Pylori Isolates Research Articles

Association of Malaysian Helicobacter pylori Virulence Polymorphisms with Severity of Gastritis and Patients’ Ethnicity

Polymorphisms of Helicobacter pylori cagA and vacA genes do exist and may contribute to differences in H. pylori infection and gastroduodenal diseases among races in the Malaysian population. This study was conducted to characterize the polymorphisms in H. pylori cagA and vacA in Malaysian population. A total of 110 H. pylori isolates were genotyped by PCR and sequenced for cagA and PCR-RFLP for vacA. East Asian cagA was predominantly detected (64.5%), whereas vacA s1m1 and s1m2 alleles were detected in 60.9 and 37.3% of strains, respectively. A statistical association between cagA type with patients' ethnicity (p < .0001) and age group >50 years old (p = .027) was identified. vacA alleles showed significant association with age group >50 years old (p = .017) and increased neutrophil activity in gastric mucosa (p = .028 and p = .016 for moderate and marked activity, respectively). Further identification of vacA polymorphism revealed that 84% of strains from Malays and Indians showed one RFLP pattern (RFLP-1), whereas more than one RFLP patterns (RFLP-2, 3, 4, 5, 6, and 8) were predominantly observed in strains from Chinese (82%) (p < .0001). Increasing severity of gastric inflammation was observed in gastric mucosa infected with strains carrying RFLP-2, 3, 4, 5, and 6 (p = .037). About 86.6% of H. pylori strains with East Asian cagA were vacA RFLP-2, 3, 4, 5, 6, and 8, and 88% of Western cagA strains were vacA RFLP-1 (p < .0001). Chinese and Indians are susceptible to different virulence genotypes of H. pylori, whereas Malays showed a mixed virulence genotypes. Marked differences in the polymorphisms of cagA and vacA were observed among strains in Malaysian population. This provides a new insight into the pathogenicity of H. pylori in multiracial population.

Is Helicobacter pylori antibiotic resistance surveillance needed and how can it be delivered?

Most patients are prescribed Helicobacter pylori treatment without culture and antibiotic susceptibility testing, as current guidance recommends that patients with recurrent dyspepsia should be tested for H. pylori using a non-invasive breath or faecal antigen test. To determine the prevalence of H. pylori antibiotic resistance in patients attending endoscopy in England and Wales, and the feasibility of an antibiotic resistance surveillance programme testing. We tested the antibiotic susceptibility of H. pylori isolates from biopsy specimens from 2063 of 7791 (26%) patients attending for endoscopy in Gloucester and Bangor, and 339 biopsy specimens sent to the Helicobacter Reference Unit (HRU) in London. Culture and susceptibility testing was undertaken in line with National and European methods. Helicobacter pylori were cultured in 6.4% of 2063 patients attending Gloucester and Bangor hospitals. Resistance to amoxicillin, tetracycline and rifampicin/rifabutin was below 3% at all centres. Clarithromycin, metronidazole and quinolone resistance was significantly higher in HRU (68%, 88%, 17%) and Bangor isolates (18%, 43%, 13%) than Gloucester (3%, 22%, 1%). Each previous course of these antibiotics is associated with an increase in the risk of antibiotic resistance to that agent [clarithromycin: RR = 1.5 (P = 0.12); metronidazole RR = 1.6 (P = 0.002); quinolone RR = 1.8 (P = 0.01)]. Helicobacter pylori infection is now uncommon in dyspeptic patients at endoscopy. A surveillance system is feasible and necessary to inform dyspepsia management guidance. Clinicians should take a thorough antibiotic history before prescribing metronidazole, clarithromycin or levofloxacin for H. pylori.

Heterogeneity of Helicobacter pylori

Although many physicians view Helicobacter pylori strains as a homogenous groupof organisms, it has become increasingly clear that populations in humans are highly diverse. This heterogeneity can be analyzed at two different levels: genotypic variation among strains and variations in H. pylori populations within an individual host. Genotypic variation includes point mutations in conserved genes (e.g. ureC), variation in the gene order on physical maps, mosaicism in conserved genes (e.g. vacAs1a), non-conserved genes (e.g. cagA) and extragenetic elements (e.g. IS605). Population differences include the observations that humans can be simultaneously infected with two or more H. pylori strains and that a single strain may represent a cluster of closely related organisms (a 'quasispecies'). The presence of multiple organisms within a host may occur as a result of recombination events leading to genetic shift, whereas ongoing mutation within a strain can lead to the formation of quasispecies by genetic drift. Over recent years it has become increasingly clear that observations on the fundamental biology of H. pylori have considerable clinical relevance. Several genotypic markers (e.g. cagA, vacA, s1a and iceA1) are associated with an increased risk of disease. Also, the multiplicity of infection and quasispecies indicates that analysis of a single H. pylori isolate is inaccurate for defining the genotype of H. pylori strains present in a patient. Global assays, such as serology, are more suitable. The aim of this paper is to review the general phenomenon of diversity in H. pylori and to describe particular heterogeneities that are related to clinical outcome.

Gemifloxacin Can Partially Overcome Quinolone Resistance of H. pylori with gyrA Mutation in Taiwan

The levofloxacin resistance caused by gyrA gene mutation is rising rapidly to limit wide application for Helicobacter pylori eradication. We investigated whether gemifloxacin has a superior antimicrobial activity to levofloxacin against H. pylori. Forty-four consecutive clinical H. pylori isolates with levofloxacin resistance and 80 randomly selected levofloxacin-sensitive controls were tested for gemifloxacin sensitivity by E-test. The resistance to levofloxacin or gemifloxacin was defined as minimal inhibitory concentration (MIC) > 1 mg/L. The clinical features and GyrA mutation patterns checked by direct sequencing were also analyzed to assess its association with the H. pylori gemifloxacin resistance. All levofloxacin-sensitive H. pylori isolates were sensitive to gemifloxacin. Eight strains (18.2%) resistant to levofloxacin could be still sensitive to gemifloxacin. Gemifloxacin achieved a 5-time lower in MIC levels against levofloxacin-resistant isolates. Nearly all levofloxacin-resistant isolates (97.7%, 43/44) had GyrA mutation at amino acid position 87 or 91. Double mutation sites may play dual roles in quinolone resistance, as N87K plus H57Y or D91N plus V77A mutations showed high-level resistance to both quinolones; whereas D91Y plus A97V or D91N plus A97V mutations showed low level levofloxacin resistance to become sensitive to gemifloxacin. In H. pylori isolates with single N87K, D91Y or D91N mutation, near 20% was gemifloxacin-sensitive and levofloxacin-resistant. The gemifloxacin-resistant rate of H. pylori was higher in patients with gastric ulcer than in those without (p <.05). Gemifloxacin is superior to levofloxacin in antimicrobial activity against clinical H. pylori isolates, and even overcome some levofloxacin resistance.

Reinfection rate of Helicobacter pylori after eradication treatment: a long-term prospective study in Japan

We previously reported that the reinfection rate with Helicobacter pylori in Japan was low despite a high prevalence of infection. In the present study, we extended our previous work to more accurately determine the reinfection rate. We enrolled 1625 patients (219 women and 1406 men, mean age 50.8 years) who had received H. pylori eradication therapy. After documentation of eradication, bacterial culture and urea breath test were carried out yearly. H. pylori strains were analyzed by using random amplification of polymorphic DNA fingerprinting. A total of 1609 patients were followed for up to 12.5 years (mean 4.7 years); H. pylori became re-positive in 26 patients. In 13 of the 26 patients, H. pylori became positive at the first-year follow up. Stored H. pylori isolates were available for analysis from ten of the 13 patients; four of the isolates were genetically different from the initial strain, but the other six were identical to the initial strain. In the other 13 patients, H. pylori became positive at later follow up (mean 4.8 years; range 1.8-8.0 years). In all of the four of these patients whose isolates could be analyzed, the H. pylori strains were different from the initial strain. Assuming that reinfection occurred in the four patients positive for different strains of H. pylori at the first-year follow up and in the 13 positive at later follow up, the reinfection rate was 0.22% per year. When probable recrudescence (H. pylori positivity with identical strains) was excluded, the reinfection rate of H. pylori in this Japanese population was very low, but we note that reinfection can occur over many years.

Sitafloxacin resistance in Helicobacter pylori isolates and sitafloxacin-based triple therapy as a third-line regimen in Japan

The third-line treatment regimen for Helicobacter pylori after failure of clarithromycin- and metronidazole-based therapies is not yet established. Sitafloxacin (STX) is a quinolone that possesses potent in vitro activity against H. pylori. In this study, the susceptibility of H. pylori isolates to STX was examined and the efficacy of STX-based triple therapy as a third-line regimen was evaluated. STX showed minimum inhibitory concentrations (MICs) of ≤1μg/mL against all 100 H. pylori isolates, and the MIC90 (MIC for 90% of the organisms) of STX was 5 log2 dilutions lower than that of levofloxacin (LVX). The MIC50 (MIC for 50% of the organisms) of STX against gyrA mutants was 0.12μg/mL and was significantly lower than that of LVX (8μg/mL). The activity of STX at pH 5.5 was significantly less than that at pH 7.0. In the clinical trial, 28 patients with two eradication failures were treated with STX-based triple therapy [rabeprazole 10mg twice daily (b.i.d.), amoxicillin 750mg b.i.d. and STX 100mg b.i.d. for 7 days]. The eradication rate was 75% using intention-to-treat analysis and 80% using per-protocol analysis. Two gyrA mutant strains were eradicated. Amongst participants, a low pepsinogen I/II ratio was associated with successful eradication. These results suggest that STX could be active against most clinical H. pylori isolates and that STX-based triple therapy is a promising and safe third-line therapy.

Mucoid Helicobacter pylori Isolates with Fast Growth under Microaerobic and Aerobic Conditions

Helicobacter pylori is microaerobic and turns into coccoid under aerobic conditions. In this study, two mucoid strains, A and D, were isolated from gastric biopsies which grew well on blood agar after 24-hour incubation under aerobic as well as microaerobic conditions. The aim of this study was to identify these strains and compare their growth under aerobic and microaerobic conditions with that of control H. pylori. The two isolates A and D were identified as H. pylori according to microscopic morphology, urease, catalase and oxidase tests. Their growth under humidified aerobic and microaerobic conditions was compared with that of control H. pylori which grew only under microaerobic conditions. They were further identified by amplification of 16S rRNA, vacA alleles, cagA and ureAB genes by PCR. Their susceptibility to current antimicrobials was also examined. The strains A and D produced mucoid colonies under aerobic and microaerobic conditions after 24-hour, exhibiting the typical spiral morphology of H. pylori. The results of urease, catalase and oxidase tests were positive. Sequencing of amplified products showed 99-100% homology with those of the reference H. pylori strains in GenBank. Both strains exhibited resistance to the high concentrations of antimicrobials. This study reports the isolation of two mucoid strains of H. pylori with confluent growth under aerobic and microaerobic conditions. It appears that production of exopolysaccharide (EXP) could serve as a physical barrier to reduce oxygen diffusion into the bacterial cell and uptake of antibiotics. EXP protected the mucoid H. pylori isolates against stressful conditions, the result of which could be persistence of bacterial infection in the stomach.

Recovery of Helicobacter Pylori from Sewage in Basrah Governorate and its Confirmation by 16S rRNA

A total of one hundred and fifty six sewage samples were collected from three regions in Basrah governorate namely Five miles, Al-Jame’eyat and Al-Ashar from February 2008 to February 2009. This study examined the possibility that sewage harbors Helicobacter pylori. Because of existing methods for the direct culture of H. pylori from sewage are unsuccessful, a modified Columbia urea agar and Monophasic-Diphasic culture set up have been exploited. As a result 76 isolates were identified as H. pylori using conventional techniques. Polymerase chain reaction technique was used to confirm the identity of H. pylori isolates using primers for 16S rRNA and Ure A gene. Only 6 isolates out of 76 gave positive results for the identification by PCR using 16S rRNA, and all of them were from Five miles region. Also, these 6 isolates have an identical biochemical profile. On other hand the 76 H. pylori isolates have not been confirmed by PCR using Ure A gene. These findings suggest that sewage may represent a reservoir for this bacterium and reveal that conventional tests for the identification of H. pylori are not sensitive enough to rely on. The fact that all the six isolates were from one region and one of them showed an RFLP pattern similar to those from clinical and drinking water may be of an epidemiologic significance.

The status of antimicrobial resistance of Helicobacter pylori in Eastern Azerbaijan, Iran: comparative study according to demographics

Helicobacter pylori-associated infections are extremely common in Iran, but few data about antibiotic sensitivity of H. pylori are available for this region. The purpose of this study was to investigate the prevalence of resistance in isolates against commonly used antibiotics in Eastern Azerbaijan, Iran, and the dependence of prevalence on the sex and age of patients. H. pylori isolates were collected by culture from gastric biopsies. Antibiotic susceptibility of isolates was determined by use of the disk agar diffusion test, and the minimum inhibitory concentration of clarithromycin was established by use of the Etest. A total 395 of biopsy specimens were studied; 112 samples of H. pylori were isolated (28.3%), 55 (49%) from males and 57 (51%) from females. The prevalence of resistance to clarithromycin, metronidazole, erythromycin, amoxicillin, ciprofloxacin, rifampin, nitrofurantoin, and tetracycline were 16 (14.3%), 86 (76.8%), 29 (26.0%), 32 (28.6%), 37 (33.0%), 32 (28.6%), 13 (11.6%), and 21 (18.7%), respectively. Antimicrobial resistance was not statistically significantly associated with sex or age. Furthermore, the prevalence of resistance to metronidazole was high and that to clarithromycin was reasonable, consistent with reported low success in H. pylori treatment in this area. Therefore, continuous surveillance of antibiotic resistance of H. pylori is essential.

H. pylori clinical isolates have diverse babAB genotype distributions over different topographic sites of stomach with correlation to clinical disease outcomes

BackgroundIntragenomic recombination between babA and babB mediates antigenic variations and may help H. pylori colonization. This study determined whether variable genotypes of babA and babB correlate to different clinical disease outcomes, and can distribute over the different gastric niches.ResultsThis study enrolled 92 clinical strains (45 from peptic ulcer, 27 from gastritis, and 20 from gastric cancer) to detect whether the babA and babB are at locus A or B by PCR reactions using the primers designed from the upstream and variable region of the babA and babB genes. Four genotypes of babA and babB (A B, AB B, A AB, AB AB) were found. The distribution of the 4 genotypes in 92 clinical strains was significantly different among patients with different gastric diseases (p < 0.05). The isolates from gastric cancer patients had a higher rate of AB AB genotype than those from non-cancer patients (40.0% vs. 9.7%, p < 0.05). The AB AB genotype was associated with a higher intensity of intestinal metaplasia (p < 0.05), but did not correlate with a higher inflammation and colonization density in gastric histology (p > 0.05). Besides, the study enrolled 19 patients to verify whether variable genotypes of babAB existed in the different gastric niches. Among the patients infected with more than one babAB genotypes over antrum and corpus, there were higher rate of genotypes as A B or AB AB in isolates from antrum than in those from corpus (75.0 % vs. 16.7%, p < 0.05).ConclusionsThe H. pylori isolate with the AB AB genotype correlates with an increased gastric cancer risk, and colonize in an antrum predominant manner.

Study ofHelicobacter pylorigenotype status in saliva, dental plaques, stool and gastric biopsy samples

To compare genotype of Helicobacter pylori (H. pylori) isolated from saliva, dental plaques, gastric biopsy, and stool of each patient in order to evaluate the mode of transmission of H. pylori infection. This cross-sectional descriptive study was performed on 300 antral gastric biopsy, saliva, dental plaque and stool samples which were obtained from patients undergoing upper gastrointestinal tract endoscopy referred to endoscopy centre of Hajar hospital of Shahrekord, Iran from March 2010 to February 2011. Initially, H. pylori strains were identified by rapid urease test (RUT) and polymerase chain reaction (PCR) were applied to determine the presence of H. pylori (ureC) and for genotyping of voculating cytotoxin gene A (vacA) and cytotoxin associated gene A (cagA) genes in each specimen. Finally the data were analyzed by using statistical formulas such as Chi-square and Fisher's exact tests to find any significant relationship between these genes and patient's diseases. P < 0.05 was considered statistically significant. Of 300 gastric biopsy samples, 77.66% were confirmed to be H. pylori positive by PCR assay while this bacterium were detected in 10.72% of saliva, 71.67% of stool samples. We were not able to find it in dental plaque specimens. The prevalence of H. pylori was 90.47% among patients with peptic ulcer disease (PUD), 80% among patients with gastric cancer, and 74.13% among patients with none ulcer dyspepsia (NUD) by PCR assay. The evaluation of vacA and cagA genes showed 6 differences between gastric biopsy and saliva specimens and 11 differences between gastric and stool specimens. 94.42% of H. pylori positive specimens were cagA positive and all samples had amplified band both for vacA s and m regions. There was significant relationship between vacA s1a/m1a and PUD diseases (P = 0.04), s2/m2 genotype and NUD diseases (P = 0.05). No statically significant relationship was found between cagA status with clinical outcomes and vacA genotypes (P = 0.65). The evaluation of vacA and cagA genes showed 6 differences between gastric biopsy and saliva specimens and 11 differences between gastric and stool specimens. Regard to high similarity in genotype of H. pylori isolates from saliva, stomach and stool, this study support the idea which fecal- oral is the main route of H. pylori transmission and oral cavity may serve as a reservoir for H. pylori, however, remarkable genotype diversity among stomach, saliva and stool samples showed that more than one H. pylori genotype may exist in a same patient.

Helicobacter pylori isolates from Greek children express type 2 and type 1 Lewis and α1,6-glucan antigens in conjunction with a functional type IV secretion system

Helicobacter pylori infection is often acquired in childhood and can persist for life. Previous studies in adult patients have shown that H. pylori isolates from North American and European hosts express predominantly type 2 Lewis x (Le(x)) and Le(y) epitopes, while Asian strains have the capacity to express type 1 Le(a) and Le(b) structures. In order to understand the influence of environmental and host factors on the expression of Le antigens, we analysed 50 Greek H. pylori isolates from symptomatic children. Both CagA-positive and -negative strains were evaluated. The expression of Le antigens was determined by whole-cell indirect ELISA (WCE), and LPS profiles were assessed by gel electrophoresis and immunoblotting. Occurrence of Le(x) and/or Le(y) antigens was confirmed in 35 of the isolates (70 %) while 15 of the isolates were non-typable. It was found that 11 of the paediatric isolates had the propensity to express type 1 Le(b) blood-group antigen (22 %), a feature relatively uncommon in H. pylori isolates from adults. One strain expressed both Le(b) and Le(a) antigens. The majority of the isolates (49/50, 98 %) expressed α1,6-glucan, an antigenic non-Le determinant present in the outer core region of H. pylori LPS. All Le(x)- and Le(y)-expressing strains also carried a functional cag pathogenicity island-encoding a type IV secretion system, capable of translocating CagA protein, as well as the vacAs1 allele, suggesting that Le(x) and Le(y) epitopes may aid the persistence of more aggressive strains. No association between bacterial virulence characteristics and the histopathological observations was evident.

Helicobacter pylori antibiotic resistance in Brazil: clarithromycin is still a good option

The antibiotic susceptibility is the cornerstone for the eradication therapies of Helicobacter pylori. To evaluate the prevalence of primary resistance of H. pylori was evaluated in an urban Brazilian population. H. pylori isolates were obtained from patients submitted to an upper gastrointestinal endoscopy for the evaluation of dyspeptic symptoms. Biopsies from antrum, corpus and fundus were taken to determine the antibiotic susceptibility of H. pylori isolates. The minimal inhibitory concentration of furazolidone and bismuth were routinely determined by agar dilution method and the minimal inhibitory for amoxicillin, clarithromycin, tetracycline, levofloxacin, and metronidazole were routinely determined with the E-test. Fifty-four patients were included. In vitro antimicrobial susceptibility of H. pylori strains were obtained from 39 patients. Resistance to metronidazole was detected in 20 patients (51%), to clarithromycin in 3 patients (8%), to levofloxacin in 9 patients (23%) and to bismuth in 2 patients (5%). There was no observed resistance to amoxicillin, tetracycline or furazolidone. Due to the low amoxicillin and clarithromycin resistance observed in this study, therapies using these antimicrobials remain appropriated first-line H. pylori therapy.

Characterization and Functional Activity of Murine Monoclonal Antibodies Specific for α1,6-Glucan Chain of Helicobacter pylori Lipopolysaccharide

The outer core region of H. pylori lipopolysaccharide (LPS) contains α1,6-glucan previously shown to contribute to colonizing efficiency of a mouse stomach. The aim of the present study was to generate monoclonal antibodies (mAbs) specific for α1,6-glucan and characterize their binding properties and functional activity. BALB/c mice were injected intraperitoneally with 10(8) formalin-fixed H. pylori O:3 0826::Kan cells 3× over 56 days to achieve significant titer. Anti-α1,6-glucan-producing hybridomas were screened by indirect ELISA using purified H. pylori O:3 0826::Kan LPS. One clone, 1C4F9, was selected for further characterization. The specificities of mAbs were determined by indirect and inhibition ELISA using structurally defined H. pylori LPS and synthetic oligosaccharides, and whole-cell indirect ELISA (WCE) of clinical isolates. They were further characterized by indirect immunofluorescent (IF) microscopy and their functional activity in vitro determined by serum bactericidal assays against wild-type and mutant strains of H. pylori. The generated anti-α1,6-glucan IgM, 1C4F9, has demonstrated an excellent specificity for the glucan chain containing 5 to 6 α1,6-linked glucose residues and showed surface accessibility by IF microscopy with H. pylori cells adherent to gastric adenocarcinoma cells monolayers. Of 38 isolates from Chile, 17 strains reacted with antiglucan mAbs in WCE (OD450 ≥ 0.2). Bactericidal activity was observed against selective wild-type and mutant H. pylori strains exhibiting OD450 values of ≥ 0.45 in WCE. Anti-α1,6-glucan mAbs could have potential application in typing and surveillance of H. pylori isolates as well as offer insights into structural requirements for the development of LPS-based vaccine against H. pylori infections.

Analysis of cagA in Helicobacter pylori Strains from Colombian Populations with Contrasting Gastric Cancer Risk Reveals a Biomarker for Disease Severity

Helicobacter pylori infection is a risk factor for the development of gastric cancer, and the bacterial oncoprotein CagA contributes to gastric carcinogenesis. We analyzed H. pylori isolates from persons in Colombia and observed that there was marked variation among strains in levels of CagA expression. To elucidate the basis for this variation, we analyzed sequences upstream from the CagA translational initiation site in each strain. A DNA motif (AATAAGATA) upstream of the translational initiation site of CagA was associated with high levels of CagA expression. Experimental studies showed that this motif was necessary but not sufficient for high-level CagA expression. H. pylori strains from a region of Colombia with high gastric cancer rates expressed higher levels of CagA than did strains from a region with lower gastric cancer rates, and Colombian strains of European phylogeographic origin expressed higher levels of CagA than did strains of African origin. Histopathologic analysis of gastric biopsy specimens revealed that strains expressing high levels of CagA or containing the AATAAGATA motif were associated with more advanced precancerous lesions than those found in persons infected with strains expressing low levels of CagA or lacking the AATAAGATA motif. CagA expression varies greatly among H. pylori strains. The DNA motif identified in this study is associated with high levels of CagA expression, and may be a useful biomarker to predict gastric cancer risk. These findings help to explain why some persons infected with cagA-positive H. pylori develop gastric cancer and others do not.

EPIYA motif patterns among Cuban Helicobacter pylori CagA positive strains.

It is known that polymorphisms in C-terminal region of CagA influence gastric disease development on Helicobacter pylori infection. Additionally, the geographic distribution of these polymorphisms has been associated with the appearance of more severe gastroduodenal pathologies. Objective. To determine the CagA phosphorylation motifs pattern (EPIYA pattern) in Cuban H. pylori isolates, and to study its association with patient´s pathologies. DNAs from 95 H. pylori cagA-positive strains were used to amplify the 3´ variable region of cagA gene by PCR using two different strategies. Additionally, new primers were designed to identify either Western or Eastern CagAEPIYA motiftype by PCR. To confirm the PCR results, PCR products from 14 representative isolates were purified and sequenced. The distribution of the EPIYA motif found was: 2 AB (2.1 %), 1 AC (1.1 %), 1 BC (1.1 %), 70 ABC (73.6 %), 19 ABCC (20 %), and 2 ABCCC (2.1 %). Sequencing analysis confirmed the PCR classification in the 14 studied strains and showed three strains with unusual nucleotide sequences, not reported before. Distribution of the EPIYA-ABC pattern was equivalent in all pathologies (78.9 % in gastric ulcer, 72.5 % in duodenal ulcer and 72.2 % in non-ulcer dyspepsia). The PCR results using the new primers confirmed that all studied strains carried the Western CagA type. No specific EPIYA motif was associated with peptic ulcer. This is the first report that shows EPIYA motif distribution in H. pylori isolates from the Caribbean region.

EPIYA motif patterns among Cuban Helicobacter pylori CagA positive strains

Introduction. It is known that polymorphisms in C-terminal region of CagA influence gastric disease development on Helicobacter pylori infection. Additionally, the geographic distribution of these polymorphisms has been associated with the appearance of more severe gastroduodenal pathologies.Objective. To determine the CagA phosphorylation motifs pattern (EPIYA pattern) in Cuban H. pylori isolates, and to study its association with patient’s pathologies. Materials and methods. DNAs from 95 H. pylori cagA-positive strains were used to amplify the 3’ variable region of cagA gene by PCR using two different strategies. Additionally, new primers were designed to identify either Western or Eastern CagA EPIYA motif type by PCR. To confirm the PCR results, PCR products from 14 representative isolates were purified and sequencedResults. The distribution of the EPIYA motif found was: 2 AB (2.1 %), 1 AC (1.1 %), 1 BC (1.1 %), 70 ABC (73.6 %), 19 ABCC (20 %), and 2 ABCCC (2.1 %). Sequencing analysis confirmed the PCR classification in the 14 studied strains and showed three strains with unusual nucleotide sequences, not reported before. Distribution of the EPIYA-ABC pattern was equivalent in all pathologies (78.9 % in gastric ulcer, 72.5 % in duodenal ulcer and 72.2 % in non-ulcer dyspepsia).Conclusion. The PCR results using the new primers confirmed that all studied strains carried the Western CagA type. No specific EPIYA motif was associated with peptic ulcer. This is the first report that shows EPIYA motif distribution in H. pylori isolates from the Caribbean region.

Infection with Helicobacter pylori strains lacking dupA is associated with an increased risk of gastric ulcer and gastric cancer development

Recently, dupA was reported as a new virulence factor in Helicobacter pylori, but its association with gastroduodenal disorders and its mode of action are still unclear. Here, an association of the dupA status with different disease groups was determined and a biological explanation for the observed associations was tested. In total, 216 H. pylori isolates were obtained from 232 presumed H. pylori-infected patients. A positive association was observed between the occurrence of duodenal ulcer (DU) and the presence of dupA [odds ratio (OR) 24.2; 95 % confidence interval (CI) 10.6-54.8]. In addition, an inverse association between the occurrence of gastric cancer (GC) [OR 0.16; 95 % CI 0.05-0.47] and gastric ulcer (GU) [OR 0.34; 95 % CI 0.16-0.68] with the presence of dupA was observed. A putative explanation for the observed associations might be a more corpus-located infection (pan-gastritis) by the dupA-positive strains due to their increased acid resistance. Indeed, a strong association between dupA-positive H. pylori isolated from gastritis patients and in vitro acid resistance was observed (P<0.05). The observed higher acid resistance of the dupA-positive strains suggests that these strains are adapted to a stomach with high gastric acid output. This may in part explain the observed associations, as an increased gastric acid output is thought to be typical for an antrum-predominant H. pylori infection and, whilst this is associated with an increased risk of DU formation, it also decreases the risk for the genesis of GUs and GC.

Mutations of Helicobacter pylori Associated with Fluoroquinolone Resistance in Korea

Fluoroquinolone resistance of Helicobacter pylori is known to be dependent on mutations in the QRDR of gyrA. This study was performed to investigate the distribution of gyrA point mutations and to evaluate the impact of the mutations on second-line H. pylori eradication therapy. After H. pylori isolation from gastric mucosal specimens, fluoroquinolone resistance was examined using the agar dilution method. DNA sequencing of the QRDR of gyrA was performed in 89 fluoroquinolone-resistant and 27 fluoroquinolone-susceptible isolates. Transformation experiments were performed to confirm mutations in the resistant strains. The eradication rates of moxifloxacin-containing triple therapy were evaluated depending on the resistance of fluoroquinolone. The gyrA mutations were detected in 75.3% (55 of 73 strains) of the primary resistant strains and 100% (16 strains) of the secondary resistant strains. The most common mutations were Asp-91 (36.0%) and Asn-87 (33.7%). The MIC values in the transformed strains differed depending on the gyrA mutations, N87, and D91. Six patients with fluoroquinolone-resistant strains received moxifloxacin-containing triple therapy as the second-line therapy, and two of three patients with Asn-87 mutations (66.7%) failed in the eradication. By contrast, three patients with Asp-91 mutations had successful eradication treatment. Fluoroquinolone resistance of H. pylori was caused by gyrA Asn-87 and Asp-91 point mutations. The Asn-87 mutation seems to be an important determinant of failure of fluoroquinolone-containing triple eradication therapy based on eradication results.

PP-005 Clarithromycin resistance assessment in Helicobacter pylori isolates by using 23S rRNA gene molecular markers

PP-005 Clarithromycin resistance assessment in Helicobacter pylori isolates by using 23S rRNA gene molecular markers