Shigella and Salmonella Lecture

This lecture dicusses Shigella and Salmonella and their lab diagnosis. It was taught to Allied Health Sciences BSc students.

1. Shigella and Salmonella Ms. Clemencia Tjazuko MSc. Medical Microbiology

2. Morphology Short gram-negative rods 0.5 by 1-3 µm in size Non-motile Non- sporing Non-capsulated Fimbriae may be present C. Tjazuko

3. Shigella Agent of bacillary dysentery Dysentery- infection of the intestines which causes the passage of bloody stools mixed with mucus. Named after Japanese microbiologist Kiyoshi Shiga , who isolated the first member  S.dysenteriae in 1886 from epidemic dysentery C. Tjazuko

4. Antigenic Structure Simple ‘Major’ antigens and ‘Minor’ somatic antigens Some have K antigens Fimbrial antigens C. Tjazuko

5. Classification Classified into four species based on combination of biochemical and serological characteristics Serotypes distinguished within the species C. Tjazuko

6. S.dysenteriae (Group A) Does not ferment mannitol 15 serotypes Serotype 1- S.shigae Only shigella producing shiga toxin ( exotoxin ) Only member of enterobacteriaceae that is catalase negative Indole negative C. Tjazuko

7. Serotype 2 - S.schmitzi Forms indole Ferments sorbitol and rhamnose Serotype 3-7 Formerly Large-Sachs group C. Tjazuko

8. S.flexneri (Group B) Based on specific type antigen 6 serotypes Complex antigenically Serotype 6 Indole negative and has three biotypes Manchester, Newcastle and Boyd 88 C. Tjazuko

9. S. Boydii (Group C) 19 serotypes Isolated least frequently Resemble S.flexneri biochemically but not antigenically Described by Boyd (1931) from India C. Tjazuko

10. S. Sonnei (Group C) Sonne (1915), Denmark Ferments lactose and sucrose late Indole negative Antigenically homogenous 2 forms: Phase I and Phase II Phase II  colonies are larger, flatter and irregular Mildest form of bacillary dysentry C. Tjazuko

11. Toxins Endotoxin May be due to the LPS of Gram-negative cell wall Has irritating effect on intestinal wall which causes diarrhoea and subsequently intestinal ulcers C. Tjazuko

12. Exotoxin Produced by S.dysenteriae type 1 Also called Shiga toxin or verotoxin Acts as enterotoxin and neurotoxin Inhibit protein synthesis C. Tjazuko

13. Verocytotoxin (VT) Cytotoxin produced by S.dysenteriae type 1 Acts on vero cells VT1 and VT2 VT1 two subunits A and B. A binds cytotoxin to cells B inhibit protein synthesis C. Tjazuko

14. Pathogenesis of Shigella Mode of transmission:  infection through contaminated fingers  contaminated food/water  flies (rare) transmitted sexually (homosexuals), gay bowel syndrome Minimum Infective Dose:  10-100 bacilli C. Tjazuko

15. Entry via M cell:  Bacilli enter mucosa via M cell  Cross the basolateral side of M cells to reach submucosa where they are engulfed by macrophages  Bacilli released by macrophages Cytokines released which attract inflammatory cells to infected site inflammation acute collitis C. Tjazuko

16. Invasion:  Inside submucosa , shigellae induce own uptake into adjacent epithelial Determined by large virulence plasmid , codes for ipa proteins and Type III secretion system Direct cell-to-cell spread:  from one host cell to the other by inducing actin polymerisation cells of host C. Tjazuko

17. Bacillary dysentery Short incubation period (1-7 days) Clinical features: Frequent passage of loose, scanty feces containing blood and mucus, with abdominal cramps and tenesmus Complications: Arthritis, toxic neuritis, conjunctivitis, parotitis , intussusception in children Hemolytic uremic syndrome in severe cases C. Tjazuko

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19. Laboratory Diagnosis Specimen Isolating bacillus from feces Fresh feces inoculated or transported in Sach’s buffered glycerol saline or Gram-negative broth @ pH7.0-7.4 Rectal swabs not satisfactory Gram-negative broth sodium deoxycholate to inhibit gram-positive bacteria, subcultured 6-8hrs after incubation Selenite F Broth/Salmonella Shigella (SS) broth C. Tjazuko

20. Microscopy- plenty of pus cells, erythrocytes and macrophages Culture- best to use mucus flakes MacConkey , DCA/ XLD Incubation @ 37 ºC, plates inspected for pale or pink-coloured colonies, identified by biochemical reactions Non-motile bacillus that is : urease , citrate, H 2 S and KCN negative further investigated by biochemical tests Confirmed by slide agglutination with sera C. Tjazuko

21. Salmonella Shigella Agar - colonies colourless Hektoen enteric (HE) Agar – contains bile salts as inhibitory agents and some dyes, colonies appear green with fading colour to the periphery C. Tjazuko

22. Treatment Acute cases infants and young children, dehydration corrected propmptly Antibiotics reserved for severe toxic cases Many strains still sensitive to Nalidixic acid and Norfloxacin C. Tjazuko

23. Salmonella Morphology Gram-negative rods,1-3 by 0.5 µm in size Fimbriae – present Motile with petrichate flagella But S.Gallinarum and S.Pullorum are non-motile C. Tjazuko

24. Clinical Classification Typhoidal Salmonella Serotypes S.Typhi and S.Paratyphi restricted to human hosts causing enteric fever (typhoid/paratyphoid fever) Non- typhoidal Salmonella Remaining serotypes are the food poisoning group, infect human beings causing gastroenteritis, septicemia or localised infections C. Tjazuko

25. Antigenic Classification Kauffmann-White scheme Based on the presence of different somatic (O) and flagellar (H) which is detected by agglutination with respective antisera Based on O antigen: Serogroup 2 Serogroup 4 Serogroup 9 Within each group further diffrentiation by Phase 1 and Phase 2 flagellar antige ns C. Tjazuko

26. Antigenic characterisation – Kauffmann White scheme SEROGRPS SEROTYPE ANTIGEN O H Phase I Phase II 2-A S. para A 1,2,12 a - 4-B S. para B 1,4,5,12 b 1,2 S. typhimurium 1,4,5,12 I 1,2 S. chester 4,5,12 e,h e,n,x 7-C S. para C 6,7(Vi) c 1,5 S. cholera – suis 6,7 c 1,5 8-C2 S. muenchen 6,8 d 1,2 9-D S . Typhi 9,12(Vi) d - S. enteritidis 1,9,12 g,m - S . Gallinarum 1,9,12 - - 10-E S. anatum 3,10 e,h 1,6 C. Tjazuko

27. Antigenic Structure Flagellar antigen H Somatic antigen O Surface antigen Vi H antigen Present on flagella, heat labile Destroyed by boiling or alcohol H suspensions agglutinate rapidly to form large, loose, fluffy clumps C. Tjazuko

28. Strongly immunogenic, induce antibody formation Occurs in two phases O antigen Phosholipid -protein-polysaccharide complex Identical to endotoxin Heat and alcohol stable 67 O antigens Less immunogenic O suspensions form compact, chalky, granular clumps with antisera C. Tjazuko

29. Vi antigen Surface polysachharide envelope or capsular antigen covering the O antigen Inhibits phagocytosis, resists complement activation, bacterial lysis Heat labile, Poorly immunogenic Present bacilli is inagglutinable with O antisera . Becomes agglutinable after boiling or heating at 100 ºC for 1hr S.Paratyphi C C. Tjazuko

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31. Antigenic Variation H-O Variation Associated with the loss of flagella. Induced by: Phenol agar: inhibits flagella Mutation: Non-motile mutant of S.Typhi 901-O strain subcultured in Craigie’s tube Phase variation: Phase 1: serotype specific (a, b,c etc) Phase 2: nospecific /group antigens (1,2, etc) C. Tjazuko

32. V-W Variation V forms of S.Typhi isolates with Vi antigens are agglutinated by Vi antisera but not O antisera After serial subcultures, Vi antigen is lost  W forms agglutinable by O antisera but not Vi antisera Also VW forms  agglutinable with both C. Tjazuko

33. S-R Variations Smooth to rough variation, due to loss of of O antigen side chain from LPS leading to exposure of core polysaccharide portion Smooth colonies produced by virulent strains Rough strains form large, round and irregular colonies, avirulent due to loss of O antigen C. Tjazuko

34. Pathogenesis of Salmonella S.Typhi and S.Paratyphi A, B, C  Typhoidal salmonellae Cause Enteric fever also known as Typhoid fever Infective Dose :10 3 – 10 6 bacilli Risk factors : stomach acidity, intestinal integrity Entry through epithelial cells (M cells): lines the mucosa. Salmonellae cause formation of membrane ruffles. Ruffles enclose adherent bacteria bacteria mediated endocytosis (BME) C. Tjazuko

35. Mechanism of BME: Type III secretion system  delivery of bacterial proteins into cytoplasm of epithelial cells alters actin cytoskeleton required for bacterial uptake Entry into macrophages: Salmonellae containing vacuoles cross epithelial layersubmucosa  phagocytosed Survival inside macrophages: S.Typhi alters its surface so that it is no longer suspectible to lysosomal enzymes of macrrophages Encode Type III secretion system that delivers bacterial proteins into macrophage cytoplasm C. Tjazuko

36. Primary bacteremia : Salmonellae inside macrophages spread via lymphatics to enter blood stream Spread: disseminate throughout liver, spleen, lymph nodes and bone marrow; and gallbladder, kidneys and lungs  further multiplication Secondary bacteremia : from clinical organs C. Tjazuko

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38. Clinical Manifestations Gradual onset  with headac he, malaise,anorexia , coated tongue and abdominal discomfort with constipation or diarrhea Soft, palpable spleen. Hepatomegally Rashes/Rose spots that fade on pressure Complications: gastrointestinal bleeding and intestinal perforation in third or fourth week of illness C. Tjazuko

39. Carriage 10% of untreated patients become carriers and excrete S.Typhi in urine or feces Two types of carriers: Fecal carriers: bacilli multiply in gall bladder and excreted in feces . Common Urinary carriers: multiplication in kidneys and bacilli excreted in urine Tyhoid Mary-chronic carrier, a New York cook who caused 1300 cases outbreaks C. Tjazuko

40. Laboratory Diagnosis Specimen and Culture Isolation Blood and Bone marrow culture: In first week of illness in blood culture bottle (BHI broth, automated BACTEC) Stool Culture (3-4 weeks of illness) Enrichment broth such as Selenite F Broth, tetrathionate broth and gram-negative broth MacConkey Agar NLF DCA, XLD agar, SS agar and Wilson Blair’s C. Tjazuko

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43. Urine Culture In 3-4 weeks of illness On MacConkey agar Culture Smear and Motility Motile, gram- neg bacilli Biochemical Identification Catalase positive and oxidase negative Nitrate reduced to nitrite Indole and Urease negative C. Tjazuko

44. Slide Agglutination Confirm the serotype C. Tjazuko

45. Serum Ab detection ( Widal Test) 2-3 weeks of illness, Abs detected against TO, TH, AH, BH antigens S.Typhi infection: high TO and TH Abs S.Paratyphi A infection: high TO and AH Abs S.Paratyphi B infection: high To and BH Abs Antigen detection (serum and urine ) ELISA, CIEP C. Tjazuko

46. Molecular Methods PCR detecting flagellin gene, iroB , and fliC gene Antimicrobial testing Done on Mueller-Hinton Agar C. Tjazuko

47. Assignment due 22 Feb 2017 To which family does Shigella and Salmonella belong to? What are the general properties of that family? Name the Non- Typhoidal Salmonellae species? Write short notes about what diseases they cause C. Tjazuko