Clustering of South African Helicobacter pylori Isolates from Peptic Ulcer Disease Patients Is Demonstrated by Repetitive Extragenic Palindromic-PCR Fingerprinting

Abstract

The present report assesses the association between clonal groupings, disease, and the virulence fingerprint of 76 South African Helicobacter pylori cagA ؉ strains isolated from 57 Cape-colored subjects. Two methods, repetitive extragenic palindromic (REP)-PCR and random amplified polymorphic DNA (RAPD)-PCR, were used to generate DNA fingerprints, and computer-assisted analysis was used to derive clusters. The two PCR techniques were only partially complementary (48%). REP-PCR fingerprints identified a distinct pathological cluster consisting of strains from 63% of the patients and was strongly associated with both disease (P < 0.00001) and the vacuolating cytotoxin A (vacA) signal sequence type (P < 0.003). RAPD-PCR fingerprinting was not associated with disease and was less strongly associated with vacA (P < 0.05) than REP-PCR was. Hierarchical analysis indicated that isolates from patients with peptic ulcer disease tended to cluster differently than isolates from patients with gastritis alone or gastric adenocarcinoma. These relationships are consistent with a loosely clonal population structure associated with disease for H. pylori in the Cape-colored population in South Africa.Genetic diversity between strains of Helicobacter pylori is more marked than for any other bacterial species (13). The main reason for this is that recombination between strains is also higher than has been described for any other bacterium (8,13). Despite this high level of recombination, comparisons between strains based both on sequence analysis and on multilocus enzyme electrophoresis have shown that clonal population structure is not entirely destroyed (1, 9). It has been hypothesized that different clonal groupings may be associated with disease, but two studies using repetitive extragenic palindromic (REP)-PCR have reached different conclusions (7,16).In contrast to genome-based strategies such as REP-PCR, studies examining the association between specific virulence marker genes and disease have shown consistent associations. Virulence markers associated with disease include the presence of the gene cagA and the cag pathogenicity island (PAI), the s1/m1 and s1/m2 types of the vacuolating cytotoxin gene, vacA, and type 1 of the epithelial contact-induced gene, iceA. Another marker studied by us and others in populations in which cagA is ubiquitous is the length of the 3Ј portion of cagA: larger fragments appear to be more closely associated with disease (10, 20). Many of these markers are usually (although not invariably) associated with each other; for example, strains possessing the cag PAI usually have vacA with an s1 signal type (4). One possible explanation for this would be the remnant of an original clonal structure.The Cape-colored population of South Africa has a high rate of gastric adenocarcinoma (GCa), has a high prevalence of H. pylori infection, and harbors an interesting range of H. pylori strains (10, 12). Sequence analysis has suggested that the clonal population structure is stronger than for European and Asian...

Análise das impressões digitais de DNA e de fatores de virulência de linhagens de Helicobacter pylori

“…O RAPD tem capacidade discriminatória razoável e é eficaz para a genotipagem do H. pylori em larga escala, mas a estabilidade desse método deve ser levada em consideração, pois a reprodutibilidade interlaboratorial é muito baixa. BURUCOA et al (10) , KUIPERs et al 31 e KIDD et al (30) sugeriram que a técnica RAPD pode ser estável e reprodutível desde que esteja altamente padronizada, incluindo métodos criteriosos de preparo do DNA, o uso constante de polimerase termoestável, os volumes e concentrações dos reagentes que nunca devem variar e o procedimento adequado e padronizado do termociclador e do método de visualização das bandas. Neste estudo todas as reações de RAPD foram repetidas por 3 vezes em tempos diferentes e por operadores distintos para a garantia da reprodutibilidade.…”

“…A análise dos padrões de bandas obtidos foi incapaz de identificar associação entre agrupamento e potencial das doenças, estando de acordo com o observado por KIDD et al (30) . Outro estudo realizado nos Estados Unidos obteve resultados distintos onde, isolados clínicos provenientes de pacientes com UD foram agrupados (24) .…”

“…Diferentemente do observado por outro estudo, que sugere haver associação entre RAPD das impressões digitais de DNA e as variações de vacA e a presença de cag PAI, porém esse mesmo estudo falhou em associar a segregação dos agrupamentos com o genótipo de iceA e da região m do gene vacA (30) .…”

In Vitro Anti-Helicobacter pylori Activity of Chinese Chive (Allium tuberosum)

“…The acidified-WCC was centrifuged at 4,100 × g for 1 min in order to remove the insoluble substances formed by acidification, and was then kept at 4℃ (Trang et al, 2009). In addition, 4 strains frequently seen world-wide, NCTC11637 (derived from USA), SS1 (derived from Australia) (Hiratsuka et al, 2005), J99 (derived from USA) (Hiratsuka et al, 2005) and 26695 (derived from United Kingdom) (Kidd et al, 2001;Akopyants et al, 1998), were employed in this study.…”

“…The acidified-WCC was centrifuged at 4,100 × g for 1 min in order to remove the insoluble substances formed by acidification, and was then kept at 4℃ (Trang et al, 2009). In addition, 4 strains frequently seen world-wide, NCTC11637 (derived from USA), SS1 (derived from Australia) (Hiratsuka et al, 2005), J99 (derived from USA) (Hiratsuka et al, 2005) and 26695 (derived from United Kingdom) (Kidd et al, 2001;Akopyants et al, 1998), were employed in this study.All H. pylori strains were grown on Brucella agar plates supplemented with 10% horse serum (HS) and 1.4% agar at 37℃ under microaerobic conditions (10% CO 2 ) (Sanyo CO 2 incubator, Osaka, Japan) for 48 h, as described previously (Trang et al, 2009). Brucella medium supplemented with 10% HS (Brucella-serum medium) was used, unless otherwise stated.…”