DNA-level characterization of Helicobacter pylori strains from patients with overt disease and with benign infections in Bangladesh

Abstract

The complex relation between the genotype of Helicobacter pylori and its association with clinical outcome is not well understood. Studies in the West have showed that strains expressing certain virulence factors (vacAs1, vacAm1, and cagA) are associated with duodenal ulcer disease. However, the H. pylori genotype is known to vary with geographic region. In the present study, we compared several virulence markers (cagA, vacA, and iceA) and neutral markers (IS605, IS606, and IS608) in H. pylori strains isolated from 65 adult patients with peptic ulcer (PU) and 50 patients with nonulcer dyspepsia (NUD). PCR tests indicated that cagA is present in 75% of the strains from patients with PU compared to 55% in patients with NUD, and 80% of the isolates from patients with PU carried potentially toxigenic vacAs1 alleles of the vacuolating cytotoxin gene (vacA) compared to 60% in isolates from patients with NUD. However, no significant difference in any other virulence marker was observed in isolates from both groups. Phylogenetic analysis of the vacA middle region and the 5' end of the cagA gene indicates that Bangladeshi isolates are more closely related to H. pylori isolates from India and are different from isolates from East Asia.

FIG. 1.

Ethidium bromide-stained agarose gel of representative PCR amplicon using primers mentioned in Table 1. (A) Tests for presence and absence of cagA gene using CagA5 and CagA2 primers. (B) Tests for presence of cag PAI empty site using Luni1 and R5280 primers. (C) Tests for presence of vacAs1 (259-bp amplicon), vacAs2 (286-bp amplicon), and both vacAs1 and vacAs2 alleles (both 259- and 298-bp amplicon) using primers VA1-F and VA1-R. (D) Tests for presence of vacAm1 allele using VAm-F and VAm-R primers and vacAm2 allele using VA4-F and VA4-R primers. (E) PCR amplicon of iceA1 obtained with primers iceAF1 and M.Hpy1R. (F) PCR amplicon of iceA2 obtained with cysF and iceA2R primers. (G) PCR amplicon demonstrating a 94-bp deletion in iceA1, obtained with primers A1F673 and A1R1174. (H) PCR typing of isolates by 3′ end of cag gene.

FIG. 2.

Phylogenetic trees of sequences within the cagA gene and vacAm1 alleles. Sequences from non-Bangladeshi strains that were used here were from public databases, as indicated. (A) Phylogenetic tree based on informative 219-bp segment of cagA of H. pylori strains determined in this study. The tree was generated by using PHYLIP (Phylogeny Inference Package), version 3.5c, of J. Felsenstein. The strains used were as follows (GenBank accession number in parentheses): 1, CHINAR48 (AJ252983); 2, JAPANF32 (AJ239726); 3, JAPANGC4 (AF198484); 4, CHINAR29 (AJ252980); 5, CHINAR59 (AJ252986); 6, CHINAR47 (AJ252985); 7, HK77 (AF198485); 8, THI88-28 (AJ239722); 9, CHINAR29 (AJ252980); 10, HK97-42 (AJ239726); 11, CHINAR40 (AJ252982); 12, HK81 (AF198486); 13, DH93 (AY169298); 14, S-AFR19 (AF198470); 15, I-9A (AF202225); 16, PERU35B (AF202221); 17, DH140 (AY169293); 18, DH37 (AY169296); 19, India 18 (AF202224); 20, DH60 (AY169297); 21, India19 (AF202225); 22, DH29 (AY169295); 23, DH200 (AY169294); 24, DUT79 (AJ252970); 25, DUT25 (AJ252968); 26, Guatemala88 (AF198472); 27, Gambia4797 (AF198472); 28, South Africa19 (AF198470); 29, Peru4A (AF198477); 30, Gambia4659 (AF198468); 31, Dutch107 (AJ252963); 32, 26695 (AE000569); 33, G31 (AY169299); 34, DH102 (AY196292); 35, Peru34B (AF198475). Isolates from Bangladesh are shown in boldface type. (B) Phylogenetic trees of sequences within the vacAm1 alleles. Sequences from non-Bangladeshi strains used here were from public databases as indicated. The tree is based on a 648-bp segment of vacA gene containing the vacAm1 allele of Bangladeshi H. pylori strains determined in this study. The tree was generated by using PHYLIP (Phylogeny Inference Package), version 3.5c, of J. Felsenstein. The sequences of East Asian, European, and Indian vacAm1 alleles were taken from GenBank (GenBank accession number in parentheses): 1, GERM19 (AJ006967); 2, DH131 (AY16928); 3, DH92 (AY169289); 4, India48 (AF220112); 5, DH60 (AY169288); 6, India89 (AF220114); 7, India19 (AFAF220111); 8, India226 (AF220115); 9, DH153 (AY169286); 10, DH 9 (DH169290); 11, DH114 (AY169291); 12, India18 (AF220110); 13, India227 (AF220116); 14, India66 (AF220113); 15, India230 (AF220117); 16, 26695 (AE000598); 17, NCTC11638 (U07145); 18, Poland492 (AF097570); 19, NCTC11637 (AF049653); 20, Poland278 (AF097571); 21 Japan99 (AE001511); 22, Kenya AFN4847 (AF191644); 23, Chile-CH1 (AF479031); 24 Mex4467 (AF159855); 25, JapanF63 (AF049635); 26, China13 (AF035610); 27, ChinaR59 (AF035611); 28, JapanF52 (AF049631); 29, JapanF55 (AF049632); 30, JapanF72 (AF049651); 31, JapanF61 (AF049645); 32, JapanF42 (AF049626); 33, JapanF36 (AF049462); 34, JapanF64 (AF049647); 35, JapanF35 (AF049625); 36, JapanF45 (AF049628); 37, JapanF47 (AF049629); 38, JapanF57 (AF049634). Isolates from Bangladesh are shown in boldface type.