Salmonella

1. Salmonella

2. General characters • Rod-shaped, non-spore-forming Gram-negative bacterium • Belongs to the family Enterobacteriaceae • close relative of E. coli • Motile by peritrichous flagella (H antigen). • nonmotile exceptions: S. gallinarum and S. pullorum

3. Cultural Characters • Nutrient agar: Large 2-3 mm, convex, translucent, moist. • MacConkey agar: Non-Lactose fermenting colonies. • Selective media: • XLD agar: Red coloured colonies • Deoxycholate Citrate agar (DCA): Colourless colonies • Wilson and Blair medium: Jet black colonies, with a metallic sheen due to H2S production. • Enrichment media: Selenite F and Tetrathinate broth

4. Biochemical reactions • Oxidase negative • Catalase positive • Ferment glucose, Mannitol and maltose with gas • Important exception is S. typhi: Anaerogenic • Do not ferment lactose • TSI K/A + gas and H2S: S. typhi produces only a small amount of H2S and no gas , and S. paratyphi A produces no H2S • Urea – • Citrate +/- • Indole -

5. Antigenic Structure • O antigens • Lipopolysaccharide units in LPS. • H antigens • flagellar antigens (protein) and may occur in one of two phase variations. • Vi antigen • a capsular polysaccharide

6. O Antigen • Integral part of cell wall • Not a single factor but a mosaic of more antigenic factors • Unaffected by boiling • When mixed with O antisera forms compact chalky granular clumps • Less immunogenic than H antigen

7. H Antigen • Present on the flagella • Heat labile protein. • When mixed with H antisera forms loose fluffy clumps • Highly antigenic • Occurs in one or two phases.

8. Vi Antigen • Surface polysaccharide Ag enveloping the O antigen • Analogous to K Ag in E. Coli • Vi Ag inhibits phagocytosis, resist bacterial lysis by alternate C pathway. • Poorly immunogenic • Persistence of Vi Ab indicates Carrier state

9. Antigenic Variation • Genotypic and phenotypic variation. • H-O variation: Associated with loss of Flagella. • Salmonella grown on agar containing phenol flagella is inhibited • Temporary change as flagella regained when subcultured on media without phenol

10. Antigenic Variation • Phase variation: • Flagella Ag present in two phases • Phase 1: Species specific. Designated as a,b,c,d.....etc after z as z1, z2, etc • Phase 2: Group specific. Termed as 1,2,3 etc • Monophasic: Possess only one phase. Eg; S. Typhi. • Biphasic: S. paratyphi A

11. Antigenic Variation • V-W variation: Fresh isolates of S.typhi have Vi Ag which masks O Ag and makes them nonagglutinable- V form. After Sub cultures Vi Ag is lost: W form. • S-R variation: Smooth to rough variation is associated with change in colony morphology by serial subcultures in lab. • Variation in O Ag: Induced by lysogenisation. S. anatum 3,10: e,h: 1,6 phage 15 S. newington 3,15: e,h: 1,6 phage 34 S. minneapolis 3,34: e,h: 1,6

12. Classification • Old: Serotyping & biochemical assays used to name individual species within genus (e.g., Salmonella enteritidis, S. choleraesuis, S. typhi) • Over 2400 O-serotypes : Kauffman-White antigenic scheme • Modern: DNA homology shows only two species Salmonella enterica (six subspecies) and S. bongori • Most pathogens in S. enterica ssp. enterica

13. Kauffmann-White classification

15. Classification Enteric fevers: S. typhi S. paratyphi A S. paratyphi B S. paratyphi C Gastroenteritis: S. typhimurium S. choleraesuis S. enteritidis Septicemia: S. choleraesuis S. typhimurium S. enteritidis

16. Salmonella infections in humans • Enteric fever • typhoid and paratyphoid fevers • S. typhi, paratyphi A, B, C • systemic infection • infects only humans • GI symptoms may not be evident

17. Salmonella infections in humans • Salmonella gastroenteritis • non-typhi serovars • zoonosis: predominantly food-borne • can be complicated by septicaemia • more common with some serovars, e.g. S. dublin (15% mortality rate when septicemic in the elderly) • Metastatic disease, e.g. osteomyelitis

18. Epidemiology Enteric fever • person-to-person spread • no animal reservoir • contamination with human faeces • usual vehicle contaminated water. • occasionally, contaminated food (usually handled by an individual who harbours S. typhi)

19. Infectious dose • typically about 1,000,000 bacteria • much lower if the stomach pH is raised • much lower if the vehicle for infection is chocolate • protects the bacteria in their passage through the stomach • an infectious dose of about 100 bacteria

20. Pathogenesis • Virulence factor Invasiveness Endotoxin Enterotoxin • Diseases Enteric fevers (typhoid / paratyphoid) Enterocolitis Septicemia transmission: gastrointestinal tract

21. Pathogenesis Enteric Fever • Bacteria invade mucosa or Peyer's patches of small intestine (?M cells), pass into mesenteric lymph nodes where they multiply and then enter the blood stream via the thoracic duct • Primary bacteraemia cleared by RES, bacteria multiply in RES cells and destroy them • Facultative intracellular parasites

22. Pathogenesis Enteric Fever • Secondary bacteraemia occurs and results in spread to other organs. • Infection of the biliary tract. • Multiplication in biliary tract leads to seeding the intestine with large numbers of bacteria. • Involvement of intestinal lymphoid tissue may lead to necrosis and ulceration. • In untreated nonfatal cases, temperature drops in 3 to 4 weeks (onset on immunity?)

23. Ingestion of S. typhi Incubation period Hemorrhage; perforation Inflammation and ulceration of Peyer’s patches Small intestine Lymph nodes Bile Mesenteric Lymph nodes Cholecystitis; Carrier state gallbladder Thoracic duct Multiplication in macrophages in liver, spleen, kidney,and bone marrow Bile Fever; Relatively slow pulse; Enlarged liver And spleen; Rose spots; Normal or low WBC count Transient (primary) bacteremia Septicemia (second) Fever, malaise Signs and symptoms

24. Pathogenesis of Salmonella Salmonella are ingested in contaminated food or water Organisms reach the terminal ileum Enteritis Organisms invade the gut wall & cause ulceration, perforation & hemorrhage Organisms spread to intestinal lymphatics & are phagocytosed by macrophages Organisms disseminate to bones, kidneys, lungs,liver, brain & blood Enteric fever or typhoid fever

25. Clinical features Enteric Fever • incubation period 7 to 14 days • Mild undifferentiated pyrexia (ambulant typhoid) • Rapidly fulminating disease • Onset gradual with • myalgia and headache • Fever (Step ladder fever) • Relative bradycardia • Soft splenomegaly with hepatomegaly • Rose spots which fade on pressure (macular rash on abdomen)

26. Complications • Intestinal perforation • Hemorrhage • Circulatory collapse • Others: cholecystitis, arthritis, nephritis, haemolytic anaemia, osteomyelitis etc. • Paratyphi A & B cause paratyphoid fever and are milder. • Paratyphi C often leads to septicemia and suppurative complications

27. Asymptomatic Carriage • Chronic carriage in 1-5% of cases following S. typhi or S. paratyphi infection • Patients who shed typhoid bacilli in feces • for 3 weeks to 3 months after clinical cure-convalescent carriers • More than 3 months- temporary carriers • Over a year- chronic carriers • Gall bladder usually the reservoir • Fecal carrier or Urinary carrier

28. Laboratory Diagnosis Laboratory diagnosis of enteric fever is done by: • Isolation of bacilli • Demonstration of antibodies in serum • Demonstration of circulating typhoid antigen

29. Laboratory Diagnosis Isolated from stool, blood and urine • During the first week of illness: blood culture (90%) • During the second week : blood culture (75%) , urine culture (25%), serology. • Serology: Widal test, a tube agglutination test using antigens of S. typhi, paratyphi A & B to be agglutinated by the patient serum • Third week: blood culture (60%), urine culture (25%), • Stool cultures are also positive throughout the course of illness of illness, and even during convalescence.

30. Blood culture • 5-10 ml blood collected and inoculated in 50- 100ml of 0.5% bile broth and incubated at 37º C. • Subcultured on MacConkey agar. • Appears as a non-lactose fermenter • on MacConkey agar or similar selective media • Biochemical tests and serological tests must be done in parallel • Phage typing done for epidemiological purposes • E.g. to find source of outbreak

31. Identification • Biochemical tests: Ferment glucose Mannitol and maltose, not lactose. • S. Typhi is anaerogenic • Slide agglutination tests: Factor D antisera (Factor 9) the phase antisera • Vi Ag should be removed if no agglutination.

32. Blood culture bottles should be subcultured daily and declared negative only after 10 days • Clot Culture: • 5ml blood is withdrawn in a sterile test tube and allowed to clot. • Serum is pipetted off and used for widal test. • Clot is broken with sterile glass rod and added to bile broth along with streptokinase (100 units/ml). • Clot culture yield higher rate of isolation

33. Stool culture • Salmonellae are shed in feces throughout the course of illness and even in convalescence • Almost as valuable as blood culture. • Selenite F broth, or Tetrathionate broth are used as enrichment media. • Fecal samples plated on MacConkey, DCA and Wilson-Blair media after enrichment for 8-12 hrs.

34. Urine culture: • Intermittent shedding of salmonella • Generally positive in second or third week of illness (only 25%). Other specimens: Bile, Bone marrow, CSF etc

35. Widal test • Test for the measurement of H and O agglutinins for typhoid and paratyphoid bacilli. • Dreyer’s tube(H) and Felix tube (O). • Equal volumes of serial dilutions of serum (1/10 to 1/640) and H &O antigens are mixed • Incubated in water bath at 37ºC overnight. • Control tubes kept to check auto-agglutination. • H- loose cottony wool clumps, O- chalky powder. • Negative- compact disc at bottom.

36. Antigens used • S. typhi O Ag • S. typhi H Ag • S. paratyphi AH Ag • S. paratyphi BH Ag • Paratyphoid O Ags cross react hence not included due to Factor 12 sharing.

37. Interpretations • Test usually positive by end of 1st week of illness. • Rises till 3rd or 4th week and declines • Rising titre in two or more serum samples is meaningful. • Single test: ≥O-1:100 and ≥ H-1:200 • Basal titre in endemic areas. • Recently vaccinated individuals have high titre of antibodies of paratyphi A and B also. • Anamnestic response:

38. Anamnestic reaction Persons who have had prior infection or immunisation may develop rise in antibody titre when suffering from unrelated fever, The anamnestic response is transient and no rising titre.

39. Demonstration of Antigens • Circulating Ags: Early phase of infection. • Sensitised Staphylococcal coagglutination test Cowan I strain Which contains protein A stabilised with formaldehyde and coated with S. typhi Ab. Agglutination seen with patients serum positive for S. typhi Ags.

40. Diagnosis of carriers • Isolation of bacillus from feces and bile • Widal test has no value • Vi Agglutinins can be demonstrated and used as a screening test • Tracing of carriers performed by sewer swab technique.

41. Prophylaxis • General measures: Improved sanitation, protested water supply etc. • Immunoprophylaxis: • Three licensed vaccines • TAB vaccine • Typhoral vaccine • Vi subunit vaccine

42. Salmonella vaccines TAB Vaccine: • heat-killed • S typhi: 1000 million, S paratyphi A and B: 750 million each. • 0.5ml S/C two doses at an interval of 4-6 weeks. Typhoral vaccine: • live oral vaccine • Stable mutant of S. typhi Ty2 1a lacking UDP-galactose-4-epimerase (Gal E mutant). initiates infection but self destructs after 4-5 cell divisions. • enteric coated capsule having 109 mutant bacilli

43. typhim Vi • Injectable vaccine • contains purified Vi polysaccharide Ag (25g/ dose) from S typhi strain Ty2 • Single subcutaneous or IM injection

44. Salmonella Gastroenteritis • Food-borne transmission by • contamination of cooked food by raw food • failing to achieve adequate cooking temperatures. • contaminated food is major vehicle, usually: • red and white meats, raw eggs, milk & dairy products • symptoms usually begin within 6 to 48 hours • Nausea and Vomiting • Diarrhoea • Abdominal pain

45. Common salmonella spp causing gastroenteritis: S typhimurium, S enteritidis, S newport etc. • Laboratory diagnosis is made • by isolating salmonella from feces • In food poisoning outbreaks from food.