An ultrastructural study of Helicobacter mustelae and evidence of a specific association with gastric mucosa.

The ultrastructure of Helicobacter mustelae, a natural inhabitant of the ferret stomach, has been studied and compared with the human gastroduodenal pathogen H. pylori. H. mustelae is a short, slightly curved rod, 2 microns x 0.5 micron, with four or more sheathed flagella. The most common flagellar configuration is a single flagellum at one terminus, bipolar arrangement at the other end and a lateral flagellum. Dense inclusion bodies were observed below the flagellar insertion sites. It is suggested that this configuration is a specialised adaptation to motility in a viscous environment. On examination of the ferret gastric mucosa, similarities to H. pylori were observed such as adherence to gastric tissue and the formation of adhesion pedestals. However, unlike H. pylori, significant numbers of bacteria were intracellular. Furthermore, a much greater proportion of H. mustelae were attached to the mucosa with few bacteria lying free in the mucus, as is seen with H. pylori. It is concluded that the ferret is an important model for the study of adherence of bacteria to gastric mucosa and their possible role in peptic ulceration.     

Helicobacter mustelae-associated gastric MALT lymphoma in ferrets.

Gastric lymphoma resembling gastric mucosa-associated lymphoid tissue (MALT) lymphoma linked with Helicobacter pylori infection in humans was observed in ferrets infected with H. mustelae. Four ferrets with ante- or postmortem evidence of primary gastric lymphoma were described. Lymphoma was diagnosed in the wall of the lesser curvature of the pyloric antrum, corresponding to the predominant focus of H. mustelae induced gastritis in ferrets. Two ferrets had low-grade small-cell lymphoma and two ferrets had high-grade large-cell lymphoma. Gastric lymphomas demonstrated characteristic lymphoepithelial lesions, and the lymphoid cells were IgG+ in all ferrets. Lymphoma was confirmed by light chain restriction, which contrasted with the 1.2:1 kappa lambda ratio observed in H. mustelae-associated chronic gastritis. H. mustelae infection in ferrets has been used as a model for gastritis, ulcerogenesis, and carcinogenesis. The ferret may provide an attractive model to study pathogenesis and treatment of gastric MALT lymphoma in humans.     

Comparison of isolates of Helicobacter pylori and Helicobacter mustelae.

On the basis of analysis of protein profiles, isolates of Helicobacter pylori and Helicobacter mustelae were less than 40% similar. Cytotoxin produced by H. pylori was not detected in isolates of H. mustelae. Both bacterial species agglutinated human erythrocytes. These results substantiate a taxonomic difference between H. pylori and H. mustelae.     

Helicobacter mustelae and Helicobacter pylori bind to common lipid receptors in vitro.

Helicobacter pylori is a recently recognized human pathogen causing chronic-active gastritis in association with duodenal ulcers and gastric cancer. Helicobacter mustelae is a closely related bacterium with similar biochemical and morphologic characteristics. H. mustelae infection of antral and fundic mucosa in adult ferrets causes chronic gastritis. An essential virulence property of both Helicobacter species is bacterial adhesion to mucosal surfaces. The aim of this study was to determine whether H. mustelae binds to the same lipids shown previously to be receptors for H. pylori adhesion in vitro. By using thin-layer chromatography overlay and a receptor-based enzyme-linked immunosorbent assay, H. mustelae was found to bind the same receptor lipids as H. pylori, namely, phosphatidylethanolamine and gangliotetraosylceramide. In addition, both H. pylori and H. mustelae bound to a deacylplasmalogen phosphatidylethanolamine. In contrast to H. pylori, H. mustelae binding to receptors was unaffected by motility or viability. Murine monoclonal and bovine polyclonal antibodies against exoenzyme S, and exoenzyme S itself (from Pseudomonas aeruginosa), inhibited binding of H. mustelae to phosphatidylethanolamine and gangliotetraosylceramide. These findings show that H. mustelae binds in vitro to the same lipid receptors as H. pylori and suggest that the adhesion of H. mustelae to such species is mediated by preformed, surface-exposed adhesins which include an exoenzyme S-like protein.     

Adherence of isogenic flagellum-negative mutants of Helicobacter pylori and Helicobacter mustelae to human and ferret gastric epithelial cells

Isogenic flagellum-negative mutants of Helicobacter pylori and Helicobacter mustelae were screened for their ability to adhere to primary human and ferret gastric epithelial cells, respectively. We also evaluated the adherence of an H. pylori strain with a mutation in the flbA gene, a homologue of the flbF/lcrD family of genes known to be involved in the regulation of H. pylori flagellar biosynthesis. H. pylori and H. mustelae mutants deficient in production of FlaA or FlaB and mutants deficient in the production of both FlaA and FlaB showed no reduction in adherence to primary human or ferret gastric epithelial cells compared with the wild-type parental strains. However, adherence of the H. pylori flbA mutant to human gastric cells was significantly reduced compared to the adherence of the wild-type strain. These results show that flagella do not play a direct role in promoting adherence of H. pylori or H. mustelae to gastric epithelial cells. However, genes involved in the regulation of H. pylori flagellar biosynthesis may also regulate the production of an adhesin.     

Purification and characterization of Helicobacter mustelae urease.

Helicobacter mustelae is a urease-rich bacterium associated with gastritis in ferrets. The ureases of H. mustelae and Helicobacter pylori, a bacterium implicated in human gastritis, share many characteristics. Helicobacter sp. ureases appear to be unique among bacterial enzymes in exhibiting submillimolar Km values and in being composed of two subunits.     

Rapid isolation of Yersinia spp. from feces.

Direct plating or cold enrichment or both have been used to isolate Yersinia spp. from feces. Freeze-shock double enrichment and KOH treatment have been recommended for recovery of Yersinia enterocolitica from surface waters and food, respectively. These techniques were evaluated as alternatives for rapid recovery of Yersinia spp. from feces. Stool samples were homogenized in buffered saline and autoclaved. Escherichia coli. Klebsiella pneumoniae, and Pseudomonas aeruginosa were each added to the suspension at a final concentration of 1.5 x 10(6) colony-forming units per ml. Yersinia cells were then added to a final concentration of 1.5 x 10(3), 1.5 x 10(4), 1.5 x 10(5), or 1.5 x 10(6) colony-forming units per ml. A total of 21 strains of Y. enterocolitica, 2 of Yersinia kristensenii, and 1 each of Yersinia intermedia and Yersinia fredriksenii were tested. For freeze-shock double enrichment, seeded stool samples were frozen overnight (-70 degrees C), transferred successively to m-tetrathionate broth (6 h. 37 degrees C) and selenite broth (2 h 37 degrees C), and plated on MacConkey, salmonella-shigella, and cellobiose-arginine-lysine agars for quantitation. For KOH treatment, seeded stool samples were mixed with 0.5% KOH at a ratio of 1:2 for 2 min and plated as described above. E. coli, K. pneumoniae, and P. aeruginosa were virtually eliminated after either method was used. All Yersinia strains were recovered after KOH treatment even at the lowest initial concentration (1.5 x 10(3) colony-forming units per ml). However, after freeze-shock double enrichment, not all strains were retrievable, and those isolates which were recovered were grown only from samples containing the highest number of Yersinia strains (1.5 x 10(6) colony-forming units per ml). KOH treatment of stool samples seems to be a viable substitute for more protracted methods of recovering Yersinia spp.     

Protein Hpn: cloning and characterization of a histidine-rich metal-binding polypeptide in Helicobacter pylori and Helicobacter mustelae.

Helicobacter pylori is a human gastrointestinal pathogen involved in gastritis, duodenal ulcers, and gastric neoplasia. This microorganism produces large amounts of a urease which, like all known ureases, has nickel in the active site. We have identified a protein in clinical isolates of H. pylori and an identical protein in the ferret pathogen Helicobacter mustelae that strongly binds Ni2+ and Zn2+. This protein has been named Hpn to emphasize its origins in H. pylori and its affinity for nickel. The encoding hpn gene, cloned and expressed in Escherichia coli ER1793, has an open reading frame (180 bp) that specifies a protein with a calculated molecular mass of 7,077 Da and with the same amino-terminal sequence as that of wild-type Hpn. The deduced sequence of Hpn consists of 60 amino acids, of which 28 (47%) are histidines. The hpn gene does not map with the urease gene cluster on the H. pylori chromosome. An Hpn-negative, isogenic H. pylori strain, generated by hpn gene deletion and grown on blood agar, had the same urease activity that wild-type cells did. Thus, the role of Hpn in helicobacters is unknown.     

Biochemical studies of Helicobacter mustelae fatty acid composition and flagella.

The fatty acid compositions of Helicobacter mustelae whole cells, isolated phospholipids, and isolated lipopolysaccharides were analyzed by gas-liquid chromatography. Major phospholipid fatty acids were C16:0, C18:0, C18:1, and C19:0 cyc. In isolated lipopolysaccharides, 3-OH-C16:0, 3-OH-C14:0, C14:0, C16:0, and C18:0 were found. The lipid composition of H. mustelae thus showed pronounced differences from that of H. pylori. Flagella were purified by mechanical shearing and centrifugation steps. In all H. mustelae strains, the flagellin had an apparent molecular mass of 53 kDa and was thus the same size as H. pylori flagellin. The flagellin of strain NCTC 12032 was further purified and subjected to N-terminal amino acid sequence analysis. The first 10 amino acids were identical to those of H. pylori flagellin, but the next 5 were different. Significant homology was also found with flagellins of other bacteria.     

Quantitative culture of Helicobacter pylori from gastric juice: the potential for transmission

The transmission of Helicobacter pylori may occur by spread of organisms from gastric juice which has been introduced into the mouth by gastro-oesophageal reflux. The aim of this study was to quantify the load of H. pylori present in gastric juice available for transmission. Gastric antral biopsy and gastric juice samples were collected from 108 adult dyspeptic patients undergoing routine upper gastroscopy and the presence of H. pylori was determined. In all, 54 (50%) of 108 patients gave positive results in the gastric antral biopsy rapid urease test and for H. pylori histology. The gastric juice of 40 (37%) of patients gave positive results for the urease A gene by PCR assay; 34 (31%) of patients were positive by these three tests and H. pylori was cultured from the gastric juice of 13 (38%) of these patients. The median count of H. pylori in gastric juice was 1.75 x 10(1) cfu/ml. Viable organisms in gastric juice may lead to transmission of H. pylori when refluxed or vomited into the mouth.     

Optimal combination of media for primary isolation of Helicobacter pylori from gastric biopsy specimens.

The aim of the present study was to compare eight media, four nonselective and four selective media, to determine the best combination of media for the primary isolation of Helicobacter pylori. Over a period of 5 months, mucosal antral biopsy specimens were obtained from 222 consecutive dyspeptic patients undergoing endoscopy. Biopsy samples were plated in parallel on all eight media. Egg yolk emulsion agar (EYE), Skirrow's medium, Dent's medium, and modified Thayer-Martin medium were used as selective media; modified chocolate agar (MCHOC), Triptycase soy agar (TSA), brucella agar, and brain heart infusion agar were used as nonselective media. Overall, by using these eight media, H. pylori was recovered from biopsy specimens from 114 of 222 patients, yielding an isolation rate of 51%. Comparison of all possible combinations of the eight media showed that the highest rate of isolation of H. pylori was 100% (114 of 114) with EYE-MCHOC, followed by 96.5% (110 of 114) when EYE-TSA was used. Conversely, it was found that none of the media used alone yielded a 100% rate of recovery (the maximum recovery rate was 95%, which was achieved with EYE). These results indicate that the association of EYE and MCHOC yielded the maximum recovery of H. pylori from gastric biopsy specimens. Therefore, the use of selective and nonselective media in parallel offers optimal recovery rates with only a slight increase in costs.     

Construction and characterization of an isogenic urease-negative mutant of Helicobacter mustelae.

Helicobacter mustelae infects the ferret stomach and provides an opportunity to study pathogenic determinants of a Helicobacter species in its natural host. We constructed an isogenic urease-negative mutant of H. mustelae which produced no detectable urease and showed a reduced acid tolerance. This mutant provides an opportunity to further evaluate the role of urease in the pathogenesis of Helicobacter infection.     

Selective medium for isolation of Clostridium botulinum from human feces.

A selective medium, Clostridium botulinum isolation (CBI) agar, was developed for the isolation of C. botulinum from human feces. This medium contains cycloserine (250 microgram/ml), sulfamethoxazole (76 microgram/ml), and trimethoprim (4 microgram/ml) as selective inhibitory agents. Qualitative tests indicated complete recovery of C. botulinum types A, B, F, and G on CBI medium. It was more difficult to recognize type G colonies on the medium because of their lack of lipase activity. Except for a few species of Clostridium, the growth of other obligate anaerobes and of the facultative anaerobes tested on CBI medium was suppressed. Quantitative studies of C. botulinum on the selective medium yielded counts comparable to those obtained on egg yolk agar control plates. Isolation of C. botulinum types A, B, and F from seeded fecal specimens was easily achieved with CBI medium. The use of CBI agar should aid the rapid isolation of C. botulinum from fecal specimens associated with foodborne and infant botulism.     

Selective medium for isolation of Clostridium butyricum from human feces.

The incidence of exfoliative toxin-producing strains of Staphylococcus aureus was studied. Samples from hospitalized patients of all ages and samples from infants less than 6 weeks old were screened; out of 2,632 coagulase-positive S. aureus strains tested, 6.2% synthesized exfoliative toxin. The clinical features could be assessed in 86 patients harboring exfoliative toxin-producing staphylococci. Skin lesions (pustules, blisters, and bullous impetigo) could be observed only when the exfoliative toxin-positive strains were isolated from the skin. Phage nongroup II strains seemed less skin pathogenic than phage group II strains. Outbreaks and sporadic cases were observed.     

Epidemiological characteristics of non-A, non-B viral hepatitis with a fecal-oral transmission mechanism

The analysis of verified cases of non-A-non-B hepatitis with the fecal-oral mode of transmission which had occurred in one of the districts of the Turkmen SSR, 1984-1985, revealed a this infection: an explosive nature of the incidence, number of epidemiological characteristics of this infection: an explosive nature of the incidence, even distribution in the territory of the district and within one residential area, predominant affection of 15-29-year-old subjects, high mortality among hepatitis-affected pregnant women, insignificant number of secondary cases in the families of index cases. The occurrence of these non-A-non-B hepatitis cases was associated with water. The results of virological and serological studies ruled out the role of hepatitis A and B viruses in the etiology of the acute hepatitis cases occurring in the area. Anti-hepatitis A IgM was detected in the blood in only 3% of the patients in 1984 and in 2% in 1985, exclusively in young patients, and HBsAg in 11% and 9%, respectively. Immune electron microscopy revealed virus-like particles 27-30 nm in diameter in fecal extracts from the patients. The antigen of non-A-non-B hepatitis virus was detected in the first days of the jaundice period in feces of 14% of the patients in 1984 and in 11% in 1985 by an enzyme-immunoassay using a test developed at the Institute of Virology of the USSR Academy of Medical Sciences.     

An ABC Transporter and a TonB Ortholog Contribute to Helicobacter mustelae Nickel and Cobalt Acquisition

The genomes of Helicobacter species colonizing the mammalian gastric mucosa (like Helicobacter pylori) contain a large number of genes annotated as iron acquisition genes but only few nickel acquisition genes, which contrasts with the central position of nickel in the urease-mediated acid resistance of these gastric pathogens. In this study we have investigated the predicted iron and nickel acquisition systems of the ferret pathogen Helicobacter mustelae. The expression of the outer membrane protein-encoding frpB2 gene was iron and Fur repressed, whereas the expression of the ABC transporter genes fecD and ceuE was iron and Fur independent. The inactivation of the two tonB genes showed that TonB1 is required for heme utilization, whereas the absence of TonB2 only marginally affected iron-dependent growth but led to reduced cellular nickel content and urease activity. The inactivation of the fecD and ceuE ABC transporter genes did not affect iron levels but resulted in significantly reduced urease activity and cellular nickel content. Surprisingly, the inactivation of the nixA nickel transporter gene affected cellular nickel content and urease activity only when combined with the inactivation of other nickel acquisition genes, like fecD or ceuE. The FecDE ABC transporter is not specific for nickel, since an fecD mutant also showed reduced cellular cobalt levels and increased cobalt resistance. We conclude that the H. mustelae fecDE and ceuE genes encode an ABC transporter involved in nickel and cobalt acquisition, which works independently of the nickel transporter NixA, while TonB2 is required primarily for nickel acquisition, with TonB1 being required for heme utilization.The genus Helicobacter comprises bacterial pathogens that colonize the alimentary tract of mammals, with the best-known example being the human gastric pathogen Helicobacter pylori (17). Other examples include Helicobacter species colonizing the gastric mucosa of big cats (Helicobacter acinonychis), cats and dogs (Helicobacter felis), and ferrets (Helicobacter mustelae) (21, 31). Gastric colonization by Helicobacter species can illicit a strong immune response, which may develop into pathologies like peptic ulcer disease and precancerous lesions (17). The lifelong colonization of the gastric mucosa suggests that these Helicobacter species are well adapted to this harsh environment and are able to combat the diverse antimicrobial activities employed by the host within the gastric mucosa, such as iron restriction and acidity (43).Transition metals like iron and nickel are both essential for gastric Helicobacter species. Iron is involved in redox reactions and functions as a cofactor of many enzymes, whereas nickel is the cofactor of two important enzymes in gastric Helicobacter species: urease and hydrogenase. The urease enzyme is the main factor allowing acid resistance, whereas hydrogenase is important for energy production, and both enzymes are essential for gastric colonization by Helicobacter species (2, 20, 32, 37). Analyses of complete genome sequences of gastric Helicobacter species allowed the prediction of many genes putatively involved in iron acquisition but surprisingly few predicted nickel acquisition genes (12, 22, 35).Ferric iron acquisition in Gram-negative bacteria is usually mediated by a TonB-dependent outer membrane receptor coupled to an ABC transporter for transport in the periplasm and over the inner membrane. Ferrous iron acquisition requires only an FeoB-like single-component system for inner membrane transport (1, 41). As for ferrous iron, the transport of nickel was until recently thought not to require an outer membrane component but only an ABC transporter or single-component NiCoT transporter (18, 19). However, it has become apparent that there is much more variation in these themes and that TonB-dependent outer membrane proteins, annotated as putative iron acquisition systems, may be involved in the transport of a range of other metals or compounds (10, 14, 28, 29, 34). Examples of these new insights stem from recent work with H. pylori, where two out of three FecA and FrpB orthologs were shown to be regulated by iron and Fur, whereas the third FecA and FrpB ortholog is NikR and nickel repressed (9, 14, 39) and has been proposed to function in nickel acquisition (10, 28). Similarly, the H. mustelae putative TonB-dependent outer membrane protein NikH contributes to urease activity, probably by mediating nickel acquisition (34). Caution also needs to be taken when annotating ABC transporters, as the proposed ferric citrate permease and ATPase FecD and FecE, respectively, were shown not to contribute to iron acquisition in H. pylori (41), suggesting a role for these H. pylori genes in the acquisition of other metals or compounds. Hence, the currently available genome annotations are potentially unreliable, and functional and mutational data are required to validate or correct the annotation of genome sequences.Here we present a study of some of the putative iron and nickel acquisition systems identified in the H. mustelae genome sequence (22) (listed in Table S1 in the supplemental material). We have determined their contribution to iron and nickel acquisition by using regulatory studies, growth promotion assays, urease activity, and intracellular metal content and demonstrate that the two TonB systems play differential roles in iron and nickel acquisition. We also show that the CeuE and the FecDE ABC transporter components are likely to be misannotated, as they function in nickel and cobalt acquisition.     

Inability of an isogenic urease-negative mutant stain of Helicobacter mustelae to colonize the ferret stomach.

Eight ferrets specific-pathogen-free for Helicobacter mustelae were given, per dose, approximately 3.0 x 10(7) CFU of either the wild-type parent strain of H. mustelae (NCTC 12032) (two ferrets) the isogenic urease-negative mutant strain of H. mustelae (10::Tn3Km) (four ferrets), or sterile culture broth (two ferrets). Infection status was monitored by endoscopic gastric biopsy for urease activity, histopathology, and culture and by serology at 3, 6, 10, and 21 weeks. All ferrets were necropsied at 25 weeks. Both negative control ferrets remained uninfected, both ferrets receiving the H. mustelae wild-type parent strain became infected after two doses of the organism, and all four ferrets given two doses of the isogenic urease-negative mutant strain of H. mustelae remained uninfected throughout the 6-month study. Histopathology correlated with infection status. H. mustelae-infected ferrets exhibited diffuse mononuclear inflammation in the subglandular region and the lamina propria of the gastric mucosa, while uninfected ferrets showed no or minimal inflammation. These results suggest that urease activity is essential for colonization of the ferret stomach by H. mustelae.     

Recombinant antigens prepared from the urease subunits of Helicobacter spp.: evidence of protection in a mouse model of gastric infection.

Urease is an important virulence factor for gastric Helicobacter spp. To elucidate the efficacy of individual urease subunits to act as mucosal immunogens, the genes encoding the respective urease subunits (UreA and UreB) of Helicobacter pylori and Helicobacter felis were cloned in an expression vector (pMAL) and expressed in Escherichia coli cells as translational fusion proteins. The recombinant UreA and UreB proteins were purified by affinity and anion-exchange chromatography techniques and had predicted molecular masses of approximately 68 and 103 kDa, respectively. Western blotting (immunoblotting) studies indicated that the urease components of the fusion proteins were strongly immunogenic and were specifically recognized by polyclonal rabbit anti-Helicobacter sp. sera. The fusion proteins (50 micrograms) were used, in combination with a mucosal adjuvant (cholera toxin), to orogastrically immunize mice against H. felis infection. Gastric tissues from H. felis-challenged mice were assessed by the biopsy urease test and by histology. In mice immunized with recombinant H. felis UreB, 60% of animals (n = 7) were histologically negative for H. felis bacteria after challenge at 17 weeks. This compared with 25% (n = 8) for mice immunized with the heterologous H. pylori UreB antigen. Neither the homologous nor the heterologous UreA subunit elicited protective responses against H. felis infection in mice. The study demonstrated that a recombinant subunit antigen could induce an immunoprotective response against gastric Helicobacter infection.     

Helicobacter mesocricetorum sp. nov., A novel Helicobacter isolated from the feces of Syrian hamsters

A spiral-shaped bacterium with bipolar, single, nonsheathed flagella was isolated from the feces of Syrian hamsters. The bacterium grew as a thin spreading film at 37 degrees C under microaerobic conditions, did not hydrolyze urea, was positive for catalase and alkaline phosphatase, reduced nitrate to nitrite, did not hydrolyze hippurate, and was sensitive to nalidixic acid but resistant to cephalothin. Sequence analysis of the 16S rRNA gene and biochemical and phenotypic criteria indicate that the novel bacterium is a helicobacter. The novel bacterium is most closely related to the recently described mouse enteric helicobacter, Helicobacter rodentium. This is the first urease-negative Helicobacter species with nonsheathed flagella isolated from feces of asymptomatic Syrian hamsters. We propose to name this novel helicobacter Helicobacter mesocricetorum. The type strain is MU 97-1514 (GenBank accession number AF072471).     

Pentatrichomonas hominis: first isolation from the feces of a dog with diarrhea in China

A trichomonad-like parasite isolated from canine fecal samples in Changchun, China was successfully cultivated in vitro using RPMI1640 medium supplemented with 10 % heat-inactivated calf serum and antibiotics. These were then subjected to scanning and transmission electron microscopy for ultrastructural study. This parasite has four anterior flagella of unequal length, one independent flagellum, and one recurrent flagellum. It exhibits an anterior nucleus, a Golgi complex, an axostyle, food vacuoles, and hydrogenosomes. These features are consistent with the ultrastructural characteristics of previously described Pentatrichomonas hominis. Polymerase chain reaction and sequence analysis of three genetic loci, including ITS1-5.8S rRNA-ITS2, 18S rRNA, and EF-1α, were also used to compare these samples with other trichomonad species. Molecular identification was also consistent with P. hominis. This is the first time that isolation of P. hominis has been isolated from dog in China, although several other strains of P. hominis have been isolated from human samples.