Helicobacter pylori and Food Products

A Public Health Problem Anavella Gaitan Herrera

1. Introduction

Helicobacter pylori is a major human pathogen causing gastritis and chronic superficial infection (CSG). It colonizes the stomach of more than 50% of humans and causes disease (1). This microorganism is associated with the gastric antral epithelium in patients with active chronic gastritis, peptic (gastric) or duodenal ulcers, and gastric adenocarcinoma (2-4) H. pylori is present in feces, sewage, and water (5) but is killed by routine chlorination. Therefore, in developing countries (6), consumption of sewage-contaminated drinking water and vegetables may pose a risk (7); properly cooking foods and chlorinating water reduces the risk of transmitting H. pylori to humans (see Note 1). In South America the consumption of raw vegetables fertilized with human feces has been found to be a risk factor for infection, and consumption of water from a municipal supply has been suggested as a risk factor for children (8).

Epidemiological studies have found that H. pylori organisms colonize the stomach and duodenum of humans and many animal species and family clusters (9); it is believed to be orally transmitted person to person (10). This transmission is the major, if not exclusive, source of infection.

H. pylori has been detected in the mouth from dental plaque. Recent observations in persons infected with H. pylori caused to vomit or have diarrhea showed that an actively unwell person with these symptoms could spread H. pylori in the immediate vicinity by aerosol, splashing of vomitus, infected vomitus, and infected diarrhea. In summary, H. pylori is usually spread by the fecal-oral route but possibly also by the oral-oral route and the spread of contaminated secretions. Thus, in developing countries, individuals catch H. pylori at a very young age from other persons (children) in their environment. In developed countries, H. pylori is more difficult to acquire and is usually transmitted from one family member to another, possibly by the fecal-oral route, or by the oral-oral route, e.g., kissing, vomitus. On occasion, transmission occurs from person to person via contaminated endoscopes. Other gastric Helicobacter-like organisms have now been observed in a variety of animals (11), including rodents, primates, swine, and ferrets, but, with the exception of primates and possibly cats, these isolates are clearly different from human isolates (12). Foodborne transmission would not be unusual.

In the developed world, up to approx 50-90% of the adult population will be infected by the microorganism at the age of 50 yr, H. pylori affects about 20% of persons younger than 40 yr old. Children acquire the infection soon after being weaned. A 10% acquisition rate per year for children between 2 and 8 yr of age, for example, is found in West Africa; 80% of the children may be infected with the organism by age 5 yr. The overriding association with H. pylori is lower socioeconomic status during childhood.

The genus Helicobacter has grown steadily in the past few years; some of the new species have been reassigned from Campylobacter. The genus now includes H. pylori, H. heilmanni, H. muridarum, H. nemestrinae, H. mustelae, H. felis, H. acinonyx, H. pamatensis, H. bilis, H. canis, H. hepaticus, H. pullorum, H. cinaedi, and H. fennelliae. However, the only human pathogenic species described so far are H. pylori and H. heilmanni.

Helicobacter cells have blunted/rounded ends in gastric biopsy specimens. H. pylori organisms are microaerophilic, nonsporulating (characteristics that facilitate penetration and colonization of mucosal environments), Gram-negative spiral bacteria or curved rods, 3.5 ^m long and 0.5-1 ^m wide, with a spiral periodicity in fresh cultures on agar medium and spherical (coccoid) forms in older or prolonged cultures (see Notes 2 and 3). H. pylori further differs from Campylobacter species in having multiple polar sheathed flagellae; H. pylori are highly motile by means of lophotrichous flagella (tufts of flagella at poles of cell). H. fennellae and H. cinaedi have a single polar flagellum (electron microscopy reveals between four and six flagella positioned on one pole), a unique composition of cell wall with unusual fatty acids, and a smooth surface (11).

Taxonomy is based on 16S rRNA sequencing, DNA hybridization, genus-specific probes, cell wall protein and lipid characterization, serological and biochemical analyses. Campylobacter and Helicobacter (formerly Campylobacter) are the most clinically important members of the rRNA superfamily Recently defined family members of Campylobacteriaceae include the genera Campylobacter, Arcobacter, Helicobacter, Wolinella, and Flexispira, now considered a phylogenetically distinct family, and as yet unnamed Helicobacter (17 species by rRNA sequencing) human pathogens. Only three species are currently considered to be human pathogens: H. pylori, H. cinaedi, and H. fennelliae, formerly called Campylobacter-like organisms (CLOs).

2. Materials

1. Oxidase test.

2. Chocolate or blood agar plates. Blood agar containing an antifungal agent.

3. Medium containing urea and phenol red indicator.

4. Bismuth salts.

5. Sensidisc of erythromycin, rifampin, tetracycline, metronidazole, nalidixic acid, and sulfonamides.

6. Samples of endoscopic antral gastric biopsy material, oral secretions, dental plaque, and human feces. Gastric tissue or samples from biopsies of esophageal or duodenal tissue containing gastric metaplasia.

7. Transport media for culture: Nutrient, thioglycolate, Brucella, brain-heart infusion, supplemented tryptic soy broths, and semisolid agars (e.g., Wang's blood-supplemented medium) have all been used for transport culture. Insensitive complex basal media (agar or broth) supplemented with whole blood, heme, serum, charcoal, cornstarch, or egg yolk emulsion (5).

8. Microaerobic conditions (C-rich atmosphere for broth culture 15-17% oxygen and 610% carbon dioxide).

9. Columbia agar with 5% horse blood and triphenyltetrazolium.

3. Methods

3.1 Culture Characteristics

1. H. pylori has been cultured from both the oral cavity and feces, but the typical site is the gastric mucosa. It can be isolated from the stomach in individuals whose fecal and oral cultures are negative, but it is always found in the stomach when the other two sites are positive.

2. Samples can be simply smeared onto blood agar containing an antifungal agent (which is required to avoid fungal overgrowth) and incubated in a humid atmosphere.

3. The culture conditions are similar to those used for Campylobacter, i.e., a reduced oxygen atmosphere of about 5% with 10% carbon dioxide and the balance either nitrogen or hydrogen (see Note 4). Hydrogen may offer better recovery of injured organisms.

4. Cultures should be incubated at 37°C for a minimum of 5 d. H. pylori produces urease and does not grow when incubated below 30°C.

5. Growth is best on chocolate or blood agar plates after incubation for 2-5 d; for liquid media, either a blood or a hemin source appears to be essential. On Columbia agar, small colonies appear after 5 d of incubation. The colonies have a yellow sheen, which can aid recognition during primary isolation using nonselective media such as chocolate agar, or antibiotic-containing selective media, such as those of Skirrow or Goodwin. Spiral organisms that are oxidase-, catalase-, and urease-positive can be identified as H. pylori. Culture allows determination of antimicrobial susceptibilities.

6. As transport media for culture, nutrient, thioglycolate, Brucella, brain-heart infusion, supplemented tryptic soy broths, and semisolid agars (e.g., Wang's blood-supplemented medium) have all been used. Insensitive complex basal media (agar or broth) supplemented with whole blood, heme, serum, charcoal, cornstarch, or egg yolk emulsion may also be used.

7. Grow optimally under microaerobic conditions (C-rich atmosphere for broth culture).

3.2. Biochemical Characteristics

H. pylori is oxidase positive but nonsaccharolytic and does not reduce nitrate. Two enzymes of H. pylori, urease (13) and catalase, are unusual among medically important bacteria because of their high activity and can be used in the identification of H. pylori.

Abundant quatities of urease are produced only by gastric strains. Abundant quantities of mucinase and catalase are produced.

3.3. Detection Methods

1. Detection of urease activity in H. pylori can be used as a bedside test by applying a gastric biopsy sample to a simple medium containing urea and phenol red indicator.

2. In the presence of H. pylori urease the ammonia released by splitting urea raises the pH of the medium, and this is detected by a color change of the phenol red.

3. The speed with which this reaction occurs in H. pylori is highly unusual, and results are obtained in minutes at room temperature.

4. In vitro susceptibility to erythromycin, rifampin, tetracycline, and metronidazole is present.

5. Resistance to nalidixic acid and sulfonamides is present.

3.4. Laboratory Identification

1. Helicobacter organisms can be recovered from or detected in endoscopic antral gastric biopsy material; multiple biopsies should be taken.

2. Characteristically shaped organisms can be seen at high magnification with silver-stained hematoxylin and eosin-stained or methylene blue-stained biopsy specimens of the gastric antrum (antral biopsies) in the mucosa adherent to the surface epithelium or pit epithelial cells deep within the crypts.

3. Organisms are always found adjacent to gastric mucosal cells.

4. Notes

1. Susceptibility to removal or inactivation by conventional water treatment processes. There is no direct evidence for the effectiveness of treatment processes for removal of H. pylori, although they are likely to be removed to a similar extent as other bacteria. On the basis of their similarity to Campylobacter organisms, they should be susceptible to inactivation by disinfectants such as chlorine and ozone.

2. A coccal form of H. pylori has been described, although speculation continues as to the function, if any, and indeed the actual viability of these forms. They may mimic the so-called nonculturable but viable forms.

3. H. pylori changes from helical to coccoid when exposed to adverse conditions (oxygen or upon prolonged culture). The coccoid forms remain viable, at least for a short time.

4. H. pylori can grow, although relatively poorly, in atmospheric oxygen.

References

1 Marshall, B. J., Barrett, L., Prakash, C., McCallum, R. W., and Guerrant, R. L. (1990) Urea protects Helicobacter (Campylobacter) pylori from the bactericidal effect of acid. Gastroenterology 99, 697-702.

2. Blaser, M. J. (1997) The versatility of Helicobacter pylori in the adaptation to the human stomach [Review]. J. Physiol. Pharmacol. 48,307-314.

3 Marshall, B. J. and Warren, J. R. (1984) Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet 1, 1311-1315.

4. Parsonnet, J., Shmuely, H., and Haggerty, T. (1999) Fecal and oral shedding of Helicobacter pylori from healthy infected adults. JAMA 282, 2240-2245.

5. Westblom, T. U., Madan, E., and Midkiff, B. R. (1991) Egg yolk emulsion agar, a new medium for the cultivation of Helicobacter pylori. J. Clin. Microbiol. 29, 819-821.

6. Shimoyama, T., Tominaga, Y., Sakagami, T., and Fukuda, Y. (1999) Epidemiological study for infection with H. pylori in Japan compared with that in USA, Europe and Asian Pacific area [Review]. Nippon Rinsho (Jpn J. Clin. Med.) 57, 11-16.

7. Hulten, K., Han, S. W., Enroth, H., et al. (1996) Helicobacter pylori in the drinking water in Peru. Gastroenterology 110, 1031-1035.

8. Webb, P. M., Knight, T., Greaves, S., et al. (1994) Relation between infection with Helicobacter pylori and living conditions in childhood: evidence for person to person transmission in early life. BMJ 308, 750-753.

9. Samuels, A. L., Windsor, H. M., Ho, G. Y., Goodwin, L. D., and Marshall, B. J. (2000) Culture of Helicobacter pylori from a gastric string may be an alternative to endoscopic biopsy. J. Clin. Microbiol. 38, 2438-2439.

10. Li, C., Ha, T., Ferguson, D. A., Jr., et al. (1996) A newly developed PCR assay of H. pylori in gastric biopsy, saliva, and feces. Evidence of high prevalence of H. pylori in saliva supports oral transmission. Dig. Dis. Sci. 41, 2142-2149.

11. Lee, L., Phillips, M. W., O'Rourke, J. L., et al. (1992) Helicobactermuridarum sp. nov., a microaerophilic helical bacterium with a novel ultrastructure isolated from the intestinal mucosa of rodents. Int. J. Syst. Bacteriol. 42, 27-36.

12. Dore, M. P., Sepulveda, A. R., Osato, M. S., Realdi, G., Graham, D. Y. (1999) Helicobacter pylori in sheep milk. Lancet 354, 132.

13. Luck, J. M. and Seth, T. N. (1924) Gastric urease. Biochem. J. 18, 1227-31.

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