Helicobacter pylori: molecular detection of vacA gene and vacuolating activity in human gastric adenocarcinoma cells.

BACKGROUND AND AIMS: Helicobacter pylori strains secrete a vacuolating cytotoxin (VacA), plays an important role for the development of peptic ulcer disease and gastro-duodenal diseases. vacA gene is responsible to regulate the activity of the vacuolating cytotoxin. The objective of this study was molecular detection of vacA gene and observes the vacuolating activity on human gastric adenocarcinoma (AGS) cells. MATERIALS AND METHODS: H. pylori vacA gene was determined by polymerase chain reaction. Vacuolation activity of VacA toxin in broth culture filtrates was assessed in AGS cells and quantified by neutral uptake assay. Different concentration dosages of VacA and incubation time were used in the measurement of the vacuolating activity on AGS cells. RESULTS: The results showed that VacA toxin could stimulate vacuolating activity on AGS cells with minimum concentration 1.0 µg/ml from both of s1m1 and s1m2 alleles (vacA gene). The toxin produced optimal reaction at 5.0 µg/ml with significant differences observed between the alleles. The results also showed that both alleles commenced the vacuolating activity at the minimum of 3 hr incubation time, and the activity showed in time-dependent manner. CONCLUSION: Optimal concentration of VacA toxin (s1m1 allele) causes more interaction with AGS cell producing more vacuolating activities. Time-dependent vaculation of both alleles might allow H. pylori for persistent infection without rapid destruction of gastric cells might promote gastro-duodenal diseases. The study provided us better understanding of the pathogenesis of the diseases associated with H. pylori infection which is an emerging problem in developing countries.

Analysis of PCR-RAPD DNA and antibiotic susceptibility profiles of antrum and corpus isolates of Helicobacter pylori from Malaysian patients.

This study was conducted to determine whether there was any genetic heterogeneity among Helicobacter pylori strains isolated from the antrum and corpus of the same individual in a Malaysian population and to determine the presence of heterogeneous susceptibility of the isolates by comparing PCR-RAPD and antibiotic profiles. Forty-four H. pylori isolates cultured from the antrum and corpus of 22 patients were analyzed. Antibiotic susceptibility testing was carried out by minimum inhibitory concentration determination, using E-Test method strips. PCR-RAPD was carried out on all the strains and the profiles generated were analysed for cluster analysis. Twenty-nine different PCR-RAPD profiles were observed in the 44 isolates. Fifteen pairs of the isolates from the same patients had the same PCR-RAPD patterns while in 7 pairs, the profiles were different. The strains were clustered into 2 separate clusters at a low coefficient of similarity, where most of the strains were in cluster 1. The degree of similarity was very low among most of the isolates. Most of the patients (16 of 22) were infected with strains that have the same antibiotic susceptibility profiles. Out of these, only 10 pairs shared the same PCR-RAPD and antibiotic profiles. Five pairs of isolates with similar PCR-RAPD profiles differed in their antibiotic profiles due to metronidazole resistance in one of the sites. A large degree of genetic heterogeneity was observed among H. pylori strains circulating among Malaysian patients. An individual patient can be infected with multiple strains and the strains can be antibiotic resistant.

Serotyping of Flavobacterium meningosepticum by co-agglutination method.

The purpose of this investigation was to evaluate the usefulness of a co-agglutination procedure for the typing of Flavobacterium meningosepticum. The sensitivity and specificity of the co-agglutination test was compared to the slide agglutination test using reference strains of the bacterial species. Antisera were characterized by both technics to determine their titer and working dilution. The specificity of the sera was assessed by performing tests which include strains of other species and serotypes. A collection of 47 strains of F. meningosepticum isolated from clinical specimens were typed by both co-agglutination and slide agglutination methods. Co-agglutination proved to be markedly more specific than the slide procedure although both methods were similar in sensitivity. It was concluded that co-agglutination proved to be an excellent method for the serotyping of F. meningosepticum.