The Shigellae

8/8/2019 The Shigellae

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THE SHIGELLAE

The natural habitat of shigellae is limited to the intestinal tracts of humans and

other primates, where they produce bacillary dysentery.

Morphology & Identification

TYPICAL ORGANISMS

Shigellae are slender gram-negative rods; coccobacillary forms occur in young

cultures.

CULTURE

Shigellae are facultative anaerobes but grow best aerobically. Convex, circular,

transparent colonies with intact edges reach a diameter of about 2 mm in 24

hours.

GROWTH CHARACTERISTICS

All shigellae ferment glucose. With the exception ofShigella sonnei, they do

not ferment lactose. The inability to ferment lactose distinguishes shigellae on

differential media. Shigellae form acid from carbohydrates but rarely produce

gas. They may also be divided into those that ferment mannitol and those that

do not (Table 163).

Table 163. Pathogenic Species of Shigella.

Present Designation Group and Type Mannitol Ornithine Decarboxylase

S dysenteriae A - -S flexneri B + -S boydii C + -S sonnei D + +Antigenic Structure

Shigellae have a complex antigenic pattern. There is great overlapping in the

serologic behavior of different species, and most of them share O antigens with

other enteric bacilli.

The somatic O antigens of shigellae are lipopolysaccharides. Their serologic

specificity depends on the polysaccharide. There are more than 40 serotypes.

The classification of shigellae relies on biochemical and antigenic

characteristics. The pathogenic species are shown in Table 163.

Pathogenesis & Pathology

Shigella infections are almost always limited to the gastrointestinal tract;


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bloodstream invasion is quite rare. Shigellae are highly communicable; the

infective dose is on the order of 103 organisms (whereas it usually is 105108

for salmonellae and vibrios). The essential pathologic process is invasion of the

mucosal epithelial cells (eg, M cells) by induced phagocytosis, escape from the

phagocytic vacuole, multiplication and spread within the epithelial cell

cytoplasm, and passage to adjacent cells. Microabscesses in the wall of the

large intestine and terminal ileum lead to necrosis of the mucous membrane,

superficial ulceration, bleeding, and formation of a "pseudomembrane" on the

ulcerated area. This consists of fibrin, leukocytes, cell debris, a necrotic

mucous membrane, and bacteria. As the process subsides, granulation tissue

fills the ulcers and scar tissue forms.

ToxinsENDOTOXIN

Upon autolysis, all shigellae release their toxic lipopolysaccharide. This

endotoxin probably contributes to the irritation of the bowel wall.

SHIGELLA DYSENTERIAEEXOTOXIN

S dysenteriae type 1 (Shiga bacillus) produces a heat-labile exotoxin that

affects both the gut and the central nervous system. The exotoxin is a protein

that is antigenic (stimulating production of antitoxin) and lethal forexperimental animals. Acting as an enterotoxin, it produces diarrhea as does

the E coliverotoxin, perhaps by the same mechanism. In humans, the exotoxin

also inhibits sugar and amino acid absorption in the small intestine. Acting as a

"neurotoxin," this material may contribute to the extreme severity and fatal

nature ofS dysenteriae infections and to the central nervous system reactions

observed in them (ie, meningismus, coma). Patients with Shigella flexnerior

Shigella sonneiinfections develop antitoxin that neutralizes S dysenteriae

exotoxin in vitro. The toxic activity is distinct from the invasive property ofshigellae in dysentery. The two may act in sequence, the toxin producing an

early nonbloody, voluminous diarrhea and the invasion of the large intestine

resulting in later dysentery with blood and pus in stools.

Clinical Findings

After a short incubation period (12 days), there is a sudden onset of

abdominal pain, fever, and watery diarrhea. The diarrhea has been attributed

to an exotoxin acting in the small intestine (see above). A day or so later, asthe infection involves the ileum and colon, the number of stools increases; they


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are less liquid but often contain mucus and blood. Each bowel movement is

accompanied by straining and tenesmus (rectal spasms), with resulting lower

abdominal pain. In more than half of adult cases, fever and diarrhea subside

spontaneously in 25 days. However, in children and the elderly, loss of

water and electrolytes may lead to dehydration, acidosis, and even death. The

illness due to S dysenteriae may be particularly severe.

On recovery, most persons shed dysentery bacilli for only a short period, but a

few remain chronic intestinal carriers and may have recurrent bouts of the

disease. Upon recovery from the infection, most persons develop circulating

antibodies to shigellae, but these do not protect against reinfection.

Diagnostic Laboratory Tests

SPECIMENS

Specimens include fresh stool, mucus flecks, and rectal swabs for culture.

Large numbers of fecal leukocytes and some red blood cells often are seen

microscopically. Serum specimens, if desired, must be taken 10 days apart to

demonstrate a rise in titer of agglutinating antibodies.

CULTURE

The materials are streaked on differential media (eg, MacConkey's or EMB

agar) and on selective media (Hektoen enteric agar or salmonella-shigella

agar), which suppress other Enterobacteriaceae and gram-positive organisms.

Colorless (lactose-negative) colonies are inoculated into triple sugar iron agar.

Organisms that fail to produce H2S, that produce acid but not gas in the butt

and an alkaline slant in triple sugar iron agar medium, and that are nonmotile

should be subjected to slide agglutination by specific shigella antisera.

SEROLOGY

Normal persons often have agglutinins against several Shigella species.However, serial determinations of antibody titers may show a rise in specific

antibody. Serology is not used to diagnose shigella infections.

Immunity

Infection is followed by a type-specific antibody response. Injection of killed

shigellae stimulates production of antibodies in serum but fails to protect

humans against infection. IgA antibodies in the gut may be important in

limiting reinfection; these may be stimulated by live attenuated strains givenorally as experimental vaccines. Serum antibodies to somatic shigella antigens


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INTRODUCTION

Vibrio, Aeromonas, Plesiomonas, Campylobacter, and Helicobacterspecies are

gram-negative rods that are all widely distributed in nature. The vibrios arefound in marine and surface waters. Aeromonas is found predominantly in fresh

water and occasionally in cold-blooded animals. Plesiomonas exists in both cold-

blooded and warm-blooded animals. 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 jejuniis a common cause

of enteritis in humans. Less commonly, aeromonas and, rarely, plesiomonas

have been associated with diarrheal disease in humans. Helicobacter pylorihas

been associated with gastritis and duodenal ulcer disease.

THE VIBRIOS

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, while other vibrios may

cause sepsis or enteritis. The medically important vibrios are listed in Table

181.

Table 181. The Medically Important Vibrios.

Organism

V cholerae serogroups O1 and O139V cholerae serogroups non-O1/non-O139V parahaemolyticusOthers

V mimicus, V vulnificus, V hollisae, V fluvialis, V damsela, V anginolyticus, Vmetschnikovii

Vibrio cholerae

The epidemiology of cholera closely parallels the recognition ofV cholerae

transmission in water and the development of sanitary water systems.

Morphology & Identification

TYPICAL ORGANISMS

Upon first isolation, V cholerae is a comma-shaped, curved rod 24 m long(Figure 181). It is actively motile by means of a polar flagellum. On


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prolonged cultivation, vibrios may become straight rods that resemble the gram-

negative enteric bacteria.

Figure 181.

Vibrio cholerae grown in broth showing slightly curved gram-negative rods.


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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, on which it produces yellow

colonies that are readily visible against the dark-green background of the agar.

Vibrios are oxidase-positive, which differentiates them from enteric gram-

negative bacteria. Characteristically, vibrios grow at a very high pH (8.59.5)

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 mediasuch 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.

GROWTH CHARACTERISTICS

V cholerae regularly ferments sucrose and mannose but not arabinose. A

positive oxidase test is a key step in the preliminary identification ofV cholerae

and other vibrios. Vibrio species are susceptible to the compound O/129 (2,4-diamino-6,7-diisopropylpteridine phosphate), which differentiates them from

Aeromonas species, which are resistant to O/129. Most Vibrio species are

halotolerant, and NaCl often stimulates their growth. Some vibrios are halophilic,

requiring the presence of NaCl to grow. Another difference between vibrios and

aeromonas is that vibrios grow on media containing 6% NaCl, whereas

aeromonas does not.

Antigenic Structure & 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 139 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


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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, while V cholerae O1 does not make

a capsule.

Vibrio cholerae Enterotoxin

V cholerae produce a heat-labile enterotoxin with a molecular weight of about

84,000, consisting of subunits A (MW 28,000) and B (see Chapter 10).

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 cAMP and results in prolonged hypersecretion of water and

electrolytes. There is increased sodium-dependent chloride secretion, and

absorption of sodium and chloride is inhibited. Diarrhea occursas much as2030 L/dwith resulting dehydration, shock, acidosis, and death. The

genes for V cholerae enterotoxin are on the bacterial chromosome. Cholera

enterotoxin is antigenically related to LT ofEscherichia coliand can stimulate the

production of neutralizing antibodies. However, the precise role of antitoxic and

antibacterial antibodies in protection against cholera is not clear.

Pathogenesis & Pathology

Under natural conditions, V cholerae is pathogenic only for humans. A personwith 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 102104 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


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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 60% of infections with classic V cholerae are asymptomatic, as are about75% of infections with the El Tor biotype. The incubation period is 14 days

for persons who develop symptoms, depending largely upon 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

SPECIMENS

Specimens for culture consist of mucus flecks from stools.

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.

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 68 hours in taurocholate-peptone broth(pH 8.09.0); organisms from this culture can be stained or subcultured.

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.

Immunity

Gastric acid provides some protection against cholera vibrios.

An attack of cholera is followed by immunity to reinfection, but the duration and


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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. Many

antimicrobial agents are effective against V cholerae. Oral tetracycline tends to

reduce stool output in cholera and shortens the period of excretion of vibrios. In

some endemic areas, tetracycline resistance ofV cholerae has emerged; the

genes are carried by transmissible plasmids.

Epidemiology, Prevention, & 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 has been 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 19921993, 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 15% of exposed susceptible persons

develop disease. The carrier state seldom exceeds 34 weeks, and the

importance of carriers in transmission is unclear. Vibrios survive in water for up

to 3 weeks.

Vibrio cholerae lives in aquatic environments. And such environments are the


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vibrios natural reservoir. Vibrio 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 foodand 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& Other Vibrios

Vibrio parahaemolyticus is a halophilic bacterium that causes acute

gastroenteritis following ingestion of contaminated seafood such as raw fish or

shellfish. After an incubation period of 1224 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 14 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 areas where people eat raw seafood. Vparahaemolyticus 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. 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 USA on the

Atlantic, Gulf, and Pacific Coasts. Infections have been reported from Korea, andthe 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), withdevelopment of bullous skin lesions, cellulitis, and myositis with necrosis.


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Several of the first deaths in Louisiana and Texas following hurricane Katrina

were caused by Vibrio 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.

Several other vibrios also cause disease in humans: Vibrio mimicus causes

diarrhea after ingestion of uncooked seafood, particularly raw oysters. Vibrio

hollisae and Vibrio fluvialis also cause diarrhea. Vibrio alginolyticus causes eye,

ear, or wound infection after exposure to seawater. Vibrio damsela also causes

wound infections. Other vibrios are very uncommon causes of disease in

humans.

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