Vibrio cholerae on TCBS Agar after Prolonged Incubation and Its Details
Vibrio cholerae on TCBS Agar After Prolonged Incubation
Vibrio cholerae on TCBS agar after prolonged incubation showing yellow to green colonies as shown above picture.
Introduction of Vibrio
Vibrios are Gram-negative, rigid, curved rods or comma-shaped and they are highly motile-single polar flagellum, non-sporulated, and non-capsulated. They are present in marine environments and surface water with a cosmopolitan distribution. The most medically important member of the genus is Vibrio cholerae. Filippo Pacini isolated micro-organisms he called “vibrions” from cholera patients in 1854, because of their motility. Robert Koch Isolated V. cholerae 1883. It was first isolated by Koch (1883) from cholera patients in Egypt. Vibrio cholerae is the causative agent of the diarrheal disease cholera. The disease is characterized by sudden effortless vomiting, profuse rice water stool followed by rapid dehydration.
Classification of V. cholerae
Species: V. cholerae
Morphology of V. cholerae
They are Gram-negative, rigid, short curved rods that are actively motile. Comma-shaped sheathed, polar flagellum having a size of about 1.5 × 0.2-0.4 µm in size. They are facultative anaerobe, non-sporulated, and non-capsulated. Growth is stimulated by sodium chloride. pH 6.4 – 9.6 (optimum 8.2). Acid labile, Temperature 16 – 40°C (Optimum 37°C)
Cultural characteristics of Vibrio cholerae
V. cholerae is a facultative anaerobic organism. 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. Selective Medium – TCBS in which V. cholerae grows well on Thiosulphate citrate bile sucrose (TCBS ) agar, on which it produces yellow colonies due to sucrose fermentation that is readily visible against the dark green background of the agar as shown above picture.
Biochemical reactions of Vibrio cholerae
Carbohydrate metabolism is fermentative, producing acid, but no gas. Vibrio cholerae ferment glucose, mannitol, maltose, mannose and sucrose but not inositol, arabinose or lactose. Indole is formed and nitrates are reduced to nitrites. These properties contribute to the ‘ cholera red reaction’. Catalase and oxidase tests are positive. Methyl red and urease tests are negative.
Vibrio cholerae is susceptible to heat, drying, and acids, but resist high alkalinity. It is destroyed at 55° C in 15 minutes. It survives in clean water for 30 days. On fruits, it survives for 1-5 days at room temperature and for a week in the refrigerator.
Cholera is an infection of the small intestine caused by the bacterium, Vibrio cholerae. The main symptoms are watery diarrhea and vomiting. This may result in dehydration and in severe cases grayish-bluish skin. Transmission occurs primarily by drinking water or eating food that has been contaminated by the feces of an infected person, including one with no apparent symptoms. The severity of diarrhea and vomiting can lead to rapid dehydration and electrolyte imbalance, and death in some cases. The primary treatment is oral rehydration therapy, typically with oral rehydration solution, to replace water and electrolytes. Worldwide, it affects 3–5 million people and causes 100,000–130,000 deaths a year as of 2010.
Pathogenesis of Cholera
Route of transmission: Feco-oral route
Water (infectious dose: 109 )
Food (infectious dose: 103)
The incubation period is 1 to 4 days.
Vibrio cholerae produces heat-labile enterotoxin with a molecular wt. of about 84,000 consisting of subunits A ( MW 28,000 ) and B. Ganglioside GM1 serves as a mucosal receptor for sub-unit B, which promotes entry of subunit A into the cell. Activation of sub-unit A1 yields increased levels of intracellular cAMP and results in prolonged hypersecretion of water and electrolytes. There is increased sodium (Na+) dependent chloride (Cl-) secretion, and absorption of sodium and chloride is inhibited. The genes for V. cholerae enterotoxin are on the bacterial chromosome. “cholera toxin” or “cholera enterotoxin” or “choleragen” binds to the cell membrane via B subunit and this subunit revert back to allow A- subunit to enter the cell membrane. A subunit dissociates into A1 and A2 activates adenyl cyclase that causes the transfer of ATP into cAMP. Increased cyclic AMP causes an outpouring of ultra cellular Na+, K+ lumen. To maintain osmotic balance, water moves from lower concentration, as a result, there will be massive loss of water causing thirst as a result patient goes on shock. The intestine is filled with an alkaline salty fluid, which is an ideal growth medium for Vibrio cholerae. Diarrhea begins when the colon can’t absorb the fluid fast enough. These factors contribute to decreasing blood pressure. The stools are rice water similar which containing mucous, epithelial cells, and a large number of vibrios. The massive loss of electrolytes from the body leads to metabolic acidosis, muscle cramps, anuria, acute tubular necrosis, shock, and sometimes death. The disease may last for 4-5 days with an average fluid loss of 15-20 liters per day.
Diarrhea occurs as much as 20 -30 Liters/Day fluids are lost which may result in dehydration, shock, acidosis, and even can lead to death. About 60% of infections are caused by classic V. cholerae and are asymptomatic, about 75% of infections are caused by the El Tor biotype.
The incubation period is 1-4 days for a person who develops symptoms, depends on the size of the inoculum ingested. Manifest with nausea, vomiting, profuse diarrhea, and abdominal cramps, rice water stool characteristic of cholera loss of fluid leads to profound dehydration, circulatory collapse, and anuria.
Laboratory Diagnosis of Vibrio cholerae
Specimen: Stool or rectal swab
Specimen collection: Collect stool samples with the help of a rubber catheter or with the help of a cholera catch device.
Venkataraman Ramakrishnan medium: This is an ideal transport medium. The organism will be viable in this medium for several weeks without multiplication
Cary- Blair medium
Bile peptone transport medium
Hanging drop preparation: for darting motility
Immobilization test: Using polyvalent Vibrio cholerae O1 antisera
If darting motility is lost after adding antisera, immobilization tests positive otherwise negative.
Alkaline peptone water (pH 8.5): Rapid growth occurs in about 6 hours with the formation of the thick surface pellicle. It is an enrichment medium. Inoculate 1 ml of stool into 10 ml of alkaline peptone water and incubate at 37 C for 4-6 hours. After incubation, subculture into TCBS agar and also perform hanging drop preparation for motility test. An immobilization test may also be performed from this.
V. cholerae is non-halophilic and can grow in nutrient broth, peptone water, and CLED agar. They fail to grow in media with more than 6% sodium chloride.
On nutrient agar, after overnight incubation round, moist, translucent, bluish colonies will appear with 1-2 mm size.
MacConkey agar: Colorless colonies will be formed after that they will change to pink color.
Blood agar: A zone of green discoloration appears around the colonies at first and later it becomes clear.
Gelatin stab culture: After three days of incubation a white line of growth appears in the medium
Alkaline bile salt agar medium: It is a modified nutrient agar medium and the colonies are similar to that appears in the nutrient agar medium.
Monsur’s gelatin taurocholate trypticase tellurite agar medium: After 24 hours of incubation small colonies will be formed with 1-2 mm in size and grayish color with black centers. The size will be increased to 3-4 mm after 48 hours of incubation.
TCBS agar: In this medium-large colonies ( 2-3 mm in diameter) will be formed in yellow color due to sucrose fermentation. Later the yellow color will be changed to green color.
Sugar fermentation test: fermentation of glucose, sucrose, and maltose will occur with the production of gas.Nitrate reduction test: positive
Indole test: positive
Catalase test: positive
Oxidase test: positive
Nitroso-indole ‘ cholera red reaction positive
Slide Agglutination Test: The specimen from a selective media is placed on a microscopic slide and add a drop of normal saline over that and then observe under a microscope after adding one drop of antiserum over the specimen. The presence of clumps indicates a positive test.
Treatment of Cholera
Adequate fluid and electrolyte replacement is necessary for such patients which are known as fluid replacement therapy. Oral rehydration therapy is very useful. Oral Tetracycline tends to reduce stool output in cholera and shortens the period of excretion of vibrios but in some endemic areas, tetracycline resistance has emerged the genes are carried by transmissible plasmids.
Epidemiology of Cholerae
Six pandemics of Cholera occurred between 1817 – 1923. Most likely V. cholerae 01 of classical type contributed to pandemics. All pandemics originated in the Indian continent. The seventh pandemic originated in Celebes Islands in Indonesia in 1961. It was spread far and wide. 8 th Pandemic is due to a spread of 0-139. Several identify that onset of 0-139 is considered as 8th pandemic started in India. V. cholerae is spread by contact with persons in early or even mild illness.
By contaminated water, food flies
Only 1 -5% of exposed will get effected
The carrier stage seldom exceeds 3- 4 weeks.
Two types of oral cholera vaccines are available:
(i) Dukoral and
(ii) Shanchol and mORCVAX.
The latter two are identical vaccines in terms of strains but formulated by different manufacturers using different methods. Characteristics of Dukoral, Shanchol, and mORCVAX are as follows-
The available oral cholera vaccines are safe 29 and provide sustained protection of >50% that lasts for 2 years in endemic populations.
30 Shanchol and mORCVAX have demonstrated longer-term protection in children aged <5 years and do not require booster doses every 6 months.
Virulence Factors Associated with Non-cholerae Vibrios
Organisms Virulence factors V. parahaemolytics: Thermostable direct hemolysin ( Kanagawa positive) V. vulnificus: Serum resistance, antiphagocytic polysaccharide polysaccharide, cytolysin, collagenase, protease, siderophore V. alginolutics: Collagenase V. bollisae: Heat-stable and heat labile, enterotoxin, hemolysin V. damsela : Cytolysin
Associated with seafood
Gastroenteritis The outbreak of food poisoning
Extra-intestinal infections- wound
Heat stable cytotoxin
Heat labile enterotoxin
Adherence to human intestinal cells
Prevention of V. cholerae
Needs improvement of Sanitation associated with water treatment and food.
Patients infected are preferably isolated.
All contacts to be followed up
Mucus flecks from stool are cultured.
Death rates from severe cholera can be decreased from ~50% to <1%.
The infective dose of V. cholerae is 106 – 108 organisms.
The use of oral cholera vaccines in emergency situations is accepted but remains a challenge. To date, there is no specific indication for use of oral cholera vaccines in endemic situations, and intervention studies are being performed to prove their effectiveness as a public health tool.
Other bacteria resembling Vibrio cholerae are Aeromonas and Plesiomonas.
2-3 loopful of a stool sample is inoculated into TCBS agar due to preferring heavy inoculum.
Symptoms of Cholera are as follows- √Diarrhea √Vomiting √Dehydration √Irritability √Drowsiness √Droopy eyes √Having a dry mouth √Dry skin √Decrease in urination √A very low blood pressure √An irregular heartbeat
Characteristics of oxidase-positive gram-negative bacilli are shown as follows-
L: Lysine decarboxylase
A: Arginine decarboxylase
O: Ornithine decarboxylase
Gardner and Venkataraman classification of Vibro cholerae-
Difference between classical and El Tor biotypes-
Vibrios are sensitive to compound 0/129 (2,4-diamino-6,7-diisopropylpteridine).
Vibrio cholerae Related Video
#Darting motility of bacteria Positive ||Vibrio cholerae ||Campylobacter jejuni
#Stool sample testing procedure for Vibrio Cholerae
#Vibrio cholerae Rapid Diagnostic Test(RDT)/ Dipstick test Procedure for Serotypes O1 and O139
#TCBS agar with Vibrio cholerae showing yellow colonies
#Cholera causing bacteria under microscope|| Vibrio cholerae|| Comma shaped bacteria
#Disease Cholera caused by Vibrio cholerae and its growth on TCBS medium, biochemical tests -TSI test, SIM test, citrate utilization test, urea hydrolyzation test, oxidation-fermentation test and decarboxylase tests like ornithine test, lysine test, and arginine test and its interpretation as shown below-
#String test positive -Vibrio cholerae as shown below-
#Vibrio cholerae antisera for serotyping
Serovar Inaba and
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