Helicobacter spp. and liver diseases

To test whether Helicobacter species play a role in the enhancement of liver necro-inflammation and fibrosis and in the development of hepatocellular carcinoma (HCC), we sought DNA sequences of Helicobacter species in liver specimens from patients with viral-related chronic hepatitis, HCC or metastatic liver carcinoma. We enrolled 28 consecutive patients with ultrasound evidence of hepatic nodule(s) on their first liver biopsy: 21 had histological evidence of HCC (Group I) and 7 of metastatic liver carcinoma (Group II). In the same period we observed 27 consecutive patients with chronic hepatitis on their first liver biopsy (Group III). Helicobacter sequences were sought by PCR using primers for the 16S rDNA of Helicobacter spp, designed to amplify a 400 base-pair fragment, and detected by 2% agarose gel and hybridization with a specific biotinylated probe. We used, as positive controls for the DNA extraction from liver tissue, hepatic biopsy sections in which HBV infection was confirmed by HBcAg positivity and in which we amplified HBV-DNA by specific primers; positive controls for the amplification of Helicobacter spp were obtained from gastric biopsy sections in which Helicobacter pylori infection was confirmed by biochemical and histochemical tests. HBV-DNA was found in all five HBcAg positive liver biopsies. Helicobacter spp 16S rDNA was detected in all five biopsy specimens of gastric mucosa and in none of liver specimens from patients in any group. Our data suggest that Helicobacter species were not involved in the pathogenesis of virus-related HCC, chronic hepatitis or liver carcinoma metastasis.     

Potential role of Helicobacter pylori in hepatocarcinogenesis

Helicobacter species can induce carcinoma in the liver of certain mice. Furthermore, Helicobacter pylori (H. pylori) exhibits hepatotoxicity in vitro. These reports indicate that H. pylori may play a role in hepatocarcinogenesis. The aim of this study was to assess the presence of H. pylori in human hepatocellular carcinoma (HCC) to determine if H. pylori may affect the development of this disease. Liver specimens from 15 HCC patients dissected into tumor and non-tumor tissues were examined for H. pylori by PCR using two sets of primers for 16S rRNA and urease B. DNA sequencing analysis was performed to confirm that PCR products with 16S rRNA primers were derived from H. pylori DNA. The specimens were also examined for H. pylori by immunohistochemistry using anti-H. pylori antibody. H. pylori was found in 13 of 15 tumor tissues, not in the non-tumor tissues. By contrast, Escherichia coli and Bacteroides fragilis, frequent colonizers of gut, were not detected by PCR in the HCC tumors. Ten cirrhotic liver tissue specimens and seven normal liver tissue specimens were also negative for H. pylori DNA by PCR. The nucleotide sequence of the amplified fragment shared 100% identity with the 16S rRNA gene of H. pylori. H. pylori was also detected in HCC tissue by immunohistochemical analysis. The presence of H. pylori in human HCC tissue was demonstrated by PCR and immunohistochemical analysis. These findings suggest that H. pylori might contribute to the development of HCC. Further study is needed to prove the pathogenetic role of H. pylori in the development of human HCC.     

Procuration and identification of bacteria in paraffin-embedded liver tissues of hepatocellular carcinoma by laser-assisted microdissection technique

This study was aimed at procuring directly and identifying the bacteria which had been found in paraffin-embedded liver tissues of hepatocellular carcinoma (HCC) patients. In our previous studies, Helicobacter spp. had been detected by polymerase chain reaction (PCR) and observed by histology in the liver tissues of HCC patients but had never been cultured successfully. To obtain and identify the uncultured bacteria, laser microdissection and pressure catapulting (LMPC) techniques were applied. Following microdissection from the liver tissue sections, these bacteria were examined by PCR using Helicobacter genus-specific 16S rRNA primers and sequence analysis. Amplified products of 16S rRNA were positive in all six microdissected samples with bacteria, and showed 99%-100% similarity with Helicobacter pylori by sequence analysis. Another H. pylori-specific 26 kDa gene (encoding one 26 kDa protein as H. pylori-specific antigen) was also tested by PCR. Four of six samples were positive. Therefore, Helicobacter spp. detected by PCR in the liver tissues of HCC patients in our previous studies are actually the bacteria observed by histology and identified as H. pylori by further sequence analysis. The laser-assisted microdissection technique can be extensively applied for identification of bacteria in tissue samples in bacteriology research.     

Helicobacter marmotae sp. nov. isolated from livers of woodchucks and intestines of cats

Woodchucks (Marmota monax) have a high incidence of hepatocellular carcinoma (HCC) associated with chronic infection with woodchuck hepatitis virus (WHV) and serve as a model of hepatitis B virus-associated HCC in humans. Helicobacter hepaticus, an enterohepatic helicobacter in mice, is known to cause hepatocellular adenomas and carcinomas in susceptible mouse strains. In long-term chemical bioassays conducted with B6C3F(1) mice, H. hepaticus has been regarded as a confounding factor because of its tumor-promoting activity. In order to determine if woodchucks harbor a Helicobacter sp. that might play a role in potentiating hepatic inflammation or neoplasia, a study was undertaken to determine whether woodchucks' livers were infected with a Helicobacter sp. Frozen liver samples from 20 (17 WHV-infected and 3 noninfected) woodchucks, 10 with WHV-associated hepatic tumors and 10 without tumors, were cultured by microaerobic techniques and analyzed by using genus- and species-specific helicobacter PCR primers. A 1,200-bp Helicobacter sp.-specific sequence was amplified from 14 liver samples. Southern hybridization confirmed the specific identity of the PCR products. Nine of the 10 livers with tumors had positive Helicobacter sp. identified by PCR, whereas 5 of the 10 livers without tumors were positive. By use of 16S rRNA species-specific primers for H. marmotae, two additional liver samples from the nontumor group had positive PCR amplicons confirmed by Southern hybridization. A urease-, catalase-, and oxidase-positive bacterium was isolated from one liver sample from a liver tumor-positive woodchuck. By 16S rRNA analysis and biochemical and phenotypic characteristics, the organism was classified as a novel Helicobacter sp. Subsequently, four additional bacterial strains isolated from feces of cats and characterized by biochemical, phenotypic, and 16S rRNA analysis were determined to be identical to the woodchuck isolate. We propose the name Helicobacter marmotae sp. nov. for these organisms. Further studies are required to ascertain if this novel Helicobacter sp. plays a tumor promotion role in hepadnavirus-associated tumors in woodchucks or causes enterohepatic disease in cats.     

Identification of Helicobacter pylori and other Helicobacter species by PCR, hybridization, and partial DNA sequencing in human liver samples from patients with primary sclerosing cholangitis or primary biliary cirrhosis

Helicobacter pylori was identified in human liver tissue by PCR, hybridization, and partial DNA sequencing. Liver biopsies were obtained from patients with primary sclerosing cholangitis (n = 12), primary biliary cirrhosis (n = 12), and noncholestatic liver cirrhosis (n = 13) and (as controls) normal livers (n = 10). PCR analyses were carried out using primers for the Helicobacter genus, Helicobacter pylori (the gene encoding a species-specific 26-kDa protein and the 16S rRNA), Helicobacter bilis, Helicobacter pullorum, and Helicobacter hepaticus. Samples from patients with primary biliary cirrhosis and primary sclerosing cholangitis (11 and 9 samples, respectively) were positive by PCR with Helicobacter genus-specific primers. Of these 20 samples, 8 were positive with the 16S rRNA primer and 9 were positive with the 26-kDa protein primer of H. pylori. These nine latter samples were also positive by Southern blot hybridization for the amplified 26-kDa fragment, and four of those were verified to be H. pylori by partial 16S rDNA sequencing. None of the samples reacted with primers for H. bilis, H. pullorum, or H. hepaticus. None of the normal livers had positive results in the Helicobacter genus PCR assay, and only one patient in the noncholestatic liver cirrhosis group, a young boy who at reexamination showed histological features suggesting primary sclerosing cholangitis, had a positive result in the same assay. Helicobacter positivity was thus significantly more common in patients with cholestatic diseases (20 of 24) than in patients with noncholestatic diseases and normal controls (1 of 23) (P = <0.00001). Patients positive for Helicobacter genus had significantly higher values of alkaline phosphatases and prothrombin complex than Helicobacter-negative patients (P = 0.0001 and P = 0.0003, respectively). Among primary sclerosing cholangitis patients, Helicobacter genus PCR positivity was weakly associated with ulcerative colitis (P = 0.05). Significant differences related to blood group or HLA status were not found.     

Helicobacter pylori: association with gall bladder disorders in Pakistan

Helicobacter species colonise the biliary tract and therefore this study explores the relationship between of Helicobacter pylori and cholecystitis. Bile and gall bladder tissue samples were obtained from 144 patients who underwent cholecystectomy. Of these, 89 had chronic cholecystitis with cholelithiasis, 44 had gall bladder carcinoma and 11 had gall bladder polyps. Histopathology examination included special staining and immunohistochemistry (IHC), while Helicobacter species (H. pylori, H. bilis and H. hepaticus) were detected by the polymerase chain reaction (PCR). Sequencing and BLAST query of PCR products was undertaken and samples were considered to contain H. pylori if both PCR and IHC were positive. Immunohistochemistry for H. pylori was positive in 22 (25%) cases compared to five (9%) in the control group (P=0.02). Testing (PCR) for 16S rDNA was positive in 23 (26%) cases compared to six (11%) controls (P=0.03). Negative PCR results were obtained for H. bilis and H. hepaticus. Twenty-four (89%) were positive by both 16S rDNA PCR and IHC for H. pylori (P<0.001). Both PCR for 16S rDNA and IHC were positive in 21 (24%) cases compared to five (9%) controls (P=0.03). Sequencing of 16S rRNA and glmM PCR products were consistent with H. pylori. In conclusion, H. pylori DNA was demonstrated in cases of chronic cholecystitis and gall bladder carcinoma associated with cholelithiasis, but this association requires further study.     

Development of two PCR-based techniques for detecting helical and coccoid forms of Helicobacter pylori

The primary mode of transmission of Helicobacter pylori, a human pathogen carried by more than half the population worldwide, is still unresolved. Some epidemiological data suggest water as a possible transmission route. H. pylori in the environment transforms into a nonculturable, coccoid form, which frequently results in the failure to detect this bacterium in environmental samples by conventional culture techniques. To overcome limitations associated with culturing, molecular approaches based on DNA amplification by PCR have been developed and used for the detection of H. pylori in clinical and environmental samples. Our results showed the glmM gene as the most promising target for detection of H. pylori by PCR amplification. Under optimal amplification conditions, glmM-specific primers generated PCR-amplified products that were specific for H. pylori and some other Helicobacter species. Genome sequence analysis revealed the existence of a conserved region linked to a hypervariable region upstream of the 16S rRNA gene of H. pylori. Selective PCR primer sets targeting this sequence were evaluated for the specific detection of H. pylori. One primer set, Cluster2 and B1J99, were shown to be highly specific for H. pylori strains and did not produce any PCR products when other Helicobacter species and other bacterial species were analyzed. In tests with 32 strains of H. pylori, 6 strains of other Helicobacter species, 8 strains of Campylobacter jejuni, and 21 strains belonging to different genera, the primers for glmM were selective for the Helicobacter genus and the primers containing the region flanking the 16S rRNA gene were selective for H. pylori species only. The combination of two sensitive PCR-based methods, one targeting the glmM gene and the other targeting a hypervariable flanking region upstream of the 16S rRNA gene, are complementary to each other. Whereas the glmM-specific primers provide a rapid, sensitive presumptive assay for the presence of H. pylori and closely related Helicobacter spp., the primers for sequences flanking the 16S rRNA gene can confirm the presence of H. pylori and locate the potential source of this bacterium.     

Comparison of four different primer sets for detection of Helicobacter pylori in gastric biopsies and oral samples by using real-time PCR

AIMS: The presence of Helicobacter pylori. in the oral cavity remains controversial and the most appropriate method for detection of oral H. pylori has yet to be established. The aim of the present study was to compare four different primer sets on the detection of H. pylori in gastric biopsies and oral samples using real-time PCR. MATERIAL AND METHODS: Gastric biopsy and oral samples were collected from eight patients with gastric symptoms. DNA from clinical samples was extracted and analyzed for the presence of H. pylori by real-time PCR (LightCycler 2.0) using four pairs of primers which targeted 16S rRNA (16S rRNA#295; 16S rRNA#120) or glmM (glmM#294; glmM#722) DNA genes. Three H. pylori strains and three clinical isolates served as reference. The specificities of the four primer pairs were examined for seven oral microorganisms and two Helicobacter non-pylori species. RESULTS: Primer pair 16S rRNA#120 showed an acceptable specificity and a high sensitivity. Primer pairs glmM#294 and glmM#722 demonstrated a high specificity but a low sensitivity and primer pair 16S rRNA#295 demonstrated a poor specificity but acceptable sensitivity. Four H. pylori positive gastric biopsies were demonstrated by culture, histology and real-time PCR with primer pairs 16S rRNA#295 or 16S rRNA#120. No H. pylori was detected in oral samples, either by culture or by real-time PCR. CONCLUSION: Of the four different primer pairs examined, 16S rRNA#120 was the most appropriate to detect H. pylori in clinical samples using real-time PCR.     

Molecular evidence of Helicobacter cinaedi organisms in human gastric biopsy specimens

One hundred twenty-six urease-negative gastric biopsy specimens were evaluated for the presence of Helicobacter genus-specific 16S ribosomal DNA (rDNA) and H. pylori-specific glmM DNA sequences by PCR. The species specificity of the glmM PCR assay was demonstrated, as H. pylori was the only Helicobacter species that yielded the expected glmM amplicon. Most urease-negative specimens (118 of 126 specimens) lacked Helicobacter DNA. However, 8 of 126 urease-negative specimens contained Helicobacter 16S rDNA. In order to identify the Helicobacter species present in urease-negative gastric biopsy specimens, 16S rDNA amplicons were cloned and sequenced. Sequence comparisons were performed by analyses of the sequences in public sequence databases. Two samples contained 16S rDNA that was identified as H. cinaedi with 100% identity and that spanned approximately 400 bp (398 and 398 bp, respectively). In contrast, multiple differences (97% identity; 390 of 398 bp) were observed with H. pylori 16S rDNA in this region. This finding was verified by sequencing an overlapping 537-bp fragment within the 5' portion of 16S rDNA. Although the clinical findings were consistent with H. pylori infection (e.g., duodenal ulcer disease), rapid urease testing and DNA sequence analyses suggested the presence of H. cinaedi organisms and the absence of H. pylori in two human antral biopsy specimens. This study represents the first report of an enteric urease-negative helicobacter in the human stomach. Although these organisms were previously associated with extragastric infections, the roles of these organisms in the pathogenesis of chronic gastritis or peptic ulcer disease remain unclear.     

Helicobacter infection and cirrhosis in hepatitis C virus carriage: is it an innocent bystander or a troublemaker?

Since it has been shown that Helicobacter hepaticus causes both chronic hepatitis and hepatocellular carcinoma (HCC) in mice, it is suggested that differences in the progression of chronic hepatitis C may be due to a cofactor stemming from co-infection by bacteria, especially Helicobacter pylori, and/or other Helicobacter species. An assessment was made of the prevalence of H. pylori infection in HCV-positive cirrhotic patients. The presence of Helicobacter species (spp). was evaluated in resected liver tissue from HCC patients. Serum anti-H. pylori IgG antibodies were determined in 70 males with a clinical and/or histological diagnosis of cirrhosis and HCV infection and in 310 age-matched male blood donors. The prevalences of H. pylori antibody were 77% (54/70) and 59% (183/310) (P 0.004). Primers identifying 26 Helicobacter species were used to determine the presence of the genomic 16S rRNA of this genus in liver tissue resected from 25 cirrhotic HCC patients. Genomic sequences corresponding to H. pylori and H. pullorum were identified in 23 of these 25 livers. Together, these findings support the proposal that H. pylori is implicated in the pathogenesis and progression of cirrhosis, particularly in HCV-infected individuals. Involvement of Helicobacter spp. in HCC also seems highly possible.     

Molecular resistance testing of Helicobacter pylori in gastric biopsies

OBJECTIVE: To evaluate simultaneous diagnosis of infection and molecular resistance testing of Helicobacter pylori. METHODS: Gastric biopsies were obtained from 26 rapid urease-positive and 51 rapid urease-negative test kits used to diagnose H pylori infection. Following glass bead-assisted DNA isolation, amplification of H pylori 16S ribosomal DNA (rDNA), glmM, and 23S rDNA target genes was performed. RESULTS: Helicobacter pylori DNA was successfully amplified from 100% (26/26) of urease-positive and 3.9% (2/51) of urease-negative gastric biopsies. Subsequent restriction enzyme-mediated digestion of 23S rDNA amplification products revealed that 17% (4/24) of urease-positive and H pylori DNA-positive biopsy specimens contained point mutations (A2142G or A2143G) associated with clarithromycin resistance. Helicobacter pylori DNA from gastric biopsies was successfully amplified 8 weeks following rapid urease testing. CONCLUSION: Helicobacter pylori genotyping may be used to detect macrolide-resistant H pylori in individuals prior to initiation of therapy or in patients refractory to anti-H pylori therapy. Two urease-negative specimens yielded Helicobacter DNA distinct from that of H pylori and indicated the need for further investigations of Helicobacter species present in the human stomach.     

Comparison of diagnostic techniques for Helicobacter cetorum infection in wild Atlantic bottlenose dolphins (Tursiops truncatus)

Helicobacter cetorum sp. nov. has been cultured from the stomach of Atlantic white-sided dolphins (Lagenorhynchus acutus) and the feces of Pacific white-sided (L. obliquidens) and Atlantic bottlenose (Tursiops truncatus) dolphins and a beluga whale (Delphinapterus leucas). H. cetorum has high homology to Helicobacter pylori as shown by 16S rRNA sequencing, and H. cetorum infection has been associated with gastritis and clinical signs in cetaceans. Because the prevalence of H. cetorum in wild populations is unknown, minimally invasive techniques for detecting H. cetorum were compared for 20 wild bottlenose dolphins sampled as part of a long-term health study. Fecal samples were tested for helicobacter by culture, Southern blotting, and PCR using genus-specific and H. cetorum-specific primers. An enzyme-linked immunosorbent assay (ELISA) was developed to measure H. cetorum immunoglobulin G (IgG). H. cetorum was cultured from 4 of 20 fecal samples, 7 samples were positive using Helicobacter sp. PCR, and 8 samples were positive for H. cetorum using species-specific primers. Two additional fecal samples were positive by Helicobacter sp. Southern blotting, suggesting infection with another helicobacter. All 20 sera contained high levels of IgG antibodies to H. cetorum that were significantly lowered by preabsorption of the sera with whole-cell suspensions of H. cetorum (P < 0.02). Until the specificity of the serum ELISA can be determined by testing sera from dolphins confirmed to be uninfected, PCR and Southern blot screenings of feces are the most sensitive techniques for detection of H. cetorum, and results indicate there is at least a 50% prevalence of H. cetorum infection in these dolphins.     

PCR detection of colonization by Helicobacter pylori in conventional, euthymic mice based on the 16S ribosomal gene sequence.

Many animal models of Helicobacter infection have been described, including infection in rhesus monkeys, ferrets, gnotobiotic piglets, and mice. These animal models utilize a combination of detection methods, including culture, urease testing, and histopathology, all of which may be unreliable, insensitive, or labor-intensive. Development of new animal models of Helicobacter pylori requires new methods of detection with increased sensitivity and specificity. We have developed sensitive and specific PCR primers based on the 16S ribosomal gene sequence of H. pylori. The primers detected single-copy 16S DNA representing 0.2 cell of pure H. pylori (2 cells in the presence of mouse stomach mucosal DNA) and did not cross-react with closely related bacteria. We were able to detect colonization by H. pylori in conventional, euthymic, outbred mice up to 4 weeks postinoculation with a high percentage of isolates tested. One isolate of H. pylori was detected by PCR in 100% of the mice at 6 months and 60% of the mice 1 year after inoculation. Approximately 10(3) to 10(4) H. pylori cells per stomach were detected by utilizing this PCR methodology semiquantitatively. These primers and PCR methodology have facilitated detection of H. pylori colonization in conventional, euthymic mice, colonization which may not have been detectable by other methods.     

Direct polymerase chain reaction test for detection of Helicobacter pylori in humans and animals.

We designed a polymerase chain reaction (PCR) for amplifying the Helicobacter pylori gene encoding 16S rRNA. Primers for the specific detection of H. pylori were designed for areas of the 16S rRNA gene in which there is the least sequence homology between H. pylori and its closest relatives. The specificity of detection was confirmed by ensuring that the primers did not amplify DNA extracts from the campylobacters H. cinaedi, H. mustelae, and Wolinella succinogenes, which are the closest relatives of H. pylori, as determined by 16S rRNA sequencing. Serial dilution experiments revealed the detection of as little as 0.1 pg of DNA by PCR and 0.01 pg by nested PCR. H. pylori DNA was detected successfully in clinical paraffin-embedded and fresh gastric biopsy specimens from patients positive for the bacterium and also in fecal suspensions seeded with the organism. The DNA from the nonculturable coccoid form of H. pylori was also identified by the primers. Universal primers designed for highly conserved areas on the 16S rRNA gene enabled large amplification products to be produced for direct sequencing analysis. Gastric bacteria resembling H. pylori have been isolated from animals. DNA of these animal gastric bacteria amplified with H. pylori-specific primers yielded PCR products identical to those from human isolates of H. pylori, as confirmed by the use of a 20-base radiolabelled probe complementary to an internal sequence flanked by the H. pylori-specific primers. The results of PCR amplification and partial 16S rRNA gene sequence analysis strongly support the contention that the gastric organisms previously recovered from a pig, a baboon, and rhesus monkeys are H. pylori.     

Development of epidemiological method for the Helicobacter pylori by polymerase chain reaction.

The polymerase chain reaction was used to develop a method for the detection of Helicobacter pylori, a causative agent of gastritis, as well as for the elucidation of its mode of transmission. A genomic library of Helicobacter pylori DNA in Escherichia coli JM109 was constructed by cloning Hind III-digested DNA fragments into plasmid vector pUC18. The nucleotide sequences from seven recombinant clones were determined and five sets of oligonucleotide primers were synthesized on the basis of the sequences from five clones (B4, B9, B10, C15 and I22). The PCR amplifications with these primers were performed using DNA samples from five strains of Helicobacter pylori, two Campylobacter spp. and eleven species of enteric bacteria. Amplifications of the target DNA fragments in all of 5 strains of Helicobacter pylori were observed from the PCR with primers derived from clone B4, B9, C15 and I22. When the specificity was checked with the DNA samples from 13 other bacteria as template DNA for the PCR, specific amplification that produced the correct size of the target DNA of Helicobacter pylori was shown only in the PCR with primers derived from clone B9 and C15. The detection limit in the PCR amplification, determined by the heat-lysis method, was 500 cells of Helicobacter pylori.     

Genotypic identification of erythromycin-resistant campylobacter isolates as helicobacter species and analysis of resistance mechanism

The correct identification of Campylobacter species remains cumbersome, especially when conventional biochemical tests and antimicrobial susceptibility patterns are used for a phenotypical identification. Correct identification is important for epidemiological purposes and for studying changes in antimicrobial resistance patterns. Six erythromycin-resistant campylobacter strains were investigated by 16S ribosomal DNA (rDNA) sequencing, 23S rDNA sequencing, and restriction fragment length polymorphism analysis of a putative heme-copper oxidase domain described as being specific for thermophilic Campylobacter species. Three erythromycin-resistant isolates from feces of human immunodeficiency virus (HIV)-seropositive patients with diarrhea and one blood isolate of from HIV-seropositive patient with cellulitis were identified by 16S rDNA analysis as Helicobacter cinaedi, whereas 23S rDNA sequencing suggested Wolinella succinogenes. The 16S rDNA sequence data of fecal isolates of two patients with travelers diarrhea revealed Helicobacter pullorum and were also in contrast with 23S rDNA sequencing. Of 4 H. cinaedi isolates, 1 contained the putative heme-copper oxidase gene thought to be specific for thermophilic species. The six erythromycin-resistant Helicobacter species had a similar point mutation A2143G in 23S rDNA resembling the macrolides resistance in Helicobacter pylori. We conclude that 16S rDNA sequencing should be preferred to 23S rDNA analysis and that macrolide-resistant campylobacter strains should be investigated by this approach for a correct identification.     

Comparison of Helicobacter spp. in Cheetahs (Acinonyx jubatus) with and without gastritis

Chronic gastritis causes significant morbidity and mortality in captive cheetahs but is rare in wild cheetahs despite colonization by abundant spiral bacteria. This research aimed to identify the Helicobacter species that were associated with gastritis in captive cheetahs but are apparently commensal in wild cheetahs. Helicobacter species were characterized by PCR amplification and sequencing of the 16S rRNA, urease, and cagA genes and by transmission electron microscopy of frozen or formalin-fixed paraffin-embedded gastric samples from 33 cheetahs infected with Helicobacter organisms (10 wild without gastritis and 23 captive with gastritis). Samples were screened for mixed infections by denaturant gel gradient electrophoresis of the 16S rRNA gene and by transmission electron microscopy. There was no association between Helicobacter infection and the presence or severity of gastritis. Eight cheetahs had 16S rRNA sequences that were most similar (98 to 99%) to H. pylori. Twenty-five cheetahs had sequences that were most similar (97 to 99%) to "H. heilmannii" or H. felis. No cheetahs had mixed infections. The ultrastructural morphology of all bacteria was most consistent with "H. heilmannii," even when 16S rRNA sequences were H. pylori-like. The urease gene from H. pylori-like bacteria could not be amplified with primers for either "H. heilmannii" or H. pylori urease, suggesting that this bacteria is neither H. pylori nor "H. heilmannii." The cagA gene was not identified in any case. These findings question a direct role for Helicobacter infection in the pathogenesis of gastritis and support the premise that host factors account for the differences in disease between captive and wild cheetah populations.     

Sensitive detection of Helicobacter pylori by using polymerase chain reaction.

A polymerase chain reaction (PCR) for the specific detection of Helicobacter pylori was developed with a single primer pair derived from the nucleotide sequence of the urease A gene of H. pylori. We achieved specific amplification of a 411-bp DNA fragment in H. pylori. After 35 cycles of amplification, the product could be detected by agarose gel electrophoresis and contained conserved single HinfI and AluI restriction sites. This fragment was amplified in all 50 strains of H. pylori tested, but it was not detected in other bacterial species, showing the PCR assay to be 100% specific. PCR DNA amplification was able to detect as few as 10 H. pylori cells. PCR detected H. pylori in 15 of 23 clinical human gastric biopsy samples, whereas culturing and microscopy detected H. pylori in only 7 of the samples found to be positive by PCR. Additional primer pairs based on the urease genes enabled the detection of H. pylori in paraffin-embedded human gastric biopsy samples. The detection of H. pylori by PCR will enable both retrospective and prospective analyses of clinical samples, elucidating the role of this organism in gastroduodenal disease.     

Identification of novel Helicobacter species in pig stomachs by PCR and partial sequencing

Evidence of infection with Helicobacter species in pig stomach was investigated by the use of a PCR with Helicobacter genus-specific primers. Forty pig stomachs, each of four different ulcer lesion grades, 0, 1, 2, and 3 in the pars esophagea area, were collected from a slaughterhouse in Minnesota. Of 160 stomach samples examined, 102 (63.8%) were positive by the PCR assay. The 40 samples each of lesion grades 0, 1, 2, and 3 showed 22.5, 52.5, 85.0, and 95.0% PCR-positive results, respectively. There was a significant trend (P < or = 0.01) in the proportions of PCR-positive cases relative to severity of the lesion. About 80% of the 16S rRNA gene was amplified, and PCR-restriction fragment length polymorphism (RFLP) patterns were analyzed. Of 102 PCR-positive samples, the PCR-RFLP patterns resulted in four different types, 32 samples being classified into type MN 1, 16 samples into type MN 2, 43 samples into type MN 3, and 11 samples into type MN 4. When the sequences of each RFLP type were compared to those reported in databases by using BLAST software, types MN 1, MN 2, MN 3, and MN 4 showed homologies of 97.3, 95.4, 96.7, and 99.5% with the 16S ribosomal DNA of Helicobacter flexispira taxon 3, Helicobacter sp. strains MIT 94-022 and MZ 640285, and Helicobacter suis, respectively. None of the 102 samples positive for the Helicobacter genus were positive with a primer set specific for Helicobacter pylori. Attempts to culture the organisms from selected stomachs in vitro were unsuccessful.     

Detection of Helicobacter DNA in bile from bile duct diseases.

Several species of Helicobacter colonize the hepatobiliary tract of animals and cause hepatobiliary diseases. The aim of this study is to investigate Helicobacter found in the biliary tract diseases of humans. Thirty-two bile samples (15 from bile duct cancer, 6 from pancreatic head cancer, and 11 from intrahepatic duct stone) were obtained by percutaneous transhepatic biliary drainage. Polymerase chain reaction analysis using Helicobacter specific urease A gene and 16S rRNA primers, bile pH measurement, and Helicobacter culture were performed. Helicobacter DNA was detected in 37.5%, and 31.3% by PCR with ureA gene, and 16S rRNA, respectively. The bile pH was not related to the presence of Helicobacter. The cultures were not successful. In conclusion, Helicobacter can be detected in the bile of patients with bile duct diseases. The possibility of pathogenesis of biliary tract diseases in humans by these organisms will be further investigated.