1. Salmonella, Shigella and Yersinia ID# 9014 By Dr. Wadha Alfouzan

2. Weekly Learning objective (s) Know the common characteristics of Salmonella, Shigella and Yersinia on culture media (WLO 2615) Objectives Description of common characteristics of Salmonella, Shigella and Yersinia on culture media Discussion on principles of laboratory diagnosis of Salmonella, Shigella and Yersinia infections Discussion on the virulence factors and disease spectrum associated with Salmonella, Shigella and Yersinia infections

3. Common characteristics All members of the family Enterobacteriaceae Aerobe and facultative anaerobe Gram-negative bacilli Motile or nonmotile Ferment glucose Oxidase-negative Reduce nitrate to nitrite Produce lactose nonfermenting colonies on MacConkey agar Cause disease within the intestinal tract and outside the intestinal tract

4. Salmonella Properties Gram-negative, motile bacteria; produce lactose nonfermenting pale colony on MacConkey agar, and black-centred pale colony on tellurite containing media (eg. Salmonella-Shigella agar) due to production of H 2 S Salmonella species on S-S agar

5. Classification of Salmonella More than 2500 serotypes of Salmonella; Serotype represented by somatic (O) antigen and phase 1 and phase 2 flagellar (H) antigens; Broadly classified into Typhoid Salmonella and Non-Typhoid Salmonella. Typhoid Salmonella Salmonella typhi Salmonella paratyphi A,B,C Non-Typhoid Salmonella Numerous other serotypes

6. Diseases Enteric fever (typhoid and paratyphoid fever) Caused by S. typhi and S. paratyphi A, B, C; source of the organisms is humans only; transmitted by faecal-oral route (by contaminated food and water) Disease: Invasive disease characterized by GI (constipation) and generalised symptoms (fever, chills, fatigue, headache, anorexia), hepato-splenomegaly, maculopapular rash (rose spots), chronic carrier state due to diseased gall bladder.

9. Virulence of enteric fever organisms ●Multiply within macrophages-intracellular pathogen ● S. typhi and S. paratyphi C possess Vi (virulence) capsular antigen ● LPS antigen contributes to production of proinflammatory cytokines (TNF, IL-1, IL-6) mediating fever

10. Non-typhoid Salmonella Common serotypes are: Salmonella typhimurium, Salmonella enteritidis Diseases: Gastroenteritis of inflammatory nature (major cause of food poisoning), septicaemia, meningitis, osteomyelitis (children with sickle cell anaemia prone), metastatic abscess (at the site of aneurysm [widening of artery] and infarct [dead tissue-thrombus, embolus]) Reservoirs: Poultry (intestine) and eggs (infection of oviduct), cattle, pet animals (dogs, snakes, turtles), lizards Virulence factors: Multiply within macrophages, invade intestinal mucosa (terminal ileum & colon) and produce enterotoxins (mediating diarrhoea)

11. Laboratory diagnosis Enteric fever Commonly diagnosed by blood culture; organisms also cultured from urine and stool; identified by biochemical reaction and confirmed by slide agglutination test with specific antisera Serological test (Widal test which measures rising serum antibody to O and H antigens of the organisms) Non-typhoid Salmonella infections Clinical specimen cultured for Salmonella on enteric media (MacConkey agar, Salmonella-Shigella agar); identification of Salmonella as above; serotyping available in reference laboratories only.

12. Epidemiology of enteric fever Prevalent in developing countries Organisms have a predilection for gall bladder with establishment of chronic carrier state and excretion in stool (about 5% of typhoid fever patients become carriers) Food handlers who are carriers can transmit the organism via contaminated food due to poor personal hygiene Live oral vaccine and Vi antigen-based injectable vaccine available against S. typhi

13. Shigella Characteristics of Shigella Gram-negative bacilli, nonmotile, non-capsulate, do not ferment lactose (S. sonnei is late lactose-fermenter after 24 h of incubation); genetically closely related to E. coli Classification Biochemically and serologically divided into 4 species or groups: Shigella dysenteriae (with 15 serotypes) Shigella flexneri (with 15 serotypes & subtypes) Shigella boydii (with 20 serotypes) Shigella sonnei (with a single serotype)

14. Diseases Causative agent of bacillary dysentery S. dysenteriae (particularly serotype 1) causes very severe disease, and S. sonnei causes mild disease

15. Virulence factors Invasion of and multiplication in colonic mucosa producing ulcer Invasion mediated by invasion plasmid which encodes invasion plasmid antigens (outer membrane proteins) Production of enterotoxins (ShET 1 & 2) which mediate watery diarrhoea preceding dysentery S. dysenteriae 1 produces Shiga toxin similar to that of enterohaemorrhagic E. coli resulting in haemolytic uraemic syndrome (HUS)

16. Diagnosis Examine stool for blood and pus by naked eye and microscopy Culture on selective media-MacConkey agar, Salmonella-Shigella agar Lactose non-fermenting colonies identified as Shigella by biochemical reaction Organism confirmed as Shigella by slide agglutination test with specific antisera Dysenteric stool with blood & pus Microscopy of stool showing blood & pus cells

17. Epidemiology Infectious dose 100-200 organisms Person-to-person transmission predominant mode of transmission Humans, the main reservoir; other reservoirs include nonhuman primates (monkeys, chimpanzees) Infection with S. dysenteriae and S. flexneri predominant in developing countries with epidemics due to S. dysenteriae 1; in developed countries, infection is mainly due to S. sonnei Transmission interrupted by improved personal hygiene like handwashing

18. Yersinia The organisms in this genus are animal pathogens transmitted to humans via food and water or insect vectors. They are Gram- negative coccobacilli. There are three species in the genus Yersinia: Yersinia enterocolitica Yersinia pseudotuberculosis Yersinia pestis

19. Yersinia enterocolitica Description Gram-negative, non-sporing, bacilli; grows on MacConkey agar producing small pink colonies; grows on selective medium CIN agar producing “bulls eye” colonies; motile at 25 o C, nonmotile at 37 o C Small pink colonies on MacConkey agar ‘Bulls eye” colonies on CIN agar

20. Diseases Bloody diarrhoea with fever and abdominal pain Pseudoappendicitis-mesenteric lymph adenitis and terminal ileitis mimicking this condition Sequelae-arthralgia (joint pain), reactive arthritis/ Reiters syndrome (inflamation of joints as a reaction to infection elsewhere in the body; autoimmune disease),erythema nodosum (erythematous nodules under the skin in front of legs below knees)

21. Virulence factors Invade terminal ileum and colon, produce enterotoxin Plasmid (pYV) encodes outer membrane proteins (Yops) that kill macrophages and neutrophils

22. Diagnosis Enrichment of stool in buffered saline at 4 o C up to 6 weeks; characteristic growth on selective media; lack of motility at high temperature and motility at low temperature Epidemiology Common in countries with cold climate; animals (pigs) main reservoir with transmission via contaminated pork; infection transmitted through transfused blood because organism multiplies in refrigerated contaminated blood.

23. Yersinia pseudotuberculosis Gram-negative bacilli with bipolar staining Causes septicaemia and visceral nodules in animals Transmitted to humans by contaminated food and water Causes acute ileitis and mesenteric lymphadenitis in humans Virulence factors include plasmid-encoded invasin and enterotoxin Isolated by cold enrichment in buffered saline

24. Yersinia pestis Gram-negative, non-sporing, non-motile, bacilli with bipolar staining resembling safety pin Wright’s stain of Y. pestis from blood film showing bipolar staining Safety pin

25. Diseases Causative agent of plague (literal meaning calamity). Epidemic form (black death, so called due to subepidermal haemorrhage [purpura] and gloom) killed millions since ancient times. There are three forms of the disease. Bubonic plague: Swollen lymph node at the area of flea bite; symptoms include rigor, fever, mental confusion, hypotension Septicamic plague: Organims spread through the body; symptoms include gastrointestinal symptoms, hypotension, shock, bleeding from orifices, high mortality Pneumonic plague: A complication of the above two or primary form acquired through aerosol; individuals develop pneumonia, almost fatal

26. Virulence factors Intracellular pathogen of phagocytes F1 capsular antigen resists phagocytosis V and W antigens contribute to intracellular survival Yops (yersnia outer membrane proteins) injected into phagocytes inhibit phagocytosis and cytokine production

27. Laboratory diagnosis Characteristic bipolar stained bacilli in tissue Culture on blood agar Demonstration of F1 antigen by immunofluorescent staining

28. Epidemiology Rat fleas transmit infection to humans in wild areas (sylvatic plague), in urban areas (urban plague) Transmission from human to human by respiratory droplet Infection prevalent in Middle-East, South and Central Asia, and South and North America Organism can be used as a weapon of biological warfare (due to ability to kill vast number of people in a short time). Formalin-killed vaccine gives partial protection against bubonic plague