Dr.Ashna Ajimsha PG Resident Department of Microbiology SGMC & RF Venjaramoodu Kerala

1. Yersinia,Pasteurella,Francisella
Dr.Ashna Ajimsha

2. YERSINIAE

3. Tribe Yersinieae genus
Yersinia  three well-
established human
pathogens
Yersinia pestis: rodent-
borne zoonotic disease –
plague
Yersinia
pseudotuberculosis and
Y.Enterocolitica—
yersiniosis, a self-limiting
gastrointestinal illness

4. Yersinia pestis
◦ 1894 - Yersinia pestis isolated for the first time
- Alexandre Yersin in in Hong Kong
◦ Agent of plague - systemic zoonosis,
transmitted from rodents by arthropod
vector (the rat flea)
◦ Gram-negative oval coccobacillus,
bipolar staining and pleomorphism in
older cultures (enhanced with 3% NaCl)
◦ Non-motile and capsulated

5. Epidemiology of Plague - Pandemics
Biotypes Nitrate
reduction
Glycerol
fermentation
Pandemics associated
Medievalis - + First pandemic Justinian,AD541
Antiqua + + Second pandemic Black death,
Europe(1347-52)
Orientalis + - Third pandemic (1894-1918)
Hong Kong, China, India

6. Timeline of
plague in India
Four potential endemic foci
1. region near Kolar, Karnataka
2. Beed-Latur belt in Maharashtra
3. Rohru in Himachal Pradesh
4. Dangud village, Uttaranchal

7. Current situation in the world
◦ Over five years (2004 to 2009), a total of 12,503 cases were reported
(with 843 deaths) worldwide, mainly confined to 16 countries of
Africa, Asia and America
◦ Africa accounts for highest number of cases (97%) worldwide.

8. Epidemiological
factors
◦ Reservoir: Wild rodents, field mice &
bandicoot
◦ Source of infection
- infected wild rodents,
-rat fleas
-cases of pneumonic plague
◦ Vector: Rat flea
- Xenopsylla cheopis - most efficient, North
India
- Xenopsylla astia - less efficient, South India
- Xenopsylla brasiliensis

10. Plague cycles: two natural cycles:
1. Domestic cycle: between humans, rat
fleas and rodents
2. Wild or sylvatic cycle: occurs in nature
among wild rodents, independent of human
beings

11. Mode of
transmission
- Bite of an infected rat flea (most common)
- Direct contact with tissues of infected
animal (rodents)
- Droplet inhalation (man to man) -
pneumonic plague
- Bite of an infected human flea (Pulex
irritans)

12. Blocked flea
Partially blocked
flea- more
dangerous –upto
4 year survival
transmission of
infection
while making
efforts to suck, it
regurgitates the
blood mixed
bacteria into the
bite
Bacilli multiply
enormously in the
gut of flea and
may block the
proventriculus

13. Contamination of bite wound with feces of infected fleas
Extrinsic incubation period – two weeks for Xenopsylla cheopis
Cheopis index - Average number of X.cheopis per rat is most
significant flea index
Cheopis index >1  outbreak likely
-Seasonality: North India - September to May, South India -
throughout the year

14. Virulence factors of Y. pestis
Yersiniae Virulence antigens
Common to all Yersinia Lipopolysaccharide- endotoxin activity
Pigmentation-
absorbs hemin,brown colonies,biofilm
Low calcium response plasmid codes for type III secretion system
Siderophore- iron acquisition, reduces ROS
Y.pestis specific Fraction 1 (F1) antigen- heat labile capsular protein, inhibits phagocytosis,
antigenic
Phospholipase D/murine toxin- survival in midgut of flea
Surface proteases– activate plasminogen, degrade complement, adhere to
laminin, dissemination of infection

15. Essentials of Medical Microbiology
Yersiniae Virulence antigens
Y. pestis & Y.pseudotuberculosis pH 6 antigen
Y.enterocolitica and
Y.pseudotuberculosis
Inv protein
Ail protein
Yersinia adhesin A (YadA)
Y.enterocolitica specific Myf antigen
Heat stable toxin (ST toxin)
Y. pseudotuberculosis specific Super antigen mitogen

16. Human plague- Clinical Types
Bubonic
pneumonicsepticemic

17. Bubonic plague
Most common type, transmitted by the bite of an infected rat flea
Bacilli pass through the local lymphatics  regional lymph nodes multiply
Incubation period: 2–7 days
Sudden onset, fever, malaise, headache & painful lymphadenitis
Buboes: Regional lymph nodes - tense, tender swellings “buboes”
Common site - inguinal
Other sites - crural, axillary, cervical, or submaxillary
Cannot spread from person to person
Without treatment, dissemination occurs leading to pneumonia (secondary) and meningitis

18. Pneumonic plague
Spread: Inhalation of bacilli in droplets
expelled from another person or an animal
with plague pneumonia
Incubation period: 1–3 days
Manifestation: Sudden onset fever,
headache and respiratory symptoms pain
Rare (<1%) but highly infectious and highly
fatal
Agent of bioterrorism

19. Septicemic plague
◦ Secondary septicemic plague is more common
than Primary
◦ From spread of bubonic or pneumonic plague
◦ Incubation period:2–7 days
◦ Hemorrhages in the skin and mucosa  gangrene
of affected site- black death

20. Lab diagnosis
◦ Specimen Collection
- Bubonic plague—pus or fluid
aspirated from buboes
- Pneumonic plague—sputum
and blood
- Septicemic plague—blood and
splenic aspirate (post mortem)
◦ Transport medium - Cary–Blair
medium

21. Direct microscopy
◦ Gram staining
Gram negative oval coccobacilli with
rounded ends and convex sides surrounded
by capsule
◦ Wayson stain or methylene blue stain
bipolar or safety pin appearance- 2 ends
darkly stained, central clear area

22. Culture
◦ Y. pestis is aerobic and facultatively anaerobic
◦ Optimum temperature - 27°C but the capsule develops best at 37°C
◦ Not fastidious
◦ Blood agar: Non-hemolytic and dark brown pigmented colonies
(absorption of the hemin)
◦ MacConkey agar: Lactose non-fermenting

23. • Nutrient broth: Granular turbidity  surface
pellicles
• Nutrient broth with oil or ghee floated on top -
stalactites-like growth
• Yersinia-specific CIN agar (cefsulodin, irgasan,
novobiocin): selective medium, useful for culture
of specimens containing normal flora, e.g.
sputum.

24. Culture smear
and motility
testing
◦ Gram stain – pleomorphic
coccid, coccobacillary, bacillary,
filamentous and giant forms
◦ Y.pestis - nonmotile both at 25°C
and 37°C

25. Biochemical
reactions
◦ Sugar fermentation:
- Ferments glucose, mannitol and maltose
with the production of acid but no gas.
- Lactose and sucrose are not fermented
◦ Catalase positive, oxidase negative
◦ Indole, urease and citrate tests are
negative
◦ MR positive, but VP test is negative
◦ Biotyping is done based on glycerol
fermentation and nitrate reduction

26. F1 Antigen Detection
From bubo aspirate or sputum by direct immunofluorescence test, ELISA or immunochromatographic
test (ICT)
Antibodies to F1 Antigen Detection
-By ELISA, passive agglutination or CFT
-Limited diagnostic value as they appear late
-Useful epidemiological markers
Molecular Methods:
-PCR - gene coding F1 antigen, pesticingene, and the plasminogen activator gene
Animal Inoculation - Guinea pigs or white rats

27. Treatment
◦ Gentamicin - DOC
◦ Alternatives:
- Streptomycin - 10 days
- Levofloxacin - for treatment and
postexposure prophylaxis
- Doxycycline and Chloramphenicol
- β-lactams and Macrolides - not
recommended as the response is poor

28. Prophylaxis
◦ Control of cases by early diagnosis, isolation
and treatment of cases
◦ Control of fleas by use of effective
insecticides - DDT or BHC (beta-
hexachloro-cyclohexane)
◦ Control of rodents
◦ Chemoprophylaxis - contacts of
pneumonic plague
- Doxycycline (100 mg bid)
- Tetracycline (500 mg qid)

29. Vaccines
◦ Only for prevention of an anticipated outbreak and not for general use
◦ Formalin killed vaccine (Sokhey’s modification of original Haffkine
vaccine):
- Subcutaneous, two doses 4 weeks apart  booster after 6 months
- Protection is short-lasting (<6 months)
- Not protective against pneumonic plague and has considerable side effects

30. Live attenuated vaccine based on strain EV76
-still used in countries of the former Soviet Union
-Has significant side effects
Subunit recombinant F1 (rF1) vaccine - under trial

31. YERSINOSIS
Zoonotic infection - by enteropathogenic Yersinia
species – Y.enterocolitica or Y. pseudotuberculosis
Usual hosts - Pigs & other wild/domestic animals
Human infection - consumption of contaminated
food (raw pork, milk)
Most common in childhood and in colder climates
Presentation - abdominal pain and sometimes diarrhea

32. Geographical Distribution
Y. Enterocolitica - found worldwide, most commonly in Northern Europe and
America
Y. Pseudotuberculosis – rare, Finland
Serogrouping
Y. Enterocolitica – six biotypes and 60 serotypes
Commonest infections with serogroups O:3 and O:9
Y. pseudotuberculosis - six serotypes (1 to 6)

33. Virulence factors- common to both species
Invasin (Inv) protein: Binds to β-1 integrins on M cells of GI mucosa
which helps in invasion
Ail protein (attachment and invasion locus): It helps in attachment,
invasion and inactivates complements
Yersinia adhesin A (Yad A): Helps in invasion & inactivates
complements

34. All Yersiniae-LPS, pigments, siderophores and low calcium response plasmid
Y. enterocolitica specific virulence factors:
- Myf antigen - fimbrial antigen, helps in adhesion
- Heat-stable toxin: Similar to that of E. coli, produced only at temperature <30°C
- pH6 antigen: Fimbrial surface protein, helps in adhesion
Y. pseudotuberculosis specific virulence factors:
- Super antigen—binds to T cells non-specifically leading to massive cytokine release

35. Clinical features
◦ Y. Enterocolitica more common than Y. pseudotuberculosis
◦ Self-limited gastroenteritis (diarrhea with or without blood) -
younger children
◦ Intestinal complications - older children, characterized by
terminal ileitis and mesenteric adenitis
- Patients present with acute pain abdomen, may mimic
pseudoappendicitis
◦ Septicemia: typically in adults - fever and leukocytosis
- with coexisting diabetes mellitus, liver disease and iron overload

36. Post infective phenomena
With Y. Enterocolitica
Autoimmune activity - deposition of bacterial non-viable components in joints and other sites
- Reactive arthritis (association with HLA-B 27)
- Erythema nodosum
- Graves’ disease - Y. enterocolitica has antigen similar to TSH binding site
Super antigen: Some strains of Y. Pseudotuberculosis - mitogen scarlet-like fever, Izumi-
fever and idiopathic acute systemic vasculitis of childhood (Kawasaki’s disease)

37. Lab diagnosis
Culture isolation
◦ From blood: BHI broth
◦ From lymph nodes aspirate: blood agar,
nutrient agar and MacConkey agar
- Blood agar: granular translucent colonies
with a beaten copper surface, non-hemolytic
colonies
- MacConkey agar: Growth of Y.
Pseudotuberculosis is poor. Y. enterocolitica grows
well and produces lactose non-fermenting
pale colonies

38. For isolation from feces, food or soil: Selective media
-Deoxycholate citrate agar
-MacConkey agar
-Yersinia CIN agar (Cefsulodin-irgasan-novobiocin): Dark red bull’s
eye colonies
Incubation: at 25°C and 37°C - differentiate from other pathogens which
grow only at 37°C
Cold enrichment - phosphate-buffered saline at 4°C for 3 weeks

39. Biochemical
Tests
Y. enterocolitica and Y. Pseudotuberculosis
can be differentiated from Y. pestis:
- Differential motility: motile at 22°C
(but not at 37°C)
- Cold enrichment: Growth improves
on refrigeration (4°C)
- Urease positive

40. Y. enterocolitica v/s Y. Pseudotuberculosis :
-Sugar fermentation - Sucrose, cellobiose and sorbitol are fermented only by
Y.Enterocolitica
-Rhamnose, salicin and melibiose are fermented only by Y. Pseudotuberculosis
- Ornithine decarboxylase-positive only for Y. Enterocolitica
-VP test is positive only for Y. Enterocolitica
Serology – Antibodies detected by agglutination or ELISA using serotype
specific O-antigen types

41. Treatment
◦ Mostly diarrhea is self-limiting.
◦ Treatment is required only for systemic
infections – septicemia
◦ Fluoroquinolone (Ciprofloxacin) or third-
generation Cephalosporins (Cefotaxime)
◦ Y. enterocolitica strains nearly always produce β-
lactamases but not Y. pseudotuberculosis strains

42. Francisella
tularensis
◦ Tularemia – plague like disease of rodents & other
small animals
◦ Source- contaminated environment, insects, other
animals
◦ Transmission-zoonotic
-interaction with biting/ blood sucking
insects(ticks/tabanid flies)
-contact with wild/ domestic animals
-contaminated food/water
-Inhalation of infective aerosols

43. Clinical features
o Ulceroglandular tularemia
-most common form
-Ulcer + regional lymohadenopathy
o Pulmonary tularemia-atypical pneumonia
o Oropharyngeal tularemia
-ingestion of contaminated undercooked meat
-membranous pharyngitis+ cervical lymohadenopathy
-Lemming fever
o Oculoglandular tularemia
-purulent conjunctivitis+pre auricular lymphadenopathy
o Typhoid like illness
o Agent of bioterrorism

44. Lab diagnosis
Culture -Highly fastidious,special media
-BCG (Blood cysteine glucose agar)
-CHAB(cysteine heart agar
supplemented with 9% sheep blood)
Specimens
-Ulcer scrapings
-LN biopsy
-Gastric washings
-Sputum
-Blood

45. Colonies
Blue-gray
Round
Smooth
Mucoid
Zone of α- hemolysis
Safety precautions – Biosafety level III

46. Identification
◦ Gram negative coccobacillus with bipolar appearance
◦ Non motile
◦ Capsulated
◦ Catalase(+) oxidase( -) 𝐻2 𝑆 (+)
◦ Produces acid from glucose, maltose, mannose
◦ Direct fluorescent antibody detection
◦ Antibody detection- ELISA, Latex & tube agglutination
◦ PCR assay
TREATMENT- Gentamycin –DOC 5 mg/kg 7-10 days
Doxycycline/Ciprofloxacin

47. Pasteurella
◦ Respiratory tract of animals, commensal in
human respiratory tract
◦ Most common- Pasteurella multocida
◦ Following animal bite- red, swollen, painful,
regional lymphadenopathy, fever
Meningitis
Appendicitis
Chronic respiratory infections

48. Lab diagnosis and
treatment
◦ Direct microscopy
Non motile Gram negative cocco-bacilli with bipolar
staining
◦ Culture
Aerobes and facultative anaerobes
Grow on NA at 370
C
Oxidase (+)
Indole (+)
Fail to grow on MA
TREATMENT
Penicillin-G / Amoxycillin clavulanate DOC