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Vibrio cholerae on nutrient agar

Vibrio cholerae on nutrient agar 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 noncapsulated. They are present in marine environments and  surface water with cosmopolitan  in 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

Domain: Bacterim

Phylum: Proteobacteria

Class: Gammaproteobacteria

Order: Vibrionales

Family: Vibrionaceae

Genus: Vibrio

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 size of about  1.5 × 0.2-0.4 µm in size. They are facultative anaerobe, non-sporulated  and noncapsulated. 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. cholrae grows well on Thiosulphate citrate bile sucrose (TCBS ) agar, on which it produces yellow colonies due to sucrose fermentation that are readily visible against the dark green background of the agar as shown above picture.

Biochemical reactions of Vibrio cholerae

Carohydrate metabolism is fermentive, 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 to properties contribute to the ‘ cholera red reaction’.  Catalase and  oxidase tests are positive . Methyl red  and urease tests are negative.

Resistance

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

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 the 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: Faeco-oral route

Water (infectious dose: 10⁹ )

Food (infectious dose: 10³)

Person-to-person

Incubation period is 1 to 4 days.

Vibrio cholerae produces heat labile enterotoxin with a molecular wt. of about 84,000 consisting of sub units 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 hyper secretion 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 cell membrane via B sub unit and this sub unit revert back to allow A- sub unit to enter cell membrane. A sub unit dissociates into A1 and A2  activates adenyl cyclase that causes transfer ATP into cAMP. Increased cyclic AMP causes 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. Diarrhoea begins when the colon can’t absorb the fluid fast enough. Theses factors contribute to decrease blood pressure. The stools are rice water similar which containing mucous, epithelial cells and large number of vibrios. The massive loss of electrolytes from the body leads to metabolic acidosis, muscle cramps, anuria, acute tubular necrosis, shock and some times death.  The disease may last for 4-5 days with an average fluid loss of 15-20 liters per day.

Clinical manifestations

Diarrhea occurs as much as 20 -30 Liters/Day fluids are lost which may results in dehydration, shock, acidosis and even can lead to death. About 60% of infections are caused with classic V. cholerae and are asymptomatic, about 75% of infections are caused by El Tor biotype.

Clinical features

The incubation period is 1-4 days for person who develop 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 sample with the help of rubber catheter or with the help of cholera catch device.

Transport Medium

Venkataraman Ramkrishnan 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

Direct Microscopy

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.

Dark field microscopy shows rapidly motile vibrios.

 Cholera Rapid Test Dipsticks

Culture

Alkaline peptone water (pH 8.5):  Rapid growth occurs in about 6 hours with formation of 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. Immobilization test may also be performed from this.

V. cholerae is non-haophilic 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 be appear with 1-2 mm size.

MacConkey agar: Colorless colonies will be form after that it will change to pink color.

Blood agar:  A zone of green discoloration appears around the colonies at first and later it become 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 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.

Biochemical Tests 

Sugar fermentation test: fermentation of glucose, sucrose and maltose will occur with the production of gas.Nitrate reduction test: positive

Iodole 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. Presence of clumps indicates the positive test.

Treatment of Cholera

Adequate fluid and electrolyte replacement is necessary for such patients which is 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 Indian continent. The seventh pandemic originated in Celebes Islands in Indonesia on 1961. It was spread far and wide. 8 th Pandemic is due to 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

Carrier stage seldom exceeds 3- 4 weeks.

Vaccination

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-

1. The available oral cholera vaccines are safe 29 and provide sustained protection of >50% that lasts for 2 years in endemic populations.
2. 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-satble and heat labile, enterotoxin, hemolysin
V. damsela : Cytolysin

Vibro parahaemolytics

• Halophilic vibrio
• Associated with seafood

Gastroenteritis
Outbreak of food poisoning

Extra-intestinal infections- wound

Virulence Factors

• Haemolysin
• Heat stable cytotoxin
• Heat labile enterotoxin
• Adherence to human intestinal cells

Prevention of V. cholerae

1. Needs improvement of Sanitation associated with water treatment and food.
2. Patients infected preferably isolated .
3. Excreta disinfected
4. All contacts to be followed up

Key Notes

• Mucus flecks from stool are cultured.
• Death rates from severe cholera can be decreased from ~50% to <1%.
• Infective dose of V. cholerae is 10⁶ – 10⁸ organisms.
• 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 stool sample is inoculated into TCBS agar due to preferring heavy inoculum.
• Vibrio cholerae-

• Symptoms of Cholera are as follows-
  √Diarrhoea
  √Vomiting
  √Dehydration
  √Irritability
  √Drowsiness
  √Droopy eyes
  √Having a dry mouth
  √Dry skin
  √Decrease in urination
  √A very low blood pressure
  √An irregular heart beat
• Characteristics of oxidase positive gram negative bacilli are shown as follows-

V: Variable

L: Lysine decarboxylase

A: Arginine decarboxylase

O: Ornithine fecarboxylase

+: Positive

-: Negative

• 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 gowth on TCBS medium, biochemial tests -TSI test, SIM test, citrtate utilization test, urea hydrolysation test, oxidation-fermentation test  and decarboxylase test like orthine test, lysine test  and arginine test and its interpretation as shon below-

#String test positive -Vibrio cholerae as shown below-

#Vibrio cholerae antisea for serotyping

1. 0139 Bengal
2. Serovar Inaba and
3. Serovar Ogawa

References

1. Cowan and Steel’s Manual for identification of Medical Bacteria. Editors: G.I. Barron and  R.K. Felthani, 3rd ed 1993, Publisher Cambridge University press.
2. Bailey and Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm and  Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
3. Clinical Microbiology Procedure Hand book, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
4. Colour Atlas and Text book of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr and Sommers H.M.
5. Jawetz, Melnick and Adelberg’s Medical Microbiology. Editors: Geo. F. Brook, Janet S. Butel & Stephen A. Morse, 21st ed 1998, Publisher Appleton & Lance, Co Stamford Connecticut.
6. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
7. Text book of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
8. https://www.slideshare.net/doctorrao/vibrio-cholera
9. https://www.who.int/cholera/task_force/cholera-rapid-diagnostic-test.pdf
10. https://www.who.int/cholera/task_force/cholera-rapid-diagnostic-test.pdf
11. https://www.cdc.gov/cholera/crystal-vc.html
12. https://www.ncbi.nlm.nih.gov/books/NBK8407/
13. https://www.cdc.gov/cholera/non-01-0139-