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。     

Cell surface characteristics of Helicobacter pylori

Abstract Helicobacter pylori is an important gastroduodenal pathogen of humans. Immunological and structural studies have been performed on the phospholipids, lipopolysaccharides (LPS) and some surface proteins of H. pylori strains. H. pylori LPS has, in general, low immunological activity and this property may aid the survival of this chronic infection. Nevertheless, H. pylori LPS has been found to influence the quality of gastric mucin and to stimulate pepsinogen secretion, thereby contributing to gastric disease. A number of putative adhesins of the bacterium have been described. This multiplicity of adhesins may reflect that H. pylori adherence is a multi-step process involving different interactions, and that different adhesins may mediate adherence to various sites in gastric tissue.     

Metronidazole resistance and virulence factors in Helicobacter pylori as markers for treatment failure in a paediatric population

The eradication rate obtained using the classical triple therapy containing metronidazole, amoxicillin and bismuth citrate, was determined in 57 paediatric patients with digestive disorders, according to the susceptibility to metronidazole of the Helicobacter pylori strains (determined by agar dilution) and the cagA and vacA status (determined by PCR). Eradication was obtained in 38 out of 43 patients (88.3%) infected by H. pylori with metronidazole MIC < or = 2 mg l(-1), in 3 out of 6 patients (50%) when MIC was 4-8 mg l(-1) and in 4 out of 8 patients (50%) when MIC was > 8 mg l(-1). Among patients infected with cagA+ and cagA- strains an eradication rate of 75% (6/8) and 75% (18/24) was found, and 50% (3/6) and 80% (21/26) among vacA s1- and vacA s2-infected subjects (P > 0.05). H. pylori eradication depends on the susceptibility of the strain to metronidazole, being higher in patients infected with susceptible H. pylori. However, according to our data the cagA or vacA status was not an important factor in treatment failure in the eradication of H. pylori.     

Importance of the host genetic background on immune responses to Helicobacter pylori infection and therapeutic vaccine efficacy

In order to investigate the role of host factors in Helicobacter pylori infection and immunity, two different strains of inbred mice, C57BL/6 and BALB/c, were infected with a standard H. pylori strain, SS1. A month later, infected mice were immunized orally with whole-cell lysates of H. pylori SS1 and cholera toxin on days 1, 3, 6, 30, and 54. Ten days after the last immunization, mice were sacrificed and the stomach was collected to assess H. pylori colonization density by quantitative culture. H. pylori SS1 colonization was significantly greater in C57BL/6 than in BALB/c (P<0.02 and P<0.003 at 2 and 13 weeks post-inoculation, respectively). Colonization in C57BL/6 persisted at equivalent levels for 13 weeks but the colonization density in BALB/c decreased significantly during this period. In contrast to the pattern of bacterial colonization, antibody responses following H. pylori SS1 infection were greater in BALB/c than in C57BL/6, suggesting that host factors may modulate the immune responses to H. pylori infection. Following therapeutic immunization, H. pylori colonization in BALB/c mice was also significantly reduced (P<0.03), while no significant differences in bacterial density were observed in C57BL/6. These observations collectively demonstrate the great importance of host factors in H. pylori infection and the development of effective immune responses.     

Adhesion of water stressed Helicobacter pylori to abiotic surfaces

AIM: The main aim of this work was to study and compare the adhesion of water exposed Helicobacter pylori to six different substrata and correlate any changes in morphology, physiology, ability to form aggregates and cultivability when in the planktonic or in the sessile phase. METHODS AND RESULTS: The number of total cells adhered for different water exposure times and modifications in the cell shape were evaluated using epifluorescence and scanning electron microscopy, and physiology assessed using Syto9 and propidium iodide (PI) cellular uptake. All abiotic surfaces were rapidly colonized by H. pylori, and colonization appeared to reach a steady state after 96 h with levels ranging from 2.3 x 10(6) to 3.6 x 10(6) total cells cm(-2). Cell morphology was largely dependent on the support material, with spiral bacteria, associated with the infectious form of H. pylori, subsisting in a higher percentage on nonpolymeric substrata. Also, sessile bacteria were generally able to retain the spiral shape for longer when compared with planktonic bacteria, which became coccoid more quickly. The formation of large aggregates, which may act as a protection mechanism against the negative impact of the stressful external environmental conditions, was mostly observed on the surface of copper coupons. However, Syto9 and PI staining indicates that most of H. pylori attached to copper or SS304 have a compromised cell membrane after only 48 h. Cultivability methods were only able to detect the bacteria up to the 2 h exposure-time and at very low levels (up to 500 CFU cm(-2)). CONCLUSIONS: The fact that the pathogen is able to adhere, retain the spiral morphology for longer and form large aggregates when attached to different plumbing materials appeared to point to pipe materials in general, and copper plumbing in particular, as a possible reservoir of virulent H. pylori in water distribution systems. However, the Syto9/PI staining results and cultivability methods indicate that the attached H. pylori cells quickly enter in a nonviable physiological state. SIGNIFICANCE AND IMPACT OF THE STUDY: This represents the first study of H. pylori behaviour in water-exposed abiotic surfaces. It suggests that co-aggregation with the autochthonous heterotrophic consortia present in water is necessary for a longer survival of the pathogen in biofilms associated to drinking water systems.     

Prevalence and risk factors for Helicobacter pylori infection in Chinese populations

Background:  The prevalence of Helicobacter pylori is higher in developing countries. The aim of this study was to investigate the prevalence and risk factors of H. pylori infection in areas with high prevalence of gastric cancer in Jiangsu Province, China.
Methods:  A prospective epidemiologic survey of H. pylori infection was accomplished in a natural population of 1457 individuals in Xiangshui and Gaoyou counties, Jiangsu Province, China. Questionnaires and laboratory tests for H. pylori infection (¹³C-urea breath test and serum IgG antibodies to H. pylori ) were used and performed, respectively.
Result:  Among 1371 subjects who completed questionnaires and H. pylori detection, 851 (62%) were H. pylori positive. The prevalence reached a peak at the age of 30–40 years (67%). There was no sex difference. The annual family income level was shown to be positively correlated with the risk of H. pylori infection. The prevalence of H. pylori infection was also associated with family size, education level, and several diet-related factors, such as the number of times cooked rice and potatoes eaten per week, and a family history of stomach diseases. Compared to nonsymptomatic individuals, people with dyspeptic symptoms (nausea, vomiting, and belching) presented a low prevalence of H. pylori infection. No association between H. pylori prevalence and smoking or drinking was found. Using multivariate logistic regression analysis, annual family income and education level were the independent predictors for H. pylori infection.
Conclusion:  High prevalence of H. pylori infection was found in areas with a high risk of gastric cancer and was related to several risk factors. The underlying mechanisms need to be further investigated.     

Potential animal models of Helicobacter pylori infection in immunological and vaccine research

Abstract Presence of Helicobacter pylori in the human gastric mucosa is associated with chronic gastritis and promotes the formation of peptic ulceration. Furthermore, long-term gastritis caused by the bacteria represents an increased risk of developing gastric cancer. Much controversy remains about the pathogenic mechanisms by which H. pylori can induce disease because of the limitations of animal models and the relevance of in vitro observations to the in vivo disease process. Studies of putative pathogenic factors such as induction of inflammatory mediators and immune evasion are required to understand how to design a vaccine against the infection. Vaccine adjuvants, delivery systems and therapeutic vaccination are likely to be the areas of major progress in the future. Data related to immunological aspects and vaccine development in potential animal models are reviewed.     

益生菌与幽门螺杆菌对黏膜屏障的影响及其机制的研究进展

消化系黏膜屏障对致病菌有防御功能,但是在根除幽门螺杆菌(H. pylori)过程中会导致黏膜屏障的破坏。大量研究表明,益生菌通过修复上皮细胞以及细胞间连接、减少黏液分泌和炎症反应以及缓解消化道菌群紊乱等方式修复黏膜屏障。本文主要综述幽门螺杆菌对消化系黏膜屏障的损害及其机制,以及益生菌对黏膜屏障损伤的保护及修复机制研究的进展。     

Identification of glycoprotein receptors within the human salivary proteome for the lectin‐like BabA and SabA adhesins of Helicobacter pylori by fluorescence‐based 2‐D bacterial overlay

Because gastric infection by Helicobacter pylori takes place via the oral route, possible interactions of this bacterium with human salivary proteins could occur. By using modified 1‐ and 2‐D bacterial overlay, binding of H. pylori adhesins BabA and SabA to the whole range of salivary proteins was explored. Bound salivary receptor molecules were identified by MALDI‐MS and by comparison to previously established proteome maps of whole and glandular salivas. By use of adhesin‐deficient mutants, binding of H. pylori to MUC7 and gp‐340 could be linked to the SabA and BabA adhesins, respectively, whereas binding to MUC5B was associated with both adhesins. Binding of H. pylori to the proline‐rich glycoprotein was newly detected and assigned to BabA adhesin whereas the SabA adhesin was found to mediate binding to newly detected receptor molecules, including carbonic anhydrase VI, secretory component, heavy chain of secretory IgA1, parotid secretory protein and zinc‐α₂‐glycoprotein. Some of these salivary glycoproteins are known to act as scavenger molecules or are involved in innate immunity whereas others might come to modify the pathogenetic properties of this organism. In general, this 2‐D bacterial overlay technique represents a useful supplement in adhesion studies of bacteria with complex protein mixtures.     

High rate of Helicobacter pylori reinfection in children and adolescents

AIMS: Primary Helicobacter pylori infection occurs predominantly in childhood. The aims of this study were to establish the rate of H. pylori reinfection after successful eradication in children and adolescents and to determine the risk factors associated with reinfection. PATIENTS AND METHODS: This retrospective study involved 45 children (20 girls, 25 boys) who met the following criteria: eradication of H. pylori confirmed at least 4 weeks after the completion of therapy, and the search for reinfection at least one year after control of eradication of H. pylori. Demographic data, socioeconomic status and living conditions were recorded. RESULTS: Forty-five children aged 1.2-17.6 years (median, 10.9 years) at the time of H. pylori treatment were reviewed 1 to 9 years after H. pylori eradication. Eight children (18%) had been reinfected (5.4% to 6% per patient-year). Six of 25 (24%) children older than 10 years at the time of diagnosis became reinfected. None of the studied risk factors was associated with reinfection. However, having a sibling younger than 5 years was found in four of seven (57%) reinfected children versus five of 24 (21%) nonreinfected children (p = .08). CONCLUSION: Children become reinfected more frequently than adults. Adolescents become reinfected, whereas acquisition of primary H. pylori infection occurs predominantly in early childhood. Close contact with young children, especially siblings, younger than 5 years could be a more important risk factor than the age of the patient at the time of treatment for the high rate of reinfection in childhood.     

Helicobacter pylori disulphide reductases: role in metronidazole reduction

Disulphide reductases play an important role in maintaining intracellular redox potential. Three disulphide reductase activities were identified in Helicobacter pylori, which used dithiobis-2-nitrobenzoic acid, glutathione or l-cystine and ferredoxin as substrates. The kinetic parameters of these activities were determined and it was demonstrated that the reductase activities were inhibited by the presence of metronidazole. Substrate competition experiments served to show inhibition of metronidazole reduction by dithiobis-2-nitrobenzoic acid, glutathione and ferredoxin in lysates from metronidazole susceptible and resistant matched pairs of strains. The study demonstrated that the activities of three disulphide reductases were modulated by the presence of metronidazole, and that metronidazole reduction was inhibited by the presence of disulphide reductase substrates.     

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.     

The role of interleukin-17 in the Helicobacter pylori induced infection and immunity

Background: Helicobacter pylori infection is regarded as the major cause of various gastric diseases and induces the production of several cytokines including interleukin‐17 (IL‐17) recently recognized as an important player in the mammalian immune system. Objective: This review deals with the role of IL‐17 on the H. pylori‐induced infection and immunity in humans and experimental animals. Results: H. pylori infection increases IL‐17 in the gastric mucosa of humans and experimental animals. In humans, IL‐17 induces the secretion of IL‐8 by activating the ERK 1/2 MAP kinase pathway and the released IL‐8 attracts neutrophils promoting inflammation. IL‐23 is increased in patients with H. pylori‐related gastritis and regulates IL‐17 secretion via STAT3 pathway. Studies in H. pylori‐infected mice indicate that IL‐17 is primarily associated with gastric inflammation. The early events in the immune response of immunized and challenged mice include the recruitment of T cells and the production of IL‐17. Neutrophil attracting chemokines are released, and the bacterial load is considerably reduced. IL‐17 plays a dual role in infection and vaccination. In infection, T regulatory cells (Tregs) suppress the inflammatory reaction driven by IL‐17 thereby favoring bacterial persistence. Immunization produces Helicobacter‐specific memory T‐helper cells that can possibly alter the ratio between T‐helper 17 and Treg responses so that the IL‐17‐driven inflammatory reaction can overcome the Treg response leading to bacterial clearance. Conclusion: IL‐17 plays an important role in H. pylori‐related gastritis and in the reduction of Helicobacter infection in mice following immunization.     

Virulence-related DNA sequences and adherence patterns in strains of enteropathogenic Escherichia coli

The presence of the genes for Escherichia coli adherence factor (EAF), attaching and effacing lesion (eae) and bundle-forming pili (bfp) in 72 strains identified as enteropathogenic E. coli (EPEC) by slide agglutination was evaluated using hybridization and PCR. The adherence property of these strains was assayed using 3h HeLa cells adherence assay. The results obtained indicated that virulence-associated genes were present in 65% of the strains but only ten (13.9%) isolates were positive for all the three markers (typical EPEC), 37 (51.4%) isolates carried either one or two of these determinants (atypical EPEC) and the remaining 25 (34.7%) were negative for all these genes. In vitro adherence assay showed that 44 (61.1%) strains adhered to HeLa cells with a defined pattern, 13 (18.1%) isolates adhered loosely with no definite pattern and the remaining 15 (20.8%) were non-adherent. Analysis of the results showed a statistically significant association between the presence of the virulence-related genes with adherence of the strains with a defined pattern (P相似文献   

Cellular mucosal defense during Helicobacter pylori infection: a review of the role of glutathione and the oxidative pentose pathway

Helicobacter pylori is the primary cause of gastritis and peptic ulcer disease and is known to infect greater than 50% of the world's population. It is also known to lead to the onset of gastric cancer and unless treated, lasts throughout life in most individuals. Mouse models of H. pylori infection have improved our ability to study this organism and can be used to investigate the host mucosal response to the infection, particularly the early events postinoculation. Previous studies have shown that H. pylori infection leads to an increased production of reactive oxygen species within the gastric mucosa which are thought to play a major role in the mediation of associated disease. Recent studies have shown differences in the availability of an important antioxidant, glutathione, during chronic H. pylori infection. The availability of glutathione is primarily controlled by the activity of the oxidative pentose pathway. This review proposes that the severity of inflammation and damage associated with H. pylori infection is dependent on the ability of mucosal cells to counteract the increased load of reactive oxygen species. It is hypothesized that the oxidative pentose pathway and glutathione availability are important factors modulating this response. It is suggested that the therapeutic regulation of glutathione availability could provide a novel method for preventing or reducing the damage caused during H. pylori infection.     

cagA gene and vacA alleles in Spanish Helicobacter pylori clinical isolates from patients of different ages

The prevalence of the cagA gene and vacA alleles in 124 Spanish Helicobacter pylori clinical isolates from patients of different ages ranging from 3 to 78 years was studied (21 patients < or = 10 years, 30 patients 11-20 years, 17 patients 21-40 years, 31 patients 41-60 years and 25 patients 61-80 years). The cagA gene and vacA s1 or vacA s2 alleles were identified by PCR from the strain. 66.9% of the isolates were cagA+ and 33.1% cagA-. vacA s1 was detected in 48.4% of the isolates and vacA s2 in 51.6%. 44.4% of patients were cagA+/vacA s1, 22.5% were cagA+/vacA s2, 4% were cagA-/vacA s1 and 29% were cagA-/vacA s2. The percentage of cagA+ isolates and the vacA s1 alleles in the different groups were as follows: 23.8% and 28.6% in 0-10 years, 40% and 30% in 11-20 years, 88.2% and 70.6% in 21-40 years, 90.3% and 70.9% in 41-60 years and 92% and 44% in the 61-78 years group. 93% (54/58) of isolates found in ulcer patients and 90.9% (10/11) of isolates from gastritis patients older than 20 years were cagA+. In patients younger than 20 years ulcer disease was rare with 60% of isolates being cagA+ (3/5) compared with 31.6% cagA+ isolates (12/38) in patients suffering from gastritis in the younger group. The prevalence of the cagA gene and vacA s1 allele increased with age, being more frequent in older patients than in younger.     

Bacteria and mucosal inflammation of the gut: lessons from Helicobacter pylori

The human gastrointestinal tract is colonized by an abundance of bacteria, which are in constant interaction with the epithelial lining usually leading to an intricate balance between tolerance and immunological response. There is ample evidence that the abundant presence of bacteria thus plays a role in the maintenance of human health, as well as in the induction of chronic inflammatory diseases of the gastrointestinal tract. Research in this field is, however, considerably hampered by the abundance of bacterial species, many of which have not even been characterized, and are difficult to culture specifically. These important limitations may to some extent be overcome by recent molecular biologic methods. Furthermore however, the adherent mucosal flora may differ largely from the luminal flora and that in excreta. These characteristics do not pertain to Helicobacter pylori, which generally colonizes the human stomach as a single strain with stable characteristics. Such colonization is stable throughout life, but can be treated. Furthermore, the association with chronic gastritis is very strong. For these reasons, H. pylori serves as an excellent model for the understanding of the processes involved in bacterial colonization and host response including mediation of immunoregulation, and the mechanisms by which this response can lead to disease.     

Reduced intracellular survival of Helicobacter pylori vacA mutants in comparison with their wild-types indicates the role of VacA in pathogenesis

The vacuolating cytotoxin VacA of Helicobacter pylori plays an important but yet unknown role in pathogenesis. We studied the impact of the vacuolating cytotoxin on H. pylori invasion of and survival within AGS cells (human gastric cell line derived from an antral adenocarcinoma). Isogenic vacA and cagA mutants were constructed in a wild-type clinical isolate H. pylori, AF4. An H. pylori VacA-deficient mutant, AF4(vacA::kan), was cultured in significantly lower numbers from AGS cells after 24 h incubation with gentamicin added to the culture medium than were the type I wild-type strain AF4 (P<0.03) and an isogenic cagA mutant (P<0.01). Complementation of the AF4 vacA mutant with broth culture supernatant from wild-type AF4 improved the intracellular survival of the vacA mutant. We conclude that H. pylori's vacuolating cytotoxin improves the intracellular survival of H. pylori within AGS cells, suggesting the role of the vacuolating cytotoxin in H. pylori pathogenesis.     

The Helicobacter pylori plasticity region locus jhp0947-jhp0949 is associated with duodenal ulcer disease and interleukin-12 production in monocyte cells

Colonization with Helicobacter pylori always results in chronic gastritis, which is controlled by infiltration of mononuclear cells and the subsequent release of cytokines like interleukin (IL)-12. To identify H. pylori factors involved in inducing cytokine production in mononuclear cells, a random H. pylori mutant library was screened for the inability to induce IL-12 production in monocyte THP-1 cells. Of the 231 random mutants screened, one mutant (M1) showed a consistent twofold decrease in the amount of IL-12 induction compared to the parental strain 1061 (P <0.01). Further characterization of mutant M1 revealed that the kanamycin resistance cassette had integrated in the jhp0945 gene, which is situated in an H. pylori strain-specific plasticity region. Three reference strains possessing this plasticity region induced significantly higher amounts of IL-12 when compared to the H. pylori 26695 reference strain, which does not possess this plasticity region. The role in disease outcome of jhp0945 as well as the neighbouring plasticity region genes jhp0947 and jhp049 was assessed in a Dutch population cohort. Firstly, the presence of jhp0947 was completely linked with that of jhp0949 and was roughly associated with jhp0945 (P=0.072), but not with the cag pathogenicity island (PAI) (P=0.464). The presence of the jhp0947 and jhp0949 genes, but not of jhp0945, was significantly associated with duodenal ulcer disease when compared to gastritis (P=0.027). Therefore, the jhp0947-jhp0949 locus may be a novel putative H. pylori marker for disease outcome independent of the cag PAI.