Vibrio,and related organisms one hour Aim: study the differentiation and clinical importance of vibrionaceae genera with its infections Topics: -classification of vibrionaceae genera - General Characteristics of Vibrio,

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Vibrio,and related organisms
one hourAim study the
differentiation and clinical importance of
vibrionaceae genera with its infectionsTopics-c
lassification of vibrionaceae genera- General
Characteristics of Vibrio, Aeromonas and
Plesiomonas- Taxonomy of Vibrio
cholerae-structures and physiology-pathogenicity
- virulence factors enzymes, toxins-mechanism
of cholera toxin -cholera and other Vibrio
infections-laboratory diagnosis-
immunity-epidemiology--predisposing factors of
Vibrio infections-treatment , prevention and
control measures-Same as above for other Vibrio
genera , Aeromonas and Plesiomonas
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General Characteristics of Vibrio, Aeromonas and
Plesiomonas

• Similarities to Enterobacteriaceae
• Gram-negative
• 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

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Morphology Physiology of Vibrio

• Comma-shaped (vibrioid) bacilli
• V. cholerae, V. parahaemolyticus, V. vulnificus
  are most significant human pathogens
• Broad temperature pH range for growth on media
• 18-37?C
• pH 7.0 - 9.0 (useful for enrichment)
• Alkaline peptone water APW (pH8.6) - rapid
  isolation
• from faeces (enrichment medium for V.
  cholerae)
• Grow on variety of simple media
  includingMacConkeys agar
• Selective mediumTCBS (Thiosulfate Citrate Bile
  salts Sucrose) agar
• V. cholerae grow without salt
• Most other vibrios are halophilic

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Vibrio spp. (Family Vibrionaceae) Associated with
Human Disease
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Taxonomy of Vibrio cholerae

• ANTIGENIC STRUCTURE
• O antigen specific H antigen less specific
• gt200 serogroups based on somatic O-antigen
• 1- O1 serogroup subdivided into
• Two biotypes El Tor and classical (or cholerae)
• Three serotypes ogawa, inaba, hikojima
• Some O1 strains do not produce cholera
  enterotoxin (atypical or nontoxigenic O1 V.
  cholerae)
• 2- non-O1 V.cholerae Other strains are
  identical to O1 strains but do not agglutinate in
  O1 antiserum (non-cholera (NCV) or
  non-agglutinating(NAG) vibrios) (O139 serogroups)
  
• O1 and O139 serogroups are responsible for
  classic epidemic cholera

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Virulence factors

• 1-Toxins and enzymes
• - heat stable endotoxin(LPS)
• - enterotoxin (exotoxin cholera toxin CT)
• - CT is antigenically and pharmacologically
  identical in all sero and bio types
• 2- Vascular permeability factor (VPF)
• CT increases capillary permeability factor
• 3- Mucinase
• Extreme cellular desquamation
• 4-Resistance
• Susceptible to heat, drying, autoclaving temp,
  high pH, salty can survive in linen for 1-3 days.
• ElTor survive longer periods than the classical
  type

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Virulence Factors Associated with Vibrio cholerae
O1 and O139
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• Laboratory diagnosis
• Specimens - From patients
• - watery stools and mucous flakes From contacts
  and carriers
• rectal swabs
• Direct microscopy
• Not a reliable method Wet preparation in
  saline/dark field Darting movement
• Movements stop on adding antisera
• Transport media
• - Cary-Blair medium(semisolid medium)
  (recommended)
• -Venkataraman-Ramakrishnan (VR)
• Vibrios do not multiply
• Bile peptone
• and pepton water medium (sea salt medium)
• Enrichment medium - alkaline peptone broth
• Vibrios survive and replicate at high pH
• Other organisms are killed or do not multiply
• Selective/differential culture medium
• TCBS agar V. cholerae grow as yellow colonies

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• Biochemical tests
• - Ferment glucose, maltose, mannitol and sucrose
  but not lactose, salicin and arabinose, with
  production of acid but not gas.
• Reduces nitrate.
• oxidase-positive .
• indole-positive.
• Main tests to be performed to defferentiate
  between
• classical and
  El Tor biotypes
• VP _
  
• Haemolysis _
  
• Polymyxin B
  _
• Phage typing
  _
• (Mukherjee phage IV)

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- Identification 1. Presumptive identification

V. Cholerae reactions Test
Small, Gram -ve curved rods. Gram stain
Small, curved rods with darting motility. Wet mount
Positive. Oxidase test
Positive. String test
Acid yellow slant/acid yellow butt, no gas produced. Triple sugar iron agar
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2. Serological test for confirmation
Microorganisms can be confirmed as cholera
vibrios by a rapid slide agglutination test with
specific antiserum. It should be noted that V.
cholerae colonies, that fail to agglutinate in
either O1 or O139 antisera, should be reported as
non-O1/O139 V. cholerae. 3. Biochemical tests
as befor
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V. Cholera on TCBS
Gelatin agar medium
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String test is useful for ruling out bacteria
that share similarities to vibrio species,
particularly Aeromonas species. - The test is
done by emulsifying a large colony in a small
drop of 0.5 sodium desoxycholate in sterile
distilled H2O. Within 60 seconds, the cells lyse
(loss of turbidity) DNA strings when a loopful
is lifted (up to 2-3 cm) from the slide. - All
V. cholerae strains, as well as most other
vibrios, are positive, whereas Aeromonas strains
are negative.
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Pathogenesis of V.cholerae

• Incubation period 2-3 days
• As more fluid is lost, feces-streaked stool
  changes to rice-water stools
• Colorless, Odorless , No protein ,Speckled with
  mucus
• Source of infection contaminated water from
  feces of human carriers or patients with cholera
  marine shellfish
• Transmitted by contaminated food water
• Predisposing factors Poor sanitation,
  Overcrowding, Malnutrition
• Infective dose Organism very sensitive to
  gastric acid
• Patients using antacid drugs-the dose is reduced
• High infectious dose gt108 CFU
• 103 -105 CFU with achlorhydria or hypochlorhydria
  
• (lack of or reduced
  stomach acid)
• Death may occur from
• 1 - Hypovolaemic shock. 2-
  Metabolic acidosis. 3 - Uraemia resulting from
  acute tubular necrosis.

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Epidemiology of Vibrio spp.

• All Vibrio spp. can survive and replicate in
  contaminated waters with increased salinity and
  at temperatures of 10-30oC
• Pathogenic Vibrio spp. appear to form symbiotic
  (?) associations with chitinous shellfish which
  serve as an important and only recently
  recognized reservoir
• Asymptomatically infected humans also serve as an
  important reservoir in regions where cholera is
  endemic

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• control (precautionary )measures
• - Carriers can be detected
• - Water and sewage can be tested for Vibrios
• - Increased sanitation
• - purification of water
• - Isolation of patients
• -Vaccines
• Immunity
• Antibodies can be detected after an infection
• However, there is only a limited protection,
• repeat attacks are not common for 6-12 months
• Treatment
• 1- Fluid and electrolytes replacement.
• 2- Acid base balance adjustment.
• 3- Antibiotics such as tetracycline or
  ciprofloxacin.

-Sodium chloride (3.5 g/L) - Potassium chloride
(1.5 g/L) -Rice flour (30-80g/L) -Trisodium
citrate (2.9 g/L)
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Mechanism of Action of Cholera Toxin
Cholera toxin (or choleragen) It contains 5
binding (B) subunits and an active (A) subunit.
The toxin binds, through its B subunits, to
specific receptors on the intestinal epithelial
cells. Then it releases the enzymatically active
(A) subunit that enters the cells and activates
the adenylate cyclase enzyme causing a rise in
cAMP production. This causes massive secretion
of electrolytes (Na, K, Cl-, and HCO3-) and
water into the lumen of the SI.
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Development of a vaccine, that provides long term
protection, is not yet available. However, 3
vaccines are now commercially available.
Protection duration No. of doses Route
3-6 months 2 1 week interval IM Cholera vaccine Containing extract of killed bacteria
2 years 3 6 weeks interval oral Whole cell/B subunit Containing killed bacteria/B subunit of CT
At least 6 months 1 oral Live attenuated vaccine
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Characteristics of V. cholera
Methods of Classification Serogroups Biotypes Se
rotypes Toxin
Epidemic Associated 01 Classical, El
Tor Inaba, Ogawa, Hikojima Produce cholera toxin
Not Epidemic-Associated Non-01 Biotypes
not applicable to non-01 strains Serotypes not
applicable to non-01 strains Usually do not
produce cholera toxin, may produce other
toxins
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Other Vibrio Infections
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• Vibrio parahaemolyticus It is a halophilic
  (salt-loving) Vibrio associated with enteritis
  and is acquired by ingestion of raw or improperly
  cooked sea foods produced by thermostable direct
  haemolysin.
• V. vulnificus it is a halophilic vibrio, which
  ferments lactose associated with wound infections
  as well as fatal septicemias.
• Non-O1/0139 serogroups of V. cholerae cause
  diarrhea

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Other Vibrionaceae genera Vibrios-related
organisms Aeromonas Plesiomonas
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Characteristics and Epidemiology of Aeromonas
(Family Aeromonadaceae)

• Gram-negative facultatively anaerobic bacillus
  resembling members of the Enterobacteriaceae
• Motile species have single polar flagellum
  (nonmotile species apparently not associated with
  human disease)
• 16 phenospecies Most significant human
  pathogens A. hydrophila, A. caviae, A. veronii
  biovar sobria
• Ubiquitous in fresh and brackish water
• Acquired by ingestion of or exposure to
  contaminated water or food

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Clinical Syndromes of Aeromonas

• Associated with gastrointestinal disease
• Chronic diarrhea in adults
• Self-limited acute, severe disease in children
  resembling shigellosis with blood and leukocytes
  in the stool
• 3 carriage rate
• Wound infections
• Opportunistic systemic disease in
  immunocompromised
• Putative virulence factors include
• endotoxin hemolysins eneterotoxin proteases
  siderophores adhesins

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Characteristics of Plesiomonas

• Formerly Plesiomonadaceae
• Closely related to Proteus now classified as
  Enterobacteriaceae despite differences
• Oxidase positive
• Multiple polar flagella (lophotrichous)
• Single species Plesiomonas shigelloides
• Isolated from aquatic environment (fresh or
  estuarine)
• Acquired by ingestion of or exposure to
  contaminated water or seafood or by exposure to
  amphibians or reptiles
• Self-limited gastroenteritis secretory, colitis
  or chronic forms
• Variety of uncommon extra-intestinal infections

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Characteristics of Aeromonas and Plesiomonas
Gastroenteritis
Epidemiological Features Aeromonas Plesiomonas
Natural Habitat Source of Infection Fresh or brackish water Contaminated water or food Fresh or brackish water Contaminated water or food
Clinical Features
Diarrhea Vomiting Abdominal Cramps Fever Blood/WBCs in Stool Present Present Present Absent Absent Present Present Present Absent Present

Pathogenesis Enterotoxin (??) Invasiveness
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Differentiating the Vibrio Group
TESTS

Growth in 5 NaCl - -
Sensitive to 01291 Vibriostat
-
Growth in NB with no added NaCl
-

Organism Vibrio cholera Other Vibrio Aeromonas Pl
eisiomonas

Growth On TCBS2 -
-
1150ug Disk 2Thiocitrate bilesalts sucrose agar