PCR-based restriction fragment length polymorphism typing of Helicobacter pylori

Abstract

We applied a molecular typing approach for Helicobacter pylori that uses restriction fragment length polymorphism (RFLP) analyses of an 820-bp PCR-amplified portion of the ureC gene in H. pylori. The PCR products were digested with restriction enzyme HhaI, MboI, or MseI, and the fragments generated were analyzed by agarose electrophoresis. Among 25 independent clinical isolates, each showed a different pattern when a combination of the three RFLP patterns was used. Using this method, we studied isolates from the antrum or the body of the stomach of 14 patients before and after antibiotic therapy. Before treatment, successful isolation of H. pylori from the two sites of the stomach was possible for 12 of the 14 patients. For 10 of these 12 patients, each pair of isolates had identical RFLP profiles. For the other two patients (16.7%), however, isolates from the antrum and the body of the stomach had different RFLP profiles. Treatment was successful for 6 of the 14 patients; of the 8 patients with treatment failures, 5 had identical isolate pairs. In each case, the isolates found posttreatment were the same as the pretreatment isolates. For one of the patients who was colonized with two different isolates pretreatment, one of the isolates was identified at both sites after unsuccessful treatment. We also studied six long-term follow-up patients who had sequential biopsies at intervals of up to 5 months. Each follow-up isolate from each patient had the same RFLP profile as the initial isolate. This typing method provides a reliable and reproducible typing scheme for the study of H. pylori infections and indicates that infection with more than one H. pylori isolate is not rare.

Differentiation of Helicobacter pylori strains directly from gastric biopsy specimens by PCR-based restriction fragment length polymorphism analysis without culture

“…Although several nucleic acid techniques, such as the restriction endonuclease analysis of genomic DNA (6,18,23,32) and Southern blot hybridization with rRNA gene probes (27), have been applied to type H. pylori clinical isolates from different patients, the patterns produced by these methods are complex and difficult to interpret, particularly for largescale analyses of clinical isolates. Recently, a PCR-based restriction fragment length polymorphism (RFLP) analysis method has been developed for typing of H. pylori clinical isolates (1,5,7,11,12,25). Schemes involving this method have been used to analyze H. pylori genes which encode urease and its accessory proteins, including the 2.4-kb ureA-ureB (1, 10), 1.7-kb ureC-ureD (1), 933-bp ureB (5), 1.1-kb ureC (25), and 820-bp ureC (12) genes.…”

Co‐Existing Gastric Cancer and Duodenal Ulcer Disease: Role of Helicobactor pylori Infection

“…One possibility is that mixed infection occurs with both duodenal ulcer and gastric cancer strains. Mixed infection with several strains has certainly been demonstrated and if disease-specific strains occur and circulate with different frequencies in different populations, Korea with its high frequency of both diseases would be one site where coinfection with strains with a propensity to lead to each of the diseases would not be unlikely [32][33][34][35]. Recent studies comparing cagA gene in H. pylori strains from Korea and the United States found that the pattern of the strains from Korea was different from that in the U.S. suggesting that the predominant circulating strain may vary markedly between geographic regions [36].…”

Restriction–modification system differences in Helicobacter pylori are a barrier to interstrain plasmid transfer

“…Helicobacter pylori, a microaerophilic, Gram-negative bacterium that colonizes the human stomach, is a causative factor in peptic ulcer disease and gastric cancer (Blaser, 1999). H. pylori strains have a high degree of genetic diversity (Akopyanz et al, 1992;Fujimoto et al, 1994;Alm and Trust, 1999;Wang et al, 1999), including highly prevalent point mutations in conserved genes such as ureB (Kansau, 1996) and flaA (Suerbaum et al, 1998), mosaicism within genes such as vacA (Atherton et al, 1995) and the presence of non-conserved genes, such as the cag island Censini et al, 1996;Akopyants et al, 1998a). H. pylori strains also vary in the presence of insertion sequences (Censini et al, 1996;Berg et al, 1997;Hook-Nikanne et al, 1998), plasmids (Kleanthous et al, 1991), homologues of restriction± modification (R±M) genes (Tomb et al, 1997;Akopyants et al, 1998b; and gene order (Jiang et al, 1996).…”