Vibrio cholerae and vibrio parahemolyticus toxins

1. Vibrio cholerae and Vibrio
parahemolyticus toxins
Dr Ravi Kant Agrawal, MVSc, PhD,
Senior Scientist (Veterinary Microbiology)
Food Microbiology Laboratory
Division of Livestock Products Technology
ICAR-Indian Veterinary Research Institute
Izatnagar 243122 (UP) India

2. 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

3. Recent Cholera Pandemics
1-6th 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

5.  500-400 BC: Sanskrit writings
 500 BC: Hippocrates
 200 AD: Galen
 900 AD: Rhazes, Islamic physician
 Sanskrit, Arabic, and Chinese writings dating back 2,000 years
Ancient Texts Describe Cholera

6. Started in by Ganges in Calcutta - Kumbh festival
Polluted water, crowded camps
10,000 in British army and hundreds of thousands of natives
dead
Spread by trade routes – Iran, Baku, Astrakhan, Russia
Cold winter kept it from reaching western Europe
1st
Pandemic: 1817-1823

7.  England’s attempt to control spread of infectious disease
 Tried to prevent international movement
 Eventually repealed (based on flawed scientific understanding)
Quarantine Act of 1825

8.  Bengal, Afghanistan, Asia, Moscow, England, US
 William Brooke O’Shaughenessy
 Industrial Revolution
 England’s Cholera Prevention Act of 1832
 Entered US through NY and New Orleans ports – spread by
railway and troop movement after Civil War
2nd
Pandemic: 1829-1852

9.  Supernatural causes
 Wrath of God
 Astrological causes
Misguided Notions

10. Caused by miasma
Misguided Notions

11.  Prevented by alcohol
 Could be spread by contact with patient or patient’s clothes
Misguided Notions

12. 1854: identified comma-shaped bacterium
Named it Vibrio cholerae
Filipo Pacini

13.  Began in Bengal
 Britain and
Europe affected
 Dr. John Snow
 Mapped cases
to find cause
 Broad Street
Pump
3rd
Pandemic: 1852-1859

14.  Map led Snow to believe that Broad Street pump was cause of
outbreak
 Those affected drank from pump
 Sewage probably contaminated well
 Removal of pump handle - end of outbreak
 Skepticism about Snow’s findings
Broad Street Pump

15.  Compared deaths from Cholera between 2 groups
 Group A: Southwark and Vauxhall Water Co. – 70 deaths per 10,
000 (London source of Thames)
 Group B: Lambeth Water Co. – 5 deaths per 10,000 (source
upstream from London
The “Grand Experiment”

16.  Massive public health reforms
 Much smaller outbreak in 1866
Results

17.  From Egypt to Europe by returning pilgrims from the
Haj at Mecca
 Imported into NY by ship
 Last time cholera in England
 Third and Fourth International Sanitary Conferences
(Paris and Vienna)
 International Health Regulations
 International Sanitary Commission – precursor of PAHO (Pan
American Health Organization)
4th
Pandemic: 1863-1879

18.  Began in India, spread east and west
 1883 - Robert Koch cultured V. cholerae
 Good sanitation – did not affect much of Europe
 Diagnosis and quarantine – kept it out of US
Prevented contact between those with exposure to
unsanitary conditions (on ships) and those on mainland
5th
Pandemic: 1881-1896

19.  Spread through Asia
 Did not affect Europe or US
6th
Pandemic: 1899-1923

20.  1959: cholera enterotoxin by S.N. De in Calcutta
Cholera bacillus is not harmful – toxin is what induces
outpouring of fluid and inhibits sodium transport
 Treatment by rehydration (oral or intravenously) of fluid and
electrolytes
 How to measure rapid fluid loss
Discoveries

21.  Caused by El Tor strain
 From Pacific Islands to Asia, Bangladesh, India, USSR, Iran, Iraq
 1970: reemerged in Africa after 100 years
 1991: Latin America (4,000 dead of 400,000 cases)
 1993: O139 serogroup (“Bengal”) – may be start of 8th
pandemic
7th Pandemic: 1961-present

22.  Aug 2000: published complete DNA sequence of V. cholerae, El
Tor strain
 Unusual - 2 distinct chromosomes
 Hope that genome will be useful in creating an effective vaccine
Genome

23. Vibrio
• Vibrio cholerae -- gastroenteritis
• Vibrio parahaemolyticus -- gastroenteritis, wound infection,
bacteremia
• Vibrio vulnificus -- wound infection, bacteremia

24.  Grows in salt and fresh water (Brackish rivers, coastal waters)
 Can survive and multiply in brackish water by infecting
copepods
 Associate with plankton and algae
 Endemic in areas of poor sanitation (India and Bangladesh)
 Transmitted by fecal-oral route
 Has over 150 identified serotypes based on O-antigen (206
serogroups)
 ONLY O1 AND O139 ARE TOXIGENIC AND CAUSE CHOLERA.
V. cholerae

25. Classification: Serogroups and Biotypes
 The species V. cholerae can be sub-classified into > 200
serogroups based on the O antigen of LPS (lipopolysaccharide).
 Only O1 and O139 strains have been implicated in the cholera
syndrome.

26. Classification: O1 Serogroup
 2 Biotypes: El Tor and Classical
 V. cholerae O1 are further divided
into 2 major sub-serotypes (Inaba
Ogawa and Hikojima ).
 The basis for sub-typing is 3
antigenic determinants of the O
antigen structure of their LPS.
 These sub-serotypes are
differentiated in agglutination
and vibriocidal antibody tests on
the basis of their dominant heat-
stable lipopolysaccharide somatic
antigens.
 The serotypes share one
determinant known as the A
antigen.
 In addition, Inaba strains express
the C antigen whereas Ogawa
strains express the B antigen .

27. Classification: O1 Serogroup
 O1 cholera almost always fall into the Heiberg I fermentation
pattern; that is, they ferment sucrose and mannose but not
arabinose, and they produce acid but not gas.
 Hiiberg (1934) divided Vibrios in 6 groups based on fermentation
of sucrose, mannose and arabinose.
 Vibrio cholera also possesses lysine and ornithine
decarboxylase, but not arginine dihydrolase.
 Freshly isolated agar-grown vibrios of the El Tor biotype, in
contrast to classical V. cholerae, produce a CELL-ASSOCIATED
MANNOSE-SENSITIVE HEMAGGLUTININ which is found active in
chicken erythrocytes.
 Strains of the El Tor biotype, however, produce less cholera
toxin, but appear to colonize intestinal epithelium better than
vibrios of the classical variety.
 Also, El Tor biotype, seem some what more resistant to
environmental factors.
 Thus, El Tor strains have a higher tendency to become endemic
and exhibit a higher infection-to-case ratio than the classical
biotype.

28. Classification: Other antigens
 O139 Serogroup
In 1993, the emergence of an entirely new serogroup (O139)
was the cause an epidemic in Bangladesh.
O139 organisms produce a POLYSACCHARIDE CAPSULE but
do not produce O1 LPS or O1 antigen.
Toxigenic O139 cholera arose through the acquisition of a
large block of genes encoding the O139 antigen by O1 El Tor.
 Non-O1, Non-O139 Serogroup
Most are CT (cholera toxin) negative and are not associated
with epidemic disease.

29. Strains Causing Epidemics
2 main serogroups carry set of virulence genes necessary for
pathogenesis
O1-two major biotypes - classical and E1 Tor - distinction based on
biochemical properties and susceptibility to bacteriophages
E1 TOR produce hemolysins
 Classical: 1 case per 30-100 infections
 El Tor: 1 case per 2-4 infections
O39 Contained /restricted to Bangladesh, India: 1993 outbreak in
Bangladesh and India
O39 genetically derived from E1 tor, but antigenic structure
different enough—no existing immunity—all susceptible
Serogroup - group of microorganims serotype having 1/more
antigens in common

30. Profile of Vibrio cholerae
 Gram-negative rods
 CCurved or comma shapedurved or comma shaped
 Highly motile; single polar flagella
 Non-spore forming
 Associated with salt water
 Oxidase positive
 Facultative anaerobe
 1.4-2.6 μm length x .5-.8 μm width
 Chemo-organotroph
 Optimal growth 20-30 degrees
 RReadily cultivatedeadily cultivated,, ssimple nutritional requirementsimple nutritional requirements
 Tolerate alkaline conditions to pH9.0Tolerate alkaline conditions to pH9.0
 Sensitive to low pH and die rapidly in solutions below pH
6
 Proliferate in summers
 Produces cholera toxin
 Pathogenic and non-pathogenic strains
 206 serogroups
 lipopolysaccharide coat which provides protection
against hydrophobic compounds
 provides a surface for immune recognition

31.  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

32. Divisions of V. Cholerae
 Biotype (biovar)
 different strains of the same bacterial species
 distinguished by a group of phenotypic or genetic traits
 Serogroup
 bacteria of the same species with different antigenic
determinants on the cell surface
 V. Cholera has more than 150 (206) different serogroups, only
two of which cause epidemic disease

33. V. Cholerae
01 serogroup
 Classic genome: 3.2-3.6 MbClassic genome: 3.2-3.6 Mb
 El Tor (El) genome: 4 MbEl Tor (El) genome: 4 Mb
 O1 antigen is divided into 3O1 antigen is divided into 3
types: A,B,Ctypes: A,B,C
• A antigen:A antigen: made of 3-deoxy-made of 3-deoxy-
L-glycerotetronic acidL-glycerotetronic acid
• B, C antigen:B, C antigen: not beennot been
characterizedcharacterized

34.  Broad temperature & pH range for growth on media
• 18-37°C
• 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

35. Vibrio cholerae: Antigenic structure
– Common heat-labile flagellar H antigen
– O lipopolysaccharide confers serologic specificity
• More than 150 O antigen serogroups
• Only O-1 and 0139 serogroups cause Asiatic cholera
• Three serotypes; Ogawa, Inaba, Hikojima
• Two biovars of O1 derogroup; classic and El Tor

36. V. choleraeV. cholerae - Transmission
food
feces
waterwater
– freshfresh
– saltsalt

37. Vibrio cholerae: Epidemiology
– Epidemic cholera - spread by contaminated water under
conditions of poor sanitation
– Endemic - consumption of raw seafood
– Copepods

38.  People with low gastric acid levels
 Children: 10 × more susceptible than adults
 Elderly
 Blood types
 O>> B > A > AB
People Most at Risk

39. Period of Communicability
 During acute stage
 By end of week, 70% of patients non-infectious
 By end of third week, 98% non-infectious
 A few days after recovery

40. Vibrio cholerae - Clinical manifestations
Asymptomatic colonization to fatal diarrhea
Onset 2-3 days after ingestion
Abrupt onset of watery diarrhea and vomiting
RICE WATER STOOLS
Severe fluid and electrolyte loss-dehydration, metabolic acidosis,
hypovolemic shock, renal failure
Death 60% if untreated, 1% if treated for fluid loss
 cause of death: Dehydration and shock

41. Symptoms
 Occur 2-3 days after consumption of contaminated food/water
 No fever-not invasive
 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
 Clever-viable even after exit body-take host’s liquid

42. Cholera Gravis
 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

43. Vibrio-Prevention and Control
 Improved sanitation
 Fluid and electrolyte replacement
 Antibiotic prophylaxis
 Improved food handling

44. Virulence factors of V. cholerae O1 and O139
Virulence factor Biological effect
Cholera toxin Hypersecretion of electrolytes and water
Co-regulated pilus Adherence to mucosal cells adhesin
Accessory colonization
factor
adhesin
Hemagglutination
protease
Releases bacteria from mucosal cells
Zona occludens Exotoxin
Accessory cholera
enterotoxin
Exotoxin
Flagellum Motility
Siderophores Iron sequestration

45. Vibrio cholerae: Pathogenesis
 Ingest 108
-1010
organisms
 Non invasive infection of small intestine
 Organisms secrete enterotoxin
 Watery diarrhea

46.  Cholera disease begins with ingestion of contaminated water
or food.
 The bacteria that survive the acidic conditions of the stomach
colonize in the small intestine.
 The cholera toxin (CT) is responsible for the severe diarrhea
characteristic of the disease.
 Other virulence factors- heat stable endotoxin
Cholera Toxin
 CT is a proteinaceous enterotoxin (exotoxin) secreted by V.
cholerae.
 CT is antigenically and pharmacologically identical in all
serotypes and biotypes.
Pathogenesis of V. cholerae

47. Horizontal Gene Transfer
1. Acquisition of VPI1. Acquisition of VPI
2. lysogenic conversion by phage2. lysogenic conversion by phage
3. exchange of genes leads to expression of O-antigen and capsule3. exchange of genes leads to expression of O-antigen and capsule

48. V. Cholera
• The 01 strain and
the recent 0139
strain have
different antigens
expressed in the
polysaccharide
capsule
• The change in
structure is
thought to have
arisen from a
recombination
event.

49. V. Cholerae
two circular chromosomestwo circular chromosomes
 Chromosome 1 is larger
(2.96 million base pairs)
and carries many genes
for essential cell
functions and
housekeeping.
 Chromosome 2 is smaller
(about 1.07 million base
pairs) and carries the
integron island.

50. Chromosome 1
 Carries many genes for essential cell
functions and housekeeping.
 It also contains important virulence
genes, most of which have been
acquired by lateral gene transfer from
other species
 Chromosome one carries two
bacteriophages.
 ONE VIRUS is called the V. cholera
pathogenicity island phage (VPI),
which infects and inserts its DNA into
the bacterial chromosome and allows
the synthesis of a pilus which the
bacteria uses to attach to the host
intestine.
 SECOND VIRUS is called the cholera-
toxin phage phi (CTXΦ). The CTX
phage inserts itself into chromosome
one and the bacterium is then capable
of secreting a powerful enterotoxin.

51. Chromosome 2
 The integron region is often found
on plasmids and serves as a "gene
capture system."
 This region may contain antibiotic
resistance genes.

52. Genetics of Cholera Toxin
 Genes encoding CT ctxAB-
recognized to be the genome
of a filamentous phage CTXΦ
(ctxA and ctxB).
 Transcription of ctxAB is
regulated by several proteins.
 CTXΦ genome can integrate
into the host genome at a
specific site, attRS.
 The CTX genetic element also
has a “core” region carrying
several phage morphogenesis
genes.
 These entire CTX gene set is
flanked repeated sequences,
the attRS1 site.
 The entire genetic element is
6.9kb.
The receptor for CTXThe receptor for CTXΦΦ is Toxin-Coregulated Pili (TCP)is Toxin-Coregulated Pili (TCP)

53. Toxin-Coregulated Pili
 Efficient colonization of V. cholera in the small intestine
requires the expression of TCP’s
• TCP’s are expressed on the surface of V. cholera
• TCP’s are long laterally associated filaments
• The major pilin subunit is TcpA
Genes for TCP production are clustered on the pathogenicity
island located on chromosome 1

54. Recap of phage movement
 V. cholerae did not always cause disease.
 Infection with the CTX phage (a temperate and filamentous
phage) gives the bacterium its toxinogenicity.
 The phage recognizes a pilus on the surface of the bacterium
and uses it to enter the cell.
 Once inside the cell, the CTX phage integrates into the
chromosome and the lysogen expresses cholera toxin.
 The CTX phage has received special attention because it is the
first filamentous phage found to transfer toxin genes to its
host.
 The important lesson from this discovery is that many different
types of phage may carry virulence factors, and transfer of
virulence genes by phage may be a major mechanism of
evolution of new bacterial diseases.

55. Genomic Structure: Bacteriophage
 In 1996 Matthew K. Waldor and John J. Mekalanos reported a
stunning discovery about the toxin.
 The toxin was for the first time shown to be not a part of the
bacterium but actually that of a virus that got integrated into
the V. cholerae genome.
 Normally this virus remains silent within V. cholerae but during
infection it gets activated.
 The major virulence factor of cholera, CT (cholera toxin) is
encoded on a filamentous phage (ctxΦ) that is capable of
transducing the ctx gene into other cholera strains.
 The released phages specifically attach to the bacterium and
enter it.
 Vigorous viral multiplication results in the production of large
amounts of toxin causing severe diarrhea.

56. Genomic Structure: Pathogenicity Islands (PAI)
 Upon transduction, the
bacteriophage (ctxΦ)
brings the toxin and a
specific pilus called toxin-
co-regulated pilus (TCP).
 The important genes
involved in intestinal
colonization (tcp) and
virulence gene regulation
(toxT) are encoded in a
40Kb pathogenicity island.
This PAI is present in
pathogenic cholera
strains.
tcp gene
ctx gene

57. Pathogenesis: Overview
 To establish disease, V. cholerae must
be ingested in contaminated food or
water and survive passage through
the gastric barrier of the stomach.
 On reaching the lumen of the small
intestine, the bacteria must overcome
the clearing mechanism of the
intestine (peristalsis), penetrate the
mucous layer and establish contact
with the epithelial cell layer.

58. Pathogenesis: Overview
 Colonization of the intestinal microvilli and the subsequent
production and release of cholera toxin, lead to the purging
diarrhea.
 This complex progression of events appears to involve tightly
regulated differential gene expression by the bacteria.
 This is because expression of intestinal colonization factors is
unlikely to be of advantage to the bacterium in its salt/fresh
water environment niche.

59. Pathogenesis: Cholera Toxin (CT)
 In 1983, by administering purified CT to volunteers, Levin et al.
were able to conclusively demonstrate that the toxin is the
major mediator of the cholera syndrome.
 Ingestion of only 5μg of purified toxin resulted in production of
1-6 L of diarrheal stool.
 CT elicits vigorous mucosal immune responses in the absence of
a conventional adjuvant.
 Direct immunomodulatory effects of CT on leukocytes include
induction of CD25 and class II MHC on B cells, apoptosis of CD8+
T cells, and activation of macrophages with release of IL-10.

60. Pathogenesis: Cholera Toxin (CT) Structure
 CT is a prototype A/B subunit toxin,
consisting of one A subunit and 5 B
subunits.
 The B subunit weighs 11.6kDa each and
multimerize to form a pentameric ring,
which binds the holotoxin to a eukaryotic
cell surface receptor.

61. Pathogenesis: Cholera Toxin (CT) Structure
• The A subunit contains an intracellular ADP-ribosyltransferase
activity.
• The mature A subunit is proteolytically cleaved to produce a
21.8kDa A1 polypeptide, which contains the intracellular
enzymatic activity, and a 5.4kDa A2 polypeptide.
• After cleavage, the A1 and A2 polypeptides remain linked by a
disulphide bond.
• The crystal structure of CT revealed that the 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.

62. Pathogenesis: Cholera Toxin (CT) Structure
 CT must be assembled for activity, as neither the A nor B
subunit individually can cause secretory diarrhea.
 CT holotoxin is assembled in the periplasmic space.
 The subunits are exported individually into the periplasm
through the cytoplasmic membrane via the general secretion
pathway; both the A and B protein subunits contain normal
sequences at their N-terminus.

63. Pathogenesis: Cholera Toxin (CT) Structure
 Once in the periplasm, both
subunits must undergo
modification by the
periplasmic enzyme DsbA,
which is responsible for
disulphide bond formation.
 Again, once the holotoxin is
secreted from the bacterium,
the A subunit must be cleaved
to generate separate A1 and
A2 peptides for maximum
toxin activity.

64. Cholera Toxin (CT) Structure
 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.
 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.

65. 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.

66. • The biological activity of CT is dependent on binding of B
pentamer to specific receptors on enterocytes: GM1 ganglioside.
• Internalization is initiated once CT-GM1 complexes cluster which
then invaginate to form apical endocytic vesicles.
How Does Cholera Toxin Work?

67. 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)).

68. 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

69. 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

70.  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-
and bicarbonates into the
intestinal contents.
How Does Cholera Toxin Work?

71. • 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?

72. Encoded by a prophage
Molecular mass of 84,000 daltons
A subunit-ADP-ribosylating toxin
B subunit-bind GM1-gangliosides on enterocytes
A subunit ADP ribosylates Gs-alpha which regulates activation
of adenlyate cyclase which inactivates GTPase function of G-
protein coupled receptors in intestinal cells
G proteins stuck in “On” position
Result is persistent increase in cAMP levels-100 fold increase in
cAMP
Activation of ion channels
Ions flow out- Hyper secretion of Na, Cl, K, bicarbonate
The change in ion concentrations leads to the secretion of large
amounts of water into the lumen, known as diarrhea
How Does Cholera Toxin (ctxAB) Work?

75. Toxin Pathway Cartoon

76. 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
 Bacterial food poisoning
 Viral gastroenteritis

77. 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)

78. Laboratory Diagnosis
Visualization by dark field or phase
microscopy
Look like “shooting stars”
Gram Stain: gram – curved rods
Isolate V. cholerae from patient’s
stool/vomitus - Specimens
Culture:
Transport medium - Cary-Blair semi-
solid agar
Enrichment medium - alkaline
peptone broth: Vibrios survive and
replicate at high pH while other
organisms are killed or do not
multiply
Selective Medium: TCBS agar plate,
sucrose agar: Yellow colonies form
(sucrose fermentation)
Quick immunological methods:
immunofluorescent “ball” test and
PCR

80. Immunity
• Strong immunity after recovery, SIgA

81. Vibrio Prevention & Control
• Disrupt fecal-oral transmission by
improved sanitation
• Fluid and electrolyte replacement
• Antibiotic prophylaxis
• Improved food handling

82. Treatment
*Even before identifying cause of disease, rehydration therapy
must begin Immediately because death can occur within hours*
Oral rehydration
Intravenous rehydration
Antimicrobial therapy

83. Treatment: Oral Rehydration
 Reduces mortality rate from over 50% to less than 1%
 Recover within 3-6 days
 Should administer at least 1.5x amount of liquid lost in stools
 Use when less than 10% of bodyweight lost in dehydration

84. Treatment: Oral Rehydration Salts (ORS)
 Reduces mortality from over 50% to less
than 1%
 Packets of Oral Rehydration Salts
Distributed by WHO, UNICEF
Dissolve in 1 L water
NaCl, KCl, NaHCO3, glucose

85. Treatment: How ORS Works
Na+
transport coupled to
glucose transport in small
intestine
Glucose enables more efficient
absorption of fluids and salts
Potassium passively absorbed

86. Treatment: ORS in United States?
 American doctors skeptical of such simple, inexpensive
treatment
 Cost
 ORS: $270/infant
 IV: $2,300/infant
$1 billion/year for IV treatment for rehydrating children
 Insurance companies do not reimburse for ORS
 600 American children die unnecessarily from dehydration each
year
 Hospitals consider IV more time efficient
Less personal attention required

87. Treatment: Intravenous Rehydration
Used when patients have lost more than 10% bodyweight from
dehydration
Unable to drink due to vomiting
Only treatment for severe dehydration

88. Treatment: Intravenous Rehydration
Ringer’s Lactate
Commercial product
Has necessary
concentrations of
electrolytes
Alternative options
Saline
Sugar and water
Do not replace
potassium, sodium,
bicarbonate

89. Treatment: Antibiotics
 Adjunct to oral rehydration
 Reduce fluid loss by half
 Reduce recovery time by half
2-3 days instead of 4-6
 Tetracycline, Doxycycline
 Not recommended
Short duration of illness
Antibiotic resistance
Limited gain from usage

90.  Boil or treat water with chlorine or iodine
 No ice
 Cook everything
 Rule of thumb: “Boil it, cook it, peel it, or forget it.”
 Wash hands frequently
Traveling Precautions

91. Vaccines
 Need localized mucosal immune response
Oral Vaccine
 Not recommended
Travelers have very low risk of contracting disease: 1-2
cases per million international trips
Not cost-effective to administer vaccines in endemic
regions
Brief and incomplete immunity
 Two types approved for humans:
Killed whole-cell
Live-attenuated

92. Vaccines: Killed Whole-cell Vaccines
 Provides antigens to evoke protective antitoxic and
antibacterial immunity
 Contains:
1 x 1011
heat inactivated bacteria
Mixture of V. cholerae O1 El Tor and classical strains
1 mg of B subunit of cholera toxin

93. Killed Whole-cell Vaccines: Disadvantages
 50% protection for 6 months to adults
 Gives less than 25% protection to children aged 2-5
 Need for multiple doses of nonliving antigens

94. Vaccines: Live-Attenuated
 Eliminates need for multiple doses of non-living antigens
 Ensures that crucial antigens potentially altered during killing
process would be retained
Expected to mimic broad immunity conferred by natural
infection
 85-90% protection against classical biovar
 65-80% protection against El Tor biovar

95. Live Attenuated Vaccines: Disadvantages
 In children, protection rapidly declines after 6 months
 In adults, only receive 60% protection for 2 years
 Live vaccine induces mild cholera symptoms
Mild diarrhea, abdominal cramping

96. Prevention
 Disrupt fecal-oral transmission
 Water Sanitation
 Water treatment

97. Precautions Taken in US
 Environmental Protection Agency (EPA) works closely with
water and sewage treatment operators
 FDA
Tests imported shellfish
Controls US shellfish sanitation program

98. 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.
 Major source: raw sea-food.

99. 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

100. Vibrio spp. (Family Vibrionaceae)
Associated with Human Disease

101. Summary of Vibrio parahaemolyticus Infections

102. Summary of Vibrio vulnificus Infections

103. Virulence Factors Associated with Non-cholerae Vibrios
(Kanagawa positive)

104. Thanks
Acknowledgement: All the presentations available online on the
subject are duly acknowledged.
Disclaimer: The author bear no responsibility with regard to the
source and authenticity of the content.