further examined by immunoblot assay. Detection of Helicobacter DNA in liver biopsies was done by denaturating gradient gel electrophoresis of PCR products (PCR-DGGE) and DNA sequence analysis. RESULTS: Six inflammatory bowel disease patients, four with ulcerative colitis and two with Crohn's disease, and one liver disease patient with autoimmune cholangitis had antibodies to non-pylori helicobacters by an immunoblot assay. Four immunoblot assay-negative patients, three with autoimmune and one with non-autoimmune liver disease, had Helicobacter DNA in liver biopsies; three of the polymerase chain reaction (PCR) products were closely related to non-pylori helicobacters. CONCLUSION: Evidence for non-pylori helicobacters was scant in Finnish patients with inflammatory bowel disease or chronic but not end stage liver disease. We cannot, however, rule out their role in these diseases.