Helicobacter pylori: Introduction, Morphology, Pathogenecity, Lab Diagnosis, Treatment and Prevention

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5 years ago

Helicobater pylori: Introduction, Morphology, Pathogenecity, Lab Diagnosis, Treatment and Prevention

Introduction of Helicobacter pylori

Helicobacter pylori are spiral bacteria from the stomach that were seen nearly 125 years ago (1896 by Salmon) and subsequently by others but had been misinterpreted. In 1983, Marshall and Warren described Camphylobacteria as organisms causing colonization in the human stomach. At the same time, Rollason et. al.( 1984), we’re also seeing these bacteria in the gastric biopsy specimen. The organism was named Campylobacter pyloridis by Marshal et. al.(1984), later changed to Campylobacter pylori by Marshal and Gardner in 1987. Now, this organism has been placed in the genus, Helicopter, and species, pylori in 1989 by the International Society of Bacteriologists.

Scientific classification

Domain: Bacteria

Phylum: Proteobacteria

Class: Epsilonproteobacteria

Order: Campylobacterales

Family: Helicobacteraceae

Genus: Helicobacter

Species: H. pylori

Definition of Helicobacter pylori

S-shaped or curved gram-negative rods, measuring 1.5-3.5 µm in length and 0.5-0.9µm in width. They are motile by a tuft of flagella up to 4-7 sheathed flagella at one end and are non-spring. They may be changed into the coccal form on exposure to air within 2 hours. Microaerophilic, non-saccharolytic, rich in oxidase, catalase, and urease enzyme. Guanine -cytosine content of DNA is 35.2 mol %.

Morphology of Helicobacter pylori

In a gastric biopsy, H. pylori appear short and spiral from and even comma (,) shaped bacilli measures 3 x 0.5-0.9µm. Upon culture in the artificial medium, organisms appear rod-shaped and contain 4-7 flagella at one end. H. pylori tend to change into a coccal form upon exposure to room temperature for 1-2 hrs and fail to recover on subculture.

Antigenic properties of Helicobacter

Whole-cell and outer membrane protein gel electrophoresis profiles of H. pylori are different from those of Campylobacter. However, a few proteins bands are common, nearly to the major flagellar antigen of C. jejuni and C. fetus. This protein reacts with antiserum raised against purified C. jejuni flagella (Perez and Blaser 1987). Although Lipopolysaccharide antigens are shared, the side chain antigens are strain-specific.

Cellular constituents of Helicobacter

The fatty acid profiles of H. pylori are distinctive and different from the general pattern of Campylobacter spp.

Pathogenicity of Helicobacter pylori

Mode of transmission: Human gastric mucosa is the only known natural reservoir of H. pylori.

Animal to humans through infected milk.
Through contaminated water
Feco- oral route
Iatrogenic infection from the stomach to the stomach through the use of inadequately sterilized endoscope between patient to patient. H. pylori live only on the gastric mucosa. Most infections are symptomless. It’s colonization with type -b antral chronic gastritis. This predisposes to gastric ulceration and gastric carcinoma. The course of infection: Incubation period: few days. Patients suffer from a mild attack of abdominal, nausea, flatulence, and bad breath. Site of infection: Gastric antrum is most favored. The bacteria are present in large numbers in the mucus overlying the mucosa (pH about 7.0) Colonization often extends to gastric glands. Peptic ulceration: In type gastritis, H. pylori are present virtually in all patients with a duodenal and gastric ulceration H. pylori is rich in urease and protease enzyme. The ammonia produced after the action of urease on urea causes an ionic change in the mucus layer which in turn causes the back diffusion of hydrogen ions in the mucosa, as a result, it contributes further damage to the mucosal cell. Moreover, mucus degrading protease and lipopolysaccharide may also play a vital role in the formation of peptic ulcers. The clouds of ammonia generated after the breakdown of urea by enzyme will neutralize the acid showers in the stomach, as a result helping to protect the H. pylori from acid showers. Ammonia produced may directly damage the cells. It has cytotoxic effects on somatostatin cells. Ammonia produced may in turn affect the performance of the ca ion-exchange mechanism of the mucosal layer thereby prevent the normal passage of the hydrogen ions from gastric glands to gastric lumen, thus permitting back diffusion. Immunity: In acute infection ( during colonization), there would be the production of IgM. In chronic infection, IgM will be substituted by IgG. To some amount, IgA would also be produced.

Model representing the role of *H. pylori* and other factors in gastric carcinogenesis, based on the cascade proposed by Correa et al.

Laboratory diagnosis of Helicobacter pylori

Specimen: It depends on invasive and non-invasive methods. A biopsy is the choice specimen for invasive while blood and stool for non-invasive methods.

Invasive methods
Histology
Culture biopsy
Rapid urease (CLO) test
Noninvasive methods
Urea breath test
Fecal antigen test
Serology

A biopsy of mucosa from the gastric antrum and the materials from the mucosal region, taken by endoscopy should be placed in a small 6.0 ml screw-capped containing 0.2 ml sterile physiological alien and should be processed within 1 hour. The second piece of biopsy for the rapid urea test: should be placed in a 20% urea broth and look for color change. Hydrolysis of urea depends upon the number of organisms present in the biopsy materials. It is said that this test will be positive if there are > 10⁵CFU/ml of biopsy.

Third biopsy for histopathology and Giemsa stain/ Warthin- Starry Silver stain/ Grams stain

Note: It has been said that H. pylori have also been cultured from gastric juices and saliva.

Culture

Crush the gastric biopsy and inoculate heavily making a carpet culture.

Media :

Non- selective media

Solid media

Brucella agar with 5% sheep blood

Tryptone soya agar

Brain Heart Infusion (BHI) agar with 5% horse blood

Chocolate agar

Transport medium: Hugh mucin

Liquid Media

Brucella broth with 5-10% horse and fetal calf serum

Selective media

To make the media selective, add the following into the above media

Vancomycin (6 µg/ml), Nalidixic acid ( 20 µg/ml ) and Amphotericin- B ( 2 µg/ml)

H. pylori are resistant to trimethoprim, vancomycin, polymyxin- B, and amphotericin -B.

Skirrow’s campylobacter medium: Suitable for isolation of H.pylori

Incubation: at 37°with additional 10% CO₂ or most suitable is a mixture of the following gases in the following proportion:

CO₂ : 10%, O₂ : 5% and N₂ : 85%

The condition is known as a microaerophilic condition. With a high level of humidity, a freshly poured plate without drying should be used. Temperature and incubation time: at 37ºC for 3-7 days and colony size: generally less than 2 mm, gray-white, flat, and watery. Spreading growth with discrete colonies may occur. Motility: Best in broth culture and darting type but weak or absent when grown on agar medium.

Biochemicals tests:

Oxidase + Catalase +, Urea hydrolysis test +,

Note: The urease activity of H. pylori is 100 times more than Proteus vulgaris.

All strains produce DNAase, Alkaline phosphatase, leucine aminopeptidase, and gamma-glutamyl aminopeptidase.

Rapid pyloric kits are available to test the above activities.

Non-invasive methods to diagnose H.pylori infection:

Urea breath test: It is very simple but very costly. Urea labeled with 14 C or 13-C is fed to the patients and the emission of isotopes 14-CO₂ in-breath is measured using a gamma counter that is very costly.

Serology: Detection of IgM is useful to diagnose acute infection.

Antimicrobial Susceptibility Testing of Helicobacter

Sensitive to penicillin, cephalosporin, tetracycline, erythromycin, rifampicin, aminoglycosides, nitrofurantoin, and ciprofloxacin has been shown. Resistant to 1 % bile, trimethoprim, vancomycin, polymixin-B, nalidixic acid, amphotericin-B. 33% strains of H. pylori have been reported resistant to metronidazole by R. Safaralizadeh et al.

Molecular diagnosis

DNA/DNA hybridization

Polymerase chain reaction (PCR)

RFLP, Ribotyping, and pulse-field gel electrophoresis for strains characterization.

Treatment of Helicobacter pylori

Use of antibiotics, bismuth salts
Ingestion of bismuth salicylate
Antibiotics, tetracyclines, and metronidazole for two weeks
Use of omeprazole
Clarithromycin
In gastritis, triple-drug therapy consisting of clarithromycin, amoxicillin, and a proton-pump inhibitor is given for 14–21 days often being considered first-line treatment while in peptic ulcer, the standard first-line therapy is a one-week “triple therapy” consisting of proton-pump inhibitors such as omeprazole and the antibiotics clarithromycin and amoxicillin.

Prevention of Helicobacter pylori

Practice good hygiene and handwashing, with food preparation in particular.
All patients with chronic symptoms of the gastrointestinal tract that might be associated with H. pylori infection should be tested and treated in order to avoid exposure to members of the family. To maximize the potential for a cure, patients should complete the full course of therapy (antibiotics and acid blockers). Keep proper nutrition to prevent.

Key Notes on Helicobacter pylori

Histology is the “Gold standard” in routine hospital diagnostics with
sensitivity and specificity >95%.
The urea breath test is an alternative gold standard also with sensitivity and specificity >95%.
People who have H. pylori and concurrent helminth infection have standard H. pylori colonization and gastritis patterns, but they display significantly less H. pylori-associated disease. This indicates that the enteric helminth infection can modulate the host’s immune system to attenuate Helicobacter-induced gastric ulceration, atrophy, and cancer.
Asymptomatic colonization treatment should be avoided.
Drug resistance is a growing problem.