Detection of the Bacteroides fragilis toxin gene in sheep with and without small intestinal adenocarcinoma.

Aims: To determine if presence of the Bacteroides fragilis toxin (bft) gene, a molecular marker of colonic carriage of entertoxigenic Bacteroides fragilis (ETBF) in humans, was associated with a finding of small intestinal adenocarcinomas (SIA) in sheep in New Zealand. Methods: Samples of jejunal tissue were collected from the site of tumours and from grossly normal adjacent tissue in 20 sheep, in different consignments, diagnosed with SIA based on gross examination of viscera following slaughter. Two jejunal samples were also collected from a control sheep in the same consignment that had no gross evidence of SIA. A PCR assay was used to detect the presence of the bft gene in the samples. Results: Of the sheep with SIA, the bft gene was amplified from one or both samples from 7/20 (35%) sheep, and in sheep that had no gross evidence of SIA the bft gene was amplified from at least one sample in 11/20 (55%) sheep (RR 0.61; 95% CI = 0.30-1.25; p = 0.34). Of 11 positive samples analysed, ETBF subtype bft-1 was detected in one, bft-2 was detected in 10, and none were bft-3. Conclusions and Clinical Relevance: There was a high prevalence of detection of the bft gene in both SIA-affected and non-affected sheep, but there was no apparent association between carriage of ETBF, evidenced by detection of the bft gene, and the presence of SIA. ETBF are increasingly implicated in the aetiology of human colorectal cancer, raising the possibility that sheep may provide a zoonotic reservoir of this potentially carcinogenic bacterium. Abbreviation: Bft: Bacteroides fragilis toxin; ETBF: Enterotoxigenic Bacteroides fragilis; SIA: Small intestinal adenocarcinoma.

Helicobacter species in the human colon.

OBJECTIVE: To determine the prevalence, frequency and colonization patterns of Helicobacter species throughout the colon. METHOD: Patients having initial colonoscopy for nonspecific gastrointestinal disturbance had colonic biopsies taken from up to four sites during colonoscopy and examined for evidence of the Helicobacteraceae family using a group-specific PCR. Serum was also collected and examined for IgG reactivity to Helicobacter pylori. RESULTS: 100 patients had colonoscopy of whom 35 were found to have DNA evidence of Helicobacter species throughout the colon, with 22 having H. pylori. Fifteen patients had a demonstrable serum IgG response to H. pylori that was not always associated with molecular evidence of H. pylori DNA in colon biopsies and vice versa. No specific association with colon disease was found in patients with H. pylori infection. CONCLUSION: We found evidence of Helicobacter infection in a significant number of patients presenting for colonoscopy but no specific association between the presence of these bacteria and colon disease. Our finding of disparity between molecular and serological techniques to detect Helicobacter species suggests that future studies should not rely on serology alone to detect these bacteria in the human colon.

Ovine small intestinal adenocarcinomas are not associated with infection by herpesviruses, Helicobacter species or Mycobacterium avium subspecies paratuberculosis.

Sheep in New Zealand more frequently develop small intestinal adenocarcinoma (SIA) than sheep in other countries. The reasons for this high rate of intestinal neoplasia are not known. In man, differences between countries in the incidence of neoplasia are often due to differences in the rate of infection by carcinogenic viruses or bacteria. Therefore, it was hypothesized that New Zealand sheep more frequently develop SIA as they are more frequently exposed to an infectious agent. This study compared rates of detection of herpesviruses, Helicobacter species, and Mycobacterium avium subspecies paratuberculosis (MAP) in ovine SIA to rates of detection in samples of intestine with non-neoplastic disease. These infectious agents were chosen as all three have been associated with human intestinal cancer. Microscopical examination did not reveal helical bacteria within sections of SIA or non-neoplastic jejunum. Polymerase chain reaction amplified herpesviral DNA more frequently from samples of non-neoplastic jejunum than samples of SIA. MAP DNA was not amplified from either neoplastic or non-neoplastic jejunum. These results suggest that the high rates of SIA in New Zealand sheep are not due to frequent infection by herpesviruses, Helicobacter species or MAP.

The effect of Helicobacter pylori infection and dietary iron deficiency on host iron homeostasis: a study in mice.

BACKGROUND: Helicobacter pylori, which requires iron to survive, may cause host iron deficiency by directly competing with the host for available iron or by impairing iron uptake as a consequence of atrophy-associated gastric hypochlorhydria. The aim of this study was to examine the effect of H. pylori infection and dietary iron deficiency on host iron homeostasis in a mouse model. MATERIALS AND METHODS: H. pylori SS1-infected and uninfected C57BL/6 mice, fed either a normal diet or an iron-deficient diet, were assessed for iron status and infection-associated gastritis over a 30-week period. RESULTS: After 10 weeks, serum ferritin values were higher in H. pylori-infected mice than in uninfected controls, irrespective of dietary iron intake (p = .04). The infection-related increase in body iron stores persisted in the iron-replete mice but diminished over time in mice with restricted dietary iron intake (p < .0001). At 30 weeks serum ferritin levels were lower in these animals (p = .063). No significant difference in bacterial numbers was detected at the 30-week time point (p > .05) and the histological changes observed were consistently associated with infection (p < .01) and not with the iron status of the mice (p = .771). CONCLUSIONS: Infection with H. pylori did not cause iron deficiency in iron-replete mice. However, diminished iron stores in mice as a result of limited dietary iron intake were further lowered by concurrent infection, thus indicating that H. pylori competes successfully with the host for available iron.

Iron influences the expression of Helicobacter pylori outer membrane vesicle-associated virulence factors.

BACKGROUND: Helicobacter pylori shed outer membrane vesicles (OMV) in vitro and in vivo. These OMV, which contain active VacA, provide a potential vehicle for the delivery of H. pylori virulence factors to the gastric mucosa. OBJECTIVE: To assess the influence of environmental iron levels on H. pylori OMV VacA and protease expression in vitro. METHODS: Three well-characterized H. pylori type-strains were grown for 72 h under normal (Brucella broth, 5% fetal calf serum) and iron-limiting (Brucella broth, 5% fetal calf serum, 50 micromol/l deferoxamine) conditions. Following harvesting by differential centrifugation, the ratio of whole cells to OMV was determined. OMV VacA levels in response to iron availability were determined by ELISA and immunolabelling of washed bacteria. Protease activity was detected by zymography of OMV in the presence and absence of enzyme inhibitors and activators. HEp-2 cells were used to assay for OMV-associated cytopathogenic toxins. RESULTS: Decreased iron availability, which limited bacterial growth but not OMV release, also influenced the expression of OMV-associated virulence factors. VacA levels were reduced, whereas two new proteolytic enzymes were expressed on these OMV. When an iron salt was added to counteract the effect of the deferoxamine, VacA levels were restored in the outer membrane and the proteolytic activity disappeared. CONCLUSIONS: These results suggest that OMV release by H. pylori is influenced by environmental iron levels, and that the qualitative changes that occur in outer membrane composition may contribute to the clinical patterns of H. pylori-associated disease.

Lack of protection following immunisation with H. pylori outer membrane vesicles highlights antigenic differences between H. felis and H. pylori.

Helicobacter pylori-induced inflammation is associated with the development of gastritis, peptic ulcer disease and gastric cancer in humans. Immunisation against this bacterium would ultimately have a major impact on H. pylori-related disease, notably global gastric cancer rates. To date, several potential H. pylori vaccine candidates have been identified. In this study, the Helicobacter felis/murine model was used to assess the immunogenicity of a previously undescribed H. pylori outer membrane vesicle fraction in immune protection.

37 kBq 14C-urea breath test and gastric biopsy analyses of H. pylori infection.

BACKGROUND: The treatment of H. pylori-associated gastroduodenal disease is increasingly aimed at bacterial eradication which requires follow-up assessment of therapeutic effectiveness and re-infection. A simplified 37 kBq 14C-urea breath test for H. pylori infection has been developed. METHODS: The 37 kBq 14C-urea breath test was compared with biopsy urease (CLO) and histological analyses of gastric-biopsies obtained from 63 patients undergoing endoscopy. RESULTS: The 30-min breath test correlated closely with biopsy findings, had a sensitivity of 100%, a specificity of 95% and a positive predictive value of 92%. CONCLUSIONS: The simplified, low-dose, 14C-urea breath test is a convenient, low-cost, transportable means of facilitating the management of H. pylori-associated diseases.

Dual silver staining to characterise Helicobacter spp. outer membrane components.

Helicobacter pylori is a bacterial pathogen, estimated to infect half the world's population. The bacterium is the aetiological cause of gastritis, the common precursor for peptic ulcer disease and gastric cancer. Immunisation of at-risk individuals is the most cost-effective means of dealing with such a widespread pathogen. Potential vaccine candidates need to be identified and characterised. Conventional silver staining is commonly used for the sensitive detection of bacterial protein components separated by SDS-PAGE. Modified silver stains employing periodate oxidation have also been developed for the analysis of purified bacterial lipopolysaccharide. By using these methods in parallel, as a dual silver stain, bacterial fractions can be characterised in terms of protein and LPS content. Strain differences can also be readily identified by comparing protein and LPS profiles. When combined with differential immunoblotting, the dual silver stain is a useful analytical tool for characterising potential vaccine candidate antigens.

Fusidic acid prophylaxis before cataract surgery: patient self-administration.

In a placebo-controlled, randomised, double-blind clinical trial, the authors evaluated the efficacy of patient-administered 1% fusidic acid viscous eye drops in clearing the commonest organisms causing pseudophakic endophthalmitis (Staphylococcus epidermidis and aureus) from the lids and conjunctivae of 79 patients before cataract surgery. The treatment group self-administered fusidic acid viscous eye drops four times daily for seven days before surgery; the placebo group received inert ophthalmic drops. Fellow eyes of both groups remained untreated as a natural control. Lower fornix and lid margin cultures were taken from both eyes before and after treatment. Before treatment, there was no statistical difference in organism counts between the groups. After treatment, eyes receiving fusidic acid were more likely to be free of clinically relevant Staphylococcus spp. than all pre-treatment eyes (for lids, P << 0.001; conjunctivae, P = 0.02). A highly significant (P < 0.001) number of lid margins were rendered 'clinically clean' (i.e., 0-49 organisms/swab) by fusidic acid when compared with untreated eyes. Treatment also effectively (P < 0.05) reduced the numbers of bacteria isolated from conjunctivae. This study indicates that there is a highly significant reduction of Staphylococcus spp. (P << 0.001), non-Staphylococcus spp. (P << 0.001) and attainment of sterile eyes (P << 0.001) at operation gained by patient self-administration of 1% fusidic acid four times daily for seven days before surgery.

Topical antibiotic prophylaxis for cataract surgery: a controlled trial of fusidic acid and chloramphenicol.

The effectiveness of topical fusidic acid 1%, in a viscous drop base, to reduce or eliminate ocular microflora in patients undergoing cataract surgery has been studied. Forty-two patients received fusidic acid on a double-blind basis and for comparison 21 patients were similarly assessed with topical chloramphenicol. A further 17 patients received no treatment other than subconjunctival cephazolin administered to all operated eyes at the time of surgery. Quantitative bacterial counts from the conjunctivae and lash lines of each patient were made 24 hours before surgery, on the morning of operation and again 48 hours after surgery. With a regimen of five administrations on the day prior to surgery, neither topical fusidic acid 1.0% nor chloramphenicol 0.5% produced clinically or statistically significant reductions of the ocular microflora. In contrast perioperative subconjunctival cephazolin effectively reduced or eliminated lid and conjunctival microflora following surgery. This study indicates that the effectiveness of a topical antibiotic preparation for overt ocular infection cannot be directly extrapolated to the effect on resident ocular microflora, at least with short-term use for presurgical prophylaxis.