VIBRIOVIBRIO

A PresentationA PresentationByBy

DR. ALPANA VERMADR. ALPANA VERMA International Medical & Technological International Medical & Technological

University, Tanzania.University, Tanzania.

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IntroductionIntroduction

• The second major group of gram-negative, facultatively anaerobic, fermentative rods are the genera Vibrio and Aeromonas.

• These organisms were at one time classified together in the family Vibrionaceae and were separated from the Enterobacteriaceae on the basis of a positive oxidase reaction and the presence of polar flagella.

• These organisms were also classified together because they are primarily found in water and are able to cause gastrointestinal disease.

• However, molecular biology techniques have established that these genera are only distantly related and belong in separate families:

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• Vibrio and Aeromonas are now classified in the families Vibrionaceae and Aeromonadaceae, respectively.

• Vibrios are found in marine and surface water.

• Aeromonas is found predominantly in fresh water and occasionally in cold blooded animals.

• Vibrio cholerae produces an enterotoxin that causes cholera, a profuse watery diarrhea that can rapidly lead to dehydration and death.

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The VibriosThe VibriosVibrios are among the most Vibrios are among the most common bacteria in surface waters common bacteria in surface waters worldwide. They are curved aerobic worldwide. They are curved aerobic rods and are motile, possessing a rods and are motile, possessing a polar flagellum. polar flagellum.

V cholerae serogroups O1 and O139 V cholerae serogroups O1 and O139 cause cholera in humans, while cause cholera in humans, while other vibrios may cause sepsis or other vibrios may cause sepsis or enteritis. enteritis.

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Definition of Genus Definition of Genus VibrioVibrio

• Short, 2 × 0.5 µm, often curved Gram negative rods.• Motile by means of a single polar, sheathed flagella.• Facultative anaerobes, producing acid from the ferrmentation

of a range of carbohydrates.

• Oxidase positive (except V. metschnikovii).• Reduce nitrate to nitrite (except V. metschnikovii).• Sensitive to vibriostatic agent O/129 (2,4-diamino-6,7-

diisopropylteridine phosphate; 150 µg disc).• Growth stimulated by Na+, an absolute requirement for most

species (except V. cholerae & V. mimicus).• Simple nutritional requirements.• Produce a wide array of enzymes including proteases,

nucleases, lipases & chitinase.• G+C content (mol % G+C) of their DNA varies from 39-51%.

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Growth Characteristics

V cholerae regularly ferments sucrose and mannose but not arabinose. A positive oxidase test is a key step in the preliminary identification of V cholerae and other vibrios. Vibrio species are susceptible to the compound O/129 (2,4-diamino-6,7-diisopropylpteridine phosphate), which differentiates them from Aeromonas species, which are resistant to O/129.

Most Vibrio species are halotolerant, and NaCl often stimulates their growth.

Some vibrios are halophilic, requiring the presence of NaCl to grow. Another difference between vibrios and aeromonas is that vibrios grow on media containing 6% NaCl, whereas aeromonas does not.

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Vibrio Vibrio Species Species Associated With Human DiseaseAssociated With Human Disease

Species Source of Infection

Clinical Disease

V. alginolyticus Seawater Wound infection, external otitis

V. cholerae Water, food Gastroenteritis

V. cincinnatiensis* Unknown Bacteremia, meningitis

V. fluvialis* Seafood Gastroenteritis, wound infection, bacteremia

V. furnissii* Seawater Gastroenteritis

V. harveyi* Seawater Wound infection (shark bite)

V. etschnikovii* Unknown Bacteremia

V. mimicus* Fresh water Gastroenteritis, wound infection, bacteremia

V. parahaemolyticus

Shellfish, seawater Gastroenteritis, wound infection, bacteremia

V. vulnificus Shellfish, seawater Bacteremia, wound infection, cellulitis

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V. choleraeV. cholerae Basics BasicsV. cholerae Gram-negative. Curved rod. Polar monotrichous flagella. Asporogenous. 2 chromosomes. Nonhalophilic. Grows readily on ordinary media at 37°C. Selected on TCBS media, producing yellow colonies. Oxidase positive. Ferments:o Glucose. o Sucrose.o Mannitol.

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Morphology & Staining ReactionsMorphology & Staining Reactions

• Short, often curved gram negative rods on first isolation (on prolonged cultivation may be straight rods resembling Enerobacteriaceae).

• S-shaped or spiral forms due to two or more cells lying end to end.

• Size – 2 - 4 × 0.5 µm.• Motile by means of a single polar, sheathed flagellum; motility

is of darting type.• Non acid fast.• Non spore forming.• Non capsulated.• Pleomorphism frequent in old cultures.• In stained films of mucous flakes from acute cholera cases,

the vibrios are seen arranged in parallel forms.

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Antigenic Structure & PropertiesAntigenic Structure & Properties

• Many vibrios show a single heat labile, flagellar (H) antigen.• Antibodies to H antigen not protective in susceptible hosts.

• The somatic polysaccharide (O) antigen used to subdivide Vibrio species into serogroups is of fundamental importance in the identification of this organism.

• There are > 140 serotypes of V. cholerae, (O1 – 140), • 7 - O groups of V. vulnificus and 13 O serogroups of V.

parahaemolyticus.• V. cholerae O1, or O139 (Bengal strain) are the causative

agents of cholera epidemics. • Most of the other serotypes are harmless.• Antigenic variation plays an important role in the

epidemiology and virulence of cholera.

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V. CholeraeV. Cholerae Serological Serological ClassificationClassification

Toxigenic V. cholerae

O1Division into 2 biotypes

inaba ogawa hikojima

A & B (A little C) Antigens

A & C

O139

A, B, C

Each O1 biotype can have 3 serotypes

Classical El Tor

Division into 2 epidemic serotypes

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V. choleraeV. cholerae Serological Serological ClassificationClassification

I define Vibrios! I’m an

O1 or O139 Strain

NON-TOXIGENIC TOXIGENIC

I may not be O1,Or O139!

(but I can still

stir up trouble)

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Genetic OrganizationGenetic Organization • The genome of V. Cholerae

consists of two circular chromosomes of 2,961,146 bp and 1,072,314 bp. 

• The vast majority of recognizable genes for essential cell function and pathogenicity are located on the large chromosome. 

• In contrast, the small chromosome carries a gene capture system (the integron island) and additional genes that are typically found on plasmids.

V. cholerae chromosomes: integration regions of pathogenicity islands and super integron are enhanced.

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Growth & Cultural CharacteristicsGrowth & Cultural Characteristics• Vibrios grow on a variety of simple media.• They have a broad temperature range of 14°C - 40°C (optimum

37°C) for growth.• The only non-halophilic (that do not require salt for growth)

Vibrio species are V. cholerae and V. mimicus.• Most other species are halophilic (require salt).• They tolerate a wide range of pH (pH of 6.5 to 9.0); optimum

of pH 8.2).• Peptone Water:

Incubated at 37°C, it forms a fine surface pellicle in 6-9 hrs.• Nutrient Agar:

Glistening and translucent colonies, 1-2 mm in diameter after 18-24 hrs.

• Horse Blood Agar: Zones of haemolysis are produced.

• MacConkey’s Agar: Most strains grow well giving pale i.e., non lactose fermenting

colonies in 24-36 hrs.• Desoxycholate citrate Agar, Xylose Lysine Deoxycholate

Agar & Other enteric Selective Media: Growth is poor. 15

Growth & Cultural CharacteristicsGrowth & Cultural Characteristics• Alkaline Peptone Water:

pH 8.6 is useful for prelimnary enrichment of vibrios from faeces or other contaminated materials.

• Thiosulphate Citrate Bile-salt Sucrose Agar(TCBS)Most widely selective media.Give good growth of V. cholerae, V,

parahaemolyticus and other vibrios within 37°CColonies are 2-5 mm in diameter.Colonies of sucrose fermenting vibrios are yellow

e.g., V. cholerae.Colonies of sucrose non-fermenting vibrios are

green e.g., V. parahaemolyticus. Inhibits most enterobacteria and gram positive

bacteria though allowing some strains of Proteus, Aeromonas, and enterococci to form small colonies (1 mm). 16

Growth & Cultural CharacteristicsGrowth & Cultural Characteristics• Monsur’s Tellurite Taurocholate Gelatin Agar:

Useful for isolation of cholera and other vibrios from faeces, rectal swabs & other contaminated materials.

Classical strains of V. cholera grow more freely on it than TCBS agar.

The high pH and potassium tellurite are inhibitary to most enterobacteria and Gram positive bacteria.

Proteus may form grey-centered colonies without a halo. Vibrios at 24 hrs show small (1-2 mm) translucent colonies

with a grey-black center and a turbid halo; at 48 hrs show larger (3-4 mm) colonies with a black center and a well defined halo.

• Alkaline Salt Transport Medium: No nutritive fluid. Helps maintain viability of V. cholera & other Vibrio species

in a specimen of faeces and prevents its overgrowth by other bacteria when there may be a delay of more than few hrs in the specimen transmission to the laboratory.

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Growth & Cultural CharacteristicsGrowth & Cultural Characteristics• Taurocholate Peptone Transport and Enrichment

Medium: Nutritive selective liquid medium. Used either as:

o Transport (holding) medium to maintain the viability of V. cholerae and other Vibrio species in specimens during delay in transmission to the laboratory or,

o Enrichment medium to promote selective outgrowth of V. cholerae from faeces before plating on a selective agar medium.

• Cary Blair Medium: Stool specimens suspected of containing Vibrio species, should be

collected and transported only in Cary-Blair medium.• Buffered Glycerol Saline:

Not acceptable, because glycerol is toxic for Vibrios.

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PathogenesisPathogenesis• V. cholerae, the causative bacterium, enters the mouth with

fecally contaminated food or drink.

• The bacteria attach to epithelial cells of the small intestine.

• V. cholerae toxin enters the cells and prevents them from , regulating secretion of water and electrolytes.

• The epithelial cells pump water and electrolytes from the blood into intestinal lumen, causing watery diarrhea.

• Shock and death occur because of fluid loss from the circulatory system, unless the fluid can be replaced.

• The bacteria exit the body with the faeces.

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Pathogenesis & Pathology

Under natural conditions, V cholerae is pathogenic only for humans. A person with normal gastric acidity may have to ingest as many as 1010 or more V cholerae to become infected when the vehicle is water, because the organisms are susceptible to acid.

When the vehicle is food, as few as 102–104 organisms are necessary because of the buffering capacity of food. Any medication or condition that decreases stomach acidity makes a person more susceptible to infection with V cholerae.Cholera is not an invasive infection. The organisms do not reach the bloodstream but remain within the intestinal tract. Virulent V cholerae organisms attach to the microvilli of the brush border of epithelial cells. There they multiply and liberate cholera toxin and perhaps mucinases and endotoxin.

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Clinical Findings

About 60% of infections with classic V cholerae are asymptomatic.

The incubation period is 1–4 days,.

There is a sudden onset of nausea ,vomiting and profuse diarrhea with abdominal cramps.

Stools, which resemble "rice water," contain mucus, epithelial cells, and large numbers of vibrios. There is rapid loss of fluid and electrolytes, which leads to profound dehydration, circulatory collapse, and anuria.

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Vibrio cholerae Enterotoxin

V cholerae produce a heat-labile enterotoxin with a molecular weight of about 84,000, consisting of subunits A (MW 28,000) and B .

ganglioside GM1 serves as the mucosal receptor for subunit B, which promotes entry of subunit A into the cell.

Activation of subunit A1 yields increased levels of intracellular cAMP and results in prolonged hypersecretion of water and electrolytes.

There is increased sodium-dependent chloride secretion, and absorption of sodium and chloride is inhibited. Diarrhea occurs—as much as 20–30 L/d—with resulting dehydration, shock, acidosis, and death. The genes for V cholerae enterotoxin are on the bacterial chromosome. Cholera enterotoxin is antigenically related to LT of Escherichia coli and can stimulate the production of neutralizing antibodies. However, the precise role of antitoxic and antibacterial antibodies in protection against cholera is still not clear.

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– – Vibrio choleraeVibrio cholerae Infections Infections• Epidemiology

Serotype O1 is responsible for major pandemics (worldwide epidemics) with significant mortality in developing countries; O139 can cause similar diseases and may cause a pandemic.

Organism found in estuarine and marine environments worldwide (including along the coast of the United States); associated with chitinous shellfish.

Organism can multiply freely in water. Bacterial levels increase in contaminated waters during the

warm months. Spread by consumption of contaminated food or water. Direct person-to-person spread is rare because the infectious

dose is high; the because most organisms are killed by stomach acids.

• Disease Cholera: Begins with an abrupt onset of watery diarrhea and

vomiting and can progress to severe dehydration, metabolic acidosis and hypokalemia, and hypovolemic shock.

Gastroenteritis: Milder forms of diarrheal disease can occur in toxin-negative strains of V. cholerae O1 and in non-O1 serotypes.

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African countries invaded and African countries invaded and affected by cholera epidemics in affected by cholera epidemics in

the seventies.the seventies.

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Clinical ManifestationsClinical Manifestations

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Clinical FeaturesClinical Features

• V. cholerae produces cholera toxin, whose action on the mucosal epithelium is responsible for the characteristic diarrhea of the disease cholera.

• In its extreme manifestation, cholera is one of the most rapidly fatal illnesses known. A healthy person may become hypotensive within an hour of the onset of symptoms and may die within 2-3 hours if no treatment is provided. More commonly, the disease progresses from the first liquid stool to shock in 4-12 hours, with death following in 18 hours to several days.

• Cholera begins with sudden onset of massive diarrhea. The patient may lose liters of protein-free fluid and associated electrolytes, bicarbonates and ions within a day or two.

• This results from the activity of the cholera enterotoxin which activates the adenylate cyclase enzyme in the intestinal cells, converting them into pumps which extract water and electrolytes from blood and tissues and pump it into the lumen of the intestine.

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Clinical FeaturesClinical Features

• This loss of fluid leads to dehydration, anuria, acidosis and shock. The watery diarrhea is speckled with flakes of mucus and epithelial cells ("rice-water stool") and contains enormous numbers of vibrios. The loss of potassium ions may result in cardiac complications and circulatory failure. Untreated cholera frequently results in high (50-60%) mortality rates.

• Treatment of cholera involves the rapid intravenous replacement of the lost fluid and ions. Following this replacement, administration of isotonic maintenance solution should continue until the diarrhea ceases.

• If glucose is added to the maintenance solution it may be administered orally, thereby eliminating the need for sterility and iv. administration.

• By this simple treatment regimen, the mortality rate of cholera can be reduced more than ten-fold.

• Most antibiotics and chemotherapeutic agents have no value in cholera therapy, although a few (e.g. tetracyclines) may shorten the duration of diarrhea and reduce fluid loss.

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– – Vibrio choleraeVibrio cholerae Infections Infections• Diagnosis

Microscopic examination of stool generally nonproductive because the organism is diluted in the large volume of watery diarrhea

Culture should be performed early in course of disease with fresh stool specimens

• Treatment, Prevention, and Control Fluid and electrolyte replacement are crucial. Antibiotics reduce the bacterial burden and exotoxin

production, as well as duration of diarrhea. Doxycycline (adults), trimethoprim-sulfamethoxazole

(children), or furazolidone (pregnant women) is administered.

Improved hygiene is critical for control. Combination inactivated whole cell and cholera toxin B

subunit vaccines provide limited protection; other vaccines are in development.

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Laboratory DiagnosisLaboratory Diagnosis

• Specimen Collection and Transport: Stool specimens suspected of containing Vibrio species should be

collected and transported only in Cary-Blair medium. Buffered glycerol medium is not acceptable, because glycerol is

toxic for vibrios. Feces is preferable, but rectal swabs are acceptable during the

acute phase of diarrheal illness.

• Direct Detection Methods: V. cholerae toxin can be detected in stool using an enzyme-linked

assay (ELISA) or or Latex agglutination test. Microscopically, vibrios are gram negative, straight or slightly

curved rods. When stool specimens are examined using dark-field microscopy,

the bacilli exibit charateristic rapid darting or shooting star motility.

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Microbiological & Microbiological & Molecular Methods of DetectionMolecular Methods of Detection

• Microbiological culture-based methods using fecal or water samples.

• Rapid Tests Dark-field microscopy. Rapid immunoassays. Molecular methods

o PCR.o DNA probes.

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Key Biochemical & Physiological Key Biochemical & Physiological Characteristics of Important VibriosCharacteristics of Important Vibrios

V. cholerae

V. parahaemolyticus

V. vulnificus

Oxidase + + +

D–Glucose (Gas) - - -

Lactose - - V

Sucrose + - V

myo-inositol - - -

Lysine decarboxylase + + +

Arginine dihydrolase - - -

Ornithine decarboxylase

+ + V

Growth in 0% NaCl + - -

Growth in 6% NaCl v + V

TCBS growth Good Good Good

Colony color on TCBS Yellow Green Green37

ERIC are highly conserved sequences present as multiple copies in the genome of bacteria, with no link to pathogenic genes. Such markers are highly discriminatory in investigating clonal origin of epidemic strains and the PCR fingerprinting method applied to these chromosomal sequences resulted highly discriminatory in investigating clonal origin of epidemic strains and a rapid and reliable technique.

In this figure it is shown ERIC types of different V. cholerae strains.

ERIC Sequences.

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Treating CholeraTreating Cholera

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Epidemic Control MeasuresEpidemic Control Measures

• Hygienic disposal of human waste.• Adequate supply of water.• Good food hygiene: Thoroughly cooking food. Eating food while it’s hot. Preventing cooked foods from contacting o Raw foods (including water or ice).o Avoiding raw fruits or vegetables.o Washing hands after defecation

& before cooking.

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Preventing Cholera: VaccinesPreventing Cholera: Vaccines

• Orochol Contains 2x108 viable cells of attenuated strain CVD 103-

HgR in a lyophilized form. Oral immunization of children older than 2. Subunit A of the cholera toxin (CT) has been removed.

• Dukoral Protects against

o O1 Inaba & Ogawa, Classical & El Tor strains.

o Contains 1x10 heat/formalin killed cells of strain WC/rBS.

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The most important part of therapy consists of water and electrolyte replacement to correct the severe dehydration and salt depletion. Many antimicrobial agents are effective against V cholerae.

Oral tetracycline tends to reduce stool output in cholera and shortens the period of excretion of vibrios. In some endemic areas, tetracycline resistance of V cholerae has emerged; the genes are carried by transmissible Plasmids.

Treatment

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Control rests on education and on improvement of sanitation, particularly of food and water.

Patients should be isolated, their excreta disinfected, and contacts followed up.

Chemoprophylaxis with antimicrobial drugs may have a place.

Repeated injection of a vaccine containing either lipopolysaccharides extracted from vibrios or dense vibrio suspensions can confer limited protection to heavily exposed persons (eg, family contacts) but is not effective as an epidemic control measure.

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