Role of fecal Clostridium difficile load in discrepancies between toxin tests and PCR: is quantitation the next step in C. difficile testing?

Direct tests for Clostridium difficile are 30-50 % more sensitive than tests for C. difficile toxins but the reasons for this discrepancy are incompletely understood. In addition to toxin degradation and strain differences, we hypothesized that C. difficile concentration could be important in determining whether toxins are detected in fecal samples. We performed standard curves on an FDA-approved real-time PCR test for the C. difficile tcdB gene (Xpert C. difficile/Epi, Cepheid) during a prospective comparison of a toxin immunoassay (Meridian Premier), PCR and toxigenic culture. Immunoassay-negative, PCR-positive samples were retested with a cell cytotoxin assay (TechLab). Among 107 PCR-positive samples, 46 (43.0 %) had toxins detected by immunoassay and an additional 18 (16.8 %) had toxin detected by the cytotoxin assay yielding 64 (59.8 %) toxin-positive and 43 (40.2 %) toxin-negative samples. Overall, toxin-negative samples with C. difficile had 10(1)-10(4) fewer DNA copies than toxin-positive samples and most discrepancies between toxin tests and PCR were associated with a significant difference in C. difficile quantity. Of the toxin-positive samples, 95 % had ≥ 4.1 log(10) C. difficile tcdB DNA copies/mL; 52 % of immunoassay-negative samples and 70 % of immunoassay and cytotoxin negative samples had <4.1 log(10) C. difficile tcdB DNA copies/mL. These findings suggest that fecal C. difficile concentration is a major determinant of toxin detection and C. difficile quantitation may add to the diagnostic value of existing test methods. Future studies are needed to validate the utility of quantitation and determine the significance of low concentrations of C. difficile in the absence of detectable toxin.

Use of real-time, label-free analysis in revealing low-affinity binding to blood group antigens by Helicobacter pylori.

Infectious diseases are often initiated by microbial adherence that is mediated by the binding of attachment molecules, termed adhesins, to cell surface receptors on host cells. We present an experimental system, oblique-incidence reflectivity difference (OI-RD) microscopy, which allows the detection of novel, low-affinity microbial attachment mechanisms that may be essential for infectious processes. OI-RD microscopy was used to analyze direct binding of the oncopathogen, Helicobacter pylori ( H. pylori ) to immobilized glycoconjugates in real time with no need for labeling tags. The results suggest the presence of additional Lewis b blood group antigen (Le(b)) binding adhesins that have not been detected previously. OI-RD microscopy also confirmed the high-affinity binding of H. pylori outer-membrane protein BabA to Le(b). The OI-RD microscopy method is broadly applicable to real-time characterization of intact microbial binding to host receptors and offers new strategies to elucidate the molecular interactions of infectious agents with human host cells.

Helicobacter spp. from gastric biopsies of stranded South American fur seals (Arctocephalus australis).

Gastrointestinal lesions with uncertain etiology have been widely described among pinnipeds. The aim of our study was to investigate the presence of Helicobacter spp. in the gastric mucosa of South American fur seals (Arctocephalusaustralis). Gastric biopsies from thirteen seals, stranded on the shores of the Southwestern Atlantic Ocean in Argentina, were evaluated for the presence of Helicobacter spp. by PCR and DNA sequence analysis. Six gastric biopsies were positive for Helicobacter spp. Pairwise sequence comparisons showed less than 95% identity to novel Helicobacter spp. described from pinnipeds from North America and Australia. However, phylogenetic analysis revealed that the South American fur seal sequences clustered with 99-100% homology with H. cetorum, a species isolated from dolphins and whales. The presence of H. cetorum in pinnipeds, if confirmed by its isolation from the gastric mucosa of these mammals, demonstrates the wide host range of this bacterium in the marine environment.

Detection of Helicobacter and Campylobacter spp. from the aquatic environment of marine mammals.

The mechanism by which Helicobacter species are transmitted remains unclear. To examine the possible role of environmental transmission in marine mammals, we sought the presence of Helicobacter spp. and non-Helicobacter bacteria within the order Campylobacterales in water from the aquatic environment of marine mammals, and in fish otoliths regurgitated by dolphins. Water was collected from six pools, two inhabited by dolphins and four inhabited by seals. Regurgitated otoliths were collected from the bottom of dolphins' pools. Samples were evaluated by culture, PCR and DNA sequence analysis. Sequences from dolphins' water and from regurgitated otoliths clustered with 99.8-100% homology with sequences from gastric fluids, dental plaque and saliva from dolphins living in those pools, and with 99.5% homology with H. cetorum. Sequences from seals' water clustered with 99.5% homology with a sequence amplified from a Northern sea lion (AY203900). Control PCR on source water for the pools and from otoliths dissected from feeder fish were negative. The findings of Helicobacter spp. DNA in the aquatic environment suggests that contaminated water from regurgitated fish otoliths and perhaps other tissues may play a role in Helicobacter transmission among marine mammals.

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.

Determination of the infectious dose of Helicobacter pylori during primary and secondary infection in rhesus monkeys (Macaca mulatta).

We sought to determine the infectious dose of Helicobacter pylori during primary and secondary infection in the rhesus monkey and to determine whether preinoculation acid suppression is necessary to produce colonization. Mixed inoculation with three human-derived strains showed that H. pylori J166 is particularly adapted to colonization of rhesus monkeys, since it outcompeted two other strains. The minimum infectious dose of H. pylori J166 was 10(4) bacteria in specific-pathogen (H. pylori)-free monkeys. Rechallenge of these monkeys after antibiotic therapy was characterized by a 10- to 100-fold decrease in bacterial load compared to primary infection, but with little change in the infectious dose. Acid suppression prior to inoculation was not necessary for colonization to occur. These results provide a basis for future animal experiments using more ecologically relevant conditions of inoculation and suggest that reduction in bacterial load rather than complete protection may be a more realistic goal for H. pylori vaccination.

Selection for urease activity during Helicobacter pylori infection of rhesus macaques (Macaca mulatta).

Helicobacter pylori strain J166 recovered from experimentally inoculated rhesus monkeys had up to a 250-fold-increased urease activity over that before inoculation. This was found to result from the selection of urease positive J166 clones from a heterogeneous inoculum, which was predominantly urease negative due to a 1-bp insertion in the ureA gene. These results confirm the importance of urease for H. pylori colonization. Strain J166 is particularly well adapted to the rhesus monkey, since it colonized preferentially despite the fact that less than 0.1% of the inoculum was urease positive.

Emergence of diverse Helicobacter species in the pathogenesis of gastric and enterohepatic diseases.

Since Helicobacter pylori was first cultivated from human gastric biopsy specimens in 1982, it has become apparent that many related species can often be found colonizing the mucosal surfaces of humans and other animals. These other Helicobacter species can be broadly grouped according to whether they colonize the gastric or enterohepatic niche. Gastric Helicobacter species are widely distributed in mammalian hosts and are often nearly universally prevalent. In many cases they cause an inflammatory response resembling that seen with H. pylori in humans. Although usually not pathogenic in their natural host, these organisms serve as models of human disease. Enterohepatic Helicobacter species are an equally diverse group of organisms that have been identified in the intestinal tract and the liver of humans, other mammals, and birds. In many cases they have been linked with inflammation or malignant transformation in immunocompetent hosts and with more severe clinical disease in immunocompromised humans and animals. The purpose of this review is to describe these other Helicobacter species, characterize their role in the pathogenesis of gastrointestinal and enterohepatic disease, and discuss their implications for our understanding of H. pylori infection in humans.

Foodborne and waterborne infectious diseases. Contributing factors and solutions to new and reemerging pathogens.

Demographic changes and complexities in food production, combined with complacency about the role of infectious diseases in general and the safety of the US food supply in particular, have brought about a resurgence in foodborne and waterborne infectious diseases and, with it, challenges that are unprecedented in recent times. A vigorous effort is already under way to ensure that food and water supplies are safe. This renewed attention to food and water safety must not be an interim response to a perceived short-term threat but, rather, a long-term effort to protect the population from pathogenic microorganisms whose wily adaptations will require constant vigilance.

Immunization with recombinant Helicobacter pylori urease in specific-pathogen-free rhesus monkeys (Macaca mulatta).

Immunization with urease can protect mice from challenge with Helicobacter pylori, though results vary depending on the particular vaccine, challenge strain, and method of evaluation. Unlike mice, rhesus monkeys are naturally colonized with H. pylori and so may provide a better estimate of vaccine efficacy in humans. The purpose of this study was to examine the effectiveness of H. pylori urease as a vaccine in specific-pathogen (H. pylori)-free rhesus monkeys. Monkeys raised from birth and documented to be free of H. pylori were vaccinated with orogastric (n = 4) or intramuscular (n = 5) urease. Two control monkeys were sham vaccinated. All monkeys were challenged with a rhesus monkey-derived strain of H. pylori, and the effects of vaccination were evaluated by use of quantitative cultures of gastric tissue, histology, and measurement of serum immunoglobulin G (IgG) and salivary IgA. Despite a humoral immune response, all monkeys were infected after H. pylori challenge, and there were no differences in the density of colonization. Immunization with urease therefore does not fully protect against challenge with H. pylori. An effective vaccine to prevent H. pylori infection will require different or more likely additional antigens, as well as improvements in the stimulation of the host immune response.

A predominant Th1 type of immune response is induced early during acute Helicobacter pylori infection in rhesus macaques.

BACKGROUND & AIMS: The immune response of gastric T cells during acute Helicobacter pylori infection has not been previously characterized. The aim of this study was to delineate the phenotypic and functional responses of gastric T cells during acute H. pylori infection of rhesus macaques. METHODS: Four monkeys were experimentally infected with H. pylori. Gastric biopsy specimens and peripheral blood samples were obtained 1 and 12 weeks after inoculation. Samples from 3 animals uninfected with H. pylori served as controls. The immunophenotypic changes and functional potential of CD4(+) and CD8(+) T cells in gastric mucosa and peripheral blood to produce cytokines (interleukin [IL]-2, IL-4, IL-13, interferon [IFN]-gamma, MIP-1beta, and tumor necrosis factor [TNF]-alpha) were determined at a single cell level using flow cytometry. RESULTS: An increase in CD4(+) T cells occurred in the gastric mucosa during acute H. pylori infection as early as 1 week after infection. Acute infection was characterized by a predominantly T helper (Th)1 (IL-2 and IFN-gamma) and proinflammatory (TNF-alpha and MIP-1beta) type of cytokine response and the absence of a Th2 type of response. CONCLUSIONS: A predominant Th1 type response was induced early during acute H. pylori infection and may contribute to the development of gastric disease.

Are infectious agents involved in primary biliary cirrhosis? A PCR approach.

BACKGROUND/AIMS: A variety of data suggest that microbial infections and, in particular, atypical mycobacteria infections, may either initiate and/or be associated with the pathogenesis of primary biliary cirrhosis. METHODS: To address this hypothesis, use was made of polymerase chain reaction techniques and primers specific for the 16s rRNA gene of Eubacteria, Archaeabacteria, Mycobacteria and Helicobacter to determine if such sequences were detectable in liver tissue specimens from 29 patients with primary biliary cirrhosis. Similar liver tissues from patients with primary sclerosing cholangitis, chronic hepatitis, alcoholic liver disease and otherwise normal donors were analyzed in parallel. Genomic DNA was extracted from each of these liver tissue specimens using sterile techniques to avoid possible laboratory contamination. The DNA was subjected to polymerase chain reaction amplification using bacterial genus specific primers and the amplified products cloned and sequenced. Sequence data were analyzed by searching for homology to existing genes. RESULTS: Sequences from primary biliary cirrhosis and control livers corresponded to those found in a variety of bacteria, but no consensus sequence was found in primary biliary cirrhosis specimens. Neither Archaeabacteria nor Mycobacteria products were detected in liver specimens of patients with primary biliary cirrhosis, and Helicobacter pylori DNA was detected in only one primary biliary cirrhosis patient. CONCLUSIONS: Although bacterial infection, particularly with intracellular organisms, has been suggested to play a role in the initiation of primary biliary cirrhosis, there is no evidence from this study to suggest an ongoing chronic infectious process.

Rhesus monkey (Macaca mulatta) model of Helicobacter pylori: noninvasive detection and derivation of specific-pathogen-free monkeys.

BACKGROUND AND PURPOSE: Development of the rhesus monkey model of Helicobacter pylori has been hampered by problems with serodetection and by the difficulty of identifying specific-pathogen (Helicobacter)-free animals. Our purpose was to determine whether detection could be improved and to determine if pathogen-free monkeys could be derived by nursery rearing. METHODS: An enzyme-linked immunoabsorbent assay (ELISA) and a [14C]urea breath test were compared to endoscopy to determine H. pylori infection status in rhesus macaques; 18 animals were hand raised in the nursery to determine whether pathogen-free animals could be selected. RESULTS: Helicobacter pylori infection was common in colony-raised young rhesus monkeys and was nearly universal by adulthood. Serodetection, using antigen from rhesus-derived H. pylori strains, was 95% sensitive and 94% specific. The [14C]urea breath test was 96% sensitive and 88% specific for detection of chronic Helicobacter infection in rhesus monkeys. Segregation of newborn animals within the first 24 h of life was a reliable method to obtain pathogen-free rhesus monkeys. CONCLUSION: Isolation of specific-pathogen-free animals, together with better detection methods, may improve the value of the rhesus monkey model for the study of H. pylori pathogenesis, immune response, and vaccine development.

Healthy cats are commonly colonized with "Helicobacter heilmannii" that is associated with minimal gastritis.

Gastric Helicobacter infection in healthy pet cats is not well characterized. We performed endoscopy with gastric biopsy on 15 healthy pet cats that were rigorously screened to exclude underlying or concurrent diseases that might affect Helicobacter colonization. Gastric mucosa biopsy specimens were examined by histology, culture, and PCR for the presence of Helicobacter infection and by histology for the presence of gastritis. Of 15 cats, all but 1 had gastric Helicobacter-like organisms (GHLOs) on examination by light microscopy, and in the one histologically negative cat, GHLOs were detected by PCR. Gastric inflammation was mild or was absent for all cats. No Helicobacter species were identified by culture. Analysis of the 16S rRNA sequence from Helicobacter strains from 10 cats showed that all bacteria were closely related to Helicobacter felis, although there was heterogeneity among the sequences. These results suggest that the gastric mucosa of healthy pet cats is commonly colonized with an uncultivated Helicobacter that is closely related to H. felis, is associated with little or no gastritis, and shows heterogeneity in its 16S rRNA sequence. The epithet "Helicobacter heilmannii" continues to be an appropriate working designation for these bacteria.

Identification of a novel enteric Helicobacter species in a kitten with severe diarrhea.

A previously undescribed Helicobacter sp. was recovered from a cat with severe diarrhea. Based upon the absence of any other identifiable cause of diarrhea, this helicobacter may be involved in the development of the disease signs. The organism could not be cultured but was described on the basis of 16S rRNA gene sequence analysis and morphology and appeared to be a new species, with Helicobacter canis being the most genetically similar species. The presence of a diarrhea-inducing helicobacter in a companion animal may pose a risk of zoonosis.

The major sigma factor (RpoD) from Helicobacter pylori and other gram-negative bacteria shows an enhanced rate of divergence.

Sequence analysis of the Helicobacter pylori major sigma factor (RpoD) shows that it is highly divergent, which may be related to the marked diversity of the H. pylori chromosome. Furthermore, the rate of divergence of RpoD among other gram-negative bacteria is much greater than that among gram-positive bacteria. This suggests that RpoD from gram-negative bacteria is functionally less constrained than that from gram-positive bacteria.

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.

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.

Molecular analysis of urease genes from a newly identified uncultured species of Helicobacter.

"Gastrospirillum hominis" is an uncultured gastric spiral bacterium that has recently been shown by 16S rDNA sequence analysis to be a newly recognized species of Helicobacter that infects humans, and it has been provisionally designated "Helicobacter heilmannii." We used PCR to directly amplify the urease structural genes of "H. heilmannii" from infected gastric tissue. DNA sequence analysis identified two open reading frames, ureA and ureB, which code for polypeptides with predicted molecular weights of 25,729 and 61,831, respectively. The urease subunit genes from "H. heilmannii" were cloned and expressed in Escherichia coli. Western blot (immunoblot) analysis showed that antiserum directed against the ureA and ureB gene products from H. pylori was cross-reactive with the corresponding polypeptides from "H. heilmannii." Analysis of the derived amino acid sequences of "H. heilmannii" UreA and UreB demonstrated that "H. heilmannii" urease is more highly related to the urease from H. felis (found in the stomachs of cats and dogs) than to the urease from H. pylori. These data are consistent with 16S rDNA sequence analysis and suggest that "H. heilmannii" is phylogenetically most closely related to H. felis.