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Salmonella basics

Salmonella basics

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Salmonella basics

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  1. Salmonellabasics Dr.T.V.Rao MD Dr.T.V.Rao MD

  2. Salmonella • Causes Infections in Humans and vertebrates, • Enteric Fever ( Typhoid fever ) • Gastroenteritis • Septicemias, • Carrier state. Dr.T.V.Rao MD

  3. Salmonella • A Very complex group • Contains more > 2,000 spp • Typed on the basis of Serotyping, and species typing • Divided into two groups 1 Enteric fever group 2 Food poisoning group – Septicemias. Dr.T.V.Rao MD

  4. Enteric FeverTyphoid Fever • Caused by Salmonella typhi, and other Groups called as Paratyphi A, B, C • Salmonella typhi - Causes Typhoid • Salmonella Paratyphi A,B,C Causes Paratyphoid fevers. • Food Poison group • Spread from Animals – Humans • Causes Gastroenteritis – Septicemias, Localized Infection Dr.T.V.Rao MD

  5. Typhoid Mary Most Dangerous Woman in America Dr.T.V.Rao MD

  6. Morphology of Salmonella • Gram negative bacilli • 1-3 / 0.5 microns, • Motile by peritrichous flagella Dr.T.V.Rao MD

  7. Bacteriology –Typhoid fever • The Genus Salmonella belong to Enterobacteriaceae • Facultative anaerobe • Gram negative bacilli • Distinguished from other bacteria by Biochemical and antigen structure Dr.T.V.Rao MD

  8. Cultural Characters • Aerobic / Facultatively anaerobic • Grows on simple media – Nutrient agar, • Temp 15 – 41ºc / 37º c • Colonies appear as large 2 -3 mm, circular, low convex, • On MacConkey medium appear Colorless ( NLF ) Selective Medium - Wilson Blair Bismuth sulphide medium. Produce Jet black colonies H2 S produced by Salmonella typhi Dr.T.V.Rao MD

  9. Enrichment MediumLiquid Medium • Selenite F medium • Tetrathionate broth • Above medium are used for isolation of Salmonella from contaminated specimens • Particularly stool specimens.. Dr.T.V.Rao MD

  10. Identifying Enteric Organisms • Isolates which are Non lactose fermenting • Motile, Indole positive • Urease negative • Ferment Glucose,Mannitol,Maltose • Do not ferment Lactose, Sucrose • Typhoid bacilli are anaerogenic • Some of the Paratyphoid form acid and gas • Further identification done by slide agglutination tests Dr.T.V.Rao MD

  11. Biochemical Characters • Glucose ,Mannitol ,Maltose produce A/G • Salmonella typhi do not produce gas • Lactose/Salicin/sucrose not fermented. • Indole – • Methyl Red + • V P - • Citrate + • Urea – • H2S – produced by Salmonella typhi • Paratyphi A do not produce H2S Dr.T.V.Rao MD

  12. Resistance of Salmonella • 55º c – 1 hour • 60º c – 15 MT • Boiling ,Chlorination, Pasteurization Destroy the Bacilli. Dr.T.V.Rao MD

  13. Antigenic structure of Salmonella • Two sets of antigens • Detection by serotyping • 1Somatic or 0 Antigens contain long chain polysaccharides ( LPS ) comprises of heat stable polysaccharide commonly. • 2 Flagellar or H Antigens are strongly immunogenic and induces antibody formation rapidly and in high titers following infection or immunization. The flagellar antigen is of a dual nature, occurring in one of the two phases. Dr.T.V.Rao MD

  14. SalmonellaAntigenic Structure • H – Flegellar antigens • O – Somatic antigen, • Vi – Surface antigen in some species only • H antigens also called flegellar antigens, heat labile protein, • Boiling destroys antigenicity • When mixed with Antiserum produces agglutination and fluffy clumps are produced • H antigens are strongly immunogenic Induces antibodies rapidly, Dr.T.V.Rao MD

  15. O Antigens Forms integral part of Cell wall, Like Endotoxin 0 Antigens unaffected by boiling. When mixed with antiserum produce chalky clumps are formed, take more time reaction, at high temp 50º – 55º c O antigens are less immunogenic. than H antigens Antigens – Salmonella ( cont ) Dr.T.V.Rao MD

  16. Antigen (Vi) – Salmonella ( contd ) • Vi antigens • Many strains in S.typhi covers the O antigens- prevents agglutination. • Resembles like K antigens • Destroyed after boiling at 60º c / 1 hour. • Vi a polysaccharide • Acts as virulence factor, protects the bacilli against Phagocytosis and activity of Complement • Poorly immunogenic • Low titer of antibodies are produced, Not diagnostic Dr.T.V.Rao MD

  17. Classification of Salmonella • Classified on the basis of Kauffmann-White Scheme • Structure of 0 and H antigens are taken into consideration, • More than 2000 species characterized. Dr.T.V.Rao MD

  18. Kauffmann – White scheme • Serotype 0 antigens H antigens Phase 1 2 1.Typhi 9,12,(Vi) d 1,2 2 Paratyphi A 1,2.12 a - 3 Paratyphi B 1,4,5,12 b 1,2 4 Typhimuruim 1,4,5,12 I 1,7 5 Enteritidis 1,9,12 g m 1,2 Dr.T.V.Rao MD

  19. Antigenic Variation in Salmonella • May be phenotypic / Genotypic • H to O = loss of Flagella May be phase variation from I to II V to W variation S to R variation Dr.T.V.Rao MD

  20. Pathogenicity • Salmonella are definite parasites to humans. • Eg S.typhi. • S.paratyphi A, B ,C • Other groups Salmonella • The important clinical syndromes 1. Enteric fever, Septicemias, gastroenteritis. Dr.T.V.Rao MD

  21. Enteric FeverTyphoid • Typhoid – caused by S.typhi • Paratyphoid Caused by Paratyphi A,B,C • Typhoid --- Like Typhus • Infective dose ID50 / 107, Dr.T.V.Rao MD

  22. Dr.T.V.Rao MD

  23. Events in a Typical typhoid Fever Dr.T.V.Rao MD

  24. Pathology and Pathogenesis • Bacilli enter through ingestion, • Bacilli attach to Microvilli,ileal mucosa, penetrate to Lamina propria and sub mucosa • Phagocytosis by Polymorphs and Macrophages • Enters the mesenteric lymph nodes • Enter the thoracic duct – Blood stream Dr.T.V.Rao MD

  25. Pathology and Pathogenesis • Bacteremia Spread to Liver, Gall bladder, Spleen, Bone marrow, Lymph nodes, Lungs, Multiply in kidneys Once again spill into Blood stream Causes clinical illness. Dr.T.V.Rao MD

  26. Pathology and Pathogenesis • Multiply abundantly in Gall bladder, • Bile rich source of Bacteria • Spill into Intestine, infects payers patches, Lymph follicles • Inflammation – Undergo necrosis, Slough off • Typhoid ulcers • Typhoid ulcers can cause perforation and hemorrhage • Duration of Illness 3 – 4 weeks • Incubation 7 -14, ( 3-56 days ) Dr.T.V.Rao MD

  27. Immunity in Typhoid • Typhoid bacilli are Intracellular pathogens • Cell mediated immunity is crucial Dr.T.V.Rao MD

  28. Clinical manifestation • Head ache, malise,anorexia ,coated tongue • Abdominal discomfort, • Constipation / Diarrhea • Step ladder type fever, • Relative bradycardia, • A soft palpable spleen • Hepatomegaly • Rose spots appear Dr.T.V.Rao MD

  29. Events in a Typical typhoid Fever Dr.T.V.Rao MD

  30. Rashes in Typhoid • May present with rash, rose spots 2 -4 mm in diameter raised discrete irregular blanching pink maculae's found in front of chest • Appear in crops of up to a dozen at a time • Fade after 3 – 4 days Dr.T.V.Rao MD

  31. Complications of Enteric fever • Intestinal perforation, • Hemorrhage, • Circulatory collapse. • Bronchitis Bronchopneumonia, • Meningitis, • Cholecystitis, • Arthritis,Periostitis / Nephritis, • Osteomyletis, Dr.T.V.Rao MD

  32. Other complications • Causes relapses in particular to patients treated with chloramphenicol. • S.paratyphi produce septicemias. Dr.T.V.Rao MD

  33. Epidemiology • Developed countries - Controlled. • Water supply/ Sanitation /Economically poor. • S.typhi and S.paratyphi are prevalent in India • Previously Typhi are more common Paratyphoid A on raise. • Age 5 – 20 years, Sanitation Dr.T.V.Rao MD

  34. Epidemiology • Sanitation has great role • Source an active patient or a Carrier shed the Bacilli. • Who are carriers. Convalescent carrier 3 weeks to 3 months Temporary carrier 3 months to 1 year Chronic carrier > 1 year, Women attain more carrier stage Dr.T.V.Rao MD

  35. Epidemiology (Contd) • Bacilli persist in the Gall bladder and kidney • Food handlers spread the infection • Cooks great role • S.typhi and S.paratyphi in humans • S.para B in Animals, • Typhoid spread through Water, Milk, Food HIV patients potentially susceptible for Typhoid disease. Dr.T.V.Rao MD

  36. Typhoid Mary • A famous example is “Typhoid” Mary Mallon, who was a food handler responsible for infecting at least 78 people, killing 5. These highly infectious carriers pose a great risk to public health. Dr.T.V.Rao MD

  37. How we Diagnose Typhoid Fever • Diagnosis is made by any blood, bone marrow or stool cultures and with the Widal test (demonstration of salmonella antibodies against antigens O-somatic and H-flagellar ). In epidemics and less wealthy countries, after excluding malaria, dysentery or pneumonia, a therapeutic trial time with chloramphenicol is generally undertaken while awaiting the results of Widal test and cultures of the blood and stool. Dr.T.V.Rao MD

  38. Laboratory Diagnosis ofTyphoid Fever • 1 Isolation of Bacilli. A Gold standard • 2 Diagnosis for presence of Antibodies, • Positive Blood culture – A gold standard • Isolation from Feces and Urine ? • Detection of Antibodies Inconclusive. • Newer methods Detection of antigen in Blood and Urine Dr.T.V.Rao MD

  39. Blood Culture 1 st week Positive in 90 % 2 nd week Positive in 75 % 3 rd week Positive in 60 % > 3 weeks positive in 25 % Draw 5 – 10 cc of Blood by venipuncture. ADD to 50 -100 ml of Bile broth. Incubate at 37 c /Subculture in MacConkey At regular intervals Dr.T.V.Rao MD

  40. Blood Cultures in Typhoid Fevers • Bacteremia occurs early in the disease • Blood Cultures are positive in 1st week in 90% 2nd week in 75% 3rd week in 60% 4th week and later in 25% Dr.T.V.Rao MD

  41. Castaneda’s method ofBlood Culture • Double medium used Solid/Liquid medium in the same Bottle. • Bottle contains Bile broth/agar slant, • For subculture the bottle is merely tilted. • A subculture into MacConkey at regular intervals, • Reduces the chances of contamination • Increases the chances of isolation. Dr.T.V.Rao MD

  42. Salmonella on Mac Conkey's agar Dr.T.V.Rao MD

  43. Salmonella on XLD agar Dr.T.V.Rao MD

  44. Clot culture Clot cultures are more productive in yielding better results in isolation. A blood after clotting, the clot is lysed with Streptokinase ,but expensive to perform in developing countries. Dr.T.V.Rao MD

  45. Bactek and Radiometric based methods are in recent use Bactek methods in isolation of Salmonella is a rapid and sensitive method in early diagnosis of Enteric fever. Many Microbiology Diagnostic Laboratories are upgrading to Bactek methods Dr.T.V.Rao MD

  46. Biochemical Characters • Non Lactose fermenter, • Motile • Indole – MR + VP - Citrate + • Ferment Glu/Mal/Man • Do not ferment Lactose/Sucrose Dr.T.V.Rao MD

  47. Slide agglutination tests • In slide agglutination tests a known serum and unknown culture isolate is mixed, clumping occurs within few minutes • Commercial sera are available for detection of A, B,C1,C2,D, and E. Dr.T.V.Rao MD

  48. Culturing other Specimens • Feces Enrichment in Tetrathionate broth and Selenite broth • Culturing in MacConkey/DCA/Wilson Blair medium – Large black colonies. • Urine Culture – positive in 25 % • Other samples Bone Marrow,Bile,CSF/Sputum Dr.T.V.Rao MD

  49. Serology • WIDAL Test – Tube agglutination test. • Detects O and H antibodies • Diagnosis of Typhoid and Paratyphoid • Testing for H agglutinins in Dryers tubes, a narrow tube floccules at the bottom • Testing for O agglutinins in Felix tubes, Chalky • Incubated at 37º c overnight Dr.T.V.Rao MD

  50. Widal Test • In 1896 Widal A professor of pathology and internal medicine at the University of Paris (1911–29), he developed a procedure for diagnosing typhoid fever based on the fact that antibodies in the blood of an infected individual cause the bacteria to bind together into clumps (the Widal reaction). Dr.T.V.Rao MD