Chapter 17: Vibrio, Campylobacter, and Helicobacter

Vibrio, Campylobacter, and Helicobacter are Gram-negative rods that are all widely distributed in nature. The vibrios are found in marine and surface waters. The campylobacters are found in many species of animals, including many domesticated animals. Vibrio cholerae produces an enterotoxin that causes cholera, a profuse watery diarrhea that can rapidly lead to dehydration and death. Campylobacter jejuni is a common cause of enteritis in humans. Helicobacter pylori is associated with gastritis and duodenal ulcer disease.

Vibrios are among the most common bacteria in surface waters worldwide. They are curved aerobic rods and are motile, possessing a polar flagellum. V cholerae serogroups O1 and O139 cause cholera in humans, and other vibrios may cause skin and soft tissue infections, sepsis, or enteritis. The medically important vibrios are listed in Table 17-1.

The epidemiology of cholera closely parallels the recognition of V cholerae transmission in water and the development of sanitary water systems.

Morphology and Identification

A. Typical Organisms

Upon first isolation, V cholerae is a comma-shaped, curved rod 2–4 μm long (Figure 17-1). It is actively motile by means of a polar flagellum. On prolonged cultivation, vibrios may become straight rods that resemble the Gram-negative enteric bacteria.

B. Culture

V cholerae produces convex, smooth, round colonies that are opaque and granular in transmitted light. V cholerae and most other vibrios grow well at 37°C on many kinds of media, including defined media containing mineral salts and asparagine as sources of carbon and nitrogen. V cholerae grows well on thiosulfate-citrate-bile-sucrose (TCBS) agar, a media selective for vibrios, on which it produces yellow colonies (sucrose fermented) that are readily visible against the dark-green background of the agar (Figure 17-2). Vibrios are oxidase positive, which differentiates them from enteric Gram-negative bacteria. Characteristically, vibrios grow at a very high pH (8.5–9.5) and are rapidly killed by acid. Cultures containing fermentable carbohydrates therefore quickly become sterile.

In areas where cholera is endemic, direct cultures of stool on selective media, such as TCBS, and enrichment cultures in alkaline peptone water are appropriate. However, routine stool cultures on special media such as TCBS generally are not necessary or cost effective in areas where cholera is rare.

C. Growth Characteristics

V cholerae regularly ferments sucrose and mannose but not arabinose. A positive oxidase test result is a key step in the preliminary identification of V cholerae and other vibrios. Most Vibrio species are halotolerant, and NaCl often stimulates their growth. Some vibrios are halophilic, requiring the presence of NaCl to grow.

Antigenic Structure and Biologic Classification

Many vibrios share a single heat-labile flagellar H antigen. Antibodies to the H antigen are probably not involved in the protection of susceptible hosts.

V cholerae has O lipopolysaccharides that confer serologic specificity. There are at least 206 O antigen groups. V cholerae strains of O group 1 and O group 139 cause classic cholera; occasionally, non-O1/non-O139 V cholerae causes cholera-like disease. Antibodies to the O antigens tend to protect laboratory animals against infections with V cholerae.

The V cholerae serogroup O1 antigen has determinants that make possible further typing; the serotypes are Ogawa, Inaba, and Hikojima. Two biotypes of epidemic V cholerae have been defined, classic and El Tor. The El Tor biotype produces a hemolysin, gives positive results on the Voges-Proskauer test, and is resistant to polymyxin B. Molecular techniques can also be used to type V cholerae. Typing is used for epidemiologic studies, and tests generally are done only in reference laboratories.

V cholerae O139 is very similar to V cholerae O1 El Tor biotype. V cholerae O139 does not produce the O1 lipopolysaccharide and does not have all the genes necessary to make this antigen. V cholerae O139 makes a polysaccharide capsule like other non-O1 V cholerae strains, but V cholerae O1 does not make a capsule.

Vibrio cholerae Enterotoxin

V cholerae produce a heat-labile enterotoxin with a molecular weight (MW) of about 84,000, consisting of subunits A (MW, 28,000) and B (see Chapter 9). Ganglioside GM₁ serves as the mucosal receptor for subunit B, which promotes entry of subunit A into the cell. Activation of subunit A₁ yields increased levels of intracellular cyclic adenosine monophosphate (cAMP) and results in prolonged hypersecretion of water and electrolytes. There is increased sodium-dependent chloride secretion, and absorption of sodium and chloride by the microvilli is inhibited. Electrolyte-rich diarrhea occurs—as much as 20–30 L/day—with resulting dehydration, shock, acidosis, and death. The genes for V cholerae enterotoxin are on the bacterial chromosome. Cholera enterotoxin is antigenically related to LT of Escherichia coli and can stimulate the production of neutralizing antibodies. However, the precise role of antitoxic and antibacterial antibodies in protection against cholera is not clear.

Pathogenesis and Pathology

Under natural conditions, V cholerae is pathogenic only for humans. A person with normal gastric acidity may have to ingest as many as 10¹⁰ or more V cholerae to become infected when the vehicle is water because the organisms are susceptible to acid. When the vehicle is food, as few as 10²–10⁴ organisms are necessary because of the buffering capacity of food. Any medication or condition that decreases stomach acidity makes a person more susceptible to infection with V cholerae.

Cholera is not an invasive infection. The organisms do not reach the bloodstream but remain within the intestinal tract. Virulent V cholerae organisms attach to the microvilli of the brush border of epithelial cells. There they multiply and liberate cholera toxin and perhaps mucinases and endotoxin.

Clinical Findings

About 50% of infections with classic V cholerae are asymptomatic, as are about 75% of infections with the El Tor biotype. The incubation period is 12 hours–3 days for persons who develop symptoms, depending largely on the size of the inoculum ingested. There is a sudden onset of nausea and vomiting and profuse diarrhea with abdominal cramps. Stools, which resemble “rice water,” contain mucus, epithelial cells, and large numbers of vibrios. There is rapid loss of fluid and electrolytes, which leads to profound dehydration, circulatory collapse, and anuria. The mortality rate without treatment is between 25% and 50%. The diagnosis of a full-blown case of cholera presents no problem in the presence of an epidemic. However, sporadic or mild cases are not readily differentiated from other diarrheal diseases. The El Tor biotype tends to cause milder disease than the classic biotype.

Diagnostic Laboratory Tests

A. Specimens

Specimens for culture consist of mucus flecks from stools.

B. Smears

The microscopic appearance of smears made from stool samples is not distinctive. Dark-field or phase contrast microscopy may show the rapidly motile vibrios.

C. Culture

Growth is rapid in peptone agar, on blood agar with a pH near 9.0, or on TCBS agar, and typical colonies can be picked in 18 hours. For enrichment, a few drops of stool can be incubated for 6–8 hours in taurocholate peptone broth (pH, 8.0–9.0); organisms from this culture can be stained or subcultured. Accurate identification of vibrios, including V cholerae, using commercial systems and kit assays is quite variable.

D. Specific Tests

V cholerae organisms are further identified by slide agglutination tests using anti-O group 1 or group 139 antisera and by biochemical reaction patterns. The diagnosis of cholera under field conditions has been reported to be facilitated by a sensitive and specific immunochromatographic dipstick test.

Immunity

Gastric acid provides some protection against cholera vibrios.

An attack of cholera is followed by immunity to reinfection, but the duration and degree of immunity are not known. In experimental animals, specific IgA antibodies occur in the lumen of the intestine. Similar antibodies in serum develop after infection but last only a few months. Vibriocidal antibodies in serum (titer ≥1:20) have been associated with protection against colonization and disease. The presence of antitoxin antibodies has not been associated with protection.

Treatment

The most important part of therapy consists of water and electrolyte replacement to correct the severe dehydration and salt depletion. Several guidelines, including those from the World Health Organization, for effective rehydration have been published and are provided in the list of references at the end of this chapter. Many antimicrobial agents are effective against V cholerae, but these play a secondary role in patient management. Oral tetracycline and doxycycline tend to reduce stool output in cholera and shorten the period of excretion of vibrios. In some endemic areas, tetracycline resistance of V cholerae has emerged; the genes are carried by transmissible plasmids. In children and pregnant women, alternatives to the tetracyclines include erythromycin and furazolidine.

Epidemiology, Prevention, and Control

Six pandemics (worldwide epidemics) of cholera occurred between 1817 and 1923 caused most likely by V cholerae O1 of the classic biotype and largely originating in Asia, usually the Indian subcontinent. The seventh pandemic began in 1961 in the Celebes Islands, Indonesia, with spread to Asia, the Middle East, and Africa. This pandemic was caused by V cholerae biotype El Tor. Starting in 1991, the seventh pandemic spread to Peru and then to other countries of South America and Central America. Cases also occurred in Africa. Millions of people have had cholera in this pandemic. Some consider the cholera caused by the serotype O139 strain to be the eighth pandemic that began in the Indian subcontinent in 1992–1993, with spread to Asia. The disease has been rare in North America since the mid-1800s, but an endemic focus exists on the Gulf Coast of Louisiana and Texas.

Cholera is endemic in India and Southeast Asia. From these centers, it is carried along shipping lanes, trade routes, and pilgrim migration routes. The disease is spread by contact involving individuals with mild or early illness and by water, food, and flies. In many instances, only 1–5% of exposed susceptible persons develop disease. The carrier state seldom exceeds 3–4 weeks, and the importance of carriers in transmission is unclear. Vibrios survive in water for up to 3 weeks.

In 2010, Haiti experienced a magnitude of 7.0 earthquake that devastated the country’s infrastructure. Peacekeepers from the United Nations were invited to Haiti to provide support. Some of those invited from countries in Southeast Asia brought V cholerae O1, serotype Ogawa, biotype El Tor with them where it was introduced into local waterways used by the populace as a source of water for drinking, cooking, and bathing. Consequently, hundreds of thousands of Haitians have been infected and cholera is now endemic in this Caribbean nation.

V cholerae lives in aquatic environments. And such environments are the vibrios natural reservoir. V cholerae lives attached to algae, copepods, and crustacean shells. It can survive for years and grow, but when conditions are not suitable for growth, it can become dormant.

Control rests on education and on improvement of sanitation, particularly of food and water. Patients should be isolated, their excreta disinfected, and contacts followed up. Chemoprophylaxis with antimicrobial drugs may have a place. Repeated injection of a vaccine containing either lipopolysaccharides extracted from vibrios or dense Vibrio suspensions can confer limited protection to heavily exposed persons (eg, family contacts) but is not effective as an epidemic control measure.

Vibrio parahaemolyticus is a halophilic bacterium that causes acute gastroenteritis after ingestion of contaminated seafood such as raw fish or shellfish. After an incubation period of 12–24 hours, nausea and vomiting, abdominal cramps, fever, and watery to bloody diarrhea occur. Fecal leukocytes are often observed. The enteritis tends to subside spontaneously in 1–4 days with no treatment other than restoration of water and electrolyte balance. No enterotoxin has yet been isolated from this organism. The disease occurs worldwide, with highest incidence in Asia and other areas where people eat raw seafood. V parahaemolyticus does not grow well on some of the differential media used to grow salmonellae and shigellae, but it does grow well on blood agar. It also grows well on TCBS, where it yields green colonies (does not ferment sucrose). V parahaemolyticus is usually identified by its oxidase-positive growth on blood agar.

Vibrio vulnificus can cause severe wound infections, bacteremia, and probably gastroenteritis. It is a free-living estuarine bacterium found in the United States on the Atlantic and Pacific Coasts and especially the Gulf Coast. Infections have been reported from Korea, and the organism may be distributed worldwide. V vulnificus is particularly apt to be found in oysters, especially in warm months. Bacteremia with no focus of infection occurs in persons who have eaten infected oysters and who have alcoholism or liver disease. Wounds may become infected in normal or immunocompromised persons who are in contact with water where the bacterium is present. Infection often proceeds rapidly, with development of severe disease. About 50% of the patients with bacteremia die. Wound infections may be mild but often proceed rapidly (over a few hours), with development of bullous skin lesions, cellulitis, and myositis with necrosis. Several of the first deaths in Louisiana and Texas after hurricane Katrina were caused by V vulnificus. Because of the rapid progression of the infection, it is often necessary to treat with appropriate antibiotics before culture confirmation of the etiology can be obtained. Diagnosis is by culturing the organism on standard laboratory media; TCBS is the preferred medium for stool cultures, where most strains produce blue-green (sucrose-negative) colonies.

Tetracycline appears to be the drug of choice for V vulnificus infection; ciprofloxacin may be effective also based on in vitro activity.

Concept Checks

• Vibrio species are halophilic, oxidase-positive, motile, curved, Gram-negative rods that are found in surface waters worldwide.

• Many Vibrio species are pathogenic for humans, but V cholerae is the species of most global importance responsible for pandemics of cholera. Although there are more than 200 serotypes of V cholerae, serotypes O1 and O139 are associated with cholera.

• V cholerae O1 can be further classified into the classic and El Tor biotypes; classic biotypes have been responsible for most of the major pandemics and are more likely to cause symptomatic infection. El Tor has caused the most recent pandemic.

• V cholerae causes an acute watery diarrhea after ingestion in high numbers in contaminated water or food by elaboration of a heat-labile enterotoxin that has the classic A-B toxin structure. The B segment binds to GM₁ ganglioside receptors, and the active A subunit induces cAMP, causing secretion of sodium chloride, while at the same time preventing the reabsorption by the microvilli.

• Diagnosis is made by culturing stool on selective medium such as TCBS or alkaline peptone broth. Treatment involves rehydration and secondarily tetracycline or doxycycline.

• Other important Vibrio species include V parahaemolyticus, the most common cause of foodborne gastroenteritis in Asia, and V vulnificus, a cause of wound infections and severe sepsis in patients with cirrhosis.

Campylobacters cause both diarrheal and systemic diseases, and are among the most widespread causes of infection in the world. Campylobacter infection of domesticated animals also is widespread. C jejuni is the prototype organism in the group and is a very common cause of diarrhea in humans.

C jejuni has emerged as common human pathogen, causing mainly enteritis and occasionally systemic infection. These bacteria are at least as common as salmonellae and shigellae as a cause of diarrhea; an estimated 2 million cases occur in the United States each year.

Morphology and Identification

A. Typical Organisms

C jejuni are Gram-negative rods with comma, S, or “gull wing” shapes (Figure 17-3). They are motile, with a single polar flagellum, and do not form spores.

B. Culture

The culture characteristics are most important in the isolation and identification of C jejuni. Selective media are needed, and incubation must be in an atmosphere with reduced O₂ (5% O₂) with added CO₂ (10% CO₂). A relatively simple way to produce the incubation atmosphere is to place the plates in an anaerobe incubation jar without the catalyst and to produce the gas with a commercially available gas-generating pack or by gas exchange. Incubation of primary plates for isolation of C jejuni should be at 42°C. Although C jejuni grows well at 36–37°C, incubation at 42°C prevents growth of most of the other bacteria present in feces, thus simplifying the identification of C jejuni. Several selective media are in widespread use. Skirrow’s medium contains vancomycin, polymyxin B, and trimethoprim to inhibit growth of other bacteria, but this medium may be less sensitive than other commercial products that contain charcoal, other inhibitory compounds, and cephalosporin antibiotics. The selective media are suitable for isolation of C jejuni at 42°C. The colonies tend to be colorless or gray. They may be watery and spreading or round and convex, and both colony types may appear on one agar plate.

C. Growth Characteristics

Because of the selective media and incubation conditions for growth, an abbreviated set of tests is usually all that is necessary for identification. C jejuni are positive for both oxidase and catalase. Campylobacters do not oxidize or ferment carbohydrates. Gram-stained smears show typical morphology. Nitrate reduction, hydrogen sulfide production, hippurate tests, and antimicrobial susceptibilities can be used for further identification of species.

Antigenic Structure and Toxins

The campylobacters have lipopolysaccharides with endotoxic activity. Cytopathic extracellular toxins and enterotoxins have been found, but the significance of the toxins in human disease is not well defined.

Pathogenesis and Pathology

The infection is acquired by the oral route from food, drink, or contact with infected animals or animal products, especially poultry. C jejuni is susceptible to gastric acid, and ingestion of about 10⁴ organisms is usually necessary to produce infection. This inoculum is similar to that required for Salmonella and Shigella infection but less than that for Vibrio infection. The organisms multiply in the small intestine, invade the epithelium, and produce inflammation that results in the appearance of red and white blood cells in the stools. Occasionally, the bloodstream is invaded, and a clinical picture of enteric fever develops. Localized tissue invasion coupled with the toxic activity appears to be responsible for the enteritis.

Clinical Findings

Clinical manifestations are acute onset of crampy abdominal pain, profuse diarrhea that may be grossly bloody, headache, malaise, and fever. Usually the illness is self-limited to a period of 5–8 days, but occasionally it continues longer. C jejuni isolates are usually susceptible to erythromycin, and therapy shortens the duration of fecal shedding of bacteria. Most cases resolve without antimicrobial therapy; however, in about 5–10% of patients, symptoms may recur. Certain serotypes of C jejuni have been associated with postdiarrheal Guillain-Barré syndrome, a form of ascending paralytic disease. Reactive arthritis and Reiter’s syndrome may also follow acute Campylobacter diarrhea.

Diagnostic Laboratory Tests

A. Specimens

Diarrheal stool is the usual specimen. C jejuni may occasionally be recovered from blood cultures usually from immunocompromised or elderly patients. Other extraintestinal infections are uncommon.

B. Smears

Gram-stained smears of stool may show the typical “gull wing”–shaped rods. Dark-field or phase contrast microscopy may show the typical darting motility of the organisms.

C. Culture

Culture on the selective media described earlier is the definitive test to diagnose C jejuni enteritis. If another species of Campylobacter is suspected, medium without a cephalosporin should be used and incubated at 36–37°C.

Epidemiology and Control

Campylobacter enteritis resembles other acute bacterial diarrheas, particularly shigella dysentery. The source of infection may be food (eg, milk, undercooked fowl) or contact with infected animals or humans and their excreta. Outbreaks arising from a common source, such as unpasteurized milk, may require public health control measures.

H pylori is a spiral-shaped, gram-negative rod. H pylori is associated with antral gastritis, duodenal (peptic) ulcer disease, gastric ulcers, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue (MALT) lymphomas.

Morphology and Identification

A. Typical Organisms

H pylori has many characteristics in common with campylobacters. It has multiple flagella at one pole and is actively motile.

B. Culture

Culture sensitivity can be limited by prior therapy, contamination with other mucosal bacteria, and other factors. H pylori grows in 3–6 days when incubated at 37°C in a microaerophilic environment, as for C jejuni. The media for primary isolation include Skirrow’s medium with vancomycin, polymyxin B, and trimethoprim, chocolate medium, and other selective media with antibiotics (eg, vancomycin, nalidixic acid, amphotericin). The colonies are translucent and 1–2 mm in diameter.

C. Growth Characteristics

H pylori is oxidase positive and catalase positive, has a characteristic morphology, is motile, and is a strong producer of urease.

Pathogenesis and Pathology

H pylori grows optimally at a pH of 6.0–7.0 and would be killed or not grow at the pH within the gastric lumen. Gastric mucus is relatively impermeable to acid and has a strong buffering capacity. On the lumen side of the mucus, the pH is low (1.0–2.0); on the epithelial side, the pH is about 7.4. H pylori is found deep in the mucous layer near the epithelial surface where physiologic pH is present. H pylori also produces a protease that modifies the gastric mucus and further reduces the ability of acid to diffuse through the mucus. H pylori produces potent urease activity, which yields production of ammonia and further buffering of acid. H pylori is quite motile, even in mucus, and is able to find its way to the epithelial surface. H pylori overlies gastric-type but not intestinal-type epithelial cells.

In human volunteers, ingestion of H pylori resulted in development of gastritis and hypochlorhydria. There is a strong association between the presence of H pylori infection and duodenal ulceration. Antimicrobial therapy results in clearing of H pylori and improvement of gastritis and duodenal ulcer disease.

The mechanisms by which H pylori causes mucosal inflammation and damage are not well defined but probably involve both bacterial and host factors. The bacteria invade the epithelial cell surface to a limited degree. Toxins and lipopolysaccharide may damage the mucosal cells, and the ammonia produced by the urease activity may also directly damage the cells.

Histologically, gastritis is characterized by acute and chronic inflammation. Polymorphonuclear and mononuclear cell infiltrates are seen within the epithelium and lamina propria. Vacuoles within cells are often pronounced. Destruction of the epithelium is common, and glandular atrophy may occur. H pylori thus is a major risk factor for gastric cancer.

Clinical Findings

Acute infection can yield an upper gastrointestinal illness with nausea and pain; vomiting and fever may also be present. The acute symptoms may last for less than 1 week or as long as 2 weeks. After colonization, the H pylori infection persists for years and perhaps decades or even a lifetime. About 90% of patients with duodenal ulcers and 50–80% of those with gastric ulcers have H pylori infection. Recent studies confirm that H pylori also is a risk factor for gastric carcinoma and lymphoma.

Diagnostic Laboratory Tests

A. Specimens

Gastric biopsy specimens can be used for histologic examination or minced in saline and used for culture. Blood is collected for determination of serum antibodies. Stool samples may be collected for H pylori antigen detection.

B. Smears

The diagnosis of gastritis and H pylori infection can be made histologically. A gastroscopy procedure with biopsy is required. Routine stains demonstrate gastritis, and Giemsa or special silver stains can show the curved or spiral-shaped organisms.

C. Culture

As above, culture is performed when patients are not responding to treatment, and there is a need to assess susceptibility patterns.

D. Antibodies

Several assays have been developed to detect serum antibodies specific for H pylori. The serum antibodies persist even if the H pylori infection is eradicated, and the role of antibody tests in diagnosing active infection or after therapy is therefore limited.

E. Special Tests

Rapid tests to detect urease activity are widely used for presumptive identification of H pylori in specimens. Gastric biopsy material can be placed onto a urea-containing medium with a color indicator. If H pylori is present, the urease rapidly splits the urea (1–2 hours), and the resulting shift in pH yields a color change in the medium. In vivo tests for urease activity can be done also. In urea breath tests, ¹³C- or ¹⁴C-labeled urea is ingested by the patient. If H pylori is present, the urease activity generates labeled CO₂ that can be detected in the patient’s exhaled breath.

Detection of H pylori antigen in stool specimens is appropriate as a test of cure for patients with known H pylori infection who have been treated.

Immunity

Patients infected with H pylori develop an IgM antibody response to the infection. Subsequently, IgG and IgA are produced, and these persist, both systemically and at the mucosa, in high titer in chronically infected persons. Early antimicrobial treatment of H pylori infection blunts the antibody response; such patients are thought to be subject to repeat infection.

Treatment

Triple therapy with metronidazole and either bismuth subsalicylate or bismuth subcitrate plus either amoxicillin or tetracycline for 14 days eradicates H pylori infection in 70–95% of patients. An acid-suppressing agent given for 4–6 weeks enhances ulcer healing. Proton pump inhibitors (PPIs) directly inhibit H pylori and appear to be potent urease inhibitors. The preferred initial therapy is 7–10 days of a PPI plus amoxicillin and clarithromycin or a quadruple regimen of a PPI metronidazole, tetracycline, and bismuth for 10 days.

Epidemiology and Control

H pylori is present on the gastric mucosa of fewer than 20% of persons younger than years 30 but increases in prevalence to 40–60% of persons age 60 years, including persons who are asymptomatic. In developing countries, the prevalence of infection may be 80% or higher in adults. Person-to-person transmission of H pylori is likely because intrafamilial clustering of infection occurs. Acute epidemics of gastritis suggest a common source for H pylori.

Concept Checks

• Campylobacters are oxidase positive, curved or spiral-shaped organisms that sometimes have a “gull’s wing” appearance. C jejuni is the major pathogen and is associated primarily with febrile diarrhea that may be bloody. Contaminated food, primarily poultry, is the major vehicle for infection.

• C jejuni grows well at 42°C in a microaerophilic environment of 5% oxygen and 10% CO₂. Selective media that contain antibiotics are generally used to recover the organisms from stool.

• Helicobacter species are also curved or spiral-shaped pathogens. H pylori is associated with upper gastrointestinal diseases such as gastric and duodenal ulcers, gastric adenocarcinoma, and MALT lymphomas. The organism is urease positive, which protects it against gastric acidity. Diagnosis is made by a variety of methods including biopsy, urea breath tests, and stool antigen. Triple and quadruple antibiotic regimens that include PPIs are necessary for successful treatment.