Discovery of Helicobacter Pylori
Helicobacter Pylori single reports about the presence of spiral microorganisms in gastric mucosa were repeated several times still from the turn of XIX and XX century.
Nonetheless, only in 1982 the Australian physician Barry Marshall and pathologist Robin Warren isolated spiral bacteria from gastric tissue biopsy of patient with chronic gastritis. By the experiment of self-infection B. Marshall and colleague proved for the first time the association between these bacteria and the development of chronic gastritis. Subsequent multiple studies completely confirmed this association, as well as established new links of these microbial agents with gastric and duodenal ulcer, gastric cancer and certain cases of lymphatic tumors.
In 1989 the novel pathogen acquired its final taxonomic name “Helicobacter pylori”. And in 2005 B. Marshall and R. Warren were awarded Nobel Prize in Physiology or Medicine for their outstanding discovery.
Classification of H. pylori
The genus Helicobacter of the family Helicobacteriacea currently comprises more than 35 microbial species (Helicobacter pylori, Helicobacter heilmannii, Helicobacter mustelae, Helicobacter felis and many others). The main agent of human diseases is H. pylori. Some relations with human pathology are reported for species H. heilmannii.
It is generally ascertained that H. pylori plays the substantial role in pathogenesis of acute and chronic gastritis, gastric and duodenal ulcer.
Furthermore, helicobacter infection predisposes to the development of stomach cancer and gastric lymphoid tumor MALT lymphoma.
Structure and Properties of H. pylori
Helicobacter pylori is a short or medium-size gram-negative bacterium of S-like spiral shapes. Microbial cells carry 2-6 flagella attached to one pole of bacterial body (lophotrichate bacteria). They have no spore or capsule.
Helicobacters are highly fastidious agents propagating only in microaerophilic (5-7% О2) and capnophilic (near 10% of СО2) gaseous conditions; in standard aerobic or anaerobic surroundings the bacteria can’t grow. Also they have a narrow temperature optimum for growth near 37°C, being completely inactivated at 25-28°C or above 410С.
H. pylori requires special and selective nutrient media with multiple growth factors. It can be cultured in blood or serum agar supplemented with broad spectrum antimicrobials (e.g., vancomycin, trimethoprim and amphotericin B) that inhibit the propagation of concomitant bacteria. Primary growth is evaluated in 5-7 days of culture.
- Biochemical properties
Helicobacters are microaerophilic bacteria. They are oxidase and catalase positive; express multiple enzymes – phosphatase, phospholipase, hyaluronidase, proteases; produce H2S, demonstrate remarkable urease activity. These bacteria utilize amino acids as nutrients; from available carbohydrates they metabolize only glucose.
- Antigenic structure
The bacteria possess somatic LPS-containing O-antigen, flagellar H-antigen and superficial outer membrane proteins (OMP), which are type-specific.
- Virulence factors
H. pylori produces the number of adhesins, aggressive enzymes and toxins. The major role in pathogenesis of helicobacter infection belongs to microbial exotoxins – cytotoxin CagA (cytotoxin-associated gene A) and vacuolating cytotoxin A (VacA).
Cytotoxin CagA is present in most of the virulent strains of H. pylori. It is encoded by the same name pathogenicity island cag. Besides CagA cytotoxin, this island codes for type IV secretion system (T4SS) of Helicobacter pylori.
Translocator proteins of T4SS deliver CagA toxin into gastric epithelial cells. The main pathogenic functions of CagA include the impairment of cellular metabolism and activation of cell-mediated inflammatory reactions.
Vacuolating cytotoxin A or VacA binds to membranes of gastric epithelial cells. It demonstrates pleiotropic pathological effects against gastric mucosal membrane. For instance, VacA elicits the secretion of proinflammatory cytokines by leukocytes. Moreover, the molecules of VacA toxin create membrane pores allowing their own entry into epithelial cells. When entered into the cells, the molecules of VacA toxin trigger cell apoptosis or at least they cause profound degenerative changes in gastric mucosa (cell vacuolization and disruption of cellular tight junctions).
Helicobacter peptidoglycan also stimulates inflammatory reactions within stomach wall. In addition, H. pylori intensively produces the number of aggression and invasion enzymes. High level of expression is essential for microbial urease that catalyzes urea decay. This leads to the production of exuberant amounts of ammonia that not only damage the mucosal tissues but also neutralize the acidity of gastric juice thus fostering microbial survival.
Hyaluronidase and microbial flagella stimulate bacterial invasion into submucous gastric layer. Microbial phospholipases destroy the membranes of epithelial cells. Siderophore proteins provide the bacteria with iron. H. pylori demonstrates primary genetic resistance to sulfonamides, glycopeptides, polymyxins and amphotericin.
Generally helicobacters are low-resistant bacteria taking into account the narrow temperature range (34-40oС) of their growth and toxic action of atmospheric oxygen. Nevertheless, there are some individual reports about helicobacter survival in dental plaque, saliva, vomits and gastric juice.
Pathogenesis and Clinical Findings of Diseases, Associated with Helicobacter pylori Infection
Helicobacter infection is regarded as one of the most common in human population. About 50% of humans are infected with H. pylori (25-40% in developed countries, where the people above the age of 50 prevail, and up to 80% of population in developing states with substantial part of young individuals).
Nevertheless, only 10-20% of H. pylori carriers finally develop gastric or duodenal ulcer; likewise, lifetime risk of stomach cancer among infected persons is about 1-2%.
Hence, the progression towards complicated helicobacter infection strongly depends on pathogen virulence, individual health state and lifestyle, nutritional habits, the safe use of certain groups of medicines like nonsteroidal anti-inflammatory drugs (NSAID), etc.
It has been established that H. pylori species is hallmarked with high genetic variability that originates from active lateral gene transfer. Up to 30% of bacterial genes are involved into infectious process. Thus, individual alterations of microbial virulence predispose to various manifestations of H. pylori infection.
The source of H. pylori infection – infected humans.
The routs of transmission are not completely elucidated yet. In most cases the infection is transmitted orally by fecal-oral mechanism or by direct contact. Iatrogenic spread of infection via contaminated endoscopic equipment also can’t be excluded.
When entered the stomach, most of the bacteria settle in gastric antrum where the local pH of mucosal tissue is higher. Next they move towards duodenum. Active locomotion of microbial cells promotes their invasion into submucous gastric layer. Here they attach to membrane glycolipid receptors.
Urease of H. pylori metabolizes urea with ammonia release that neutralizes the acidity of gastric juice, supports long-time microbial survival and directly damages gastric mucosa. The most virulent are helicobacter strains with parallel production of both bacterial cytotoxins – CagA VacA. The protein apparatus of T4SS injects toxin CagA and the fragments of peptidoglycan into gastric epithelial cells.
CagA interferes in normal life cycle of epithelial cells; peptidoglycan fragments stimulate inflammatory response via activation of transcription factor NF-kB. Together with VacA toxin they promote the development of acute gastritis and/or duodenitis. This is followed by local hyperproduction of proinflammatory cytokines (IL-8 and others) that stimulates neutrophil and lymphocyte infiltration of stomach wall.
Incubation period of acute gastritis doesn’t exceed several days. Without proper management acute helicobacter gastritis has evident chances for transformation into chronic disease especially under the action of other predisposing factors (smoking, alcohol consumption, treatment with NSAIDs, etc.)
The next course of infection largely depends on predominant localization of inflammatory process. If chronic gastritis affects mainly the pyloric part of the stomach, it leads to permanent hyperproduction of gastrin and HCl that finally results in development of ulcer of duodenal or antral localization.
If chronic helicobacter gastritis progresses into chronic pangastritis with damage of cardia, fundus and body of stomach, it causes the gradual but irreversible destruction of gastric epithelial cells. The production of hydrochloric acid declines resulting finally in chronic atrophic gastritis with achlorhydria.
Chronic atrophic gastritis is the significant risk factor of stomach cancer. That’s why helicobacter infection is regarded as biological carcinogen.
The influence of helicobacter virulence factors on proliferation of immune cells may cause the emergence of rare cancer disease MALT lymphoma – the tumor originated from gastric lymphoid follicules. Despite intensive activation of local cell-mediated immunity, inflammatory response is unable to eliminate the infection resulting in lifelong helicobacter carriage. Only efficient complex antimicrobial therapy results in eradication (complete removal) of helicobacter infection.
Laboratory Diagnosis of Helicobacter pylori Infection
As helicobacter infection is common among individuals, specific laboratory examination is usually required for the cohort of patients with gastric and duodenal pathology.
Two groups of laboratory methods are used for detection of H. pylori – non-invasive and invasive tests; the latter need gastric biopsy specimens. Rapid non-invasive carbon urea breath test discovers urease activity of H. pylori. The test is convenient for mass screening of people attending medical offices and clinics. When tested, the examined person drinks urea solution radioactively labelled with [14С] or [13C]. Under the action of microbial urease labeled CO2 is released that is registered in expired air.Other non-invasive tests include determination of Ags of H. pylori in feces by ELISA test and detection of microbial DNA by PCR.
Invasive tests presume the examination of gastric biopsy specimens taken during endoscopy. For instance, rapid urease test detects helicobacter urease in gastric biopsy by placement of the specimen into urea solution. The decay of urea is followed by ammonia accumulation that elevates pH of the medium and changes the color of indicator dye.
The most reliable test for direct detection of H. pylori in biopsy specimen is microscopy with histological hematoxylin-eosin staining or Warthin-Starry’s silver stain that is more sensitive. Also luminescent stain can be used, e.g. with acridine orange dye. Typical morphology of bacteria is observed.
For isolation of microbial culture, the tissue specimen is inoculated into special media supplemented with antibiotics and multiple growth factors. Incubation is performed in microaerophilic conditions (5-7% О2) with increased concentration of CO2 (5-10%). Primary growth should be assessed in 5-7 days.
The isolated culture is further examined by microscopy, biochemical testing (e.g., for oxidase and urease), serological and molecular genetic tests. Serological diagnosis uses ELISA test for evaluation of specific antibodies (Abs) in patient serum directed against H. pylori Ags.
Treatment and Prophylaxis of H. pylori Infection
To prevent unfavorable consequences of H. pylori infection, complete eradication of this pathogen is required. Recommended first-line antimicrobial treatment (so-called “triple therapy”) includes proton pump inhibitor (e.g., omeprazole) and two antibiotics amoxicillin and clarithromycin. The efficacy of this regimen is more than 85%.
Microbiological confirmation of eradication is performed after the end of treatment course. In case of first-line treatment failure, quadruple therapy is used expanded with colloidal bismuth salts. In the light of growing antimicrobial resistance of H. pylori, antibiotics of other groups can be administered – metronidazole, tetracycline, and fluoroquinolones. Despite the high frequency of successful eradication, the cases of reinfection with H. pylori are common mainly due to the broad spread of this agent among human population.
Prophylaxis of H. pylori infection remains non-specific. It is based on general measures for efficient sterilization of medical instruments, antisepsis and disinfection. Various kinds of candidate vaccines against H. pylori are under clinical trials now.