Agglutination of Helicobacter pylori coccoids by lectins.

AIM:To study the agglutination pattern of Helicobacter pylori coccoid andspiral forms.METHODS:Assays of agglutination and agglu-tination inhibition were applied using fifteen commercial lectins.RESULTS:Strong agglutination was observed with Mannose specific Concanavalin A(Con A), fucose specific Tetragonolobus purpureas(Lotus A) and N-acetyl glucosamine-specific Triticum vulgaris (WGA) lectins. Mannose and fucose specific lectins were reactive with all strains of H. pylori coccoids as compared to the spirals. Specific carbohydrates, glyco-proteins and mucin were shown to inhibit H. pylori lectin agglutination reactions.Pre-treatment of the bacterial cells with formalin and sulphuric acid did not alter the agglutination patterns with lectins. However, sodium periodate treatment of bacterial cells were shown to inhibit agglutination reaction with Con A, Lotus A and WGA lectins. On the contrary, enzymatic treatment of coccoids and spirals did not show marked inhibition of H. pylori-lectin agglutination. Interes-tingly, heating of H. pylori cells at 60° for 1 hour was shown to augment the agglutination with all of the lectins tested.CONCLUSION:The consi-derable differences in lectin agglutination patterns seen among the two different-tiated forms of H. pylori might be attributable to the structural changes during the events of morphological trans-formation, resulting in exposing or masking some of the sugar residues on the cell surface. Possibility of various sugar residues on the cell wall of the coccoids may allow them to bind to different carbohydrate receptors on gastric mucus and epithelial cells. The coccoids with adherence characteristics like the spirals could aid in the pathogenic process of Helicobacter infection. This may probably lead to different clinical outcome of H. pylori associated gastro-duodenal disease.

Serum IgG response to differentiated antigens of Helicobacter pylori.

AIM:To detect antibodies against Helicobacter pylori spiral and coccoid antigens in human sera.METHODS:Blood samples were collected from 278 patients with gastric diseases. A 3-day-old culture of H. pylori on chocolate blood agar was used to providespiral form. Synchronous coccoids were cultured in (BHY) (brain heart infusion supplemented with 10% horse serum and 0.4% yeast extract) medium in a chemostat.Antigens from spiral and coccoid form were prepared using acid glycine extraction.Enzyme-linked immunosorbent assay (ELISA) was performed to detect serum IgG anti-bodies against spiral and coccoid forms of H. pylori.RESULTS:Seroprevalence of H. pylori infection was higher in patients with gastric ulcer (79%) and gastric cancer (83%) than those with non-ulcer dyspepsia (NUD)(44%) and other diseases (45%) (P <0.05). IgG antibodies against spiral and coccoid antigens were detected in 50.7% (141/278) and 49.6% (138/278), respectively.CONCLUSION:The spiral and coccoid forms of H.pylori coexist in patients infected with the bacterium.

Helicobacter pylori acquistion of metronidazole resistance by natural transformation in vitro.

AIM:To study whether Helicobacter pylori is naturally transformable.METHODS:Transformation was performed in BHI broth supplemented with horse serum and yeast extract. Genomic DNA extracted from a metronidazole resistant H.pylori strain was added to H. pylori broth culture. The mixture was incubated at microaerophilic atmosphere. The DNA-treated cells were plated on blood agar containing 8mg/L metronidazole to select for transformants. Sterile distilled water was used as a negative DNA control.The DNA profiles of transformants were compared with that of their parent strains by randomly amplified polymorphic DNA (RAPD) fingerprinting.RESULTS:Transformation ofH. pylori with DNA from a metronidazole resistant strain as a marker was demonstrated. Out of the 12 strains of H. pylori tested, 9 (75%) strains were found to be transformable. The transformation frequencies ranged from 3.4?10(-6) to 2.4 10(-4). By RAPD, DNA fingerprints of the transformants and their parent strains showed no change in DNA profiles though transformants were all resistant to metronidazole as compared with their metronidazole-sensitive parent strains.CONCLUSION:Helicobacter pylori is naturally transformable which might be one of the ways that H. pylori develops resistance to metronidazole.

Coexistence of Helicobacter pylori spiral and coccoid forms in experimental mice.

AIM:To infect mice with Helicobacter pylori and detect immune response against two form of H. pylori.METHODS:An isolate of H. pylori obtained from a patient with gastric cancer was used to infect mice. Fifty mice were divided into eight groups. Two groups served as negative control without any inoculation and internal negative control with 0.5M NaHCO(3) and brain heart infusion (HBI), respectively. Mice in each experimental group were first inoculated with 0.5M NaHCO(3) and then H. pylori suspension for 3 times at a 2-day interval. Mice from controls and infectious groups were sacrificed at a weekly interval postinfection. Gastric samples were trimmed, inoculated onto chocolate blood agar and then incujbated in microaerophilic atmosphere at 37° for 14 days. Sera were examined for immunoglobulins against H. pylori spiral and coccoid antigens by ELISA.RESULTS:After inoculation H. pylori was isolated in one mouse from one week postinfection.No H. pylori was detected in control mice. However,urease test was positive in 50% (5/10) control mice, 70% (7/10) mice inoculated with NaHCO(3) and BHI and 77% (23/30) mice infected with H. pylori. The systemic immune responses of the mice to H. pylori strain were determined by ELISA. The mice showed immune responses to both H. pylori spiral and coccoid antigens one week after infection with H. pylori. The peak mean absorbances of antibodies against spiral and coccoid forms were four weeks postinfection which showed 6 and 18 times higher than that of negative control group respectively (P < 0.01).CONCLUSION:Spiral and coccoid forms of H. pylori coexist in experimental mice studied.