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 of Shigella 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 16–3).

Table 16–3. 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 16–3.

Pathogenesis & Pathology

Shigella infections are almost always limited to the gastrointestinal tract;
bloodstream invasion is quite rare. Shigellae are highly communicable; the
infective dose is on the order of 103 organisms (whereas it usually is 105–108
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.

Toxins
ENDOTOXIN
Upon autolysis, all shigellae release their toxic lipopolysaccharide. This
endotoxin probably contributes to the irritation of the bowel wall.
SHIGELLA DYSENTERIAE EXOTOXIN
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 for
experimental animals. Acting as an enterotoxin, it produces diarrhea as does
the E coli verotoxin, 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 of S dysenteriae infections and to the central nervous system reactions
observed in them (ie, meningismus, coma). Patients with Shigella flexneri or
Shigella sonnei infections develop antitoxin that neutralizes S dysenteriae
exotoxin in vitro. The toxic activity is distinct from the invasive property of
shigellae 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 (1–2 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, as
the infection involves the ileum and colon, the number of stools increases; they
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 2–5 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 given
orally as experimental vaccines. Serum antibodies to somatic shigella antigens
are IgM.

Treatment
Ciprofloxacin, ampicillin, doxycycline, and trimethoprim-sulfamethoxazole are
most commonly inhibitory for shigella isolates and can suppress acute clinical
attacks of dysentery and shorten the duration of symptoms. They may fail to
eradicate the organisms from the intestinal tract. Multiple drug resistance can
be transmitted by plasmids, and resistant infections are widespread. Many
cases are self-limited. Opioids should be avoided in shigella dysentery.

Epidemiology, Prevention, & Control

Shigellae are transmitted by "food, fingers, feces, and flies" from person to
person. Most cases of shigella infection occur in children under 10 years of age.
S dysenteriae can spread widely. Mass chemoprophylaxis for limited periods of
time (eg, in military personnel) has been tried, but resistant strains of shigellae
tend to emerge rapidly. Since humans are the main recognized host of
pathogenic shigellae, control efforts must be directed at eliminating the
organisms from this reservoir by (1) sanitary control of water, food, and milk;
sewage disposal; and fly control; (2) isolation of patients and disinfection of
excreta; (3) detection of subclinical cases and carriers, particularly food
handlers; and (4) antibiotic treatment of infected individuals.


 


INTRODUCTION
Vibrio, Aeromonas, Plesiomonas, Campylobacter, and Helicobacter species are
gram-negative rods that are all widely distributed in nature. The vibrios are
found 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 jejuni is a common cause
of enteritis in humans. Less commonly, aeromonas and, rarely, plesiomonas
have been associated with diarrheal disease in humans. Helicobacter pylori has
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
18–1.

Table 18–1. The Medically Important Vibrios.

Organism
V cholerae serogroups O1 and O139
V cholerae serogroups non-O1/non-O139
V parahaemolyticus
Others
V mimicus, V vulnificus, V hollisae, V fluvialis, V damsela, V anginolyticus, V
metschnikovii
Vibrio cholerae
The epidemiology of cholera closely parallels the recognition of V 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 2–4 m long
(Figure 18–1). 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.

Figure 18–1.

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

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 (8.5–9.5)
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.
GROWTH CHARACTERISTICS
V cholerae regularly ferments sucrose and mannose but not arabinose. A
positive oxidase test is a key step in the preliminary identification of V 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

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 occurs—as much as
20–30 L/d—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 & 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 60% of infections with classic V cholerae are asymptomatic, as are about
75% of infections with the El Tor biotype. The incubation period is 1–4 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 6–8 hours in taurocholate-peptone broth
(pH 8.0–9.0); 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

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 of V 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 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.

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