The Vibrio Dr Ali Abdulwahid - PowerPoint Presentation

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The Vibrio

Dr Ali Abdulwahid

University of MustanisiriyaCollege of MedicineDepartment of Microbiology Third stage

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The Vibrio

There are more than 35 species recognized for the genus vibrio, only 12 of them are responsible for human infection (gastrointestinal and extra-intestinal infections).

Vibrio cholerae: is the most medically important species which is responsible for the cholera disease They are Gram-negative curved rods aerobic with a wide distribution in the nature (Found in marine and surface waters)Actively motile using polar flagellum.The organisms produce an enterotoxin that lead to cause cholera ( which is a watery diarrhea that can rapidly led to dehydration and even death if it is not treated)Only two serogroups (O1 and O139) cause cholera in humanOther serogroups (more than 200 ) may cause enteritis and sepsis Other species that can cause infection to human : V. parahaemolyticus, V. vulnificus, V. minicus and V. alginolyticus

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Vibrio cholerae

Morphology and Identification

Typical Organisms V cholerae is a curved, comma-shaped, gram-negative bacterium Actively motile using polar flagellum. The organism can revert to straight rods due to subculturing or prolonged cultivationThey are non-capsulated and non-spore forming Culture V. cholerae on agar plates forms moist, smooth, translucent round colonies They grow at temperature range from 16 -40 0C , but their optimum growth at 37 0CGrow well on alkaline pH (between 7.4 and 9.6) (optimum pH 8.2). Sensitive to acidic pH (below 6),Not halophilic

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Source of the picture:

Tom Kirn, Ron Taylor, Louisa Howard - Dartmouth Electron Microscope Facility / Public domain

https://commons.wikimedia.org/wiki/File:Vibrio_cholerae.jpg

Transmission electron microscope image of Vibrio cholerae

Source of the picture

CDC / Public domain

https://upload.wikimedia.org/wikipedia/commons/b/b0/Vibrio_cholerae_gram_stain_CDC.jpg

Gram-stained

Vibrio cholera

in a

specimen

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B. Culture

The bacteria grow well on a wide range of media including

Ordinary media such as (nutrient agar, MacCkonkey agar and blood agar).Selective media : such as thiosulfate-citrate-bile-sucrose (TCBS) agar : the bacteria form Yellow colonies as they ferment sucrose, while other vibrios that do not ferment sucrose produce green colonies.The yellow colonies can be easily recognised on the media which has dark-green background Enrichment media : Alkaline peptone water (pH 8.6)Useful for preliminary culturing of the bacteria from stool or other specimens Source of picture:

Microrao, JJMMC, Davangere, Karnataka, India / Public domain

https://commons.wikimedia.org/wiki/File:Vibrio_cholerae_on_TCBS_agar.jpg

yellow colonies of 

Vibrio cholerae

 on TCBS agar

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String test:

A single colony or A loopful of vibrio liquid growth is mixed with a drop of 0.5 % sodium deoxycholate or sodium taurocholate on a slide or petri dish.

If the test is positive, the sodium deoxycholate or sodium taurocholate will lysis the bacteria cells, leading to release the DNA from the sells making the suspension mucoid and form a ‘string’ when taken by loop.This test differentiate V. cholera (string +ve) from  Aeromonas spp (string –ve) (Both are isolated from diarrheal stool, show similar biochemical properties) and other vibrio species (string –ve).

String test of

Vibrio cholerae

http://

www.publicdomainfiles.com/show_file.php?id=13539910812826

Source of image : CDC, Courtesy: 

Public Health Image Library

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Antigenic Structure and Biological Classification

Flagellar (H)antigen:

Many Vibrios share a single (H)antigen , which is heat labile and it is not immunogenic (antibodies against this antigen is not protect the host from vibrio cholera infections) Somatic (O) antigen: Depending on variability in O antigen , Vibrio cholerae were divided into 206 different O serogroupsThese serogroups can be devided to three main groups:O group 1 (O1) O group 139 (O139) non-O1/non-O139 groupsOnly V. cholera strains of (O1) and (O139) cause classic cholera Other non-O1/non-O139 strains may cause cholera-like disease.

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Strains of O1 serogroup

of V. cholera are also divided into two different biotypes :

classic and El Tor. These two biotypes also divided into different serotypes depending on some determinant in the O antigen These serotypes include : Ogawa, Inaba, and Hikojima. The biotypes of V. cholerae O1 group differ in some tests as in the following table:Vibrio biotypes Tests Vibrio biotypes Voges-Proskauer

hemolysin

resistant to polymyxin B

Group

IV phage susceptibility

Classical

-ve

-ve

+ve

+ve

El Tor+ve+ve-ve-ve

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Gardner and Venkatraman Classification.

Biotypes

Vibrios

with common flagellar (H) antigen, share similar biochemical characteristic

(V. cholerae)

Somatic (O) antigen:

Vibrio

Group A

O1 group

Classical

Inaba

Ogawa

Hikojima

El Tor

Inaba

Ogawa

Hikojima

Non O1 group (involves O139)

Group B

A heterogeneous collection of vibrios

Serotypes,

d

epending on minor antigenic variation

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Vibrio cholerae Enterotoxin

It is heat-labile enterotoxin Multimric

, composed of (1 A subunit + 5 B subunits )B subunits bind to the GM1 ganglioside receptors on the intestinal epithelial cells.This will promotes the entry of subunit A in to the epithelial cellsThe subunit A then will be cleaved into A1 and A2, A1 is the active portion of the A subunit that enter the cell and activate of adenylate cyclase (which is an enzyme that responsible for production of cyclic adenosine monophosphate (cAMP) in the cell ) leading to overproduction of cAMP.This consequently lead to inhibition of the absorption of the sodium and chloride by the cells lining the microvilli, along with increased secretion of sodium-dependent chloride.This results in blocking the absorption of water, Increased outflow of water and electrolytes into the gut lumen, with consequent diarrhea.

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Pathogenesis

This bacteria is restricted pathogen for human, but it also growth in brackish water and marine waterThe infection can happen by ingestion the organism via contaminated water or food

In human with normal gastric acidity, the infection dose should be about 1010 Cells or more ( as the bacteria is sensitive to the acidity of the stomach) , while persons with achlorhydria or hypochlorhydria , as few as 102–104 bacterial cells is enough to cause the infection.Reducing the acidity of the stomach due to taking any medication leading to increase the sensitivity to infection with V. cholerae Vibrio cholerae is not invasive and do not reach the bloodstream, it attach to the microvilli of the brush border of intestinal epithelial cells.At this site, the bacteria multiply and produce cholera toxin which responsible for developing the clinical manifestation of the cholera

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Clinical Findings

The incubation period is 12 hours–3 days (this depends mainly on the number of ingested organisms)The symptoms involve a sudden onset of nausea, vomiting and profuse watery diarrhea

with abdominal cramps. The colourless watery stools “rice water stool” is a characteristic feature for the Cholera.The stool contains flecks of mucus, epithelial cells, and large numbers of vibrios. in sever cholera, the volum of fluid loos is about 1 L/h , and such rapid loss of fluid and electrolytes leads to profound dehydration, circulatory collapse, and anuria as well as renal failar. The infection without treatment is correlated with a high rate of mortality ( between 25% and 50%), but with treatment it can be decreased to 1% or less. Infection with El Tor biotype tends to be milder than the infection caused by classic biotype.

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Diagnostic Laboratory Tests

Specimens Watery stool: should be collected as early as possible in the course of the

diarrihea and inculcated within 2-4 hrs after collection into the suitable medium If that will take time, the stool specimens can be mixed with transport medium under cooling conditions (refrigeration)Smears Direct microscopic examination as well as Gram staining film for smears made from stool samples is not recommended as the results is difficult to be interpreted . The rapid diagnosis can be achieved by using dark-field or phase contrast microscopy to monitor rapid motility of vibrios (shooting star)This also can be achieved by inhibition the motility of the vibrios by adding the anti- cholera diagnostic antisera

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Culture

Specimens can be plated on different media including peptone agar, blood agar ( pH near 9.0), and TCBS agar

After 18 hrs of incubation , V. cholerae typical colonies can be seen on the agar.Serological tests V. cholerae organisms can be also identified by slide agglutination tests using anti-O group 1 or group 139 antisera.Further serological tests can also be used for differentiation between the different serotypes (Inaba, Ogawa and Hikojima)Biochemical tests :The organisms can be further identified by a variety of biochemical reactions such as IMViC, TSI and others

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Treatment

The treatment is mainly depends on the water and electrolyte replacement to correct the severe dehydration and salt depletion. This can be achieve by Oral rehydration therapy (ORT) or intravenous rehydration (in sever cases)

The antimicrobial thereby is of a secondary importance, but it can be affective to reduce the duration and the amount of the shedding vibrios in the stool.Oral tetracycline is vary affective antibiotic therapy Erythromycin and / or azithromycin are effective therapy in children and in pregnant women Other antibiotics : fluoroquinolones and doxycycline. Resistance of V cholera to antibiotics such as resistance to tetracycline has been noticed and it emerged, especially in area where cholera is endemic or epidemic.Genes responsible for such feature carried on transmissible plasmids

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Epidemiology, Prevention, and Control

Cholera is considered as both endemic and epidemic disease There were six cholera pandemics occurred between 1817 and 1923The causative agents for these pandemics were

V. cholerae O1 of the classic biotype They were mainly originated in Asia, particularly in Indian subcontinent. A seven pandemic (1961) in Indonesia, and spread to Asia, the Middle East, and Africa. The causative V cholerae was El Tor biotype. The eight pandemic (1992–1993), a spread of the serotype O139 strains in the Indian subcontinent and to other Asian countries was considered to be the eighth pandemic of cholera.

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Epidemiology, Prevention, and Control

The disease is endemic in India and Southeast Asia. It spreads to other geographical sites in the world by the means of shipping, trade, and pilgrim migration.

It spread by water, food, flies as well as contact with individuals that have mild or early infection.In majority, only 1–5% of susceptible people who exposed to V. cholerae can develop disease. People can rarely carry the bacteria asymptomatically ( carriers state) more than 3–4 weeks, and there is no long term carriage state in humans .There is no another animal host is known for V. cholerae

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Epidemiology, Prevention, and Control

The bacteria can survive in different aquatic habitats for a given periods (up to 3 weeks)Such aquatic habitats are considered as the natural reservoir for

V. cholera, as the bacteria can attach to algae, copepods, and crustacean shells and then can survive for years if the environment conditions is suitable. Control measures can be: Improvement of sanitation such as sewage treatment , water purification and prevent of food contamination Isolation of patients, disinfection and disposal of their excretes. Chemoprophylaxis with antibiotics (to household contacts) can limit the spread of the bacteria

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Vaccination

Many different vaccines have been developed against V. cholerae include:

Single dose , living cholera oral vaccine. Killed oral cholera vaccines. Whole cell V. cholerae O1 with recombinant B subunit of cholera toxin vaccine.One whole-cell V. cholerae O1 and O139 used in VietnamAnother one whole-cell V. cholerae O1 and O139 for global use Injectable phenol-inactivated strains of V. cholerae still used in some countries (not recommende by WHO, lower effectivity with short period of protection)

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Other vibrios

Vibrio parahaemolyticus It is halophilic and marine microorganism

Gram –ve , curved shape , motileCauses acute gastroenteritisThe infection is acquired by ingestion of contaminated seafood such as raw fish or shellfishThe incubation period is 12–24 hoursThe main clinical manifestations are : nausea, fever, and watery diarrhea The infection is self-limited (subside spontaneously in 1–4 days), and only water and electrolyte replacement is required . The bacteria grow well on TCBS and produce green colonies as it does not ferment sucrose.

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V. vulnificus

It is a medically important species of vibrioCausing the second serious infection caused by Vibrios after

V. cholerae It is halophilic marine microorganism, specifically apt to be found in oystersDoes not ferment the sucrose and form green colonies on TCBS agar. Lactose fermentation +ve ( it is the key differentiation feature of this species , as most other vibrio species are –ve for lactose fermentation). Diseases caused by this species : Severe wound infections, Bacteremia, Gastroenteritis

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References

Reidle, S., Morse, S. A., Meitzner, T., and Miller, S. 2019. Jawetz, Melnick & Adelberg’s Medical Microbiology , Twenty-Eight Edition. The McGraw-Hill education, Inc. USA

Kumar, S. 2012. Textbook of microbiology. Jaypee Brother Medical Publishers (P) Ltd. New Delhi, India. https://commons.wikimedia.orghttp://www.publicdomainfiles.com Public Health Image Library

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Thank you