Helicobacter pylori / seminar

Abstract
H. pylori is a Gram-negative bacteria that colonizes the mucus layer of the gastric epithelium, by the means of interaction between surface proteins and gastric epithelial receptors. The most common way of transmission is oral-to-oral and fecal-to-oral contact. Its infection utilizes chemotaxis and release of cytokines, and induces both, humoral and cellular immune responses. H. pylori diagnosis methods are variable, being mainly highlighted by endoscopy, histology and several tests. The treatment of such infection is difficult, and requires a triple antibiotic therapy. Nowadays, probiotics are being significantly used in such treatment.

 
Introduction 
Helicobacter pylori has been the subject of intense investigation since its culture from a gastric biopsy in 1982. From the beginning, this gram-negative bacterium has provoked the interest of bacteriologists, gastroenterologists, infectious disease specialists, cancer biologists, epidemiologists, pathologists, and pharmaceutical scientists. The possibility that a bacterium could cause gastritis, peptic ulcers, and, over time, cancer was a concept that was difficult to put forward.
Owing to the unique characteristics of H. pylori, such as its microaerophily, nitrogen metabolism, and ecological niche, a sound understanding of its physiology and genetics is of interest to fundamental and applied microbiology, taxonomy, molecular biology, microbial ecology, and medical, veterinary, and agricultural microbiology.














I. An overview of Helicobacter pylori
Helicobacter pylori was previously named Campylobacter pyloridis. It migrated out of Africa along with the ancestors. It is a microaerophilic Gram-negative bacterium, having a curved spiral shape, characterized by a high motility since it has 2-6 flagella. It colonizes mainly the mucus layer of the gastric epithelium of the lumen in the upper half of the gastric pit, but not too closely related mucus-secreting neck cells in the lower half of the gastric pit. It does not adhere to chief cells, parietal cells, or endocrine cells in the gastric pit. The most common way of transmission is oral-to-oral and fecal-to-oral contact, eliciting both humoral and cellular immune responses. The treatment of H. pylori is difficult, since it requires a combined intake of a proton pump inhibitor with amoxicillin and one of the two antibiotics, clarithromycin or metronidazole (Johnson et al., 2017).
I-1. Colonization
Colonization takes place upon several strains, and these strains evolve rapidly. So, the infected individual not only carries his own H. pylori strain, but this strain can undergo genetic alteration, driven by an elevated mutation rate, leading to free recombination of H. pylori genes. Therefore, plasticity of H. pylori is revealed, affecting surface proteins, such as the outer membrane proteins (OMPs), housekeeping genes, virulence genes and lipopolysaccharides (LPS), thus contributing to host adaptation, persistence and immune response evasion (Johnson et al., 2017).

I-1.1. Surface proteins

I-1.1.1. Lipopolysaccharides (LPS)

   H. pylori is characterized by having several lipopolysaccharides (LPS). LPS is the major component of the bacterial cell wall of Gram-negative bacteria. In H. pylori, it is consisted of an O-specific polysaccharide chain, a core oligosaccharide, and a lipid part called lipid A, embedded in the outer membrane. The O-antigen of H. pylori contains different human Lewis-like antigens, including Lewis (Le)^x, Le^y, Le^a and Le^b. These antigens play a role in the adherence to gastric epithelial cells in a Lewis-antigen-dependent manner, more specifically via Le^x. The receptor recognized by the O-antigen side-chain of LPS is a host β-galactoside-binding lectin, galectin-3. The expression of galectin-3 is up regulated by gastric epithelial cells following adherence of H. pylori. So, in addition to colonization, this lectin plays a role in the host response to infection (Johnson et al., 2017).

I-1.1.2. Outer membrane proteins (OMPs)

   H. pylori is also characterized by having several outer membrane proteins (OMPs). OMPs are divided into five gene families. The largest family is Family 1, comprised of Hop and Hor proteins. Families 2 and 3 comprise Hof and Hom proteins respectively. Families 4 and 5 are composed of iron-regulated OMPs and efflux pump OMPs respectively (Johnson et al., 2017).

·         BabA: the first blood group antigen-binding adhesin A to be identified, also named HopS. It mediates the binding of H. pylori to the fucosylated Lewis b blood group antigen, Le^b. Thus, the T4SS secretion system is activated, triggering the transcription of genes that enhance inflammation, development of intestinal metaplasia, and associated precancerous transformations.

·         SabA: the second well known adhesion, sialic acid-binding adhesin, also named HopP or OMP17. Upon infection and mucosal inflammation, the sialyl-dimeric-Lewis x (Le^x) glycosphingolipid is up regulated, and SabA mediated the binding of H. pylori to the sialyl-dimeric-Le^x, thus strengthening the epithelial attachment needed to achieve successful colonization. SabA binds to neutrophils, so as a consequence, these neutrophils produce reactive oxygen species causing oxidative damage of gastric epithelium.

·         AlpA, AlpB: adherence associated lipoproteins AlpA, also named HopC or OMP20, and AlpB, also named HopB or OMP21. They induce gastric injury by modulating pro-inflammatory intracellular signaling cascades upon infection, such as interleukin-8 (IL-8), which is an important T cell and neutrophil chemoattractant.

·         OipA: outer inflammatory protein A, also named HopH or OMP13. It also promotes IL-8 secretion, inducing inflammation. Even more, it is associated with more severe gastric diseases such as duodenal ulcer, gastric cancer and neutrophil infiltration. It is involved in H. pylori-induced focal adhesion kinase activation and cytoskeletal reorganization of gastric epithelial cells.

·         HopZ: also named OMP1. It is associated with the development of MALT lymphoma.

·         HomB: the smallest family of OMP. As the previous proteins, it is associated with IL-8 secretion, and contributes to bacterial adherence and more severe clinical outcomes (Oleastro et al., 2013).

I-1.2. Gastric epithelial receptors

   H. pylori has major gastric epithelial receptors which are the Lewis blood group antigens and sialylated glycans (Oleastro et al., 2013).

I-1.2.1. Lewis blood group antigens

   The major component of gastric mucus layer are the highly glycosylated mucin proteins. In healthy mucosa, mucins produced are MUC1, MUC5AC and MUC6. MUC5AC plays a role in the adherence of H. pylori to the gastric mucosa, while MUC6 exhibits an antimicrobial activity. The major receptor of H. pylori is the Le^b carbohydrate structure present in the normal gastric tissue. MUC5AC is the most important carrier of Le^b, being attached through BabA (Oleastro et al., 2013).

I-1.2.2. Sialylated glycans

   Under the clinical conditions caused by a severe H. pylori infection, Le^b is weakly expressed while there is an upward migration of Le^x. Moreover, there is an expression of sialylated glycans, such as sialyl-Le^a and sialyl-Le^x, where H. pylori binds to sialyl-Le^x via SabA adhesins.

   During an established infection, H. pylori colonization favors the acquisition of nutrients released from the damaged host cells, and the delivery of effector molecules into the gastric cell, such as CagA oncoprotein and VacA cytotoxin (Oleastro et al., 2013).

I-2. Chemotaxis

  H. pylori utilizes chemotaxis, driven by a conserved signal transduction system. Chemotaxis allows H. pylori to sense an array of environmental and bacterial signals within the stomach, guiding its motility towards its preferred niche within the gastric mucosa and glands, away from harmful conditions (Arachchi et al., 2017).

   The chemotaxis system of H. pylori contains core chemotaxis proteins found in all chemotaxis systems, and auxiliary ones found in only some. The core chemotaxis proteins are the chemoreceptors (TlpA, TlpB, TlpC and TlpD), the CheW coupling protein, the CheA kinase, and the CheY response regulator. The auxiliary chemotaxis proteins include three CheV-type coupling proteins (CheV1, CheV2 and CheV3), the CheZ phosphatase, and the unique chemotaxis protein ChePep, which localizes CheZ to the poles. Environmental signals are sensed either directly or indirectly by chemoreceptors, and are relayed to the histidine kinase CheA via the CheW or CheV1 coupling proteins (Arachchi et al., 2017).

   Chemicals sensed as repellents activate CheA's auto-phosphorylation, and this phosphoryl group is passed then to CheY via histidine-to-aspartate. Phosphorylated CheY interacts with the flagellar motor, causing it to rotate clockwise, so the bacteria change its direction (Arachchi et al., 2017).

   Mutants in any one of these proteins are non-chemotactic (Che^-). Mutants of CheW, CheA, CheY, CheV1 and CheV2 display straight swimming phenotypes, since they do not produce phosphorylated CheY. While mutants of CheZ and CheV3 display hyper-reversal phenotypes, since they produce high amounts of phosphorylated CheY (Arachchi et al., 2017).

   H. pylori senses specific chemicals via three transmembrane chemoreceptors (TlpA, TlpB and TlpC), and one cytoplasmic receptor (TlpD) (Arachchi et al., 2017).

   It faces several repellent conditions. Many of these are host generated, including acidic pH, reactive oxygen species (ROS) and bile. The acidity of the lumen of the stomach is toxic to H. pylori. It acts as a chemo-repellent with TlpA, TlpB, TlpD and TlpC. Another signal is the ROS, produced by host epithelial and immune cells (Arachchi et al., 2017).

   It is also repelled by the self-generated, quorum-sensing molecule autoinducer-2 (AI-2), promoting dispersion of H. pylori. It also responds to its own electron transport chain (ETC) (Arachchi et al., 2017).

   H. pylori senses several beneficial chemo-attractants, such as urea and Arginine, an important amino acid for H. pylori. In non-infected individuals, urea is available within the stomach, and is hydrolyzed by H. pylori urease into ammonia and bicarbonate. Both chemo-attractants help H. pylori to find key nutrients, and direct it towards the epithelial surface (Arachchi et al., 2017).

   Chemotaxis is required for wild-type (WT) level colonization of the stomach. Che^- do not colonize at all, or poorly colonize, since they require 100-fold more bacteria to initiate colonization (Arachchi et al., 2017).

Chemotaxis during early infection: Chemotaxis is critical during the first three months of an infection. During this period, Che^- mutants display significant colonization defects (Arachchi et al., 2017).

Chemotaxis during chronic infecton: Che^- H. pylori are able to achieve WT levels of colonization as early as one month post infection. During the chronic stage of infection, Che^- colonizes gastric glands in the corpus and antrum. While WT colonizes the mucus layer of the corpus too (Arachchi et al., 2017).

I-3. Cytokine profile

   Inflammation occurs upon recognition of microbial-associated molecular patterns (MAMPs), or damage associated molecular patterns (DAMPs) by local monocytes, macrophages and epithelial cells. These cells release pro-inflammatory cytokines and chemokines that recruit neutrophils and antigen presenting cells, including macrophages and dendritic cells, to the site of infection. This primes T-cell differentiation and induces the differentiation of T-helper (Arachchi et al., 2017).  

   Th17 cells are important in immunity to extracellular bacterial and fungal infections. The cytokine profile of Th17 cells includes IL-17A, IL-17F, IL-21 and IL-22, where the maintenance of Th17 cells population is mediated by IL-23 and IL-21. Upon infection of the gastric mucosa by H. pylori, a local high level of IL-17A, IL-23, IL-21 and TNF-α were detected. Specifically, IL-17 recruits neutrophils, and stimulate TNF-α secretion and other pro-inflammatory cytokines like IL-1 and IL-6. It also stimulates the fibroblasts to produce matrix metalloproteinases, which further contributes to mucosal damage and carcinogenesis. Regarding TNF-α, it specifically acts on endothelial cells to stimulate expression if adhesion molecules, facilitating the extravasation of neutrophils into the site of infection on the mucosal tissue. It can further activate T cells and stimulate cytokine production by macrophages and monocytes (Arachchi et al., 2017).

  So, the elevation of these cytokines among H. pylori infection suggests that in the Th17 pathway, IL-21 helps to maintain the Th17 cells and thus regulate Th17 effector responses during chronic H. pylori infection. This supports the role of Th17 in the pathogenesis of H. pylori infection, and the severity of gastritis (Arachchi et al., 2017).

 

I-4. H. pylori and bottled drinking water

   Bottled drinking water is contaminated with dangerous pathogens such as H. pylori. Colonization and invasion of H. pylori to gastric mucous is due to a number of virulence factors, such as vacuolating cytotoxin (vacA), induced by contact with the epithelium antigen (iceA), cytotoxin associated gene (cag), blood group antigen-binding adhesion (babA) and outer inflammatory protein (oip). The vacA gene is polymorphic, comprising variable signal regions (type s1 or s2) and mid-regions (type m1 or m2). The s1 type is further subtyped into s1a, s1b and s1c subtypes and the m1 into m1a and m1b subtypes. The iceA gene has two main allelic variants iceA1 and iceA2. CagE is 1 of 6 genes located within the pathogenicity island shown to induce secretion of chemokines, such as IL-8 and induce inflammation (Ranjbar et al., 2016).

   Bottled mineral water samples are specifically contaminated with resistant and virulent strains of H. pylori. They are resistant to amoxicillin, metronidazole, clarithromycin, furazolidone, quinolones and tetracycline. Drinking water can pose a substantial threat for transmission of H. pylori because of several important criteria. These criteria include the ability of H. pylori to adhere to different materials and to co-aggregate with other bacteria and form complex structures on pipes or other surfaces in contact with water. Notion about the disability of H. pylori to survive alone in running water, but to develop a symbiotic relationship and form complex structures on contact surfaces, makes it rational to assume that groundwater is a reservoir for H. pylori due to its stagnant nature (Ranjbar et al., 2016).

   Other possible reasons for the high prevalence of H. pylori in bottled mineral water samples are (i) lack of competent approaches for water sanitization; (ii) expending the distrustful groundwater for producing the bottled mineral water; (iii) the opportunity of presence of bacterial colonies as a biofilm in the in pipes used for water transfer; (iv) the opportunity of leakage of household, industrial and agricultural wastewater to the sources of mineral water; and finally (v) lack of personal hygiene of refinery room’s staffs (Ranjbar et al., 2016).

I-5. Diagnosis of infection

I-5.1. Traditional diagnosis

   H. pylori traditional diagnosis is made using a combination of tests. Considering the broad spectrum of diagnostic methods, only highly accurate tests should be used in clinical practice under specific circumstances and currently, the sensitivity and specificity of such tests should exceed 90% (Lopes et al., 2014).

I-5.1.1. Endoscopy

Three paired biopsies for histology were taken at the antrum, corpus lesser (CLC), and greater curve (CGC). Additional specimens were obtained at the antrum and CGC for a rapid urease test (RUT). Collecting venules, seen as numerous minute red dots in the gastric corpus, were a characteristic finding in the normal stomach without H. pylori infection. This finding was termed “regular arrangement of collecting venules” (RAC) (Lopes et al., 2014).

I-5.1.2. Histology

Recognizing that the number and distribution of H. pylori organisms vary in patients taking proton pump inhibitors (PPIs), it has been recommended to discontinue PPIs two weeks before endoscopy and to take biopsies from both the body and the antrum. Antigen stool detection can also give false-negative results in these circumstances. In contrast, serology is not influenced to such an extent by a lower density of the microorganism, and is reliable even in advanced gastric body atrophy. Pathologists have used different diagnostic techniques, including immunohistochemical (IHC) methods and special stains, such as Giemsa and Warthin-Starry. On the other hand, it is clear that IHC staining is highly sensitive and specific for H. pylori, with the lowest rate of inter observer variation and is much faster than conventional histology. H. pylori detection has increasingly focused on specific clinical settings and patient groups (bleeding peptic ulcer, gastric cancer) (Lopes et al., 2014).

I-5.2. Tests

I-5.2.1. Rapid Urease Test (RUT)

The RUT is based on the production of large amounts of urease enzyme by H. pylori, which splits the urea test reagent to form ammonia, enabling its detection by a rapid indirect test. Many commercial RUTs are available, including gel-based tests, paper-based tests and liquid-based tests, providing a result in 1-24 h, depending on the format of the test and the bacterial density in the biopsy specimen. An important conclusion of several studies is that enhancing the number of biopsy fragments and/or collecting them from various regions of the stomach (antrum and body, from example), achieves a higher sensibility of the RUT. Moreover, it was shown recently that combining tissues prior to RUT increased the detection of H. pylori, compared with testing separate specimens, and produced faster results (Lopes et al., 2014).

I-5.2.2. Culture

The most commonly used media include Brucella, Columbia Wilkins-Chalgren, brain-heart infusion or trypticase agar bases, supplemented with sheep or horse blood. An alternative to blood is supplementation of the agar base with β-cyclodextrin or yolk emulsion. Supplementation of media with cholesterol instead of serum was a viable option for H. pylori growth. Another original approach used liquid culture medium for the rapid cultivation and subsequent antibiotics susceptibility testing of H. pylori directly from biopsy specimens, with a final detection step by an enzyme linked immunosorbent assay (ELISA). It also requires an atmosphere enriched with CO₂ and O₂. Besides the issue concerning bleeding peptic ulcers, for which culture has a lower sensitivity than in non-bleeding cases, other host-related factors, such as high activity of gastritis, low bacterial load, drinking alcohol and the use of histamine H₂ receptor blockers, have been recently described as the cause of failed H. pylori culture from gastric mucosa in the infected subjects (Lopes et al., 2014).

I-5.2.3. Urea Breath Test

The 13 C-urea breath test (¹³C-UBT) is one of the most reliable tests for diagnosing H. pylori infection. It is a non-invasive, simple and safe test that provides excellent accuracy both for the initial diagnosis of H. pylori infection and for the confirmation of its eradication after treatment. Excellent accuracy is obtained when breath samples are collected as early as 10-15 min after urea ingestion. The ¹³C-UBT in adults has a high sensitivity and specificity. However, the test has shown heterogeneous accuracy in the pediatric population, especially in young children (Lopes et al., 2014).

I-5.2.4. Stool Antigen Test

The stool antigen test is a non-invasive method to detect H. pylori, usually recommended when the UBT is not available. There are two types of stool antigen tests used for H. pylori detection, the EIA and an assay based on immunochromatography. These tests are the Testmate pylori antigen EIA, in which plastic 96-well EIA microtiter plates are coated with monoclonal antibody (Mab) 21G2, and the Testmate rapid pylori antigen, which is based in immunochromatography and is presented as a test strip. For the EIA test, a drop of the suspended stool sample or a sample of the diluted bacterial antigen sample is mixed with the peroxidase-conjugated MAb 21G2. After proper incubation and washing, the optical density is measured. For the test strip, a drop of stool sample is applied in the specimen application of the test strip. When H. pylori antigens are present, they form immune complexes with the red latex-labeled MAb 21Ge and migrate by capillarity action until captured by the solid phase anti-mouse rabbit polyclonal antibodies and form a visible red test line (Lopes et al., 2014).

I-5.2.5. Antibody-Based Test

Serology was one of the first methods used for diagnosis of H. pylori infection. Currently, serology is recommended for initial screening, requiring further confirmation by histology and/or culture before treatment. Detection of antibodies is useful for detecting past or present exposure. In fact, a limitation of serology tests is the failure to distinguish between past and current H. pylori infection. Blood samples are used for serology testing, detecting anti-H. pylori antibodies (IgG) by ELISA. Several H. pylori immunogenic proteins have been presented as candidates to detect infection, such as the FlidD protein; multiple recombinant (CagA, VacA, GroEL, gGT, HcpC and UreA) proteins; CagA or Omp18 (Lopes et al., 2014).

Figure 9 - Antibody-Based Test

I-5.3. Detection of H. pylori In Other Specimens

Other specimens have been evaluated to determine their usefulness to detect H. pylori infection. These include saliva, sub-gingival biofilm, dental plaque, gastric juice, gastro-esophageal biopsies and adenotonsillar tissue. The ability to detect H. pylori antibodies in saliva is lower than in blood-based serology. However, the use of molecular techniques for the detection of H. pylori infection in saliva or dental plaque may make these specimens attractive because they are easier to collect. The enterotest or string test was designed decades ago specially for children. The string test consists of a gelatin capsule attached to a long nylon string that unwinds during ingestion. Upon reaching the stomach, the gelatin capsule dissolves and the string absorbs gastric secretions. The extraction of the string occurs 30-180 min later and should avoid contact with teeth and tongue to prevent contamination. The string may be used for culture of bacteria for H. pylori detection (Lopes et al., 2014).

I-6. Treatment from infection by H. pylori

I-6.1. Antibiotics       

  H. pylori is involved in vitamin B₁₂ and iron deficiency. The recommended treatment for H. pylori eradication is the standard triple therapy, using a proton pump inhibitor or ranitidine bismuth citrate, combined with clarithromycin and amoxicillin or metronidazole. However, due to the increase of the antibiotic resistance, some studies have started to focus on probiotics, as a therapeutic approach (Goderska et al., 2017).

I-6.2. Probiotics

   Probiotics are defined as living microbial species that can include anti-inflammatory and anti-oxidative mechanisms. They can improve microbial balance in the intestine and exert positive health effects on the host, such as preventing intestinal infections, cardiovascular diseases, cancer and anti-allergic effects. The most used probiotic bacteria are the Lactic acid bacteria, including Gram (+) cocci and rods Lactobacillus and Bifidobacterium.

    A common feature of these bacteria is the ability of anaerobic digestion of saccharides and production of lactic acid. These microorganisms are characterized by a resistance to los pH and tolerance to a wide range of temperatures (Goderska et al., 2017).

I-6.2.1. Mechanisms of action of probiotics 

   The mechanism of action of these probiotics include:

a-     Non-immunological mechanism: The first line of defense against pathogenic bacteria is acidity of the stomach and the gastric mucosa barrier. It was suggested that, by taking probiotics, this first line of defense could be stronger due to the production of antimicrobial substances competing with H. pylori for adhesion receptors, stimulating mucin production and stabilizing the gut mucosal barrier (Goderska et al., 2017).

b- Antimicrobial substances: Probiotics may inhibit H. pylori growth by secreting short chain fatty acids and antibacterial substances. Short chain fatty acids such as acetic, propionic, and lactic acids are produced during the carbohydrates metabolism by probiotics and as consequence, a pH reduction are found. Certain Lactobacillus species synthesize antimicrobial compounds (Goderska et al., 2017).

c- Competition for adhesion: Probiotic bacteria can inhibit the adhesion of H. pylori (Goderska et al., 2017).

d- Mucosal barrier: Probiotics increase the expression of MUC2 and MUC3 genes, and therefore extracellular secretion of mucin by colon cell cultures can inhibit the adherence of pathogenic bacteria. This ability of these strains restores the mucosal permeability of gastric mucosa and inhibits the adherence of pathogenic bacteria such as H. pylori (Goderska et al., 2017).

e- Immunologic mechanisms: The inflammatory response to gastric H. pylori infection is characterized by the release of various inflammatory mediators such as chemokines and cytokines. Probiotics could modify the immunologic response by the modulation of anti-inflammatory cytokines secretion, which would result in reduction of gastric activity and inflammation (Goderska et al., 2017).

   So, probiotics could not be recommended to be used by a single agent for eradication therapy. However, their use is associated to standard treatment as an adjunct will improve the eradication rates and decrease treatment-related side effects (Goderska et al., 2017).

Conclusion and perspectives

   H. pylori is a virulent bacteria that leads to severe pathogenic cases. To prevent infection by this bacteria, Lactobacilli have been introduced into several fermented dairy products. There is a large body of evidence showing the positive role of exogenous lactobacilli in the prevention and treatment of GI disorders. Lactobacilli adhere to cells of the GI tract by secreting a proteinaceous component, which serves as a bridge between the bacteria and eukaryotic cell receptors, although its precise nature still remains to be elucidated. This bacterium has been reported to inhibit the adhesion of many kinds of pathogenic enterobacteria to intestinal cells. Vaccination in addition to antimicrobial therapy will thus be needed in the campaign against H. pylori. The principle of probiotics, which involves feeding live bacteria to the host as a prophylactic or treatment for many intestinal diseases, is a great attempt too.

 References

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