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1. FRANCISELLA TULARENSIS
ARYA J
MSc
mICROBIOLOGY

2. • Class : Gamma proteobacteria
• Order :Thiotrichabes
• Family :Francisellacia
• Genus : Francisella
• Species : Francisella tularensis
Morphology
• Small , Gram negative , non motile , capsulated ,intracellular
coccobacilli filterable .
• Multiply by binary fission

3. • Francisella tularensis : causative agent of ‘tularemia‘ ; a plague-like
disease of rodents and other small animals.
EPIDEMIOLOGY
• Source: contaminated environments, insects, and animal carriers.
• Transmission: Human infection is zoonotic and usually results from:
 lnteraction with biting or blood-sucking insects (especially ticks
and tabanid flies)
 Contact with wild or domestic animals
 Ingestion of contaminated water or food
 lnhalation of infective aerosols

4. PREVALENCE
Francisella tularensis has four subspecies:
o tularensis
o holarctica
o novicida
o mediasiatica..
• The first three subspecies found in North America,
• Subspecies mediasiatica is found in central Asia.
• Subspecies tularensis : most common and the most virulent among all
isolated only from North America.

5. RESERVOIRS
• Rabbits
• Aquatic rhodents
• Rats
• Lemmings
• Mice
VECTORS
• Ticks
• Mosquitoes
• Bitinf flies

6. CLINICAL MANIFESTATIONS
Tularemia is characterized by various clinical syndromes:
 Also known as Deer-fly disease,Glandular tick fever,etc…
• Ulceroglandular tularemia: most common form, accouts 75-85% of
total cases, characterized by ulcerative lesion at site of inoculation,
with regional lymphadenopathy.
• Pulmonary tularemia: from aerosol inhalation (laboratory workers)
or can spread to lungs following bacteremia. Patients present with
atypical pneumonia.
• Oropharyngeal tularemia: occurs following ingestion of
contaminated undercooked meat & is characterized by membranous
pharyngitis with cervical lymphadenopathy.

7. o Lemming fever in Norway results from consumption of water
contaminated with excreta of infected lemming
• Oculoglandular tularemia: characterized by purulent conjunctivitis
with preauricular lymphadenopathy
• Typhoid-like illness
 Agent of bioterrorism: Because of highly infectious nature,
F. tularensis is currently classified as category A agent of
bioterrorism

8. LABORATORY DIAGNOSIS
• Specimen: Ulcer scraping , lymph node biopsy, gastric washings,
sputum, and blood
• Gram staining
F. tularensis is a small gram-negative coccobacillus with bipolar
appearance, non motile and capsulated.
• Culture: Isolation is very difficult as F. tularensis is highly fastidious.
needs special media, such as:
 BCG agar (blood cysteine glucose agar)
 CHAB agar (cysteine heart agar supplemented with 9% heated
sheep blood)
 Francis Blood Dextrose Cystein Agar [FBDCA]

9.  are inoculated on to the media and incubated a 37°C for 2-4 days
aerobically as F. tularensis is an obligate aerobe.
 Colonies are blue-gray, round, smooth, and slightly mucoid with
small zone of α-hemolysis.
• Biochemical Identification
 weakly catalase positive,
 oxidase negative
 H2S positive.
 produces acid but no gas from glucose, maltose and mannose.
 Direct fluorescent antibody tests can be done with commercially
available antisera, directly from the culture for subspecies
identification.

10. Gram staining
Growth in CHAB agar

11.  Antibody detection : is the mainstay of diagnosis as isolation is
difficult.
Agglutination tests (latex and tube agglutination) and ELISA formats are
available.
 PCR assay has been used.
Safety precautions such as biosafety level lll must be used to handle
clinical specimens to avoid the risk of laboratory-acquired infection
TREATMENT
Gentamicin : given 5 mg/kg for 7-10 days
Doxycyclin / ciprofloxacin : alternative

12. YERSINIA

13. • Tribe : Yersinieae
• 3 important species :
Yersinia pestis : cause plague [rhodent borne zoonotic]
Yersinia pseudotuberculosis
Yersinia enterocolitica Yersiniosis

14. YERSINIA PESTIS
• Isolated by : Alexandre Yersin
• Agent of plague
• Short , Gram negative , ovoid , coccobacillus.
• Pleomorphic
• In short chains / singly / groups
• Bipolar stained : in Giemsa & Methylene blue
• Appear as safety pin appearance
• Non motile , non sporing , capsulated , non acid fast & body
surrounded by slime layer

15. EPIDEMIOLOGY OF PLAGUE
• One of the greatest killer to mankind
 Plague Pandemics
Mainly 3 pandemics ; each associated with different biotypes or Y.pestis
& differentiated by glycerol fermentation and nitrate reduction

16.  Time Line Of Plague In India
• 1896 – 1918 : Hong Kong pandemic [third pandemic] entered India
and millions were died
• 1918 – 1967 : plague gradually declined and occational cases
continued to report from endemic foci
• 1967 – 1994 : no plague cases reported
• 1994 [Surat epidemic] : started as bubonic plague from Mharastra and
become pneumonic plague and spread to Surat and Gujarat
• 2002 [Shimla outbreak] : short outbreak near Shimla; 4 death reported
• 2004 [Uttarkashi outbreak] : localized outbreak of bubonic plague ;
8 cases and 3 deaths
• Four potential endemic foci : in India at present include

17.  Region near Kolar , Karnataka
 Beed – Latur belt in Maharashtra
 Rohru in Himachal Pradesh
 Dangud village ,Uttaranchal
 Epidemiological Factors
o Reservoir : wild rhodents like gerbils [Tatera indica] , field mice ,
bandicoot are main in India than domstic rats
o Source of infection : infected wild rhodents , rat fleas , cases of
pneumonic plague
o Vector : rat flea {Y.pestis}species include Xenopsylla cheopis ,
Xenopsylla astia , Xenopsylla brasiliensis
Human flea [Pulex irritans] : rare vector

18. o Plague cycles:
Plague exists in two natural cycles:
1. Domestic cycle: It occurs between humans, rat fleas and rodents.
2. Wild or sylvatic cycle: occurs in nature among wild rodents,
independent of human beings.
o Mode of transmission:
Human plague is frequently from:
• Bite of an infected rat flea (most common)
• Direct contact with tissues of infected animal (rodents)
• Droplet inhalation (man to man) from cases of pneumonic plague
• Bite of an infected human flea (Pulex irritans).

19. o Blocked flea:
In a blood meal, the fleas suck about 0.5 ml blood containing 5000
bacilli from infected rodents.
In flea gut bacilli multiply & block proventriculous
While sucking it regurgitates the blood mixed bacteria into bite
Transmits the infection
• Infection also transmitted : contamination of bite wounds with feace
of infected flea
• Partially blocked flea more dangerous than completely blocked ones
as they survive longer in burrows upto 4 years

20. • Extrinsic incubation period : interval between the flea acquiring
infection through blood meal and becoming a blocked flea; usually
about two weeks for Xenopsylla cheopis.
o Cheopis index (Average number of X. cheopis per rat) is the most
significant flea index.
• Plague outbreak is likely to occur in places having cheopis index of
more than 1.
o Seasonality
• North lndia : seasonal (September to May)
• South India it occurs throughout the year :climatic conditions
favoring the rodents to breed.

21. VIRULENCE FACTORS
 Fraction 1 antigen
heat labile plasmid encoded capsular protein antigen
inhibits macrophage phagocytosis
highly antigenic
use as immunodiagnostic marker of infection
 Phospholipase D/ Yersinia murine toxin
promotes survival of organism in infected flea gut
 Surface proteases
activate mammalian plasminogen , degrade complements
adhere to extracellular matrix component promote organism
to dissiminate from inoculation site

22.  pH 6 antigen
 Lipopolysaccharides : possess endotoxin activity
 Pigmentation :
virulent strains : brown colonies on hemin containing media
pigments promote biofilm formation
 Low calcium response plasmids
codes for type 111 secretion system {inject F1 protein} and adhesins
 Siderophore : decrease bacterial killing
 Toxins
endotoxin and exotoxin
 Ability to produce purine
 Secretion of fibrinolysin and coagulase

23. PATHOGENESIS
• Cause plague
• Zoonotic disease
• 14 th century it is called “black death”
• Human plague 3 types
1. Bubonic plague
2. Pneumonic plague
3. Septicemic plague
Bubonic plague
 Most common type
 Incubation period : 2 - 7 days
 Onset sudden,characterised by fever, malaise, headache,lymphadinitis

24. Flea bites the leg
Organism entered into localized site
Inguinal lymph node
Organisms multiply
Regional lymph nodes are enlarged & appear as tense tender swelling
buboes
Suppurates

25. Enter into blood stream
septicemia
Produces toxins
Causes haemorrahages in skin and mucosa
Lesions on skin , penis , fingers
 Buboes can also seen in axillary , cervical , submaxillary
 Children most present with cervical / axillary buboes

26.  This plague donot spread fron person to person: bacilli lock in buboes
 Sometimes infection remains in localized site of flea bite with minor
symptoms : pestis minor
 If not treated it may also cause meningial plague
Pneumonic plague
 Highly infectious
 Spread : droplet inhalation
 Incubation period : 1-3 days
 Onset sudden , characterised by fever , headache , and also produce
haemorrhagic pneumonia with production of blood,mucoid sputum
with numerous bacilli
 If untreated , is a fatal condition and agent of bioterrorism

27. Septicemic plague
 Secondary to bubonic and pneumonic plague
 Primary septicemic plague is rare
 Incubation period : 2-7 days
 Meningitric involvement occurs
 Involvement of blood vessels results inhaemorrhage in skin and
mucous , leads to gangrene of affected site
 Hence called black death

29. LABORATORY DIAGNOSIS
 Specimens
• Bubonic plague-pus or fluid aspirated from buboes
• Pneumonic plague-sputum and blood
• Septicemic plague-blood and splenic aspirate (post mortem)
Transport medium (e.g. Cary-Blair medium) can be used if delay in
transportation is expected.
 Direct Microscopy
• Gram staining: pus cells and gram-negative oval coccobacilli with
rounded ends surrounded by capsule.
• Wayson stain/methylene blue staining : typical bipolar or safety pin
appearance.Two ends are darkly stained with clear central area

30. Wayson staining
Gram stain

31.  Culture
• Aerobic and facultative anaerobes
• pH : 5 – 9.6
• Temperature :27°C but capsule developes best at 37°C
• Non fastidious so grows in ordinary medias
o Nutrient agar : small , delicate , transparent , 7 becomes opaque on
continous incubation
o Blood agar : non hemolytic and dark. brown pigmented due to
absorption of the hemin pigment.

32. o MacConkey agar: Lactose non-fermenting colourless colonies
o Nutrient broth: Granular turbidity occurs with surface pellicles
o Nutrient broth with oil or ghee floated on top characteristic stalactites-
like growth occurs hanging down from the under surface of the oil.
o Yersinia-specific CIN agar (cefsulodin, irgasan, novobiocin): is a
selective medium, useful for culture of specimens containing normal
flora, e.g. sputum

33. Stalactites growth in ghee broth

34.  Culture Smear and Motility Testing
• Gram staining of culture smear reveals pleomorphism coccid,
coccobacillary, bacillary, filamentous and giant forms.
Involution forms are seen in older cultues.
• Y. pestis is non motile both at 25°C and 37°C but other Yersinia
species which are motile at 25°C and non motile at 37°C.

35.  Biochemical Reactions
• Sugar fermentation: ferments glucose, mannitol and maltose with
production of acid but no gas. Lactose and sucrose are not fermented.
• catalase positive, oxidase negative.
• lndole, urease and citrate tests : negative.
• MR positive, but VP test is negative.
• Esculin : hydrolysed
• Gelatin liquifaction : negative
 Biotyping is done based on glycerol fermemation and nitrate
reduction

36.  F1 Antigen Detection : detected from bubo aspirate or sputum by
direct immunofluorescence test, ELISA or immunochromatographic test
(ICT) by using monoclonal antibodies
 Antibodies to F1 Antigen Detection : Antibodies may be detected by
passive hemagglutination or complement fixation test or ELISA
 Molecular Methods : PCR
 Animal inoculation : exudate / pus from buboes is injected
subcutaneously to guinea pig / white mice : disease manifestations or
death may occur

37. TREATMENT
• Choice of drug : Streptomycin 10 days
• Currently used : Gentamicin
• Post exposure prophylaxis : Levofloxacin
• Alternative drugs : Doxycyclin and chloramphenicol
PROPHYLAXIS
• Control of flea and rhodents
• Immunoprophylaxis by vaccination : 2 vaccines are used
killed vaccine and live vaccine

38.  Killed vaccine
• Widely used for active immunization
• Plague bacilli is grown in casein hydrolase and whole bacterial cell is
killed by 0.05% formaldehyde
• Preserved in phenyl mercuric nitrate
• Vaccine contains 2000 millions/ml bacilli
• 2 doses are given subcutaneously ; interval of 1-3 months
• 3rd dose after 6 months
• Immunity last for 6 months
• Side effects : fever , headache , lymphadenopathy , erythema at site of
injection

39.  Live vaccine
• Not used
• Give severe reaction
• 2 strains are used :
Otten’s Tjiwidej
Girad’s EV76
 Subunit recombinant F1 : is under trial
CHEMOPROPHYLAXIS
• Cotrimoxazole
• Deoxycyclin
• Chloramphenicol
• Streptomycin

40. YERSINIOSIS
• A zoonotic disease
• Caused by : enteropathogenic Yersinia sp ; Y.pseudotuberculosis /
Y. Enterocolitica
• Usual host : pigs and domestic animals
• Transmission : consumption of contaminated food raw pork , milk
• Yersiniosis is most common in childhood
• Patients present with abdominal pain and sometimes diarrhea
Serogrouping
• Y. enterocolitica is characterized
biochemically : six biotypes
antigenically : 60 serotypes , based on somatic O antigen

41. • Most clinical infections : O:3 & O:9
• Y. pseudotuberculosis differentiated : 6 serotypes [ 1-6 ] on somatic
and flagellar antigens
Virulence factors
• Y. enterocolitica specific virulence factors:
Myf antigen is a 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.

42. • Y. Pseudotuberculosis specific virulence factors:
Super antigen - binds to T cells non-specifically massive
cytokine release
• Virulence factors common to both species:
lnvasin (Inv) protein: helps in invasion
Ail protein : in attachment , invasion , inactivates complements
Yersinia adhesin A (Yad A): invasion and inactivates coplements
• Virulence factors common to all Yersiniae : LPS , pigments ,
siderophores , low calcium response plsmids

43. CLINICAL MANIFESTATIONS
• Self limited gastroenteritis : diarrhea with/ without blood ; in young
children
• Intestinal complications : in older children ; terminal ileitis ,
mesenteric adenitis
Patients have abdominal aute pain may mimic pseudoappendicitis
• Septicemia : in adults ; fever and leukocytosis
• Post infective phenomena : occurs commonly with Y.enterocolitica
Occurs as a result of autoimmune activity
Deposition of bacterial non viable componenets in joints etc, form
Reactive arthritis
Erythema nodosum , Grave’s disease

44. • Super antigen :
Some strains of Y.pseudotuberculosis express super antigen mitogen,
cause Scarlet fever in Russia and lzumi fever in Japan
Also cause idiopathic acute systemic vasculitis of childhood:
Kawasaki’s disease
LABORATORY DIAGNOSIS
• Specimens : blood , lymph node aspirates , faeces , food, soil
• For blood isolation : blood culture bottles (BHI broth) are used
• For isolation from lymph nodes aspirates : blood agar and MacConkey
agar is used
BA : granular , translucent colonies with beaten copper surface
non haemolytic colonies

45. MA : Y.enterocolitica grow well ;lactose non fermenting pale colonies
Y.pseudotuberculosis : poor growth
Selective media : DCA , Yersinia CIN agar (Cefsulodin –irgasan-
novobiocin):typical dark red bull’s eye :24 hours
incubation
• Incubation : plates incubated at 25°C and 37°C to differentiate from
other pathogens that grows only at 37°C
• Cold enrichment : incubating in phosphate buffered saline at 4°C for 3
weeks

46. dark red bull’s eye in CIN

47.  Biochemical tests
• Y.enterocolitica and Y.pseudotuberculosis can be differentiated from
Y.pestis
• Differential motility : motile at 22°C
• Cold enrichment : growth improves on 4°C
• Urease : hydrolysed
Tests to differentiate Y. enterocolitica from Y. pseudotuberculosis
Sucrose, cellobiose and sorbitol are fermented : Y. enterocolitica
Rhamnose. salicin and melibiose are fermented Y.
pseudotuberculosis
Ornithine decarboxylase : positive for Y. euterocolitica
VP is positive : Y. enterocolitica

48. Serology
Antibodies detection : agglutination or ELISA using specific O antigen
TREATMENT
• Most cases of diarrhea are self-limiting
• Treatment is required only for systemic infections such as in case of
septicemia
• Fluoroquinolone (ciprofloxacin) or third-generation cephalosporins
(cefotaxime) are effective.