Vibrionaceae

2. Vibrionaceae

3. Vibrio • Comma-shaped, curved gram-negative rods • Most vibrios have polar flagella • Vibrious have various pili that is important for virulence (toxin co-regulated pilus in V.cholerae) • O polysaccharide of LPS is used to subdivide vibrio species into serogroups (140 serogroup of V.cholerae,13 O serogroups of V.parahaemolyticus,7 O serogroups of V.vulnificus) • V.vulnificus and non-O1 V.cholerae produce acidic polysaccharide capsules that is important for disseminated infections.

5. vibrio • Grow on a variety of simple media within a broad temperature range (14◦C -40 ◦C) • All species require salt for growth (halophilic) • V.cholerae can grow on most media without added salt • Vibrio tolerate a wide range of pH (6.5-9) • Are susceptible to stomach acids

7. Vibrio cholera • Structure : Comma-shaped, curved gram-negative rods,(2-4µm long), • motile by a polar flagellum, • toxin co-regulated pilus (type IV pilus) • polysaccharide capsules (only in non-O1 groups ), • unusual LPS (absence of KDO) , • facultative anaerobe

9. Vibrio cholerae • Culture : grows well at 37°C for 24 hours and produces yellow colonies on TCBS (thiosulfate –citrate -bile-sucrose ) agar ,grows in alkaline peptone water (6-8 hours), grows on TTGA ( telluride –tourocolate- gelatin agar) • Growth characteristics: Simple nutritional requirements ; don’t require salt for growth but can tolerate it. Grows at very high PH (8.5-9.5), optimum pH: 7-9, are killed by acid. - Ferments sucrose and mannose . - Is oxidase (+) ,susceptible to O/129 (2,4-diamini-6,7-diisopropylpteridine)

10. Vibrio cholerae (Antigenic structure ) • heat-labile flagellar H antigen • O cell wall antigens ( strains subdivided into more than 140 serogroups) • Biotypes : Cholera (O1 serotype), El -Tor (O1 serotype) , Albensis (NAG), Proteus(NAG) • Strains of O group1 and 139 cause classic and El-Tor cholera; non-O1/O139 causes cholera-like disease. • V.cholerae serogroup O1 is further subdivided into serotypes (inaba, ogawa, hikojima) serotype ogawa (express A and b antigen), serotype Inaba (express A and C antigens), Hikojima (express A, B and C antigens) • strains can shift between Inaba and Ogawa, with Hikojima a transitional state in which both Inaba and Ogawa antigens are expressed

12. Vibrio cholerae • Antigenic structure(cont.) • Classic cholera (responsible for first 6 pandemics) and El Tor ( responsible for 7th pandemic) • The El Tor biotype produces a hemolysin, is VP(+), resistant to polymyxin B and resistant to mukerjee type IV bacteriophage. • V.cholera O139 have a polysaccharide capsule like non-O1.

13. Virulence factors of V. cholerae O1 and O139 • Cholera toxin :a heat-labile enterotoxin, interacts with G protein that control adenylate cyclase, leading to the catabolic conversion of ATP to cAMP. This results in a hyper secretion of water and electrolytes. • Toxin co-regulated pilus: adherence to intestinal mucosal cells; binding site for CTXø bacteriophage ( encoded genes for cholera toxin) • Accessory cholera enterotoxin : increases intestinal secretion • Zonula occludens toxin :loosens the tight junctions of small intestine increases intestinal permeability • Chemotaxis protein: adhesin factor • Neuraminidase : modifies cell surface to increase GM1 binding sites for cholera toxin

14. Pathogenesis of V.cholerae - Bacteriophage CTXΦ encodes the genes for : • Two subunits of cholera toxin (ctxA and ctxB genes) • Gene for accessory cholera enterotoxin(ace gene) • Gene for zonula occludens (zot gene) • Gene for chemotaxis proteins ( cep gene) • Chromosomal CTX virulence cassette and ToxT (a regulating protein) are controlled by ToxR protein that responds to PH and temperature. - Lysogenic Bacteriophage CTXΦ binds to the toxin co-regulate pilus (tcp) and moves into the bacterial cell and integrates into V.cholerae genome. • Adherence of V.cholerae to mucosa cell layer: 1- toxin co-regulated pili (controlled by ToxT protein) 2- chemotaxis proteins

16. Pathogenesis of V.cholerae • Cholera toxin is a complex A-B toxin (similar to E.coli LT toxin) • 5 B subunits bind to GM1 gangliosides receptor on epithelial cells of intestinal epithelial cells, after endocytosis A subunit translocates across the membrane of vacuole. A subunit has ADP-ribosyltransferase activityand interact with Gs membrane protein that regulateadenylatecyclase. Leading to the catabolic conversion of ATP to cAMP, increase in cAMP levels, with enhanced hypersecretion of water , chloride and decrease absorption of Na and Cl (watery diarrhea) • zonulaoccludens and accessory cholera enterotoxin produce significant diarrhae in absence of cholera toxin (in serotype O1)

17. Mechanism of Cholera toxin • Chromosomally-encoded; Lysogenic phage conversion; Highly conserved genetic sequence • Reduction of disulfide bond in A-subunit activates A1 fragment that ADP-ribosylates guanosine triphosphate (GTP)-binding protein (Gs) by transferring ADP-ribose from nicotinamide adenine dinucleotide (NAD) • ADP-ribosylated GTP-binding protein activates adenyl cyclase leading to an increased cyclic AMP (cAMP) level and hypersecretion of fluids and electrolytes

18. 1 2 Mechanism of Action of Cholera Toxin 3 4 NOTE: In step #4, uptake of Na+ and Cl- from the lumen is also blocked. HCO3- = bicarbonate which provides buffering capacity.

19. Mechanism of Action of Cholera Toxin

21. Vibrio cholera ( epidemiology) • Serotypes O1 is responsible for major pandemics (7 worldwide epidemics) with significant mortality in developing countries; O139 can cause similar diseases and may cause a pandemic (1992-1993 in Bengal) • Is endemic in India and southeast Asia. • Organism found in estuarine and marine environments • Organism can multiply freely in water • Bacterial levels increase in contaminated waters during the warm months • Spread by consumption of contaminated food or water, asymptomatically infected humans are reservoir ( spread vibrio for 4 weeks after infection by stool) • Direct person-to-person spread is rare because the infectious dose is high ( because most organisms are killed by stomach acids

22. Vibrio cholera • Pathogenesis : - Is pathogenic only for humans. - When vehicle is water, V.cholera>1010 is need for disease , and when vehicle is food >102-104. - Cholera is not a invasive infection and do not reach to bloodstream. - Attach microvilli of the brush border of epithelial cells.

24. V.Cholerae (clinical findings ) • About 60-75% 0f infections are asymptomatic • Incubation period is 1-4 days Cholera : begins with an abrupt onset of • watery diarrhea (rice water stool) • vomiting • and can progress to severe dehydration, • metabolic acidosis (bicarbonate loss) • hypokalemia, and hypovolemic shock (potassium loss), • cardiac arrhythmia • renal failure • the mortality rate is between 25-60% in untreated patients and less than 1% in treated patients.

26. V.Cholerae (clinical findings ) • Gastroenteritis : milder forms of diarrheal disease can occur in toxin-negative strains of V.cholerae O1 and in non-O1 serotypes • Extraintestinal infections :septicemia ( in patients with liver disease or hematologic malignancies

27. V.cholera(Diagnosis) • Specimens : stool on Carry Blair transport medium; Cary-Blair transport media must be refrigerated in delayed specimen transports • Microscopic examination of stool : nonproductive because of dilution of water diarrhea, darkfield or phase contrast shows motile vibrios. • Culture : should be performed early in course of disease with fresh stool specimens ; alkaline peptone broth (PH 8.6), TCBS agar , MacConkey agar, blood agar • Identification, serotyping and biotyping: by selective biochemical tests and by serotyping using polyvalent anti O group 1 antisera ,anti O group 139 antisera, serogrouping Ogawa,Inaba and Hikojima

29. V.CholeraTreatment, prevention, and control • Treatment : fluid and electrolyte replacement are crucial Sodium chloride (3.5 g/L) • Potassium chloride (1.5 g/L) • Rice flour (30-80g/L) • Trisodium citrate (2.9 g/L) • Antibiotics reduce the bacterial burden and exotoxin production , as well as duration of diarrhea • Doxycicline (adults) ,SXT (children), or furazolidone (pregnant women) is administrated • Improved hygiene is critical for control • Prevention: Combination inactivated whole cell and cholera toxin B subunit vaccines provide limited protection; other vaccines are in development

31. Vibrio parahemolyticus • Structure : Curved gram-negative , facultative anaerobe, fermenter, simple nutritional requirements but requires salt for growth • Virulence factor :thermostable direct hemolysin (TDH or kanagawa hemolysin): an entertoxin that induces chloride ion secretion in epithelia cells by increasing intracellular calcium ;produces β-hemolytic colonies on agar media with human blood . • Epidemiology : Organism found in estuarine and marine environment - Associated with consumption of contaminated shellfish - a major pathogen in countries where raw fish is eaten

32. Vibrio parahemolyticus (clinical diseases) • Gastroenteritis: range from self- limited diarrhea to a mild, cholera-like illness. • Incubation period : 5-72 hours ( mean 24 hours) • Symptoms: an explosive onset of watery diarrhea ( non blood or mucus except in severe cases) and nausea ,vomiting, abdominal cramps ,headache , and low- grade fever for 72 hours • Wound infection : Associated with exposure to contaminated seawater

33. Vibrio parahemolyticus • Diagnosis: culture on TCBS • Treatment ,prevention ,and control: - self-limited disease ,although antibiotics can shorten length of symptoms and fluid loss - Disease prevented by proper cooking of shellfish - No vaccines are available

34. Vibrio vulnificus • Structure : curved gram-negative facultative anaerobe, fermenter, simple nutritional requirements but requires salt for growth • Virulence :Antiphagocytic polysaccharide capsule ( important for the ability to produce severe, disseminated infections), cytolysins, collagenase, protease, siderophores • Epidemiology : infection associated with exposure of a wound to contaminated salt water or ingestion of improperly prepared shellfish.

35. Vibrio vulnificus(Diseases) • primary septicemia after consumption of contaminated raw oysters or rapidly progressive wound infection after exposure to contaminated seawater. • Wound infections: potentially fatal infections characterized by fever , chills, erythema, pain, vesicles or bullae formation , tissue necrosis, (mortality range 20%-30%) • Primary septicemia: sudden onset of fever, chills, vomiting, diarrhea, abdominal pain. Secondary skin lesions with tissue necrosis. Mortality rate 50% • Infections are most severe in patients with hepatic disease, hematologic diseases, chronic renal failure and those receiving immunosuppressive drugs

36. Vibrio vulnificus • Diagnosis : culture of wound and blood on TCBS • Tratment, prevention, and control: - life-threatening illness that must be promptly treated with antibiotics, - Minocycline combined with fluoroquinolone or cefotaxime is the treatment of choice - No vaccine is available

37. Other vibrios • V.mimicus: diarrhea after ingestion of uncooked seafood (raw oyster) • V.holisae: diarrhea • V.fluvialis : diarrhea • V.alginolyticus: eye, ear, wound infections after exposure to seawater • V.damsela: wound infection

39. Aeromonadaceae

40. Aeromonas • Are 1-4µm long and Motile species have single polar flagellum (nonmotile species apparently not associated with human disease) • Facultatively anaerobic fermented rods. • 21 species have been described • Produce large zones of hemolysis on blood agar. • Virulence factors: endotoxin, hemolysins, heat-labile and heat-stable enterotoxin

41. Afimbriated Aeromonas hydrophila Nonadherent Afimbriated Bacterial Cells and Buccal Cells

42. Fimbriated Aeromonas hydrophila Adherent Fimbriated Bacterial Cells and Buccal Cells

43. Aeromonas • Grow on differential media used for g(-) enteric rods and Their colony morphology are similar to that of them. • Are oxidase (+) and resistant to O/129, not growth on media containing 6% NaCl • A.hydrophila comlex ,A.caviae complex , A.veronii biovar sobria

44. Aeromonas (Diseses) • diarrheal disease (in otherwise healthy people) : acute watery diarrhea, dysenteric diarrhea ( severe abdominal pain, blood, leukocytes in the stool), chronic illness with intermittent diarrhea. • Occur after ingestion of contaminated water or food (fresh meat, diary products) • wound infections :opportunistic systemic disease ( immunocompromised patients, hepatobiliary disease or underlying malignancy patients) • Occur after a traumatic injury associated with exposure to contaminated water • Treatment: are resistant to penicillin, most cephalosporins and erythromycins • Are susceptible to tetracycline, aminoglycosides, and ciprpfloxacin

45. Characteristics of Aeromonas and Plesiomonas Gastroenteritis

46. Plesiomonas • Is a g(-) rod with Multiple polar flagella (lophotrichous) • Is common in tropical and subtropical areas. • Is isolated from freshwater fish and many animals. • Most isolates are from stool cultures from patients with diarrhea • Grows on differencial media used to isolate salmonella and shigella from stool specimens. • Share antigens with S.sonnei and cross-reaction with Shigella antisera occur • Is oxidase (+) ,DNase (+) • Plesiomonas shigelloides

48. Campylobacter

49. Campylobacter • Comma shaped ,gram (-) rods,0.2-0.5μm wide ₓ 0.5-5.0 μm • Motile by a polar flagellum • Most species microaerophilic • 25 species and 11 subspecies • Different O antigens, heat-labile capsular and flagellar antigens are used for epidemiological classification of clinical isolates • C.jejuni : gastroenteritis • C.coli : gastroenteritis • C.upsalensis: gastroenteritis • C.fetus: systemic infections (bacteremia, septic thrombophlebitis, arthritis, septic abortion, meningitis)

50. Campylobacter jejuni • Structure :Thin ,gram (-) rods with comma , S or gull wings shapes, motile with a single polar flagellum. • Culture: Charcoal or blood (remove toxic oxygen radicals) with antibiotic added media is required • grows at 37-42°C in a microaerophilic conditions ( 5%-7% oxygen) and 5%-10% CO2 on selective Skirrow`s medium contains vancomycin ,polymyxin B , and trimethoprim . Colonies are colorless to gray. • Growth characteristics : oxidase (+), catalase (+) • Small size of Campylobacter is used for recovering by filtration of stool specimen ( 0.45 μm filter) • Do not oxidize or ferment carbohydrates • Antigenic structure : LPS , enterotoxins