Al-Mustansiriyah University

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1 Al-Mustansiriyah University Monday 27/2/2017 College of Medicine Small Group Learning Committee/ Department of Microbiology Year 3/ Semester 2 / Case#2 An 18-year-old lady living in a rural area outside Baghdad develops severe profuse watery (about 8 L /day) diarrhea simulating "rice water", associated with vomiting, generalized weakness and oliguria. Learning Objectives/ Questions: 1. What is the most likely cause of her diarrhea? 2. Describe the Morphology & staining reaction, Cultural Characteristics, Viability, Classification, Antigenic structure & Toxins for these bacteria. 3. Analyze the disease Pathogenicity. 4. Outline the Laboratory Diagnosis for these bacteria. 5. Talk about the prevention, control, Treatment, Prognosis & Vaccine for these bacteria. Vibrio cholerae the causative agent of cholera Morphology & staining reaction: It was first discovered byfilippopacini in Italy in 1854 Gram ve non spore-forming, serotype O1 is non capsulated, while serotype O139 is capsulated, short curved rods comma-shape, x µ. After repeated subculture, It has 2 chromosomes, one large (~3m. base pairs) and one small (~1.1m. base pairs). The vibrios are very actively motile due to single long terminal polar flagellum. The movement is of a darting type On repeated subculture,the vibrios frequently lose their curved shape and are not different from coliform bacilli, involution also occurs readily and globular club-shaped or irregular forms may be observed, but resume their characteristic curved form when passed through animal host. During the logarithmic phase, the organism do not show any change in form, but at the end of the logarithmic phase and at the beginning of the stationary phase; the cell starts producing toxins and accompanied by internal swelling of the cell.

2 Cultural Characteristics: Strongly aerobic, temperature range C, no growth at 16 0 C Grow best at alkaline ph; which is sufficient high to inhibit pathogenic organisms. While, on the other hand it is highly sensitive to acidic ph, as ph 6.0 or less will sterilize cultures. Resistant to bile salts, bismuth sulphite, tellurite and high alkalinity making advantage for preparing selective media. Sensitive to 7% NaCl. The enrichment media used for cultivation are: 1. Alkaline peptone water; ph , lead to formation of pellicle. 2. Alkaline Bile Salt Agar BSA ; ph 8.2, contains 0.5% sod. tourocholate. 3. Mansurs medium; contains gelatin, tourocholate, trypticase, tellurite agar GTTA, with ph Thiosulphate-citrate-bile salt-sucrose medium TCBS ; widely used, colonies appear large, yellowish due to sucrose fermentation. Viability: o Killed at 55 0 C for 20 minutes, drying may kill the organism in about 2 3 hours. o In feces at high temperatures die within 1 2 days, but longer at lower temperatures. o In stagnant water, with alkaline reaction, can survive for 5 10 days or longer. o On surface of fresh vegetables and fruits remain viable for 2 5 days if stored in a cool moist environment. o The organism does not live long in river water due to the presence of large numbers of bacteriophages. o Survive for 3-4 days in ice. o Killed immediately in acidic stomach. Biochemical Reactions: Ferment glucose, mannitol, maltose, mannose and sucrose, and may be late lactose. It produceindole from peptone water medium, and also reduce nitrate to nitrite. Therefore it gives a red colour (nitroso-indole) after the addition of a drop of conc. H 2SO 4 to a 24 hour old culture of peptone water, this is called; cholera red test. Catalase and oxidase are +ve. Classification: It is mainly based on the minor differences in the O-antigens;

3 Antigenic structure: On the basis of O-antigens, cholera and cholera-like vibrios are classified into 8 serogroups. All pathogenic strains belong to the serogroup 1. Heat labile H-antigens shared by all vibrios whether pathogenic or non pathogenic, therefore it is of no significant value. Cross reaction between Brucella and Vibrio choleraeis caused by a specific O-antigen shared by both, this has given rise to a positive test of brucella agglutination test in persons who are not infected with brucella but have been immunized with cholera vaccine. El Tor was isolated in 1905 from Mekka pilgrims at the El Tor quarantine station in Saudi Arabia from mild cases of intestinal disease and was considered not dangerous. Its cultural characteristics, morphology, biochemical reactions andserological characters all are resemble V. cholerae except as shown in the table: Sensitivity to Mukerjee phage IV Polymyxin sensitivity Chicken RBC s agglutination V.P. reaction Soluble hemolysin Classical V. cholerae El Tor vibrios (+ -)

4 Toxins: Besides endotoxin, the following toxins and enzymes have been described: 1. Enterotoxin 2. Vascular permeability factor 3. Sodium pump inhibition 4. Cytotoxin 5. Hemolysin 6. Mucinase Enterotoxin or Cholera toxin: The cholera toxin (CTX or CT) is an oligomeric complex made up of six protein subunits: a single copy of the A subunit (part A), and five copies of the B subunit (part B), connected by a disulfide bond. The five B subunits form a five-membered ring that binds to GM1 gangliosides on the surface of the intestinal epithelium cells. The A1 portion of the A subunit is an enzyme that ADP-ribosylates G proteins, while the A2 chain fits into the central pore of the B subunit ring. Upon binding, the complex is taken into the cell via receptor-mediated endocytosis. Once inside the cell, the disulfide bond is reduced, and the A1 subunit is freed to bind with a human partner protein called ADP-ribosylation factor 6 (Arf6). Binding exposes its active site, allowing it to permanently ribosylate the Gs alpha subunit of the heterotrimeric G protein. This results in constitutive camp production, which in turn leads to secretion of H 2O, Na+, K+, Cl, and HCO 3 into the lumen of the small intestine and rapid dehydration. The gene encoding the cholera toxin is introduced into V. cholerae by horizontal gene transfer. Virulent strains of V. cholera carry a variant of lysogenic bacteriophage called CTXf or CTXφ. In amore simplified diagram, the mechanism is shown as follows:

5 Pathogenicity: Incubation period is 6 hours to 3 days The stool has a characteristic appearance and contains flakes of mucus floating in it, for which it is called rice water stool, which contains ~ 10 8 vibrios/ gm. The massive loss of isotonic fluid is accompanied by severe griping pain, vomiting, and muscular cramps. Then, dehydration occurs leading to hemo-concentration, anuria, acidosis and hypovolemic shock. Delay in rehydration may result in renal failure due to acute tubular necrosis. Hypokalaemia occurs due to excess loss of K + is likely to occur in children Laboratory Diagnosis: Stool and swab samples collected in the acute stage of the disease, before antibiotic administration, are the most useful specimens for laboratory diagnosis. If an epidemic of cholera is suspected, the most common causative agent is Vibrio cholerae O1. If V. cholerae serogroup O1 is not isolated, the laboratory should test for V. cholerae O139. However, if neither of these organisms is isolated, it is necessary to send stool specimens to a reference laboratory. Infection with V. cholerae O139 should be reported and handled in the same manner as that caused by V. cholerae O1. The following examination and tests should be performed for confirmation of cholera infection: 1. Microscopical Examination: a. Light microscopy b. Fluorescent Microscopy c. Immobilization 2. Cultural examination: BSA, GTTA & TCBS 3. Biochemical examination: fermentation, cholera red test, VogesProskuer test, catalase & oxidase. 4. Serological examination. Treatment: 1- Fluids In most cases, cholera can be successfully treated with oral rehydration therapy (ORT),which is highly effective, safe, and simple to administer. Rice-based solutions are preferred to glucose-based ones due to greater efficacy. In severe cases with significant dehydration, intravenous rehydration may be necessary. Ringer's lactate is the preferred solution. Large volumes and continued replacement until diarrhea has subsided may be needed. Ten percent of a person's body weight in fluid may need to be given in the first two to four hours. If commercially produced oral rehydration solutions are too expensive or difficult to obtain, solutions can be made. One such recipe calls for 1 liter of boiled water, 1 teaspoon of salt, 8 teaspoons of sugar, and added mashed banana for potassium and to improve taste. 2- Electrolytes As there frequently is initially acidosis, the potassium level may be normal, even though large losses have occurred. As the dehydration is corrected, potassium levels may decrease rapidly, and thus need to be replaced. 3- Antibiotics Antibiotic treatments for one to three days shorten the course of the disease and reduce the severity of the symptoms. People will recover without them, however, if sufficient hydration is maintained. Doxycycline is typically used first line, although some strains of V. cholerae have shown resistance. Testing for resistance during an outbreak can help determine appropriate future choices. Other antibiotics that have been proven effective include cotrimoxazole, erythromycin, tetracycline, chloramphenicol, and furazolidone.fluoroquinolones, such as norfloxacin, also may be used, but resistance has been reported.

6 Prognosis If people with cholera are treated quickly and properly, the mortality rate is less than 1%; however, with untreated cholera, the mortality rate rises to 50 60%. For certain genetic strains of cholera, such as the one present during the 2010 epidemic in Haiti and the 2004 outbreak in India, death can occur within two hours of the first sign of symptoms. Vaccine A number of safe and effective oral vaccines for cholera are available. Dukoral, an orally administered, inactivated whole cell vaccine, has an efficacy of 85%, with minimal side effects. It is available in over 60 countries. However, it is not currently recommended by the Centers for Disease Control and Prevention (CDC) for most people traveling from the United States to the third world. One injectable vaccine was found to be effective for two to three years. However, as of 2010, it has limited availability. Work is under way to investigate the role of mass vaccination. The World Health Organization (WHO) recommends immunization of high risk groups, such as children and people with HIV, in countries where this disease is endemic. If people are immunized broadly, herd immunity results, with a decrease in the amount of contamination in the environment. Vibrio parahemolyticus It is a marine organism transmitted by ingestion of raw or undercooked seafood, especially shellfish such as oysters. They can grow on medium with 8% NaCl, but V. cholerae cannot. The clinical picture varies from mild to severe watery diarrhea, nausea, vomiting, abdominal cramps, and fever. The illness is self-limited, lasting for about 3 days. Vibrio vulnificus It is also a marine organismfound in warm salt waters. It may cause severe skin and soft tissue infections (cellulitis) especially in shellfish holders.