Discovery of unique African Helicobacter pylori CagA-multimerization motif in the Dominican Republic.

BACKGROUND: Helicobacter pylori (H. pylori) colonizes the human stomach and is a major cause of peptic ulcer disease and gastric cancer. However, although the prevalence of H. pylori is high in Africa, the incidence of gastric cancer is low, and this phenomenon is called to be African enigma. The CagA protein produced by H. pylori is the most studied virulence factor. The carcinogenic potential of CagA is associated with the Glu-Pro-Ile-Tyr-Ala (EPIYA) patterns and CagA-multimerization (CM) motifs. AIM: To better understand the EPIYA patterns and CM motifs of the cagA gene. METHODS: Gastric mucosal biopsy specimens were obtained from 258 patients with dyspepsia living in the Dominican Republic, from which 120 H. pylori strains were cultured. After the bacterial DNA extraction, the EPIYA pattern and CM motif genotypes were determined using a polymerase chain reaction-based sequencing. The population structure of the Dominican Republic strains was analyzed using multilocus sequence typing (MLST). Peptic ulcer disease and gastric cancer were identified via endoscopy, and gastric cancer was confirmed by histopathology. Histological scores of the gastric mucosa were evaluated using the updated Sydney system. RESULTS: All CagA-positive strains carried the Western-type CagA according to the identified EPIYA patterns. Twenty-seven kinds of CM motifs were observed. Although the typical Western CM motif (FPLKRHDKVDDLSKVG) was observed most frequently, the typical East Asian CM motif (FPLRRSAAVNDLSKVG) was not observed. However, "FPLRRSAKVEDLSKVG", similar to the typical East Asian CM motif, was found in 21 strains. Since this type was significantly more frequent in strains classified as hpAfrica1 using MLST analysis (P = 0.034), we termed it Africa1-CM (Af1-CM). A few hpEurope strains carried the Af1-CM motif, but they had a significantly higher ancestral Africa1 component than that of those without the Af1-CM motif (P = 0.030). In 30 cagA-positive strains, the "GKDKGPE" motif was observed immediately upstream of the EPIYA motif in the EPIYA-A segment, and there was a significant association between strains with the hpAfrica1 population and those containing the "GKDKGPE" motif (P = 0.018). In contrast, there was no significant association between the CM motif patterns and histological scores and clinical outcomes. CONCLUSION: We found the unique African CM motif in Western-type CagA and termed it Africa1-CM. The less toxicity of this motif could be one reason to explain the African enigma.

Comparative study between Helicobacter pylori and host human genetics in the Dominican Republic.

BACKGROUND: Helicobacter pylori, a bacterium that infects the human stomach, has high genetic diversity. Because its evolution is parallel to human, H. pylori is used as a tool to trace human migration. However, there are few studies about the relationship between phylogeography of H. pylori and its host human. METHODS: We examined both H. pylori DNA and the host mitochondrial DNA and Y-chromosome DNA obtained from a total 119 patients in the Dominican Republic, where human demography consists of various ancestries. DNA extracted from cultured H. pylori were analyzed by multi locus sequence typing. Mitochondrial DNA and Y-chromosome DNA were evaluated by haplogroup analyses. RESULTS: H. pylori strains were divided into 2 populations; 68 strains with African group (hpAfrica1) and 51 strains with European group (hpEurope). In Y-chromosomal haplogroup, European origin was dominant, whereas African origin was dominant both in H. pylori and in mtDNA haplogroup. These results supported the hypothesis that mother-to-child infection is predominant in H. pylori infection. The Amerindian type of mtDNA haplogroup was observed in 11.8% of the patients; however, Amerindian type (hspAmerind) of H. pylori was not observed. Although subpopulation type of most hpAfrica1 strains in Central America and South America were hybrid (hspWAfrica/hpEurope), most Dominican Republic hpAfrica1 strains were similar to those of African continent. CONCLUSIONS: Genetic features of H. pylori, mtDNA, and Y haplogroups reflect the history of colonial migration and slave trade in the Dominican Republic. Discrepancy between H. pylori and the host human genotypes support the hypothesis that adaptability of hspAmerind H. pylori strains are weaker than hpEurope strains. H. pylori strains in the Dominican Republic seem to contain larger proportion of African ancestry compared to other American continent strains.

Five alternative Helicobacter pylori antibiotics to counter high levofloxacin and metronidazole resistance in the Dominican Republic.

The prevalence of Helicobacter pylori resistance to levofloxacin and metronidazole was high in the Dominican Republic. We used two-fold agar dilution method to determine the minimum inhibitory concentration of five alternative antibiotics in 63 Dominican strains. We also assessed the genetic mutations associated with the antibiotic resistance using next-generation sequencing. We revealed that all 63 strains were sensitive towards sitafloxacin, furazolidone, and rifabutin. In contrast, the prevalence of rifaximin and garenoxacin resistance were high (82.5% and 34.9%, respectively). Patients more than or equal to 60 years old had the highest risk of double-antibiotic resistance (7/9, 77.8%, OR = 31.5, P = 0.009) and garenoxacin resistances (8/9, 88.9%, OR = 45.33, P = 0.002) with an increasing risk simultaneously by age (P = 0.004, r = 0.357). Almost all rifaximin resistant strains possessed multiple mutations with more than three mutations within rpoB including the most frequent novel mutations of S352L, I2726L, and V2465A. There was a significant association between vacA genotype and rifaximin resistance (P = 0.042). Among 23 levofloxacin-resistant strains, 82.6% (19/23, P <0.001) were also resistant to garenoxacin, and 39.1% (9/23) had a high minimal inhibitory concentration ≥8 µg/mL with positive trend correlation (P = <0.001, r = 0.84). Among 19 garenoxacin resistant strains, 16 (84.2%) contained mutations at D91 and N87 of gyrA. In conclusion, sitafloxacin, rifabutin, and furazolidone might be considered as alternative antibiotics to be included in H. pylori eradication regimen in regions with high prevalence of levofloxacin and metronidazole resistance, such as the Dominican Republic.

Interleukin-17C in Human Helicobacter pylori Gastritis.

The interleukin-17 (IL-17) family of cytokines (IL-17A to IL-17F) is involved in many inflammatory diseases. Although IL-17A is recognized as being involved in the pathophysiology of Helicobacter pylori-associated diseases, the role of other IL-17 cytokine family members remains unclear. Microarray analysis of IL-17 family cytokines was performed in H. pylori-infected and uninfected gastric biopsy specimens. IL-17C mRNA was upregulated approximately 4.5-fold in H. pylori-infected gastric biopsy specimens. This was confirmed by quantitative reverse transcriptase PCR in infected and uninfected gastric mucosa obtained from Bhutan and from the Dominican Republic. Immunohistochemical analysis showed that IL-17C expression in H. pylori-infected gastric biopsy specimens was predominantly localized to epithelial and chromogranin A-positive endocrine cells. IL-17C mRNA levels were also significantly greater among cagA-positive than cagA-negative H. pylori infections (P = 0.012). In vitro studies confirmed an increase in IL-17C mRNA and protein levels in cells infected with cagA-positive infections compared to cells infected with either cagA-negative or cag pathogenicity island (PAI) mutant. Chemical inhibition of IκB kinase (IKK), mitogen-activated protein extracellular signal-regulated kinase (MEK), and Jun N-terminal kinase (JNK) inhibited induction of IL-17C proteins in infected cells, whereas p38 inhibition had no effect on IL-17C protein secretion. In conclusion, H. pylori infection was associated with a significant increase in IL-17C expression in human gastric mucosa. The role of IL-17C in the pathogenesis of H. pylori-induced diseases remains to be determined.

Clarithromycin-Based Triple Therapy is Still Useful as an Initial Treatment for Helicobacter pylori Infection in the Dominican Republic.

AbstractHelicobacter pylori antibiotic susceptibility in the Dominican Republic has not been monitored. We assessed H. pylori antibiotic susceptibility in the Dominican Republic, and analyzed H. pylori mutations associated with antibiotic resistance. We recruited 158 dyspeptic patients in Santo Domingo and used agar dilution to test susceptibility to five antibiotics. Polymerase chain reaction-based sequencing was used to assess gyrA, gyrB, rdxA, frxA, and 23S rRNA mutations; next-generation sequencing was used to identify other metronidazole resistance-associated genes. Among 64 H. pylori strains isolated, we identified two (3.1%), one (1.6%), and no strains with clarithromycin, amoxicillin, and tetracycline resistance, respectively. Moreover, high frequency of metronidazole resistance (53/64, 82.8%) was observed, whereas levofloxacin resistance is emerging (23/64, 35.9%). We identified many rdxA and frxA mutations in metronidazole-resistant strains, but no synergistic effect was apparent. We revealed novel mutations in dppA, dppB, fdxA, and fdxB, irrespective of rdxA and frxA mutations. Novel mutations at Ser-14 of trx1 and Arg-221 of dapF were associated with different levels of metronidazole resistance. Most levofloxacin-resistant strains had a substitution at Asn-87 of gyrA, including the strain with the highest levofloxacin resistance, whereas only three substitutions were found at Ser-479 of gyrB with no synergistic effect. Besides the 23S rRNA A2142G mutation, we observed another mutation at T1958G in both clarithromycin-resistant strains. We confirmed high metronidazole and levofloxacin resistance associated with genetic mutations in the Dominican Republic. However, prevalence of clarithromycin resistance was low, suggesting that standard clarithromycin-based triple therapy remains useful as initial treatment of H. pylori infection.

Differences in interleukin 8 expression in Helicobacter pylori-infected gastric mucosa tissues from patients in Bhutan and the Dominican Republic.

The outcomes of Helicobacter pylori infection vary geographically. H pylori strains, disease presentation, and environments differ markedly in Bhutan and Dominican Republic. The aims were to compare the strains, histology, and expression of interleukin (IL) 8 and IL-10 from gastric mucosa from the 2 countries. H pylori status was assessed by the combination of rapid urease test, culture, and histology. Histology was evaluated using the updated Sydney System, and cytokines in gastric biopsies were measured using real-time polymerase chain reaction (PCR). There were 138 subjects from Bhutan and 155 from Dominican Republic. The prevalence of H pylori infection was 65% and 59%, respectively. The genotype of cagA was predominantly East Asian type in Bhutan versus Western type in Dominican Republic. Gastritis severity was significantly higher in H pylori-infected subjects from Bhutan than those from Dominican Republic. IL-8 expression by H pylori infection was 5.5-fold increased in Bhutan versus 3-fold in Dominican Republic (P < .001); IL-10 expression was similar. IL-8 expression levels among H pylori-infected cases tended to be positively correlated with polymorphonuclear leucocyte and monocyte infiltration scores in both countries. IL-8 expression among those with grade 2 and 3 polymorphonuclear leucocyte and monocyte infiltration was significantly higher in Bhutan than in Dominican Republic. The difference in IL-8 expression in the 2 countries is reflected in the different disease pattern between them. Whether the dominant factor is differences in H pylori virulence, in host-H pylori-environmental interactions, genetic factors or all remains unclear. However, severity of inflammation appears to be a critical factor in disease pathogenesis. We compared IL-8 messenger RNA levels between the high gastric cancer risk country, Bhutan (mainly East Asian-type H pylori), and the lower gastric cancer risk country, Dominican Republic (mainly Western-type H pylori).

Helicobacter pylori from gastric cancer and duodenal ulcer show same phylogeographic origin in the Andean region in Colombia.

BACKGROUND: A recent report has shown that the phylogenetic origin of Helicobacter pylori based on multi-locus sequence typing (MLST) was significantly associated with the severity of gastritis in Colombia. However, the potential relationship between phylogenetic origin and clinical outcomes was not examined in that study. If the phylogenetic origin rather than virulence factors were truly associated with clinical outcomes, identifying a population at high risk for gastric cancer in Colombia would be relatively straightforward. In this study, we examined the phylogenetic origins of strains from gastric cancer and duodenal ulcer patients living in Bogota, Colombia. METHODS: We included 35 gastric cancer patients and 31 duodenal ulcer patients, which are considered the variant outcomes. The genotypes of cagA and vacA were determined by polymerase chain reaction. The genealogy of these Colombian strains was analyzed by MLST. Bacterial population structure was analyzed using STRUCTURE software. RESULTS: H. pylori strains from gastric cancer and duodenal ulcer patients were scattered in the phylogenetic tree; thus, we did not detect any difference in phylogenetic distribution between gastric cancer and duodenal ulcer strains in the hpEurope group in Colombia. Sixty-six strains, with one exception, were classified as hpEurope irrespective of the cagA and vacA genotypes, and type of disease. STRUCTURE analysis revealed that Colombian hpEurope strains have a phylogenetic connection to Spanish strains. CONCLUSIONS: Our study showed that a phylogeographic origin determined by MLST was insufficient for distinguishing between gastric cancer and duodenal ulcer risk among hpEurope strains in the Andean region in Colombia. Our analysis also suggests that hpEurope strains in Colombia were primarily introduced by Spanish immigrants.

Virulence genes of Helicobacter pylori in the Dominican Republic.

Although the incidence of gastric cancer in the Dominican Republic is not high, the disease remains a significant health problem. We first conducted a detailed analysis of Helicobacter pylori status in the Dominican Republic. In total, 158 patients (103 females and 55 males; mean age 47.1±16.2 years) were recruited. The status of H. pylori infection was determined based on four tests: rapid urease test, culture test, histological test and immunohistochemistry. The status of cagA and vacA genotypes in H. pylori was examined using PCR and gene sequencing. The overall prevalence of H. pylori infection was 58.9 %. No relationship was found between the H. pylori infection rate and the age range of 17-91 years. Even in the youngest group (patients aged <29 years), the H. pylori infection rate was 62.5 %. Peptic ulcer was found in 23 patients and gastric cancer was found in one patient. The H. pylori infection rate in patients with peptic ulcer was significantly higher than that in patients with gastritis (82.6 versus 54.5 %, P<0.01). The cagA-positive/vacA s1m1 genotype was the most prevalent (43/64, 67.2 %). Compared with H. pylori-negative patients, H. pylori-positive patients showed more severe gastritis. Furthermore, the presence of cagA was related to the presence of more severe gastritis. All CagA-positive strains had Western-type CagA. In conclusion, we found that H. pylori infection is a risk factor for peptic ulcer in the Dominican Republic. Patients with cagA-positive H. pylori could be at higher risk for severe inflammation and atrophy.

Association between Helicobacter pylori cagA-related genes and clinical outcomes in Colombia and Japan.

BACKGROUND: Specific genotypes of several virulence factors of Helicobacter pylori (eg, cagA-positive, vacA s1, oipA "on" and babA-positive) have been reported to be predictors of severe clinical outcomes. Importantly, the presence of these genotypes correlates with each other. We hypothesized that novel virulence genes correlate with the presence of cagA. Therefore, we aimed to find novel candidate virulence genes that correlate with cagA and examined the association of these genes with clinical outcomes in Colombian and Japanese populations. METHODS: cagA-associated genes were selected based on previous H. pylori genome microarray data. A total of 343 strains (174 from Colombia and 169 from Japan) were examined for the status of cagA, vacA, and candidate genes by polymerase chain reaction and dot blot. RESULTS: Microarray data showed that 9 genes were significantly correlated with the presence of cagA. Among the 9 genes, the functions of 4 were known, and we selected these 4 genes as candidate genes (hp0967, jhp0045, jhp0046, and jhp0951). The prevalences of cagA, vacA s1/m1 genotype, and hp0967 were significantly higher in Japan than Colombia, whereas those of jhp0045 and jhp0046 were more prevalent in Colombia than Japan. The prevalences of jhp0045 and jhp0046 in cagA-positive cases of gastric cancer were significantly higher than those from gastritis in Colombia (P = 0.015 and 0.047, respectively). In contrast, the prevalence of 4 candidate genes was independent of clinical outcomes in Japan. CONCLUSIONS: jhp0045 and jhp0046 might be novel markers for predicting gastric cancer in cagA-positive cases in Colombia, but not in Japan.

homB status of Helicobacter pylori as a novel marker to distinguish gastric cancer from duodenal ulcer.

The hom family of Helicobacter pylori outer-membrane proteins, especially the homB gene, has been suggested as a novel virulence factor; however, the clinical association and function of this gene are still unclear. We evaluated the presence of the homA, homB, and cagA genes in 286 strains isolated from patients in the U.S. and Colombian populations (126 with gastritis, 96 with duodenal ulcer, and 64 with gastric cancer) by PCR. The results were compared with the clinical presentation and gastric injury. The prevalence of the homB gene was significantly higher in strains isolated from gastric-cancer patients (71.9%) than in those from duodenal ulcer patients (52.1%) (P = 0.012). In a multivariate analysis, the presence of the cagA gene significantly increased the risk for developing gastric cancer and duodenal ulcer, with the presence of the homB gene acting as a factor that could distinguish gastric cancer from duodenal ulcer (adjusted odds ratio, 3.033; 95% confidence interval, approximately 1.37 to approximately 6.73). cagA status was correlated with homB status (r = 0.323; P < 0.01). A histological analysis showed that cagA status was associated with inflammation and atrophy both in the antrum and in the corpus, while homB status was associated with inflammation and atrophy in the corpus. homB gene status might be susceptible to gastric-cancer development such that the homB gene is used as a factor for discriminating the risk of gastric cancer from that of duodenal ulcer.

Proteomic Helicobacter pylori biomarkers discriminating between duodenal ulcer and gastric cancer.

Protein patterns of 129 Helicobacter pylori strains isolated from Korean and Colombian patients suffering from duodenal ulcer or gastric cancer were analyzed by the high-throughput methodology SELDI-TOF-MS. Eighteen statistically significant candidate biomarkers discriminating between the two clinical outcomes were selected by using the Mann-Whitney test. Three biomarker proteins were purified and identified as a neutrophil-activating protein NapA (HU HPAG1_0821), a RNA-binding protein (HPAG1_0813), and a DNA-binding histone-like protein HU, respectively (jhp0228). These novel biomarkers can be used for development of diagnostic assays predicting the evolution to gastric cancer in H. pylori-infected patients.

Relationship between Helicobacter pylori hopQ genotype and clinical outcome in Asian and Western populations.

BACKGROUND AND AIMS: Outer membrane proteins of Helicobacter pylori mediate important pathogen-host interactions such as colonization, adhesion and the inflammatory response. hopQ genotypes have been suggested to be associated with increased risk of peptic ulcer. The aim of this study was to test the relation of hopQ genotype to H. pylori-related disease and histological changes in Asian and Western countries. METHODS: hopQ genotype, cagA status and vacA genotype of H. pylori isolated from patients from Asian and Western countries were determined and the results were compared with the clinical presentation and gastric histology. RESULTS: Most Asian strains possessed virulent genotypes (hopQ type I, vacA s1-m1 and cagA-positive). In Western countries, hopQ type I genotype was significantly linked with vacA s1 and m1 genotypes and cagA-positive status. Inflammatory cell infiltration and atrophy scores were significantly higher in patients with hopQ type I strains than those with type II in Western patients. However, the hopQ type I genotype was not associated with an increased risk for peptic ulcer or gastric cancer, and had no additive effects to vacA genotypes or cagA-positive status. CONCLUSION: The expression of multiple putative virulence factors in Asian strains likely explains the relatively high incidence of clinical outcomes including gastric cancer compared with other parts of the world. Although hopQ genotype did not improve the predictive value above other genotyping for development of H. pylori-related gastroduodenal diseases, the hopQ genotype might be able to add a useful virulence marker for gastroduodenal diseases.