Detection of Helicobacter in the liver of patients with chronic cholestatic liver diseases.

Helicobacter species were identified in human liver tissues by PCR. Biopsies were obtained from patients with primary sclerosing cholangitis, primary biliary cirrhosis and noncholestatic liver cirrhosis. One set of Helicobacter genus-specific primers and two different primer sets for Helicobacter pylori were used in the PCR-assays. Using Helicobacter genus-specific primers 80% (8/10) of patients with primary sclerosing cholangitis and 90% (9/10) of patients with primary biliary cirrhosis were positive. Seven of these 17 samples were positive using two different primers for H. pylori and Southern blot hybridization. Among the non-cholestatic liver cirrhosis controls, only one sample was positive in the Helicobacter genus-specific PCR-assay. Significantly higher values of alkaline phosphatases and prothrombin complex was found for the patients positive for Helicobacter genus. In conclusion, gene sequences of Helicobacter species and H. pylori were detected in human liver tissue using PCR and DNA hybridization in patients with a cholestatic liver disease, but rarely in noncholestatic liver cirrhosis.     

A PCR-DGGE method for detection and identification of Campylobacter, Helicobacter, Arcobacter and related Epsilobacteria and its application to saliva samples from humans and domestic pets

AIMS: To develop a PCR-denaturing gradient gel electrophoresis (PCR-DGGE) method for the detection and identification of Campylobacter, Helicobacter and Arcobacter species (Epsilobacteria) in clinical samples and evaluate its efficacy on saliva samples from humans and domestic pets. METHODS AND RESULTS: A semi-nested PCR was developed to allow sensitive detection of all Epsilobacteria, with species separation undertaken by DGGE. A database was constructed in BioNumerics using 145 strains covering 51 Campylobacter, Arcobacter and Helicobacter taxa; Nineteen distinct DGGE profile-groups were distinguished. This approach detected Epsilobacteria in all saliva samples collected from humans, cats and dogs, and identified Campylobacter concisus and/or Campylobacter gracilis in the human samples. The pet animal samples were taken from individuals with oral/dental diseases; PCR-DGGE identified up to four different species in each sample. The most common species detected included Wolinella succinogenes, Arcobacter butzleri and two hitherto uncultured campylobacters. The enteropathogen Campylobacter lari was also found. CONCLUSIONS: PCR combined with DGGE is a useful tool for direct detection and preliminary identification of Epsilobacteria in the oral cavity of humans and small animals. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCR-DGGE method should allow determination of the true prevalence and diversity of Epsilobacteria in clinical and other samples. Contact with the oral cavity of domestic pets may represent a route of transmission for epsilobacterial enteric diseases.     

Prevalence of Helicobacter and Acanthamoeba in natural environment

Aims:  We examined whether the presence of Helicobacter is related to that of Acanthamoeba in river and soil environments.
Methods and Results:  The samples (river n  =   51, soil n  =   75) were collected in Sapporo City, Japan. PCR with primers for Helicobacter genus-specific and standard culture techniques were used to detect helicobacter. Prevalence of acanthamoeba was also evaluated by genus-specific PCR. The prevalence of Helicobacter genus-specific DNA in river water samples and in soil samples was 88% and 0%, respectively. No successful culture of helicobacter was achieved. The prevalence of Acanthamoeba genus-specific DNA in river samples and in soil samples was 61% and 96%, respectively. No statistical correlation between the prevalence of helicobacter and either that of acanthamoeba or water quality parameters (pH, turbidity and coliform group) except for temperature was found.
Conclusions:  We revealed the presence of helicobacter in river water and non-existence of helicobacter in soil. However, the distribution of helicobacter did not overlap with that of acanthamoeba in rivers.
Significance for Impact of the Study:  The role of acanthamoeba on the survival of helicobacter might be limited as the both are coincidentally present in the environment.     

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.     

The role of Helicobacter spp. in the pathogenesis of primary biliary cirrhosis and primary sclerosing cholangitis

Helicobacter species DNA has been detected in liver tissue of patients affected by primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC). To investigate a potential causative relation between Helicobacter species and PBC/PSC, we compared the presence of Helicobacter species-specific DNA in liver tissue of patients with PBC/PSC (n=18/n=13) with those of a control group of patients with various liver diseases with known cause (n=29). A PCR with Helicobacter genus-specific 16S rRNA primers was performed on DNA isolated from paraffin embedded liver tissue. Control patients had hepatitis-B (n=9), alcoholic cirrhosis (n=14), or non-cirrhotic metabolic liver disease (n=6). There was no significant difference between the incidence of Helicobacter spp.-specific DNA in PBC/PSC (9/31; 29%) and the control group (10/29; 34%). Sequence analysis confirmed Helicobacter spp. DNA. Because Helicobacter spp. DNA can be found in approximately one-third of all samples tested, it is unlikely that PSC and PBC are caused by Helicobacter infection.     

Detection of Helicobacter ganmani-like 16S rDNA in pediatric liver tissue

BACKGROUND: To determine the presence of Helicobacter species in the liver biopsy specimens from children with various chronic liver diseases as data in adult literature suggests a possible role of these bacteria in their pathogenesis. MATERIALS AND METHODS: Paraffin sections of 61 liver biopsies of pediatric patients with miscellaneous diseases and autopsy liver tissue from 10 control subjects with no evidence of preexisting liver disease were examined for the presence of Helicobacter species by a genus-specific seminested polymerase chain reaction (PCR) assay. PCR-products of positive samples were further characterized by denaturing gradient gel electrophoresis (DGGE) and DNA-sequence analysis. Based on those results, a seminested PCR assay for H. ganmani was developed and applied to the samples. RESULTS: On analysis, 40/61 patient samples were positive in the genus-specific Helicobacter PCR and 4/10 from the control group. The nucleotide sequences of 16S rDNA fragments were 99-100% similar to mainly Helicobacter sp. 'liver' and H. ganmani. PCR-products similar to H. canis and H. bilis were also found. The 16S rDNAs of control specimens showed similarity to Helicobacter sp. 'liver'. In the H. ganmani-specific PCR analysis 19 patients, but none of the controls, were positive. CONCLUSIONS: Amplified Helicobacter 16S rDNAs were related to Helicobacter sp. 'liver' or H. ganmani in liver biopsy specimens of pediatric patients. The possible significance of Helicobacter species in pediatric liver diseases needs to be evaluated further in prospective studies.     

Detection of Helicobacter colonization of the murine lower bowel by genus-specific PCR-denaturing gradient gel electrophoresis

Helicobacter genus-specific PCR and denaturing gradient gel electrophoresis can detect and speciate the helicobacters that colonize the lower bowel of laboratory mice. The method's sensitivity is comparable to that of species-specific PCR and may detect unnamed Helicobacter species. This approach should prove useful for commercial and research murine facilities.     

Natural colonization with Helicobacter species and the development of inflammatory bowel disease in interleukin-10-deficient mice

BACKGROUND: The interleukin-10-deficient (IL-10-/-) mice maintained in specific-pathogen-free (SPF) conditions develop typhlocolitis when experimentally infected with Helicobacter species. However, there is limited information regarding the role of Helicobacter species that naturally colonize IL-10-/- mice in typhlocolitis development. The aim of this study was to examine in SPF IL-10-/- mice the association between natural colonization specific Helicobacter species and typhlocolitis development. MATERIAL AND METHODS: Cecum and proximal colon from 72 C57BL/6 x 129/Ola IL-10-/- mice (8-20 weeks old) were removed for DNA extraction and histologic evaluation. Genus-specific polymerase chain reaction- denaturing gradient gel electrophoresis (PCR-DGGE) and species-specific PCR were used to detect Helicobacter species. Mice were grouped by age, sex, and Helicobacter colonization status, and their histologic scores were compared. The development of clinical typhlocolitis was observed in a further 12 mice. RESULTS: Species-specific PCR showed that mice were colonized with Helicobacter ganmani and/or Helicobacter hepaticus. The PCR-DGGE detected H. ganmani, H. hepaticus and an H. ganmani-like organism. The histologic scores in mice colonized with H. hepaticus were significantly higher than that in mice colonized with H. ganmani. Male mice showed significantly higher histologic scores than female mice. Four of the 12 mice developed clinical typhlocolitis in 38 weeks. CONCLUSION: Natural colonization with different Helicobacter species was found in IL-10-/- mice within the same breeding colony. The severity of typhlocolitis differed according to the colonizing Helicobacter species. Furthermore, the rate of typhlocolitis development in IL-10-/- mice naturally colonized with Helicobacter species was significantly slower than that reported in experimentally infected mice.     

Detection, isolation, and characterization of helicobacter species from the gastrointestinal tract of the brushtail possum

The presence of Helicobacter species in Australian marsupials was examined systematically using microscopy, culture, and PCR in different regions of the gastrointestinal tract (GIT) and in the liver of brushtail possums (BTPs) (Trichosurus vulpecula), a common Australian marsupial that feeds on eucalyptus leaves. The spatial distribution of Helicobacter species in the GIT sections also was examined microscopically in silver-stained sections and by fluorescent in situ hybridization (FISH) using a Helicobacter genus-specific probe. Helicobacter species were found colonizing the lower bowel of all BTPs studied. Good agreement was observed between the detection of Helicobacter species using culture and PCR, which was supported by the microscopic examination of silver-stained sections and FISH. The lower bowel of BTPs were colonized by one to three morphologically different (a comma-shaped species with no apparent flagella, a fusiform-shaped species entwined with periplasmic fibers and a bipolar sheathed flagella, and an S-shaped species with bipolar sheathed flagella) and potentially novel Helicobacter species, as well as in one case with a potentially novel Campylobacter species, which was a tightly coiled rod with bipolar unsheathed flagella. The isolation and characterization of these Helicobacter species in BTPs provides important information regarding the specific natural niche of these bacteria and their corelationship within their host, and it increases our understanding of the ecology of Helicobacter species.     

Visualization of Helicobacter species within the murine cecal mucosa using specific fluorescence in situ hybridization

BACKGROUND: Members of the genus Helicobacter have been associated with colitis development in a number of immunodeficient animal models. While it is known that these organisms can initiate colitis development, the location and spatial distribution of these bacteria within the intestinal tract is currently unknown. In this study, we developed and optimized fluorescence in situ hybridization (FISH) probes specifically for Helicobacter species. MATERIALS AND METHODS: Based on 16S-RNA gene alignments, two probes specific for the entire family Helicobacteraceae and two probes specific for Helicobacter ganmani and Helicobacter hepaticus were designed. Evaluation of these probes was determined using ATCC reference strains and cecum samples from ten IL-10 knockout mice. The presence of Helicobacter species was determined using FISH and verified using PCR-DGGE and microscopic examination of silver stained sections. RESULTS: Analysis of the ATCC reference strains revealed that the probes HEL274/HEL717 were specific for the family Helicobacteraceae, while HEP642 was specific for H. hepaticus and GAN1237 for H. ganmani. Using these probes, a pattern of spatial localization of the two different Helicobacter species was observed in the cecum tissues of IL-10 knockout mice. This consistently showed that H. ganmani was localized to the lower regions and H. hepaticus to the mid-upper regions of the crypts. CONCLUSION: We have developed FISH probes specific for the family Helicobacteraceae as well as two individual Helicobacter species. This study will allow the future use of the FISH to better understand host-pathogen interactions in vitro.     

Detection of Helicobacter pylori in bile of cats

Lymphocytic cholangitis (LC) in cats is a biliary disease of unknown etiology. Helicobacter spp. were recently implicated in human primary sclerosing cholangitis (PSC) and primary biliary cirrhosis (PBC). Because of the similarities between PSC/PBC with LC, we hypothesized that Helicobacter spp. are involved in feline LC. A PCR with Helicobacter genus-specific 16S rRNA primers was performed on DNA isolated from feline bile samples. Four of the 15 (26%) LC samples were positive, whereas only 8/51 (16%) of non-LC samples were PCR positive (p=0.44). Sequence analysis of the amplicons revealed a 100% identity with the Helicobacter pylori specific DNA fragments. Our data suggest an etiological role of H. pylori in feline LC and that cats are a potential zoonotic reservoir.     

Detection of Candida species by polymerase chain reaction (PCR) in blood samples of experimentally infected mice and patients with suspected candidemia

In this study, we have established and evaluated a genus-specific polymerase chain reaction (PCR) and species-specific nested PCRs for the detection of Candida species in blood samples of neutropenic mice and patients suspected of candidemia. DNA segments of the gene encoding cytochrome P450 L1A1 were targeted for amplification by using genus and species-specific primers. As compared to the genus-specific PCR, the species-specific nested PCRs improved the sensitivity by 10 times with the detection limit < 10 yeast cells. Of the 18 blood samples tested daily over a period of 8 days following Candida albicans infection in neutropenic mice, four samples were positive by genus-specific PCR and 11 were positive by species-specific nested PCR. The PCR results were correlated with culture findings obtained on blood samples. Two of the three blood culture-positive samples were positive by genus-specific PCR and all the three with species-specific nested PCR. Among 15 mice, which were negative by blood culture but had C. albicans isolated from visceral organs, 2 and 8 mice yielded positive results by genus-specific PCR and species-specific nested PCR, respectively. Consistent with the results of the animal study, species-specific nested PCR yielded much higher positivity as compared to culture (52.2% versus 21.2%) in patients suspected for candidemia. Moreover, 8 specimens which were negative for Candida by genus-specific PCR became positive by species-specific nested PCR. No correlation was apparent between PCR positivity and Candida antigen titers. The results suggest that nested PCR is a sensitive technique for the detection of Candida species from blood samples, and thus it may have application in the diagnosis of suspected cases of candidemia and candidiasis.     

Helicobacter species colonizing pig stomach: molecular characterization and determination of prevalence.

The infection rate of 60 pigs (10 pigs from each of six farms) by Helicobacter species was studied by two techniques. Histological examination of the cardiac area of the stomach yielded a 58% positive result versus an 80% positive result by PCR with genus-specific primers. Eighty percent of the 16S rRNA gene was amplified, classified in four groups by PCR-restriction fragment length polymorphism, and sequenced. Isolates from all farms except one (farm C) were identified as Helicobacter heilmannii type 1, while those from farm C were identified as H. heilmannii type 2. Attempts to culture this organism in vitro failed. Helicobacter pylori was not found in these animals.     

PCR-denaturing gradient gel electrophoresis and two feces antigen tests for detection of Helicobacter pylori in mice

PCR-denaturing Gradient Gel Electrophoresis (PCR-DGGE), a method suitable for the detection of microbial species in complex ecosystems, was evaluated for the detection and identification of Helicobacter spp. in feces and stomach tissue of mice. Two commercially available stool antigen tests for clinical diagnostics in humans were also evaluated in the C57Bl/6 mouse model of H. pylori infection. PCR-DGGE detected only Helicobacter ganmani in feces from H. pylori-infected as well as control animals, whereas in stomach specimens it demonstrated the presence of H. pylori in challenged and H. ganmani in control animals. Hence, the method detected DNA only of the predominant Helicobacter spp., which was also shown in cell dilution experiments. The Amplified IDEIA Hp StAR feces antigen test detected H. pylori in feces from all infected animals and generated no false-positive results, whereas the Premier Platinum HpSA-test also detected H. pylori in all infected animals but generated false-positive or equivocal results in 50% of the control animals. Premier Platinum HpSA, as opposed to Hp StAR, cross-reacted with non-pylori Helicobacter spp. in vitro.     

Change in the community structure of ammonia-oxidizing bacteria in activated sludge during selective incubation for MPN determination

We investigated the changes in the community structure of ammonia-oxidizing bacteria (AOB) in activated sludge during incubation of the sludge in a medium selective for AOB. The number of AOB present in the activated sludge sample was enumerated by the most-probable-number (MPN) method. Both the activated sludge sample and the incubated samples for MPN determination were analyzed by polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE). Universal PCR-DGGE indicated that even after 40-d incubation in a medium selected for AOB, the MPN samples were predominantly composed of heterotrophic bacteria and not AOB. Denitrification by heterotrophic bacteria might lead to the underestimation of the MPN count of AOB. Not dominated in whole bacteria, one species of AOB was detected in both original activated sludge and samples after MPN incubation by PCR-DGGE targeting AOB. Furthermore, two new species of AOB were detected only after incubation. Therefore, the community structure of AOB in the MPN samples partially resembled that in the original activated sludge.     

Detection of gastric Helicobacter species in free-ranging lynx (Lynx lynx) and red foxes (Vulpes vulpes) in Sweden

Specimens of gastric mucosa and liver of 25 free-ranging Eurasian lynx (Lynx lynx), and four red foxes (Vulpes vulpes) shot in Sweden during 1999-2000, were investigated for the presence of Helicobacter species. Histopathology, bacteriologic culture and urease test, Helicobacter genus-specific 16S rDNA PCR analysis, and DNA sequence analysis were applied. Numerous Helicobacter-like organisms were observed histologically in the gastric mucosa of one fox. Helicobacter spp. were detected in the stomach by PCR analysis in 17 (68%) of the lynx and in three (75%) of the foxes. Seven of the positive lynx were also positive in the urease test. PCR fragments, amplified from lynx and foxes, were sequenced and compared with those of known Helicobacter species. PCR products from lynx were closely related (>or=98% homology) to H. heilmannii, and PCR fragments from foxes demonstrated close homology to H. heilmannii and H. salomonis. No Helicobacter spp. or Helicobacter-like organisms could be cultured. The PCR analysis of the liver was negative for all animals. The pathologic significance of the presence of Helicobacter spp. in the stomach of free-ranging lynx and foxes remains uncertain.     

PCR-DGGE Comparison of Bacterial Community Structure in Fresh and Archived Soils Sampled along a Chihuahuan Desert Elevational Gradient

The polymerase chain reaction coupled with denaturing gradient gel electrophoresis (PCR-DGGE) has been used widely to determine species richness and structure of microbial communities in a variety of environments. Researchers commonly archive soil samples after routine chemical or microbial analyses, and applying PCR-DGGE technology to these historical samples offers evaluation of long-term patterns in bacterial species richness and community structure that was not available with previous technology. However, use of PCR-DGGE to analyze microbial communities of archived soils has been largely unexplored. To evaluate the stability of DGGE patterns in archived soils in comparison with fresh soils, fresh and archived soils from five sites along an elevational gradient in the Chihuahuan Desert were compared using PCR-DGGE of 16S rDNA. DNA from all archived samples was extracted reliably, but DNA in archived soils collected from a closed-canopy oak forest site could not be amplified. DNA extraction yields were lower for most archived soils, but minimal changes in bacterial species richness and structure due to archiving were noted in bacterial community profiles from four sites. Use of archived soils to determine long-term changes in bacterial community structure via PCR-DGGE appears to be a viable option for addressing microbial community dynamics for particular ecosystems or landscapes.     

Detection of filamentous genus Gordonia in foam samples using genus-specific primers combined with PCR--denaturing gradient gel electrophoresis analysis

A nested-PCR amplification combined with denaturing gradient gel electrophoresis (PCR-DGGE) approach was used to detect and identify Gordonia populations from wastewater treatment plant foam samples. The PCR-amplified region (position 722-1119) by specifically designed primers G699F and G1096R covered the hypervariable region of the Gordonia 16S rRNA gene sequence. This approach successfully distinguished Gordonia species to the interspecies level. The differential ability of PCR-DGGE analysis was effectively used to separate 12 Gordonia species belonging to different 16S rRNA gene-based phylogenetic lineages into 8 groups. Based on this method, the minimum limit of Gordonia detection was 5 x 10(4) CFU.g -1 in the seeded soil samples. The PCR-DGGE bands obtained were excised and identified by sequence analysis. Gordonia polyisoprenivorans, Gordonia amicalis, DGGE type II Gordonia species, and an uncertain Gordonia species dominated the activated sludge foam samples. Results of this study indicate that the detection and analyses of genus Gordonia within a complex microbial community could be accomplished using the PCR-DGGE approach to a larger extent, with certain limitations. Detection of diverse Gordonia populations in foam samples from wastewater treatment plants revealed the significant role of Gordonia in biological foaming during wastewater treatment. The nested-PCR amplification and DGGE can be used as a diagnostic tool for the early detection of foaming incidents in wastewater treatment plants using Gordonia as indicator organism.     

Development of a microelectronic chip array for high-throughput genotyping of Helicobacter species and screening for antimicrobial resistance

A microelectronic array assay was developed to specifically genotype Helicobacter pylori versus Helicobacter heilmannii and to determine antimicrobial resistance. Helicobacter 16S rRNA and 23S rRNA genes were specifically generated with Helicobacter genus-specific primers, respectively. The single-nucleotide polymorphisms (SNPs) in 16S rRNA, 268T specific in the H. pylori sequence, and 263A specific in H. heilmannii were used as molecular markers for identification of H. pylori and H. heilmannii, respectively. A triple-base-pair resistant mutation, AGA965-967TTC in 16S rRNA, is known to be responsible for H. pylori tetracycline resistance and was detected to identify resistant strains. H. pylori macrolide resistance was determined by the identification of 3 defined mutations in the 23S rRNA gene using the same method. The assay could be directly used to detect H. pylori in feces. The assay performs multiple determinations, including identification of Helicobacter species and antibiotic resistances, on the same microelectronic platform and is highly amenable to the development of other DNA-based assays.