Helicobacter pylori in Pediatrics

This review summarizes important pediatric studies published from April 2011 up to March 2012. Proteomics profile of ulcerogenic Helicobacter pylori strains was defined in the most interesting study of the last year. The antigen stool test is becoming the "gold standard" in prevalence studies, and according to the last epidemiologic studies, the prevalence of H.?pylori infection in childhood is not decreasing any more in the developed world. The resistance rate of H.?pylori strains is high in children. Therefore, among other important issues concerning H.?pylori in pediatrics, guidelines published by ESPGHAN and NASPGHAN last year also recommended culture and susceptibility testing before first-line treatment in areas with high or unknown antibiotic resistance rates.     

Monocyte and Macrophage Killing of Helicobacter pylori: Relationship to Bacterial Virulence Factors

Background:  Helicobacter pylori infection is an important health problem, as it involves approximately 50% of the world's population, causes chronic inflammatory disease and increases the risk of gastric cancer development. H. pylori infection elicits a vigorous immune response, but this does not usually result in bacterial clearance. We have investigated whether the persistence of H. pylori in the host could be partly due to an inability of macrophages to kill this bacterium.
Materials and Methods:  Monocytes and macrophages isolated from the peripheral blood of normal human controls were infected in vitro with five H. pylori isolates. The isolates were characterized for known H. pylori virulence factors; vacuolating cytotoxin (VacA), the cag pathogenicity island ( cag PAI), urease, and catalase by Western blot and polymerase chain reaction analysis. The ability of primary human monocytes and macrophages to kill each of these H. pylori strains was then defined at various time points after cellular infection.
Results:  The five H. pylori strains showed contrasting patterns of the virulence factors. There were different rates of killing for the bacterial strains. Macrophages had less capacity than monocytes to kill three H. pylori strains. There appeared to be no correlation between the virulence factors studied and differential killing in monocytes.
Conclusions:  Primary human monocytes had a higher capacity to kill certain strains of H. pylori when compared to macrophages. The VacA, cag PAI, urease, and catalase virulence factors were not predictive of the capacity to avoid monocyte and macrophage killing, suggesting that other factors may be important in H. pylori intracellular pathogenicity.     

上海市某三甲医院幽门螺杆菌耐药性和毒力与其生物膜形成能力的相关性研究

为探讨复旦大学附属华东医院(以下简称本院)分离培养的幽门螺杆菌(Helicobacter pylori,H. pylori)的耐药性、毒力和感染特征与其生物膜形成能力的相关性,收集2014年12月-2015年6月于本院消化内镜中心的胃活检组织标本及相应临床病例资料,分离培养获得幽门螺杆菌,分析菌株的耐药性、毒力基因型、临床病例特征。结果显示,从胃活检组织样本中共分离培养28株幽门螺杆菌,对左氧氟沙星(levofloxacin,LEV)、甲硝唑(metronidazole,MTZ)和克拉霉素(clarithromycin,CLA)的耐药率分别为32%、75%和11%,未发现阿莫西林(amoxicillin,AMX)耐药。单一药物耐药17株(17/28,61%),双重耐药10株(10/28,36%)。毒力基因cagA、oipA和vacAs1检出率为100%,未检出vacAs2。基因型vacAs1m1占39%(11/28),vacAs1m2占61%(17/28);iceA1占54%(15/28),iceA2占21%(6/28),iceA1A2占25%(7/28);dupA^＋占36%(10/28)。28株菌株均能形成生物膜,但能力不尽相同。单因素及独立样本t检验分析显示,45～59岁、iceA1^＋dupA^－基因型和甲硝唑敏感菌株形成生物膜的能力较强。结果提示,本院分离的幽门螺杆菌对甲硝唑耐药率最高,双重耐药不容忽视。菌株主要毒力基因型为cagA、oipA、vacAs1m2。幽门螺杆菌的生物膜形成能力与患者年龄有关,45～59岁组较强;携带毒力基因iceA1的菌株生物膜形成能力强;dupA基因型及甲硝唑耐药与菌株生物膜形成呈负相关。     

Helicobacter pylori infection and childhood

Pediatric-based Helicobacter?pylori research continues to contribute significantly to our understanding of both clinical and pathophysiological aspects of this infection. Here, we review the published pediatric H.?pylori literature from April 2009-March 2010. Analysis of pediatric H.?pylori strains continues to suggest that cagA(+) and cagPAI competent strains are less prevalent than in adult isolates. Studies from the Middle East report a high H.?pylori prevalence and intrafamilial transmission. Data continue to show a lack of association between H.?pylori and recurrent abdominal pain of childhood, gastroesophageal reflux disease, and growth retardation. Recent probiotic trials have not shown a benefit on H.?pylori eradication in children, while sequential therapy remains an attractive therapeutic eradication strategy in children, which requires validation in different geographic regions.     

Helicobacter acinonychis: genetic and rodent infection studies of a Helicobacter pylori-like gastric pathogen of cheetahs and other big cats

Insights into bacterium-host interactions and genome evolution can emerge from comparisons among related species. Here we studied Helicobacter acinonychis (formerly H. acinonyx), a species closely related to the human gastric pathogen Helicobacter pylori. Two groups of strains were identified by randomly amplified polymorphic DNA fingerprinting and gene sequencing: one group from six cheetahs in a U.S. zoo and two lions in a European circus, and the other group from a tiger and a lion-tiger hybrid in the same circus. PCR and DNA sequencing showed that each strain lacked the cag pathogenicity island and contained a degenerate vacuolating cytotoxin (vacA) gene. Analyses of nine other genes (glmM, recA, hp519, glr, cysS, ppa, flaB, flaA, and atpA) revealed a approximately 2% base substitution difference, on average, between the two H. acinonychis groups and a approximately 8% difference between these genes and their homologs in H. pylori reference strains such as 26695. H. acinonychis derivatives that could chronically infect mice were selected and were found to be capable of persistent mixed infection with certain H. pylori strains. Several variants, due variously to recombination or new mutation, were found after 2 months of mixed infection. H. acinonychis ' modest genetic distance from H. pylori, its ability to infect mice, and its ability to coexist and recombine with certain H. pylori strains in vivo should be useful in studies of Helicobacter infection and virulence mechanisms and studies of genome evolution.     

Prevalence of Helicobacter pylori in Georgian patients with dyspepsia

BACKGROUND: Georgia has showed a high prevalence of peptic ulcer disease (PUD), but the prevalence of Helicobacter pylori in this country is practically unknown. The purpose of this study was to determine the prevalence of H. pylori and specific genotypes in different populations in Georgia. MATERIALS AND METHODS: We studied 62 patients from several hospitals in Tbilisi, Georgia. More than 55% of patients had PUD. We determined H. pylori presence as well as specific genotypes cagA and vacA by polymerase chain reaction. In addition, we studied serum samples from 94 healthy persons to determine H. pylori and CagA prevalence by ELISA. RESULTS: We found a high prevalence of H. pylori and CagA in the healthy population (70.2 and 57.4%, respectively) and a high prevalence of CagA among the H. pylori-positive persons (71.2%). Prevalence increased with age as reported in other countries (p = .05). Among symptomatic persons, we found nearly the same high prevalence of H. pylori (64.5%) as in the asymptomatic population. Furthermore, in symptomatic H. pylori patients, we found 65.0 and 67.5% prevalence of cagA and vacA, respectively. For 33 patients with PUD, 24 patients (72.7%) were H. pylori positive and 66.7% of them were cagA positive. In contrast, among the patients with non-ulcer dyspepsia (NUD), 16 (55.2%) were H. pylori positive and 62.5% of them were colonized with cagA-positive strains. H. pylori and cagA prevalence were not significantly different between PUD and patients with NUD. CONCLUSIONS: We confirmed that among individuals in Georgia, the prevalence of H. pylori is high and cagA-positive strains were equally present among H. pylori-positive patients with PUD and NUD and asymptomatic persons.     

Clinical relevance of the cagA,vacA and iceA genotypes of Helicobacter pylori in Brazilian clinical isolates

Infection with Helicobacter pylori strains harboring determinants of pathogenicity may lead to a strong inflammatory response in gastric mucosa. In this work, we examined the frequency of the cagA, vacA and iceA genotypes in H. pylori strains isolated from Brazilian patients and correlated these with the clinical manifestations. H. pylori was isolated from 165 patients [30 with non-ulcer dyspepsia cases (NUD); 93 peptic ulcer disease (PUD): 31 gastric ulcers (GU) and 62 duodenal ulcer disease (DU); 18 with erosive gastritis (EG); and 24 gastroesophageal reflux disease (GERD)]. Allelic variants of cagA, vacA and iceA were identified using the polymerase chain reaction. More than one H. pylori strain was detected in 28 cases (17%), and these were excluded from the statistical analysis. We were unable to confirm an association between iceA status and clinical outcome. There was a strong association between the genotype cagA-positive vacA s1 and PUD. However, logistic regression analysis showed that vacA s1 was the only predictive factor for PUD (OR=4.19; 95% CI 1.95-8.98). The presence of the less virulent strain vacA s2 was related to GERD (OR=8.59; 95% CI 2.85-25.91). Our results support the hypothesis that virulent strains may protect against the development of GERD.     

Comparative Transcriptomics of H. pylori Strains AM5, SS1 and Their hpyAVIBM Deletion Mutants: Possible Roles of Cytosine Methylation

Helicobacter pylori is an important human pathogen and one of the most successful chronic colonizers of the human body. H. pylori uses diverse mechanisms to modulate its interaction with the host in order to promote chronic infection and overcome host immune response. Restriction-modification genes are a major part of strain-specific genes present in H. pylori. The role of N(6) - adenine methylation in bacterial gene regulation and virulence is well established but not much is known about the effect of C(5) -cytosine methylation on gene expression in prokaryotes. In this study, it was observed by microarray analysis and RT-PCR, that deletion of an orphan C(5) -cytosine methyltransferase, hpyAVIBM in H. pylori strains AM5and SS1 has a significant effect on the expression of number of genes belonging to motility, adhesion and virulence. AM5ΔhpyAVIBM mutant strain has a different LPS profile and is able to induce high IL-8 production compared to wild-type. hpyAVIBM from strain 26695 is able to complement mutant SS1 and AM5 strains. This study highlights a possible significance of cytosine methylation in the physiology of H. pylori.     

Experimental colonization of mice by fresh clinical isolates of Helicobacter pylori is not influenced by the cagA status and the vacA genotype

Developing murine models of infection by Helicobacter pylori is quite useful but not all the strains are able to colonize the mouse. In order to study the influence of the two main virulence factors, CagA and VacA, on the establishment of H. pylori in mice, we have inoculated C57BL/6 mice with 15 strains randomly chosen among clinical strains freshly isolated from biopsy specimens of infected patients and five reference strains. Only six of the clinical strains and two of the reference strains could infect the animals regardless of the cagA status and the vacA genotype. We concluded that 40% of the H. pylori strains are able to infect mice and that the capacity of colonization is not influenced by the cagA status and the vacA genotype. These factors cannot be used to predict the success of an experimental infection.     

Effect of cagA status on the sensitivity of enzyme immunoassay in diagnosing Helicobacter pylori-infected children

Background. The aims of our study were twofold. First, we sought to evaluate in symptomatic children the influence of the Helicobacter pylori genotype on gastritis, abdominal pain, and circulating anti– H. pylori IgG antibodies (anti– H. pylori IgG) or pepsinogen A (PGA) and C (PGC). Additionally, we sought to assess anti– H. pylori IgG, PGA, and PGC patterns in a large cohort (N = 921) of asymptomatic children.
Materials and Methods. In 183 symptomatic children, H. pylori infection and the presence of gastritis were evaluated by histology. In a subgroup of 20 H. pylori –positive children, the H. pylori genotype was evaluated also by polymerase chain reaction. Nine hundred and twenty-one asymptomatic children, aged 11 to 14 years, were studied by anti– H. pylori IgG, PGA, and PGC serum determination.
Results. The infection was found in 33 of 183 symptomatic children; among the 20 H. pylori –positive children for which the H. pylori genotype was available, cag A was present or absent in equal percentages. H. pylori infection was associated with more severe gastritis and higher serum levels of anti– H. pylori IgG and PGC but not with abdominal pain. In infected children, higher levels of anti– H. pylori IgG and the presence of abdominal pain were associated with infections caused by cag A-positive strains. In the cohort of 921 asymptomatic children, raised levels of anti– H. pylori IgG, PGA, and PGC were found in approximately 5% of the cases.
Conclusions. Infection with cag A-positive H. pylori strains can be associated with increased frequency of reported abdominal pain and higher circulating levels of anti– H. pylori IgG. The serological assessment of H. pylori IgG using H. pylori antigens containing significant amounts of cagA protein may, therefore, underestimate the true prevalence of infection.     

Helicobacter pylori Interactions with Host Serum and Extracellular Matrix Proteins: Potential Role in the Infectious Process

Helicobacter pylori, a gram-negative spiral-shaped bacterium, specifically colonizes the stomachs of humans. Once established in this harsh ecological niche, it remains there virtually for the entire life of the host. To date, numerous virulence factors responsible for gastric colonization, survival, and tissue damage have been described for this bacterium. Nevertheless, a critical feature of H. pylori is its ability to establish a long-lasting infection. In fact, although good humoral (against many bacterial proteins) and cellular responses are observed, most infected persons are unable to eradicate the infection. A large body of evidence has shown that the interaction between H. pylori and the host is very complex. In addition to the effect of virulence factors on colonization and persistence, binding of specialized bacterial proteins, known as receptins, to certain host molecules (ligands) could explain the success of H. pylori as a chronically persisting pathogen. Some of the reported interactions are of high affinity, as revealed by their calculated dissociation constant. This review examines the binding of host proteins (serum and extracellular matrix proteins) to H. pylori and considers the significance of these interactions in the infectious process. A more thorough understanding of the kinetics of these receptin interactions could provide a new approach to preventing deeper tissue invasion in H. pylori infections and could represent an alternative to antibiotic treatment.     

Helicobacter pylori interactions with host serum and extracellular matrix proteins: potential role in the infectious process.

Helicobacter pylori, a gram-negative spiral-shaped bacterium, specifically colonizes the stomachs of humans. Once established in this harsh ecological niche, it remains there virtually for the entire life of the host. To date, numerous virulence factors responsible for gastric colonization, survival, and tissue damage have been described for this bacterium. Nevertheless, a critical feature of H. pylori is its ability to establish a long-lasting infection. In fact, although good humoral (against many bacterial proteins) and cellular responses are observed, most infected persons are unable to eradicate the infection. A large body of evidence has shown that the interaction between H. pylori and the host is very complex. In addition to the effect of virulence factors on colonization and persistence, binding of specialized bacterial proteins, known as receptins, to certain host molecules (ligands) could explain the success of H. pylori as a chronically persisting pathogen. Some of the reported interactions are of high affinity, as revealed by their calculated dissociation constant. This review examines the binding of host proteins (serum and extracellular matrix proteins) to H. pylori and considers the significance of these interactions in the infectious process. A more thorough understanding of the kinetics of these receptin interactions could provide a new approach to preventing deeper tissue invasion in H. pylori infections and could represent an alternative to antibiotic treatment.     

A simple multiplex PCR assay for diagnosing virulent Helicobacter pylori infection in human gastric biopsy specimens from subjects with gastric carcinoma and other gastro-duodenal diseases

AIM: To evaluate and develop a multiplex polymerase chain reaction (PCR) assay for diagnosing and specific identification of virulent Helicobacter pylori strains and their main virulence genes cagA, cagE, cagT, vacA and hrgA. METHODS AND RESULTS: Genomic DNA from 82 gastric tissues was screened. A master pool of all the ingredients of multiplex reaction was prepared for amplification. Amplicons were sequenced to confirm the amplification of each target genes. Multiplex PCR assay was able to detect all the five target genes in 81.7% and deletions in one or more loci among 18.3%. Genotype cagT +ve/hrgA +ve/cagA +ve/cagE +ve/vacAs1 +ve was more predominant in this study population (67.07%). hrgA, cagT, cagE and cagA genes were present in 100%, 92.7%, 85.4% and 81.7% of the subjects, respectively. The vacAs1 subtype had higher prevalence frequency in patients with overt gastrointestinal disease (78.57%) than with GERD (gastro-esophageal reflux disease) and NUD (non-ulcer dispepsia) (50%). CONCLUSIONS: The multiplex PCR assay developed herein was able to genotype H. pylori isolates based on the main virulence genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to identify H. pylori and the majority of their virulence gene markers by multiplex PCR assay represents a considerable advancement over other PCR-based methods for genotyping H. pylori from large population, and can be explored to gain insights at the genotypic variability exhibited by this pathogen.     

HIV protease inhibitors attenuate adherence of Candida albicans to epithelial cells in vitro

The cagA gene is a key marker for the Helicobacter pylori cag pathogenicity island (PAI), which may vary in composition in different strains with insertion sequence mediated interruptions and deletions of genes. While presence of cagA has been associated with increased risk for peptic ulcer disease and gastric cancer, the precise link with virulence is controversial. We investigated H. pylori from dyspeptics in one location in England (mid-Essex) with reference to the prevalence and distribution by age cohort of different cag PAI forms to determine if presence of the insertion element IS605 had a modifying effect on the severity of associated disease. H. pylori isolated from gastric biopsies over a 4-year period were screened by specific PCR assays for the presence of cagA, cagD, cagE and virD4 genes in the cag PAI, and for the presence of IS605 in the PAI and elsewhere in the genome. Most (68%) of the 166 isolates of H. pylori contained a PAI based on detection of cagA whereas 29% had no detectable PAI using multiple loci. The cagA+ genotype frequencies were similar in the peptic ulcer and non-ulcer dyspepsia-gastritis groups (79% vs. 74%) whereas frequencies in the NUD-oesophagitis and normal mucosa groups were lower (58%) but not significantly different (P>0.41). Genomic IS605 inserts were present at an overall frequency of 32% and were widely distributed with respect to patient age and disease severity. The combined cagA+/IS- strain genotype was common but not significantly associated with PUD compared to endoscopically normal mucosa (P> or =0.807). We concluded that presence of the IS605 element, whether in cagA+ or cagA- strains of H. pylori, did not systematically modify the severity of associated disease in the study population.     

Helicobacter pylori inhibits phagocytosis by professional phagocytes involving type IV secretion components

Gastric infections by Helicobacter pylori are characteristically associated with an intense inflammation and infiltration of mainly polymorphonuclear lymphocytes (PMNs) and monocytes. The inflammatory response by infiltrated immune cells appears to be a primary cause of the damage to surface epithelial layers and may eventually result in gastritis, peptic ulcer, gastric cancer and/or MALT-associated gastric lymphoma. Our analysis of the interaction between H. pylori and PMNs and monocytes revealed that H. pylori inhibits its own uptake by these professional phagocytes. To some degree, this effect resembles antiphagocytosis by Yersinia enterocolitica. Increasing numbers of bacteria associated per cell are more efficient at blocking their own engulfment. In H. pylori, bacterial protein synthesis is necessary to block phagocytic uptake, as shown by the time and concentration dependence of the bacteriostatic protein synthesis inhibitor chloramphenicol. Furthermore, H. pylori appears broadly to inhibit the phagocytic function of monocytes and PMNs, as infection with H. pylori abrogates the phagocytes' ability to engulf latex beads or adherent Neisseria gonorrhoeae cells. This antiphagocytic phenotype depends on distinct virulence (vir) genes, such as virB7 and virB11, encoding core components of a putative type IV secretion apparatus. Our data indicate that H. pylori exhibits an antiphagocytic activity that may play an essential role in the immune escape of this persistent pathogen.     

Potential role of CagA in the inhibition of T cell reactivity in Helicobacter pylori infections

The pathogenicity of chronic gastroduodenal diseases is very often related to Helicobacter pylori infections. Most H. pylori strains carry the cagA gene encoding an immunodominant 120- to 128-kDa protein which is considered a virulence marker. The majority of CagA-positive H. pylori isolates also produce a 95-kDa protein cytotoxin (VacA) causing vacuolation and degradation of mammalian cells. In our previous study we have shown that live H. pylori bacteria and their sonicates inhibit PHA-driven proliferation of human T lymphocytes. The H. pylori CagA and VacA proteins were suspected of a paralyzing effect of H. pylori on T cell proliferation. In this report, by using isogenic H. pylori mutant strains defective in CagA and VacA proteins, we determined that CagA is responsible for the inhibition of PHA-induced proliferation of T cells.     

Genomics of Helicobacter pylori

During this review period, we have definitely entered into the genomic era. The Helicobacter pylori studies reported here illustrate the use of most of the technologies currently available to globally interrogate the genome of a pathogen. Global analysis of the gene content of H. pylori strains gives insight into the extent of its genetic diversity and its in vivo evolution. Our understanding of the particularities of H. pylori as a gastric pathogen colonizing a unique niche has been improved by studies aimed at: (i) the identification of H. pylori-specific genes; (ii) the establishment of correlations between the presence of one or a group of genes (or proteins) with clinical outcome; and (iii) the analysis of global regulatory circuits or responses to the extracellular signals. The response of host cells to H. pylori infection will be developed in the chapter 'H. pylori and gastric malignancies' by Sepulveda and Coehlo. Despite our knowledge of the H. pylori genome, the function of about one third of its total proteins is still unknown. Functional genomics are straightforward approaches for the identification of new gene functions or metabolic pathways as well as for the understanding of cellular processes and the detection of new virulence factors. In silico studies combined with experimental work will undoubtedly continue to develop. To date, the expansion of proteomics with refinements in mass spectrometry technology has illustrated that through immunoproteomics and comparative studies, relevant novel antigens can be identified. Genomics not only provides invaluable information on H. pylori but also opens new perspectives for diagnostic or therapeutic applications.     

Characteristics of a clinical isolate of urease-negative Helicobacter pylori and its ability to induce gastric ulcers in Mongolian gerbils

BACKGROUND: We clinically obtained urease-negative mutant strains of Helicobacter pylori. The goal of this study was to investigate the ability of the urease-negative strain to colonize and subsequently damage the gastric mucosa in Mongolian gerbils. In addition, the genes encoding the urease production in the test strain were analyzed, and other genes encoding the virulence factors, cytotoxin-associated protein and vacuolating-cytotoxin were evaluated. MATERIALS AND METHODS: The character of urease-negative isolates of H. pylori was defined. The identification of H. pylori was confirmed by polymerase chain reaction (PCR). The H. pylori isolate was transfected into Mongolian gerbils as previously described, which were followed up to 42 weeks, and the changes in their gastric mucosa were examined histologically. RESULTS AND CONCLUSION: Fifteen Mongolian gerbils orally infected with 10(7) colony forming units of urease-negative H. pylori were killed at 4, 12, 24, 36 and 42 weeks (n = 3) after infection. Culture medium without urease-negative H. pylori was given to the Mongolian gerbils as control. H. pylori continued to exist in the subject's stomach and gastric ulceration was observed and compared with the control. Clinically obtained urease-negative H. pylori continued to exist for at least 42 weeks in the subject's stomach and it induced gastric ulcers. These data demonstrated that the urease in H. pylori was not a necessary factor in the formation of gastric ulcers in the Mongolian gerbil model.