Vibrio cholerae Introduction History Cultural characters Biochemical properties

Vibrio cholerae § § § § Introduction History Cultural characters Biochemical properties Pathogenesis Epidemiology/Clinical Manifestation Diagnosis Treatment and Prevention

Morphology • Vibrios are highly motile, gramnegative, curved or commashaped rods with a single polar flagellum. • In stained mucus flakes from acute cases, vibrios appear in parallel rows resembling ‘School of fish’ appearance.

Cultural characters • Broad temperature & p. H range for growth on media ü 18 -37 C p. H 7. 0 - 9. 0 (useful for enrichment) • V. cholerae grow without salt üMost other vibrios are halophilic.

• Nutrient agar: Transparent, moist, with bluish tinge. • Mac. Conkey agar: NLF earlier, later turn to LF colonies. • Blood agar: Initially greening around colonies later clears due to haemodigestion.

• Transport media: ü Venkatraman-Ramakrishnan(VR) medium ü Carry-Blair medium ü Autoclaved sea water • Enrichment media: Alkaline peptone water ü Vibrios survive and replicate at high p. H ü Other organisms are killed or do not multiply • Plating media ü Thiosulphate, citrate, bile salts, sucrose (TCBS)agar ü Alkaline bile salt agar(BSA) ü Monsur’s GTTA medium

Biochemical reactions • Catalase and Oxidase positive. • Fermentative producing acid, but no gas • Ferment glucose maltose mannose, and sucrose, but not arabinose, and lactose. • Indole is produced and nitrates are reduced to nitrites. • Cholera red reaction: few drops of conc. sulphuric acid added to 24 hr peptone water culture produces red color.

Classification • Heiberg classification: ü Based on fermentation of Mannose, Sucrose and Arabinose. ü Classified into VIII groups ü Cholera vibrios belong to group I • Gardner and Venkatraman’s classification ü Based on major O antigen and flagellar antigen.

Gardner and Venkatraman’s classification Genus Vibrio V. cholerae Non O 1 O 2 -O 139 O 1(Division into 2 biotypes) Classical Serogroups El Tor inaba A&C ogawa A&B hikojima A, B, C Other species Vibrio parahemolyticus Vibrio vulnificus Vibrio alginolyticus

Differences between classical and EITor vibrios Test classical EITor Hemolysis - + Chick erythrocyte agglutination - + Voges-Proskauer test - + Polymyxin B sensitivity + - Mukherjii IV phage susceptibility + - Phage 5 susceptibility - +

V. cholerae • Grows in salt and fresh water • Can survive and multiply in brackish water by infecting copepods • Only O 1 and O 139 are toxigenic and cause Cholera disease • 2 categories of O 1 serotypes – Classical and El Tor

Cholera • A life-threatening secretory diarrhea induced by enterotoxin secreted by V. cholerae • Water-borne illness caused by ingesting water/food contaminated by copepods infected by V. cholerae • A major epidemic disease

Seven Recorded Pandemics • • 1 st pandemic in 1817 -1823 2 nd pandemic in 1826 -1837 3 rd pandemic in 1846 -1863 4 th pandemic in 1863 -1879 5 th pandemic in 1881 -1896 6 th pandemic in 1899 -1923 7 th pandemic in 1961 -1991 ü 7 th pandemic: · V. cholerae O 1 biotype El Tor · Began in Asia in 1961 at Tor quarantine · Dysentery like illness with gangrene of colon

• • • Reservoir Source Carriers Age group affected Habitat Resistance

Transmission • Contaminated food or water • Inadequate sewage treatment • Lack of water treatment • Improperly cooked shellfish Hanging latrine on Meghna River, Nepal

• Incubation period: 2 -3 days • High infectious dose: >108 CFU ü 103 -105 CFU with achlorhydria or hypochlorhydria • Abrupt onset of vomiting and lifethreatening watery diarrhea (15 -20 liters/day) • As more fluid is lost, feces-streaked stool changes to rice-water stools: ü Colorless ü Odorless ü No protein ü Speckled with mucus

Virulence factors 1. 2. 3. 4. 5. Cholera toxin- Enterotoxin Toxin co-regulated pilus Adhesin factor Mucinase neuraminidase

How Does Cholera Toxin Work? • B-subunit binds to GM 1 ganglioside receptors in small intestine • Reduction of disulfide bond in A-subunit activates A 1 fragment that ADP-ribosylates guanosine triphosphate (GTP)-binding protein (Gs) • ADP-ribosylated GTP-binding protein activates adenyl cyclase leading to an increased cyclic AMP (c. AMP) level • 100 fold increase in c. AMP • Activation of ion channels • Ions flow out and water follows

• • • CT binds to receptor by B subunit A subunit enters inside Cleaves into A 1 & A 2 A 1 ribosylation of G protein Upregulates adenylate cyclase Intracellular accumulation of c. AMP Inhibits absorption of sodium, activates secretion of chloride Water moves passively to gut lumen Watery diaarhoes Loss of fluid and electrolytes Shock and acidosis

Symptoms • Occur 24 -48 hours after consumption of • • • contaminated food/water Watery diarrhea Rice water stools Vomiting Cramps Without treatment, death in 18 hours-several days

Ø profuse loss of fluids and electrolytes (sodium, potassium, bicarbonate) · Hypokalemia (low levels of K in blood) · Cardiac arrhythmia and renal failure Ø Cholera toxin blocks uptake of sodium & chloride from lumen of small intestine Ø Death attributable to: · Hypovolemic shock (due to abnormally low volume of circulating fluid (plasma) in the body) · Metabolic acidosis (p. H shifts toward acid side due to loss of bicarbonate buffering capacity)

Laboratory Diagnosis • Samples: Rice watery stools, rectal swabs, blotting paper soaked in stool. • Specimens transported in suitable transport medium. • Hanging drop preparation: bacilli with darting type of motility. • Visualization by dark field or phase microscopy • Look like “shooting stars” • Gram Stain • Gram negative, curved rods

Laboratory Diagnosis: contd. . • Sample plated on semi selective and selective media. • Media also plated after enrichment of sample for 6 -8 hrs. • Isolates identified by colony characters, biochemical reactions. • Confirmation of isolates done by agglutination test with appropriate antisera (O 1, O 139, Ogawa, Inaba).

• Enrichment broth: APW, Monsur’s taurocholate telluritr peptone water • Selective media

Biochemical reactions • • • Catalase Oxidase Indole Citrate Urease MR VP TSI Nitrate reduction Cholera red reaction Sugar fermentation String test

STRING TEST String test used to separate Vibrio spp. From Aeromonas spp. and P. shigelloides

Treatment *Even before identifying cause of disease, rehydration therapy must begin Immediately because death can occur within hours* • Oral rehydration • Intravenous rehydration • Antimicrobial therapy •

Treatment: Oral Rehydration Salts (ORS) • Reduces mortality from over 50% to less than 1% • Packets of Oral Rehydration Salts

Treatment: Intravenous Rehydration • Used when patients have lost more than 10% bodyweight from dehydration • Unable to drink due to vomiting • Only treatment for severe dehydration

Treatment: Antibiotics • Adjunct to oral rehydration • Reduce fluid loss by half • Reduce recovery time by half ü 2 -3 days instead of 4 -6 • Tetracycline, Doxycycline

Traveling Precautions • Boil or treat water with chlorine or iodine • No ice • Cook everything • Rule of thumb: “Boil it, cook it, peel it, or forget it. ” • Wash hands frequently

Vaccines • Need localized mucosal immune response üOral Vaccine • Two types approved for humans: üKilled whole-cell üLive-attenuated

Vaccines: Killed Whole-cell Vaccines • Provides antigens to evoke protective antitoxic and antibacterial immunity • Contains: ü 1 x 1011 heat inactivated bacteria üMixture of V. cholerae O 1 El Tor and classical strains ü 1 mg of B subunit of cholera toxin • 50% protection for 6 months to adults • Gives less than 25% protection to children aged 2 -5

Vaccines: Live-Attenuated • Eliminates need for multiple doses of nonliving antigens • Ensures that crucial antigens potentially altered during killing process would be retained • Expected to mimic broad immunity conferred by natural infection • 85 -90% protection against classical biovar • 65 -80% protection against El Tor biovar

Non O 1/O 139 vibrios • • • O 2 - O 138 Differ clinically Cause gastroenteritis Never cause epidemic cholera Extraintestinal manifestations like otitis media, wound infection, bacteremia • Occupational or recreational exposure to seawater

Halophilic vibrios • Higher salt concentration • Vibrio parahemolyticus • Clinically causes gastroenteritis, wound infection, sepsis • Capsulated, bipolar staining, Pleomorphism, motile but not darting • Sucrose non fermentor • Swarming on blood agar • Beta hemolysis on Wagatsuma agar

• Vibrio vulnificus- rare but severe • Sepsis • Wound infection- painful swelling or cellulitis • Only vibrio which ferments lactose

• • Vibrio alginolyticus Causes eye ear and wound infection Ferments sucrose Most salt tolerant

Aeromonas • • • A. hydrophila, A. caviae, A. veronii Gastroenteritis Wound infection RTI Bacteremia HUS