What is Cholera?

A life-threatening secretory diarrhea induced by enterotoxin

secreted by V. cholerae

Water-borne illness caused by ingesting water/food

contaminated by copepods infected by V. cholerae

An enterotoxic enteropathy (a non-invasive diarrheal disease)

A major epidemic disease

Cholera

Recent Cholera Pandemics1-6th pandemic: V. cholerae O1 biotype classical 1817-1923, Asia, Africa, Europe, America and Australia

7th pandemic: V. cholerae O1 biotype El Tor Began in Asia in 1961 Spread to other continents in 1970s and 1980s Spread to Peru in 1991 and then to most of South & Central

America and to U.S. & Canada By 1995 in the Americas, >106 cases; 104 dead

1993: Cholera in Bengal caused by O139

may be cause of 8th pandemic

VibrioVibrio

• Vibrio cholerae --gastroenteritis

• Vibrio parahaemolyticus -- gastroenteritis,

wound infection, bacteremia

• Vibrio vulnificus -- wound infection, bacteremia

Grows in salt and fresh water

Endemic in areas of poor sanitation (India and

Bangladesh ), transmitted by fecal-oral route

Can survive and multiply in brackish water by infecting

copepods

Has over 150 identified serotypes based on O-antigen

Only O1 and O139 are toxigenic and cause Cholera

disease

V. cholerae

Classification: O1 Antigen

小川型 稻叶型 彦岛型

Classification: Other antigens

O139 Serogroup

In 1993, an entirely new serogroup (O139) cause an

epidemic in Bangladesh.

O139 organisms produce a polysaccharide capsule but do

not produce O1 LPS or O1 antigen.

Non-O1, Non-O139 Serogroup

Most are CT (cholera toxin) negative and are not

associated with epidemic disease.

Profile of vibrio cholerae

• G-, curved or comma-shaped rods

• Highly motile; polar flagellum

• Sensitive to low pH and die rapidly in

solutions below pH 6

• Proliferate in summers

• Cholera toxin

• Pathogenic and nonpathogenic strains

• 206 serogroups

Similarities to Enterobacteriaceae

G-, Facultative anaerobes

Fermentative bacilli

Differences from Enterobacteriaceae

Polar flagella

Oxidase positive

Formerly classified together as Vibrionaceae

Primarily found in water sources

Cause gastrointestinal disease

Shown not closely related by molecular methods

Profile of vibrio cholerae

Broad temperature & pH range for growth on media

18-37C

pH 7.0 - 9.0 (useful for enrichment)

Grow on variety of simple media including:

MacConkey’s agar

TCBS (Thiosulfate Citrate Bile salts Sucrose) agar

V. cholerae grow without salt

Most other vibrios are halophilic

Physiology of Vibrio

People with low gastric acid levels (103 -105 CFU )

Children: 10 × more susceptible than adults

Elderly

Blood types

O>> B > A > AB

People Most at Risk

Period of Communicability

During acute stage

A few days after recovery

By end of week, 70% of patients non-infectious

By end of third week, 98% non-infectious

Symptoms

Occur 2-3 days after consumption of contaminated food/water

Usually mild, or no symptoms at all

• 75% asymptomatic

• 20% mild disease

• 2-5% severe Vomiting Cramps Watery diarrhea (1L/h) Without treatment, death in 18 h-several days

Cholera Gravis (霍乱肌无力 )

More severe symptoms

Rapid loss of body fluids

6 liters/hour

107 vibrios/mL

Rapidly lose more than 10% of bodyweight

Dehydration and shock

Death within 12 hours or less

Death can occur within 2-3 hours

Virulent factors

• Toxins and enzymes

- heat stable endotoxin

- enterotoxin (exotoxin [cholera toxin CT])

CT is antigenically and pharmacologically

identical in all sero and bio types

Pathogenesis of V. cholerae

Cholera Toxin (CT) Structure

• The A subunit contains an intracellular ADP-ribosyltransferase activity.

• The mature A subunit is proteolytically cleaved to produce an A1 polypeptide, which contains the intracellular enzymatic activity, and an A2 polypeptide.

• CT is a prototype A/B subunit toxin, 1A+5B

• The B subunit form a pentameric ring, which binds the holotoxin to a eukaryotic cell surface receptor.

Cholera Toxin (CT) Structure

• After cleavage, the A1 and A2 polypeptides remain

linked by a disulphide bond.

• A and B subunits are connected through the C-terminus of the A2 subunit, which is inserted through the central pore of the B pentamer.

• The biological activity of CT is

dependent on binding of B

pentamer to specific receptors

GM1 ganglioside.

How Does Cholera Toxin Work?

• Internalization is initiated once

CT-GM1 complexes cluster which

then invaginate to form apical

endocytic vesicles.

How Does Cholera Toxin Work?

• These vesicles enter cellular

trafficking pathways leading to the

trans-Golgi network (TGN).

• The toxin then moves retrograde

via the Golgi cistern to the ER.

• Once in the ER, CT is processed to

activate the A1 peptide, which then

targets the basolateral membrane

(heterotrimeric GTPase and

adenylate cyclase (AC)).

How Does Cholera Toxin Work?

• Adenylate cyclase (AC) is

activated normally by a

regulatory protein (GS) and

GTP; however activation is

normally brief because

another regulatory protein

(Gi), hydrolyzes GTP.

NORMAL CONDITION

How Does Cholera Toxin Work?

• A1 fragment catalyzes the attachment of ADP-Ribose (ADPR) to the regulatory protein forming Gs-ADPR from which GTP cannot be hydrolyzed.

• Since GTP hydrolysis is the event that inactivates the adenylate cyclase, the enzyme remains continually activated.

CHOLERA

• Thus, the net effect of the

toxin is to cause cAMP to be

produced at an abnormally

high rate which stimulates

mucosal cells to pump large

amounts of Cl- into the

intestinal contents.

How Does Cholera Toxin Work?

• H2O, Na+ and other electrolytes follow

due to the osmotic and electrical gradients

caused by the loss of Cl-.

• The lost H2O and electrolytes in mucosal

cells are replaced from the blood.

• Thus, the toxin-damaged cells become

pumps for water and electrolytes causing

the diarrhea, loss of electrolytes, and

dehydration that are characteristic of

cholera. 

How Does Cholera Toxin Work?

• Inactivates GTPase function of G-protein coupled

receptors in intestinal cells

• G proteins stuck in “On” position

• 100 fold increase in cAMP

• Activation of ion channels

• Ions flow out and water follows

How Does Cholera Toxin Work?

Diagnosis

• Cholera should be suspected when patients present

with watery diarrhea, severe dehydration

• Based on clinical presentation and confirmed by

isolation of vibrio cholera from stool

• No clinical manifestations help distinguish

cholera from other causes of severe diarrhea: Enterotoxigenic E. coli

Viral gastroenteritis

Bacterial food poisoning

Diagnosis: Visible Symptoms

• Decreased skin turgor

• Sunken eyes, cheeks

• Almost no urine production

• Dry mucous membranes

• Watery diarrhea consists of:• fluid without RBC, proteins• electrolytes• enormous numbers of vibrio cholera (107 vibrios/mL)

Laboratory Diagnosis

• Visualization by dark field or phase microscopy• Look like “shooting stars”

• Gram Stain• Red, curved rods of bacteria

• Isolate V. cholerae from patient’s stool• Plate on sucrose agar• Yellow colonies form

Vibrio Prevention & Control

• Disrupt fecal-oral transmission

Improved sanitation

• Fluid and electrolyte replacement

• Antibiotic prophylaxis

• Improved food handling

Vibrio parahemolyticus

• One kind of halophilic vibrios;

• optimal NaCl concentration contained in culture media is 3.5%;

• hemolysin related to its pathogenicity, can be detected by human or rabbit RBC test (Kanagawa test);

• cause food poisoning in human beings.

• raw sea-food

• Clinical manifestations– Self-limiting diarrhea to mild cholera-like illness

– 24 hours after ingestion-explosive water diarrhea

• Headache, abdominal cramps, nausea, vomiting, low grade fever for 72 hours or more

• Uneventful recovery

• Wound infections in people exposed to seawater-containing vibrios

Vibrio parahemolyticus

Helicobacter pylori

Helicobacter pylori

•G- with S or spiral-shaped

•Very Motile ---corkscrew motion

•2~6 flagella at one end of HP

•5% O2+10% CO2 at 37 oC

•Reaction of urea hydrolysis and

creates an ammonia cloud

Pathogenesis of Hp

CagA (Cytotoxin associated)

VacA (vacuolationg associated)

LPS also play great importance

Flagellum and urease is necessary

for its adhesion and inhabitation

Adhesin

Duodenal Ulcer (DU) Gastric Ulcer (GU)

Campylobacter jejuni

•G- rods with comma, S, or “gull-wing”

shapes.

•Motive, with a single polar flagellum

•No spore & no capsule

•5% O2+10% CO2

•Two types of colonies:

watery and spreading

round and convex

• Produces a toxin called Cytolethal Distending Toxin (CDT).

• CDT activity requires activation of three genes: cdtA, cdtB, and cdtC.

• CdtB is nuclease that damages DNA and causes cell cycle arrest.

• Causes cell death.

Pathogenesis of Campylobacter

What Are the Symptoms?

• Diarrhea

– Usually watery and sticky

– Can contain blood and fecal leucocytes

• Fever

• Abdominal pain

• Nausea and vomiting

• Headache

• Muscle pain

Who is affected?

• All warm-blooded animals can become affected.

Some animals carry the disease without exhibiting

symptoms.

• Any person can become infected.

• Children under 5 and young adults ages 15-29 are

most often affected.

• Most deaths occur among the elderly and the

immune-suppressed.

Summary

• Properties of Vibrio (stain, culture,

biochemical reaction, antigens and

virulence factors)

• Pathogenesis of V. parahemolyticus, H.

pylori, C. jejuni