Vibrio cholerae. Genera Vibrio. Treatment of cholerae

1. THEME: VIBRIO CHOLERAE BIOLOGICAL CHARACTERISTICS OF GENERA
VIBRIO. BIOLOGICAL DIAGNOSTICS, CONTROLAND TREATMENT OF THE
CHOLERAE.
I. THEORETICAL QUESTIONS
1. General characteristics of the genus Vibrio.
2. Morphology and cultural characteristics.
3. Biochemical reactions and antigenic structure.
4. Epidemiology and pathogenesis of the cholerae.
5. Laboratory diagnostics of disease.
6. Prophylaxis and treatment of the disease.
TAXONOMIC POSITION OF THE PATHOGENIC VIBRIOS
Family Vibrionaceae, genus Vibrio; medical important species: V.cholerae,
V.parahaemolyticus, V.vulnificus, V.alginolyticus
Classification of the vibrios: Biochemical classification by Heiberg (1934): Heiberg (1934)
classified vibrios into six groups based on the fermentation of mannose, sucrose and
arabinose. Cholera vibrios belong to Group I (man+, suc+, ara -)
Serological classification: All vibrios possess common H-antigen and group-specific O-Ag.
Cholera vibrios possessing a common flagellar (H) antigen were classified as Group A
vibrios, and the rest as Group B vibrios comprising a heterogeneous collection. Based on the
major somatic (O) antigen, Group A vibrios were classified into 'subgroups' (now called O
serogroups or serovars), 139 of which are currently known.
VIBRIO CHOLERAE
Morphology. The cholera vibrio is a short, slightly curved rod about 1.5 μm x 0.2-0.4 μm in
size, with rounded or slightly pointed ends (the cell is typically comma shaped) In stained
films of mucous flakes from acute cholera cases, the vibrios are seen arranged in parallel
rows, as the 'fish in stream' appearance. It is actively motile, with a single polar flagellum.
The vibrios stain readily with aniline dyes and are Gram negative and nonacid fast
Cultural characteristics : The cholera vibrions is strongly aerobic. It grows within a
temperature range of 16-40 °C (optimum 37 °C).Growth is better in an alkaline medium the
range of pH being 6.4-9.6 (optimum 8.2)
It grows well on ordinary media. On nutrient agar, after overnight growth, colonies are moist,
translucent, round disks, about 1-2 mm in diameter, with a bluish tinge in transmitted light.
In peptone water, growth occurs in about six hours as a fine surface pellicle, which on
shaking breaks up into membranous pieces.
Special media. They may be classified as follows:
1) Alkaline peptone water at pH 8.6;
2) Monsur's taurocholate tellurite peptone water at pH 9.2.
3) Alkaline bile salt agar (BSA) pH 8.2. This simple medium has stood the test of time and is
still widely used. The colonies are similar to those on nutrient agar.
4) Monsur's gelatin taurocholate trypticase tellurite agar (GTTA) medium: Cholera vibrios
produce small, translucent colonies with, a greyish black centre and a turbid halo. The
colonies become 3-4- mm in size in 48 hours.
5) TCBS medium: This medium, containing thiosulfate, citrate, bile salts and sucrose, is
available commercially and is very widely used at present. Cholera vibrios produce large
yellow convex colonies which may become green on continued incubation.
Resistance Cholera vibrios are susceptible: to heat, drying and acids.It resists high alkalinity.
They are destroyed at 55 °C in 15 minutes. Dried on linen or thread, they survive for 1-3

2. days but die in about three hours on cover slips. Survival in water is influenced by its pH,
temperature, salinity, presence of organic pollution and other factors. They are killed in a few
minutes in the gastric juice of normal acidity but they may survive for 24 hours in
achlorhydric gastric juice.
Classification of the V. cholerae. According to their biological properties V.cholerae is
divided into 2 biovars:
V. cholerae b/v classical, V. cholerae b/v El-Tor.
The classical and El Tor vibrios share the same O-Ag and is agglutinated by O1- antiserum
(O-1 serogroup).
According to structure of the O1-Ag species V. cholerae is subdivided into 3 serotypes:
Ogawa (AB)
Inaba (AC)
Hikojima (ABC)
All isolates from epidemic cholera (till 1992) belonged to serogroup 0-1. Other vibrio
isolates which were not agglutinated by the 0-1 antiserum came to be called nonagglutinable
or NAG vibrios. (nonpathogenic and hence also called non-cholera vibrios (NCV).
Factors of virulence. 1. Exotoxin (choleragen, cholera enterotoxin, cholera toxin, CT, or
CTX). The toxin molecule, consists of one A and B subunits. The B (binding) units attach to
the ganglioside receptors on the surface of jejunal epithelial cells. The A (active) unite causes
prolonged activation of cellular adenylate cyclate and accumulation of cAMP, leading to
outpouring into the small intestinal lumen, of large quantities of water and electrolytes and
the consequent watery diarrhea.
2. Endotoxin. Cholera vibrios also possess the lipopolysaccharide O antigen (LPS,
endotoxin), as in Gram negative intestinal bacilli. This apparently plays no role in the
pathogenesis of cholera but is responsible for the immunity induced by killed vaccines.
3. Adherence factors (pili)
4. Proteolytic enzymes (gelatinase, mucinase)
Epidemiology and pathogenesis Cholera is an exclusively human disease. Infection is
acquired through fecally contaminated water or food. Direct person-to-person spread by
contact may not be common but hand contamination of stored drinking water has been
shown to be an important method of domestic spread of infection.
Pathogenesis
In the small intestine, vibrios are enabled to cross the protective layer of mucus and reach the
epithelial cells by chemotaxis, motility, mucinase and other proteolytic enzymes. Adhesion
to the epithelial surface and colonisation may be facilitated by special fimbria such as the
'toxin co-regulated pilus' (TCP).
The massive loss of water and electrolytes by action of enterotoxin, leads to
1. dehydration causing hemoconcentration, anuria and hypovolaemic shock
2. base-deficience acidosis
3. muscle cramps due to hypokalaemia.
Laboratory diagnosis. Specimens: Watery stool, rectal swab, water, food, vomiting
Microscopy
a. Stained smears by Gram
b. Wet drop smears to determine vibratory motility .
Bacteriological method . It is the most reliable to make diagnosis.
The major steps are:
Inoculation of the collected samples into alkaline peptone water and spread a large loop of
feces over a plate of TCBS medium.
After incubation for 5 h subculture from first peptone water is transmitted into second
alkaline PW and on the second plate of TCBS agar. Microscopy of wet smears from PW,
make a agglutination with O-1 antiserum.
After incubation for 12 h grown colonies from TCBS are investigated with agglutination

3. test, microscopy of stained smear. Suspected colony is transferred onto slant alkaline MPA.
Identification of vibrio pure culture (biochemical typing, serological and phage typing)
Serological method: detection vibriocidal antibody or agglutinins
For rapid diagnosis, the characteristic motility of the vibrio and its inhibition by antiserum
can be demonstrated under the dark field or phase contrast microscope, using cholera stool
from acute cases
Prophylaxis includes general measures (purification of water supplies, better provision for
sewage disposal, microbiological control of sewage and drinking water). Infected patients
should be isolated, their excreta disinfected. Contacts and carriers are followed up.
Specific measures
Killed parenteral vaccine – composed of equal number of Inaba and Ogava strains
Killed oral vaccine – B subunit whole cell vaccine. The vaccine contains cholera toxin B
subunit, heat killed classical vibrio and formalin killed El- Tor vibrio
Live oral vaccine – recombinant DNA vaccine
Treatment. Oral rehydration therapy, antibiotics
Vibrio parahaemolyticus is an enteropathogenic halophilic vibrio originally isolated in
1951 in Japan as the causative agent of an outbreak of food poisoning due to sea fish.
Morphology is resembles the cholera vibrio, except that it is capsulated, it shows bipolar
staining and has a tendency to pleomorphism, especially when grown on 3% salt agar and in
old cultures.
Culture properties. It grows only in media containing NaCl. It can tolerate salt
concentration up to 8 per cent but not 10 per cent. The optimum salt concentration is 2-4 per
cent. On TCBS agar, the colonies are green with an opaque, raised centre and flat translucent
periphery.
Not all strains of V. parahaemolyticus are pathogenic for human beings. It has been found
that strains isolated from environmental sources (such as water, fish, crabs or oysters). No
enterotoxin has been identified.
The vibrio is believed to cause enteritis by invasion of the intestinal epithelium.V.
parahaemolyticus causes food poisoning associated with marine food. It also causes acute
diarrhea, unassociated with food poisoning.
Laboratory diagnostics made with microscopy and pure culture isolation. Treatment is like at
cholera.
II Students practical activities
1. Microscopy smear prepared from pure culture of vibrio. Sketch the image.
2. To estimate the cultural characteristics of NAG vibrio has been grown
onto the alkaline nutrient media.
3. Write down the principal scheme of cholera and cholera-like diarrhea
laboratory diagnostics.