1. Enterobacteriaceae II: Intestinal Pathogens
Salmonella, Shigella, Yersinia

2. Salmonella Animals main reservoir of human disease: Transmission:
Gastroenteritis
Typhoid (Enteric) fever
Septicemia
Asymptomatic carriage
Transmission:
Ingest contaminated food (poultry, eggs, dairy products)
Direct fecal-oral spread in children

3. Salmonella Genera Describe >2500 serotypes DNA homology studies:
One Genus-species - Salmonella enterica
Seven subspecies (1, 2 ,3a ,3b ,4 ,5, and 6)
Subgroup 1 causes most human infections
Clinically Salmonella isolates often still reported as serotypes by Kauffman-White classification:
Based on O (cell) and H (flagella) antigens
H antigens occur in two phases (antigenic variation)
Polyvalent antisera used, followed by group specific antisera (A, B, C1, C2, D, and E)
Salmonella Typhi (capitalize, not italics to designate Genus-Serotype) - also has capsular Vi antigen

4. Salmonella: Biochemical Test ID
TSI = K/A + gas, H2S
Salmonella Typhi small amount of H2S, no gas
Salmonella Paratyphi no H2S
LIA = K/K (+)lysine DC, +H2S
Salmonella Paratyphi = K/A
Urea(–)
PA(-)
Citrate(±)
Indole(-)

5. Salmonella: Infection
MO found in all animals – poultry, reptiles, livestock, rodents, domestic animals, birds, and humans
Highly adapted strict human pathogen:
Salmonella Typhi
Salmonella Paratyphi
Peak incidence of infections in warm months via ingesting contaminated food at outdoor social gatherings
~50,000 cases reported annually in U.S

6. Salmonella: Infection of GI Tract
Ingest contaminated food
Infect GI cells
Blood (systemic infection, target organs)
RES (Reticuloendothelial System) – macrophages, liver, spleen, bone marrow

7. Infection of GI Tract MO adheres to intestinal mucosa epithelium
Invasion of enterocytes, M cells (microfold cells of Peyer patches)
Induced endocytosis by bacteria effector proteins
Salmonella multiplies within phagosome, released; may be phagocytosed by macrophages

8. Salmonella: Gastroenteritis
Salmonellosis most common form of gastroenteritis
Due to Salmonella Enteritidis in U.S.
Symptoms 6-48 hours after consumption contaminated food:
Nausea, vomiting, non-bloody diarrhea
Also common with elevated temperature, abdominal cramps, myalgia, headache
Persist 2-7 days, spontaneous resolution
Supportive care, no antibiotics required
Incidence greatest children <5 yrs. and adults >60 yrs.

9. Gastroenteritis MOs multiply, induce strong inflammatory response
Causes disease symptoms (fever, diarrhea, abdominal cramps)
Inflammatory response prevents spread beyond GI tract, eventually kills MOs

10. Salmonella: Typhoid ( Enteric) Fever
Typhoid fever - Salmonella Typhi
Paratyphoid fever (milder) - Salmonella Paratyphi
10-14 day incubation period following ingestion of contaminated food
Fever, headache, myalgia, malaise, anorexia
Persist 1 week, followed by GI symptoms (GI→blood→GI)
Mortality ~20% if untreated

11. Typhoid Fever
MO disseminate before high enough levels to stimulate inflammatory response
Initial symptoms low fever, constipation
MO move via lymphatics & bloodstream to liver & spleen; phagocytosis & multiplication occurs
MO re-enter bloodstream, disseminate to all organs; fever, headaches, myalgia, GI problems
Rose spots (erythema, maculopapular lesions) on abdomen
Osteomyelitis, cystitis, gall bladder infections may occur

12. Salmonella: Septicemia
Most frequently:
Salmonella Typhimurium
Salmonella Choleraesuis
Salmonella Paratyphi
At risk: children, elderly, AIDS patients
Infection presents like Gram(-) bacteremia
10% develop osteomyelitis, endocarditis, arthritis

13. Salmonella: Asymptomatic Infection
Salmonella Typhi
Salmonella Paratyphi
MO’s from enteric fever maintained by human carriage
~1-5% infected patients chronic carrier >1 year
Reservoir in gall bladder

14. Lab Diagnosis: Typhoid Fever
Blood cultures positive during first week, after second week
Stool culture, sometimes urine culture positive after second week
Widal Test (serology):
Antibodies against Salmonella Typhi
Look for 4-fold rise in titer between acute and convalescent stage (~one month)

15. Salmonella: Prevention
Gastroenteritis:
Public Health education
Improved hygiene, especially food handlers
Safe preparation of poultry & eggs, refrigerate food
Antibiotics not recommended, may prolong disease
Enteric fevers:
Treat with fluoroquinolone (ciprofloxacin), chloramphenicol ( decrease mortality to <2%)
Live oral vaccine (attenuated Salmonella Typhi) for travelers to endeminc countries
Clean water supply
ID, treat chronic carriers

16. Shigella: Gastroenteritis
Shigellosis (Bacillary dysentery)
~150 million cases annually worldwide; 450,000 in U.S.
Humans only reservoir, transmitted person-to-person by fecal-oral route
Outbreaks in communities where sanitary standards, level of hygiene low
Common in daycare centers, nurseries, custodial institutions

17. Shigella Genera Four species by O antigen serotype:
S. dysenteriae (Group A) – most severe; Shiga toxin
S. flexneri (Group B) – developing countries
S. boydii (Group C) – not commonly isolated
S. sonnei (Group D) – developed countries; U.S.
By DNA analysis, now shown to be biogroup within E. coli

18. Shigella: Biochemical Test ID
TSI = K/A with NO gas
LIA = K/A (-)lysine DC
Urea (–)
Motility(–)
S. sonnei may show delayed lactose fermentation

19. Shigella: Virulence Factors - Enterotoxin
Shiga toxin - S. dysenteriae
Smaller amounts by S. flexneri, S. sonnei
Inhibit protein synthesis by inactivating 60S ribosomal subunit
Role in ulceration intestinal mucosa

20. Shigella: Virulence Factors - Cell Wall Proteins
Expressed at body temperature
Upon contact with intestinal mucosa M cells (microfold cells, Peyer’s patches), induce phagocytosis of bacteria into vacuoles
Shigella destroys vacuoles, escape into cytoplasm
Spread laterally (actin filaments propel MO through cytoplasm) to epithelial cells; multiply
Do not usually disseminate beyond epithelium

21. Shigella: M Cell Attachment

22. Shigella: M Cell Penetration

23. Shigella: Epithelial Cell (CCEC) Invasion

24. Shigellosis Fecal-oral route, primarily contaminated hands
Infective dose very low ( MO)
Incubation 1-7 days
Fever, cramping, abdominal pain, watery diarrhea for 1-3 days (due to exotoxin)
Followed by frequent, scant stools with blood, mucous, pus (invasion of intestinal mucosa)
Rare for MO to disseminate, generally self-limited but may lead to death
Severity of disease depends upon species:
S. dysenteriae most pathogenic
Followed by S. flexneri, S. sonnei, S. boydii

25. Shigella: Treatment and Prevention
Trimethoprim-sulfamethoxazole (SXT) or fluoroquinolone (ciprofloxacin) - shortens course of disease & fecal shedding; recommended to reduce spread to contacts
Resistant strains becoming increasingly common, so antibiotic sensitivity testing required
Infection control by proper hand washing and disposal of soiled diapers/linens
Public Health monitoring for clean water supply

26. Comparison of Invasion: Salmonella versus Shigella

27. Yersinia Genera Infections are zoonotic, humans accidental host
Three species important pathogens in humans:
Yersinia pestis – plague (bubonic or pneumonic)
Yersinia enterocolitica – gastroenteritis, transfusion-related sepsis
Yersinia pseudotuberculosis – mainly disease of animals, uncommon human gastroenteritis

28. Yersinia: Infection Y. pestis: Y. enterocolitica:
Mammalian reservoir: rats, squirrels, rabbits, domestic animals
Humans infected by:
Fleas
Direct contact with infected animal tissue
Aerosols from patient with pulmonary disease
Y. enterocolitica:
Ingest contaminated food products
Infusion of contaminated blood products (growth in blood stored at 4°C)

29. Yersinia pestis: Lab ID
Non-motile - 37°C & 25°C
Bipolar staining
Slow growth, small colonies at 37°C (grows better 25°C)
TSI = K/A
Urea(-)
ODC(-)
Guinea pig LD50<10
Direct fluorescent antibody test
New DNA probe test

30. Y. pestis: Bubonic Plague
By flea bite infected animal (rat, squirrel, rabbit) to human
Endemic in local So Cal mountains
MO travel to nearest lymph node, engulfed by macrophage
High fever, buboe (enlarged lymph node), MO proliferate, stimulate inflammatory response
MO multiply in lymph node, leak into bloodstream
Lysis of bacteria releases LPS causing septic shock, DIC
Subcutaneous hemorrhage, disease named Black Death in Middle Ages
High mortality rate (30-40%) if untreated

31. Pneumonic Plague
Eventually bacteria reach lungs, engulfed by lung macrophages, cause pneumonic plague
Transmit directly to others via aerosol
Direct inhalation produces disease that progress more rapidly
Mortality rate close to 100%

32. Y. pestis: Treatment and Prevention
Streptomycin, tetracycline, SXT
Control by reducing rodent population
Vaccine (formalin killed bacteria) for individuals at risk; no longer available

33. Yersinia enterocolitica: Lab ID
Non-motile at 37°C
Motile at 25°C
Bipolar staining
Slow growth, small colonies at 37°C (grows better at 25°C)
TSI = A/A (sucrose fermentation)
Urea(+)
ODC(+)

34. CIN Agar
Isolation of Y. enterocolitica from stool specimen (also Aeromonas, Plesiomonas)
Selective - antimicrobials (Cefsulodin-Irgasan-Novobiocin), bile, crystal violet inhibit NF
Differential - mannitol, neutral red
Incubate room temperature
After 48 hours at RT, Y. enterocolitica produce typical pink colonies (mannitol fermentation) with bulls-eye appearance

35. Y. enterocolitica: Gastroenteritis
Ingestion of contaminated food or water
Common cause of human disease (mostly in children) involving fever, abdominal pain, watery diarrhea
Intestinal epithelium invasion of M cells, transcytosed through M cells, released at basal surface
Bacteria penetrate into underlying lymphoid tissue, multiply both inside and outside host cells

36. Gastroenteritis
Inflammatory response to MO responsible for extreme pain (~acute appendicitis)
Fever due to LPS endotoxin
Sometimes drain into adjacent mesenteric lymph nodes, causing lymphadenitis
Reactive arthritis may occur in some people; thought cross reacting T cells or antibodies that attack joint
Usually self-limited
If treatment indicated, susceptible to broad-spectrum cephalosporins

37. Summary: Yersinia Infections

38. Lab: Enterobacteriaceae II Group Unknowns
TSI LIA
Salmonella K/A, H2S K/K, H2S
Proteus K/A, H2S R/A, H2S
Citrobacter K/A, H2S K/A, H2S
Yersinia K/A K/A

39. Lecture Exam I Thursday, Feb. 2, 2012
Introduction thru Enterobacteriaceae
Lecture
Reading (Chap. 14, 15, 17, 19)
Key Terms, Learning assessment Questions
Case Study 1,2,3
Exam Format:
Multiple Choice
Terms
True/False Statements
Short Essay
Review, Review, Review!