Salmonella basics

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Salmonella basics

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Salmonella basics

  1. 1. Salmonella basics Dr.T.V.Rao MD Dr.T.V.Rao MD 1
  2. 2. Salmonella• Causes Infections in Humans and vertebrates,• Enteric Fever ( Typhoid fever )• Gastroenteritis• Septicemias,• Carrier state. Dr.T.V.Rao MD 2
  3. 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 3
  4. 4. Enteric Fever Typhoid 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 4
  5. 5. Typhoid Mary Most Dangerous Woman in America Dr.T.V.Rao MD 5
  6. 6. Morphology of Salmonella• Gram negative bacilli• 1-3 / 0.5 microns,• Motile by peritrichous flagella Dr.T.V.Rao MD 6
  7. 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 7
  8. 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 8
  9. 9. Enrichment Medium Liquid 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 9
  10. 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 10
  11. 11. Biochemical Characters• Glucose ,Mannitol ,Maltose produce A/G• Salmonella typhi do not produce gas• Lactose/Salicin/sucrose not fermented.• Indole –• Methyl Red +• VP -• Citrate +• Urea –• H2S – produced by Salmonella typhi• Paratyphi A do not produce H2S Dr.T.V.Rao MD 11
  12. 12. Resistance of Salmonella• 55º c – 1 hour• 60º c – 15 MT• Boiling ,Chlorination, Pasteurization Destroy the Bacilli. Dr.T.V.Rao MD 12
  13. 13. Antigenic structure of Salmonella• Two sets of antigens• Detection by serotyping• 1 Somatic 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 13
  14. 14. Salmonella Antigenic 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 14
  15. 15. Antigens – Salmonella ( cont )• 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 Dr.T.V.Rao MD 15
  16. 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 16
  17. 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 17
  18. 18. Kauffmann – White scheme• Serotype 0 antigens H antigens Phase 1 21.Typhi 9,12,(Vi) d 1,22 Paratyphi A 1,2.12 a -3 Paratyphi B 1,4,5,12 b 1,24 Typhimuruim 1,4,5,12 I 1,75 Enteritidis 1,9,12 g m 1,2 Dr.T.V.Rao MD 18
  19. 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 19
  20. 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 20
  21. 21. Enteric Fever Typhoid• Typhoid – caused by S.typhi• Paratyphoid Caused by Paratyphi A,B,C• Typhoid --- Like Typhus• Infective dose ID50 / 107, Dr.T.V.Rao MD 21
  22. 22. Dr.T.V.Rao MD 22
  23. 23. Events in a Typical typhoid Fever Dr.T.V.Rao MD 23
  24. 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 24
  25. 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 25
  26. 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, Dr.T.V.Rao MD days ) ( 3-56 26
  27. 27. Immunity in Typhoid• Typhoid bacilli are Intracellular pathogens• Cell mediated immunity is crucial Dr.T.V.Rao MD 27
  28. 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 MD Dr.T.V.Rao 28
  29. 29. Events in a Typical typhoid Fever Dr.T.V.Rao MD 29
  30. 30. Rashes in Typhoid• May present with rash, rose spots 2 -4 mm in diameter raised discrete irregular blanching pink maculaes 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 30
  31. 31. Complications of Enteric fever• Intestinal perforation,• Hemorrhage,• Circulatory collapse.• Bronchitis Bronchopneumonia,• Meningitis,• Cholecystitis,• Arthritis,Periostitis / Nephritis,• Osteomyletis, Dr.T.V.Rao MD 31
  32. 32. Other complications• Causes relapses in particular to patients treated with chloramphenicol.• S.paratyphi produce septicemias. Dr.T.V.Rao MD 32
  33. 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 33
  34. 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 34
  35. 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, FoodHIV patients potentially susceptible for Typhoid disease. Dr.T.V.Rao MD 35
  36. 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 36
  37. 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 37 Dr.T.V.Rao MD stool.
  38. 38. Laboratory Diagnosis of Typhoid 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 38
  39. 39. Blood Culture1 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 MacConkeyAt regular intervals Dr.T.V.Rao MD 39
  40. 40. Blood Cultures in Typhoid Fevers• Bacteremia occurs early in the disease• Blood Cultures are positive in1st week in 90%2nd week in 75%3rd week in 60%4th week and later in 25% Dr.T.V.Rao MD 40
  41. 41. Castaneda’s method of Blood 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 41
  42. 42. Salmonella on Mac Conkeys agar Dr.T.V.Rao MD 42
  43. 43. Salmonella on XLD agar Dr.T.V.Rao MD 43
  44. 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 Dr.T.V.Rao MD 44 countries.
  45. 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 45
  46. 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 46
  47. 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 47
  48. 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 48
  49. 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 49
  50. 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 50
  51. 51. WIDAL Testland Mark In Diagnosis • The Widal test is an old serologic assay for detecting IgM and IgG antibodies to the O and H antigens of Salmonella. The test is unreliable, but is widely used in developing countries because of its low cost. Newer serologic assays are somewhat more sensitive and specific than the Widal test, but are infrequently available. Dr.T.V.Rao MD 51
  52. 52. Widal test• S.typhi O and H tubes• Paratyphi A/B H agglutinins only• Common antigens O in all Factor sharing 12• Significance• I st week negative.• Titers raise in 2nd week Raise of titers diagnostic Dr.T.V.Rao MD 52
  53. 53. Widal Test• Single test not diagnostic.• Paired samples tests• Diagnostic. O > 1 in 80 H > 1in 160H agglutinins appear firstFalse positives in Unapparent infection, Immunization Previously infected Dr.T.V.Rao MD 53
  54. 54. Widal test• Anamnestic response previous infection and responding to unrelated infection• Other Diagnostic tests CIE and ELISADetection of Circulating antigensCo agglutination test. Dr.T.V.Rao MD 54
  55. 55. Limitation of Widal Test• The Widal test is time consuming and often times when diagnosis is reached it is too late to start an antibiotic regimen.• In spite of several limitation many Physicians depend on Widal Test Dr.T.V.Rao MD 55
  56. 56. False Positive and Negative Reactions with WIDAL Test• The Widal test should be interpreted in the light of baseline titers in a healthy local population. This is especially important when there is a high local prevalence of non-typhoid salmonellosis. The Widal test may be falsely positive in patients who have had previous vaccination or infection with S typhi. Dr.T.V.Rao MD 56
  57. 57. False Positive and Negative Reactions with WIDAL Test• Widal titers have also been reported in association with the dysgammaglobulinaemia of chronic active hepatitis and other autoimmune diseases.64 8 9 False negative results may be associated with early treatment, with "hidden organisms" in bone and joints, and with relapses of typhoid fever. Occasionally the infecting strains are poorly immunogenic. MD Dr.T.V.Rao 57
  58. 58. Diagnosis of Carriers and Environments• Fecal carriers by isolation from specimens. or Bile aspirated.• Sewer swabs• Bacteriophage typing Dr.T.V.Rao MD 58
  59. 59. Prophylaxis• TAB vaccine S.typhi 1,000 millions S Paratyphi A,B 750 millions. Injected subcutaneously 0.5 ml at 4 – 6 weeks.Live Oral Vaccine TyphoralMutant S.typhi strain Ty 2 1a Lacking enzyme UDP galctose 4 epimerase 10 to9Viable bacilliGiven orally 1 – 3 – 5 days Dr.T.V.Rao MD 59
  60. 60. Vaccines• An Inject able vaccine Typhium Vi• Contains purified Vi polysaccharide antigen from S.typhi strain Ty2• A single dose, subcutaneous route• Given to children > 5 years• Immunity lasts for 2- 3 years.• Follow a booster Dr.T.V.Rao MD 60
  61. 61. Treatment• Chloramphenicol 1948 /1970 resistance.• Other Important drugs Ampicillin Amoxicillin, Furazolidine Cotromoxazole Chloramphenical resistance /Mexico Kerala Dr.T.V.Rao MD 61
  62. 62. Other Drugs• Fluroquinolones Ciprofloxacillin, Pefloxacillin Ofloxacillin Ceftazidime Ceftriaxone / Cefotoxaime Dr.T.V.Rao MD 62
  63. 63. Coalition against Typhoid• Since May 2011, the Coalition against Typhoid (CaT) has featured monthly articles in the WHO’s Global Immunization Newsletters (GIN). The articles, written by CaT members from around the world, highlight important work being done to accelerate adoption of typhoid vaccines. Dr.T.V.Rao MD 63
  64. 64. Salmonella Gastroenteritis• Zoonotic disease• S.enteritidis• S.typhimurium• S.halder• S. agana• S.indiana• Contaminated poultry, Meat Milk, Milk products.• Enters the shells of the Intact eggs – Chicken feed, and Fecal droppings. Dr.T.V.Rao MD 64
  65. 65. Nontyphoidal Salmonella• General Incubation: 6 hrs-10 days; Duration: 2-7 days• Infective Dose = usually millions to billions of cells• Transmission occurs via contaminated food and water• Reservoir: a) multiple animal reservoirs b) mainly from poultry and eggs (80% cases from eggs) c) fresh produce and exotic pets are also a source of contamination (> 90% of reptile stool contain salmonella bacterium); small turtles ban.• General Symptoms: diarrhea with fever, abdominal cramps, nausea and sometimes vomiting Dr.T.V.Rao MD 65
  66. 66. Nontyphoidal Salmonella: Gastroenteritis• Incubation: 8-48 hrs ; Duration: 3-7 days for diarrhea & 72 hrs. for fever• Inoculum: large• Limited to GI tract• Symptoms include: diarrhea, nausea, abdominal cramps and fevers of 100.5-102.2ºF. Also accompanied by loose, bloody stool; Pseudo appendicitis (rare)• Stool culture will remain positive for 4-5 weeks• < 1% will become carriers MD Dr.T.V.Rao 66
  67. 67. Nontyphoidal Salmonella: Bacteremia and Endovascular Infections• 5% develop septicemia; 5-10% of septicemia patients develop localized infections• Endocarditis: Salmonella often infect vascular sites; preexisting heart valve disease risk factor• Arteritis: Elderly patients with a history of back/chest + prolonged fever or abdominal pain proceeding gastroenteritis are particularly at risk. - Both are rare, but can cause complications that may lead to death Dr.T.V.Rao MD 67
  68. 68. Salmonella Gastroenteritis• Can occur as cross infection• 24 hours• Manifest with Diarrhea, omitting• Abdominal pain mucous and blood in stools• Last for 2 – 4 days• Some times may lead to septicemias Dr.T.V.Rao MD 68
  69. 69. Diagnosis and Treatment• Isolation by culturing• Rarely need antibiotics.• More frequent in Developed nations. Dr.T.V.Rao MD 69
  70. 70. Salmonella septicemias• S.cholera suis• Deep abscess, Endocarditis• Isolation from Blood and Pus.• Chloramphenicol highly effective Dr.T.V.Rao MD 70
  71. 71. Programme created by Dr.T.V.Rao MD forMedical and Paramedical Students in the Developing World Email doctortvrao@gmail.com Dr.T.V.Rao MD 71

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