Hans-Olof Nilsson

Effect of Cold Starvation, Acid Stress, and Nutrients on Metabolic Activity of Helicobacter pylori

ABSTRACT Helicobacter pylori can transform, in vivo as well as in vitro, from dividing spiral-shaped forms into nonculturable coccoids, with intermediate forms called U forms. The importance of nonculturable coccoid forms of H. pylori in disease transmission and antibiotic treatment failures is unclear. Metabolic activities of actively growing as well as nonculturable H. pylori were investigated by comparing the concentrations of cellular ATP and total RNA, gene expression, presence of cytoplasmic polyphosphate granules and iron inclusions, and cellular morphology during extended broth culture and nutritional cold starvation. In addition, the effect of exposing broth-cultured or cold-starved cells to a nutrient-rich or acidic environment on the metabolic activities was investigated. ATP was detectable up to 14 days and for at least 25 days after transformation from the spiral form to the coccoid form or U form in broth-cultured and cold-starved cells, respectively. mRNAs of VacA, a 26-kDa protein, and urease A were detected by using reverse transcription-PCR in cells cultured for 2 months in broth or cold starved for at least 28 months. The ATP concentration was not affected during exposure to fresh or acidified broth, while 4- to 12-h exposures of nonculturable cells to lysed human erythrocytes increased cellular ATP 12- to 150-fold. Incubation of nonculturable cold-starved cells with an erythrocyte lysate increased total RNA expression and ureA mRNA transcription as measured by quantitative real-time reverse transcription-PCR. Furthermore, the number of structurally intact starved coccoids containing polyphosphate granules increased almost fourfold (P = 0.0022) under the same conditions. In conclusion, a specific environmental stimulus can induce ATP, polyphosphate, and RNA metabolism in nonculturable H. pylori, indicating viability of such morphological forms.

Helicobacter pylori and extragastric diseases--other Helicobacters.

The involvement of Helicobacter pylori in the pathogenesis of extragastric diseases continues to be an interesting topic in the field of Helicobacter‐related pathology.

Infection of BALB/c A mice by spiral and coccoid forms of Helicobacter pylori

Helicobacter pylori exists in two different morphological forms, spiral and coccoid. This study demonstrated that both forms can infect BALB/c A mice. The animals were inoculated orally three times at 2-day intervals with 10(8) cfu of both spiral and coccoid forms of strain CCUG 17874 (NCTC 11637), strain 25 and strain 553/93. Infection was followed over a 30-week period by histological scoring of the grade of inflammation in gastric biopsies. At each time point sera were collected for analysis in ELISA and immunoblot analysis. Both spiral and coccoid forms of all H. pylori strains gave significantly higher inflammation scores than a control group of animals 1 week after inoculation. The histological evidence persisted throughout the entire 30 weeks. The inflammation was most severe in the pylorus and duodenum. Infection with strain 553/93 displayed the most severe gastritis. The spiral form of strain CCUG 17874 gave an immune response after only 4 weeks, whereas its coccoid form as well as strains 25 and 553/93 (spiral and coccoid forms) gave a significant increase in antibody response in ELISA and immunoblot after 16 weeks. It is concluded that both spiral and coccoid forms of H. pylori can cause acute gastritis in BALB/c A mice.

Two-dimensional electrophoretic and immunoblot analysis of cell surface proteins of spiral-shaped and coccoid forms of Helicobacter pylori.

Cell surface proteins of the human gastric pathogen Helicobacter pylori extracted during different in vitro growth phases were analyzed by one‐ and two‐dimensional gelelectrophoresis (1‐DE and 2‐DE) and by 2‐DE immunoblot. Broth‐cultured H. pylori cells were stained with an acridine‐orange dye to monitor the morphological status of the organism. In 2‐day‐cultures, 96% of the bacterial cells were spiral‐shaped and four days later a morphological switch to coccoid forms occurred. In 10‐day cultures spiral‐shaped forms were not found. By 1‐DE, proteins with the molecular masses of 87 and 120 kDa were detected in the 2‐day cultures that disappeared in cells of 12‐day cultures. A protein corresponding in size to the heat shock protein (GroEl homolog, Hsp60) and a 62 kDa protein, the ureaseB‐subunit, were identified in extracted proteins of 2‐, 8‐, and 12‐day cultures. 2‐DE revealed an increased a number of silver‐stained spots of 8‐day cultures (in average 250 spots) compared with protein extracted from 2‐day cells (in average 160 spots). 2‐DE immunoblots performed with sera containing antibodies to major H. pylori proteins such as the A‐ and B‐subunits of urease and the Hsp60 showed similar reactivity to surface proteins extracted from 2‐, 8‐, and 12‐day cultures, suggesting that these proteins remain immunologically intact. Pooled sera from infected patients absorbed with spiral‐shaped cells showed an almost total blocking of the antibody reactivity to extracted coccoid proteins in 2‐DE immunoblot. Eighteen spots were still visible, but this reactivity probably represents a solid overexpression by the coccoid cells of Hsp60 and ureaseB proteins and is thus difficult to block.

Bovine anti-Helicobacter pylori antibodies for oral immunotherapy

Background: Passive immunization with orally administered antibodies against specific pathogens has previously been successfully used therapeutically in both animal and human studies. We employed a similar strategy for experimental treatment of mice infected with the gastric pathogen Helicobacter pylori. Methods: An anti- H. pylori bovine colostral hyperimmune immunoglobulin preparation (BIC) was generated and its efficacy was tested in different in vitro experiments, such as binding to the Lewis b blood group antigen, inhibition of adherence of H. pylori to human gastric mucosa tissue sections in situ and in a haemagglutination assay. The BIC preparation was also given in the drinking water to H. pylori -infected mice. Results: An inhibition of 95% of the binding of H. pylori to Lewis b glycoconjugate was observed in vitro. Furthermore, a blocking activity of almost 90% was observed when the BIC was preincubated with H. pylori bacteria. Finally, the BIC preparation inhibited the haemagglutination of H. pylori and human red blood cells. Seven of 40 (17.5%) mice remained infected in the treatment group as compared with 25 of 45 (55.5%) in the control group. Hence, the cure rate was 66%, P = < 0.001. The mean number of colonies in the antibody-treated mice where eradication was not successful was also reduced ( P < 0.05). In trials using FVB/N transgenic Lewis b expressing mice, a cure rate of 50%-66% was observed. Conclusion: Bovine colostral antibodies against H. pylori can be generated in high titres, inhibit binding in vitro and can eradicate or reduce the number of bacteria in infected mice.

High Prevalence of Helicobacter Species Detected in Laboratory Mouse Strains by Multiplex PCR-Denaturing Gradient Gel Electrophoresis and Pyrosequencing

ABSTRACT Rodent models have been developed to study the pathogenesis of diseases caused by Helicobacter pylori, as well as by other gastric and intestinal Helicobacter spp., but some murine enteric Helicobacter spp. cause hepatobiliary and intestinal tract diseases in specific inbred strains of laboratory mice. To identify these murine Helicobacter spp., we developed an assay based on PCR-denaturing gradient gel electrophoresis and pyrosequencing. Nine strains of mice, maintained in four conventional laboratory animal houses, were assessed for Helicobacter sp. carriage. Tissue samples from the liver, stomach, and small intestine, as well as feces and blood, were collected; and all specimens (n = 210) were screened by a Helicobacter genus-specific PCR. Positive samples were identified to the species level by multiplex denaturing gradient gel electrophoresis, pyrosequencing, and a H. ganmani-specific PCR assay. Histologic examination of 30 tissue samples from 18 animals was performed. All mice of eight of the nine strains tested were Helicobacter genus positive; H. bilis, H. hepaticus, H. typhlonius, H. ganmani, H. rodentium, and a Helicobacter sp. flexispira-like organism were identified. Helicobacter DNA was common in fecal (86%) and gastric tissue (55%) specimens, whereas samples of liver tissue (21%), small intestine tissue (17%), and blood (14%) were less commonly positive. Several mouse strains were colonized with more than one Helicobacter spp. Most tissue specimens analyzed showed no signs of inflammation; however, in one strain of mice, hepatitis was diagnosed in livers positive for H. hepaticus, and in another strain, gastric colonization by H. typhlonius was associated with gastritis. The diagnostic setup developed was efficient at identifying most murine Helicobacter spp.

Helicobacter species DNA in liver and gastric tissues in children and adolescents with chronic liver disease

Abstract Objective. Enterohepatic Helicobacter species (EHS) have previously been found in adults with hepatobiliary diseases. Here, we report the prevalence of Helicobacter pylori and EHS in liver and gastric tissue in children and adolescents with chronic liver disease (CLD). Material and methods. Seventy-seven consecutive children and adolescents with CLD with or without ulcerative colitis or Crohn’s disease (UC/CD) were investigated. Tissue samples were analysed using a Helicobacter genus-specific 16S rDNA polymerase chain reaction (PCR) assay and DNA-sequence analysis. Sera from 61 subjects were also analysed using enzyme immunoassay and immunoblotting. Results. The Helicobacter PCR was positive in 3/23 (13%) livers from patients with primary sclerosing cholangitis and UC, and in 1/2 livers from patients with autoimmune hepatitis (AIH) and UC. Sequenced PCR products matched the 16S rDNA of H. hepaticus, H. muridarum, H. canis, and H. pylori, respectively. H. ganmani and H. bilis were detected in gastric tissues from two AIH patients. H. hepaticus and H. pullorum were found in livers from two patients with acute liver failure and intrahepatic cholestasis. Antibody reactivity to Helicobacter cell-surface proteins was negative. Conclusions. H. pylori and EHS can be detected in the livers of some patients with UC and concomitant liver disease, as well as in other children with liver diseases. Multicentre studies from different locations are needed to find out whether these bacteria play a pathogenetic role or whether their presence is an epiphenomenon.

Immunomagnetic bead enrichment and PCR for detection of Helicobacter pylori in human stools

Abstract An immunomagnetic bead-based polymerase chain reaction assay (IMS-PCR) was developed for the detection of Helicobacter pylori in experimentally inoculated human stools and human clinical stool samples. Magnetic beads coated with anti- H. pylori rabbit antibodies were used for enrichment and concentration of H. pylori from faecal samples. Taq polymerase inhibitors, found in human faeces, are efficiently removed by the immunomagnetic separation (IMS) and subsequent washing of the magnetic beads. Conditions of the assay were developed and optimised with faeces from a healthy, H. pylori seronegative, individual. Faeces was inoculated with serial dilutions of either the spiral or the coccoid form of H. pylori . These two morphologic forms could be detected at similar concentrations when inoculated in faeces using an optimised IMS-PCR method. In 1 g of faeces less than 2.5×10 4 H. pylori cells were detected as measured with two separate sets of PCR-primers, based on a urease A subunit genesequence and a genesequence encoding a 26 kDa surface protein of H. pylori . Previously no report has shown a sensitivity below 10 6 H. pylori in faeces PCR. Preliminary analysis of stool samples from 17 patients with symptoms of gastritis and esophagitis by IMS-PCR showed a good correlation with EIA-analysis of H. pylori serum-antibodies from these patients. The results indicate that H. pylori cells are shed in faeces of infected patients and that immunomagnetic bead PCR might be an appropriate method for clinical diagnosis and studies involving immunoprophylaxis, antibiotic treatment as well as vaccine candidates.

Influence of activated charcoal, porcine gastric mucin and beta-cyclodextrin on the morphology and growth of intestinal and gastric Helicobacter spp.

Bile-tolerant Helicobacter spp. are emerging human and animal pathogens. However, due to their fastidious nature, which requires nutrient-rich complex media to grow, infection with these bacteria may be underestimated. The accumulation of toxic metabolites in cultures may be one of the main obstacles for successful culture of these organisms. The present study examined various potential growth-enhancing substances for Helicobacter spp. and, furthermore, how they may affect spiral to coccoid conversion. Five Helicobacter spp. were cultured on agar and in broth media supplemented with activated charcoal, beta-cyclodextrin, or porcine gastric mucin. Growth was determined by estimating the numbers of colony-forming units and colony diameter, as well as bacterial cell mass. Coccoid transformation was estimated every 24 h by both Gram and acridine-orange staining. Activated charcoal was superior in supporting growth and increased cell mass on agar and in broth media. beta-Cyclodextrin delayed spiral to coccoid conversion by Helicobacter pylori and Helicobacter canis, whereas activated charcoal delayed the conversion to coccoid forms of Helicobacter hepaticus and Helicobacter bilis. The progression to coccoid forms by Helicobacter pullorum on agar media was not influenced by any growth supplement. The spiral to coccoid conversion was more rapid in broth media than on agar media. The growth enhancement observed is probably related to the capacity of activated charcoal to remove toxic compounds in culture media.

Possible Clinical Importance of the Transformation of Helicobacter pylori into Coccoid Forms

Although spiral-shaped microorganisms have been observed in the stomach since the beginning of this century (1, 2), they have largely been ignored as pathogens and dismissed as contaminants (3). Not until Warren & Marshall (4, 5) culturedHelicobacter pylori (Campylobacter pylori ) in 1982 did the significance of these organisms become appreciated and their presence on the surface of the gastric mucosa linked to gastritis and peptic ulcer disease. H. pylori is a widespread common microorganism with a worldwide prevalence of 30%–90%, and it is estimated to have infected more than one billion people (6). The prevalence of H. pylori is higher in developing countries than in developed countries (7), with an increase up to 60 years of age (7, 8). No significant difference in sex has been found (7–9).H. pylori is able to convert into coccoid forms, which cannot yet be cultured and diagnosed by conventional diagnostic methods. H. pylori is found almost entirely in humans, and oral–oral transmission from parents to children and between persons in crowded institutions seems to be common (10, 11). A faecal–oral transmission through drinking water has been proposed, especially in areas with poor water supply (12–14). It has been speculated that the coccoid forms of H. pylori play a role in the survival of the bacterium outside the human host (3, 15, 16). Experimental data have shown that coccoid forms ofH. pylori are able to survive in river water for more than 1 year and subsequently be cultured (17). H. pylori may transform into coccoid forms under conditions of poor nutrition and starvation, which may contribute to the transmission ofH. pylori from an environmental source (18, 19). Coccoid forms of H. pylori have not been culturable in all studies despite prolonged culture for up to 4 weeks (19, 20). Several different antiH. pylori regimens have been established, and eradication of H. pylori infection decreases the relapses of peptic ulcers dramatically (21). H. pylori may be more easily eradicated by antibiotic treatment than other chronic infectious agents, such as Mycobacterium tuberculosis andPseudomonasin cystic fibrosis patients. However, 5%–70% treatment failures have been reported in a various studies (21–23). Some of these treatment failures are caused by poor patient compliance or by H. pylori being resistant to the antibiotics used. Some of these treatment failures remain unexplained (22). Coccoid forms of H. pylori can be induced by various compounds, including some anti-ulcer drugs and some antibiotics (24–27) and may remain in the gastric mucosa after antiH. pylori treatment of the patients in three forms: a degenerative dead form, a viable non-culturable form, and a viable culturable form (15, 20, 28, 29; H. O. Nilsson, J. Blom, M. Stollenwerk, LP. Andersen, T. Wadström. Unpublished observations). Whether the two viable forms represent different developmental stages from spiral to non-culturable coccoid forms is uncertain. The aims of this study were to critically evaluate our current understanding of the possible pathogenic potential of the coccoid forms of H. pylori in comparison with that of the spiral forms and to evaluate the clinical role of coccoid forms in transmission and relapse of H. pylori.