Presentation on Yersinia bacteria

Introduction
 The genus Yersinia is now assigned to the family
Enterobacteriaceae. Eairlers, the plague bacillus
was included in genus pasteurella.

 Has 11 species, only 3 species are pathogenic to

human. Yersinia pestis, Yersinia enterocolitica,
Yersinia pseudotuberculosis.
Morphology
 Y. pestis is a Gram-negative coccobacillus that
measures 1.5-3 0.7 µm in size, with rounded ends
and convex sides.
 The bacilli are arranged in single, in pairs, or in
short chains. Pleomorphism is very common.
 Giemsa or methylene blue stained smears of clinical
specimens show characteristic bipolar staining
(safety pin appearance).
 It is a nonmotile, nonsporing, and non–acid fast but
capsulated bacterium.
Culture
 Y. pestis is aerobic and facultatively anaerobic.

 It grows at a temperature range of 2–45°C with an

optimum temperature of 27°C (unlike most
bacteria), but it grows better at 37°C in culture.

 The bacteria grow at a wide range of pH (5–9.6),

with an optimum pH of 7.2.
Solid media
 Y. pestis can grow on a variety of media including
Mueller–Hinton agar, nutrient agar, blood agar, and
MacConkey agar.
 On nutrient agar, Y. pestis produces small, delicate, and
transparent colonies becoming opaque on prolonged
incubation.
 Colonies on blood agar (containing sodium azide) are
dark brown due to the absorption of the hemin
pigment.
 The bacteria grow poorly on MacConkey agar and
deoxycholate citrate agar, producing pinpoint reddish
colonies after 24 hours of incubation.
Growth on CIN Agar (selective medium)
Liquid media:
 In broth, Y. pestis produces a flocculent growth with
granular deposit at the bottom and along the sides
of the tube, with little or no turbidity.
Biochemical reactions
 Y. pestis is catalase positive, aesculin positive,
coagulase and MR positive.

 It ferments glucose, maltose, and mannitol with

production of acid but no gas. Lactose, sucrose, or
rhamnose are not fermented.

 It is oxidase, urease and citrate negative.

Other properties
 Susceptibility to physical and chemical agents:

 Y. pestis is sensitive to heat, sunlight, drying, and

chemical disinfectants.

 The bacilli are killed at a temperature of 55°C in

30 minutes.
Cell Wall Components
 The cell wall of Y. pestis contains a
lipopolysaccharide that functions as an endotoxin
and
 Is an important component of the virulence of the
bacteria.
Antigenic Structure
 V and W antigens:
 These two antigens are always present together.
 Production of V and W antigens is mediated by
plasmid.
 F1 envelope antigen:
 F1 envelope antigen (fraction I or F–I antigen) is a
heat-labile envelope protein generally present only
in virulent strains.
 Production of F–I antigen is mediated by plasmid.
Pathogenesis and Immunity
 Y. pestis produces many toxins and enzymes, all of
which contribute to pathogenesis of the diseases.
 Plague toxin: The term “plague toxins” refers to a
complex of at least two different types of toxins:
(a) endotoxin and
(b) murine toxin, found in culture filtrates or in cell
lysates.
Contd…
A. The endotoxin
 is a lipopolysaccharide found in the cell wall and is
responsible for many of the systemic manifestations of
the disease.
B. Murine toxins
 exhibit some properties of both exotoxins and
endotoxins.
F1 envelope antigen:
 F1 envelope antigen is a major virulence factor, which
inhibits phagocytosis and plays an important role in
conferring protective immunity in humans and in mice.
Contd….
 V and W antigens:
 These two antigens inhibit phagocytosis and
intracellular killing of the bacillus inside
macrophages.
 Other virulence factors:
 Yersinia produces the enzymes, such as coagulase,
fibrinolysin, and plasminogen activator (pIa)
protease, which contributes to virulence of the
bacterium.
Pathogenesis of plague
 Bubonic plague:
 It is a zoonotic disease transmitted by rat flea
Xenopsylla cheopis from animals to humans.


 When rat flea bites an infected and diseased rat, it

sucks nearly 0.5 mL of blood per feed containing
nearly 5000–50,000 plague bacilli.
Contd….
 Bacilli multiply in the stomach of the flea and
caused blockage of proventriculus (2-3wks).
 To overcome the starvation flea biting to other
rodents or man. And caused bubonic plague.
 They produce pathognomic lesion that is ‘bubo’ (
inflamed, necrotic and haemorragic lymph node).
Pneumonic plague
 It is transmitted from humans to humans. It occurs
following direct inhalation of the bacilli by droplet
infection due to close contact with infected hosts.
 producing a severe and rapidly progressive
multilobar bronchopneumonia, subsequently
leading to bacteremia and septicemia.
 Not zoonotic disease.
Septicemic plague:
 It is usually the terminal stage of the bubonic or
pneumonic plague.
 It may sometimes occur primarily.
 Primary septicemic plague may occur when the
plague bacilli are deposited directly in the
circulation, bypassing the lymphatics.
Clinical Syndromes
 Y. pestis causes plague, which occurs in three forms:
 (a) bubonic plague,
 (b) pneumonic plague, and
 (c) septicemic plague
Bubonic plague
 Bubonic plague is the most common clinical form of the
disease.
 The incubation period varies from 2 to 6 days.
 Sudden onset of high fever, chills,headache, body
aches, extreme exhaustion, abdominal pain, and
diarrhea.
 Presence of painful, swollen lymph glands (buboes),
usually in the groin, axilla, or neck, is the characteristic
manifestation.

 Mortality rate for untreated plague is 40–70%.

Pneumonic plague
 Abrupt onset of fever and chills, lymphadenopathy,
chest pain, dyspnea, purulent sputum, or hemoptysis
are the manifestations of pneumonic plague.

 Buboes may or may not be present in pneumonic

plague. Pneumonic plague is highly infectious and
transmitted by aerosol droplets.
Lungs of a pneumonic
plague patient
Septicemic plague
 It is usually the terminal event in the bubonic or
pneumonic plague, but may sometimes occur
primarily in elderly patients.
 The condition is associated with a rapid onset of
symptoms, such as nausea, vomiting, abdominal
pain, and diarrhea.
 Diarrhea may be the predominant symptom.
Hypothermia is common. Generalized purpura
leading to necrosis and gangrene of the distal
extremities may be observed.
26
Epidemiology
 Plague is worldwide in distribution with most of the
human cases reported from developing countries.
 India is one of the few countries that have been
worst hit by the pandemics of plague. Plague first
appeared in Bombay in 1896 and spread all over
the country, causing more than 10 million deaths by
1918.
Urban or domestic plague:
 Urban plague is maintained in rat populations and
is transmitted among rats or between rats and
humans by infected rat fleas.
Wild or sylvatic plague:
 This occurs between animals and wild rodents
independent of human beings.
 More than 200 different animal species have been
identified as hosts.
 These include domestic cats and dogs, squirrels,
deer, mice, rabbits, hares, rock squirrels, camels,
and sheep.
To 2°
only

1°
Or only
Septicemic
Laboratory Diagnosis
 Specimens
 These include bubo aspirates (in bubonic plague),
sputum (in pneumonic plague), and blood and
cerebrospinal fluid (CSF) (in septicemic plague).

 Microscopy
 Bubo aspirates is collected by injecting 1 mL of sterile
saline into the bubo with a 20-G needle.
 The bubo aspirate smears are stained with Gram,
Wright, Wayson, or Giemsa stain for demonstration of
the typical bipolar (safety pin) morphology of Y. pestis.
Culture
 The organism can be isolated from blood, CSF, sputum,
and bubo aspirates, depending on the clinical
presentation, whether it is bubonic, pneumonic, or
septicemic.

 Clinical specimens are inoculated on blood or nutrient

agar and on specialized selective media for isolation of
Y. pestis. Selective medium, such as blood agar with
sodium azide or CIN Agar is used for selective
isolation of Y. pestis from sputum and bubo aspirates
containing numerous other bacteria.
Identification of colonies
 Dark-brown colonies on blood agar and pinpoint
colonies on MacConkey agar are the characteristic
features of colonies of Y. pestis.
Animal inoculation
 Y. pestis can be isolated from bubo aspirate or
sputum by inoculating in guinea pigs or white rats.
The bubo aspirate is injected subcutaneously into
the animal. Y. pestis causes death of the animal
within 2–5 days.
 Postmortem of the dead animal shows necrosis and
edema at the site of inoculation.
Serodiagnosis
 Enzyme-linked immunosorbent assay (ELISA) and
 Indirect Hemagglutination (IHA) tests are employed
to detect specific antibodies in acute and
convalescent sera.
Treatment
 Streptomycin, doxycycline, and chloramphenicol are
used effectively for treatment of plague.
 Treatment with antibiotics is usually given for a
duration of 10 days. A two-drug regimen should be
used in severe cases.
 Streptomycin is the drug of choice to treat plague.
Prevention and Control
 These include;
 Immunoprophylaxis by plague vaccine,
 Chemoprophylaxis by antibiotic therapy, and
 Environmental sanitation.
Yersinia enterocolitica
 Enterocolitis is the most common clinical form of the
disease, which occurs primarily in young children.
 The incubation period is short, varies from 4 to 6
days.
 The condition manifests as watery, mucoid diarrhea
in majority of patients; fever, abdominal pain,
bloody stools; and leukocytes in the stool.
Contd….
 Y. enterocolitica is a Gram-negative coccobacillus
that is motile at 22°C but not at 37°C.
 This bacillus resembles Y. pseudotuberculosis.
 It in fermenting sucrose and cellobiose and
decarboxylating ornithine.
 Y. enterocolitica are VP and indole positive.
 They do not ferment rhamnose.
 They are oxidase negative and nonlactose
fermenting.
Y. pseudotuberculosis
 Y. pseudotuberculosis primarily causes
gastroenteritis.
 The incubation period varies from 5 to 10 days.
 Gastroenteritis is characterized by a self-limited
mesenteric lymphadenitis simulating acute
appendicitis. Fever, abdominal and rash are a major
triad.
Contd…
 Y. pseudotuberculosis is a small, ovoid, and bipolar
stained
 Gram-negative bacillus. It is nonsporing,
noncapsulated, and slightly acid fast. It is motile at
22°C but not at 37°C. The
 bacterium is oxidase negative, catalase positive,
and urease positive.
Differentiating features of Yersinia
species
Properties Yersinia pestis Yersinia Yersinia
pseudotuberculosis enterocolitica
Growth on
Growth on + + +
MacConkey agar
Motility at 22°C _ + +
Oxidase test _ _ +
Urease test _ + +
Indole test + _ +
Acid from sucrose + _ +
Acid from maltose _ + +
Acid from rhamnose _ + _

Acid from malibiose _ + _

Ornithine _ _ +
decarboxylase test
Shigella
 Shigella is the most common cause of bacillary
dysentery, which occurs worldwide.
 The disease is spread through fecal–oral
transmission, and humans are the only natural
reservoir of the bacteria.
Morphology
 Shigella are short, Gram-negative rods, about 0.5 x
1–3 m in size.
 They are non motile, non sporing, and non
capsulated.
 Shigella species with exceptions of S. flexneri,
serotype 6, and some strains of other serotypes
possess fimbriae.
 Shigellae are classified into four species or
subgroups, consisting of more than 45 O antigen-
based serogroups and each species consisting of
different serotypes.
Shigella dysenteriae (group A):
▪ This is subdivided into 12 serotypes. Each serotype is
characterized by the presence of a different type
of antigen.
▪ S. dysenteriae serotype 1 is the bacillus originally
described by Shiga, hence known as Shiga’s
bacillus.
▪ S. dysenteriae serotypes 3–7 were described by Large
and Sachs in India, and hence were known as the
Large–Sachs group.
Shigella flexneri (group B):
 This group is named after Flexner (1900), who

described the first of the mannitol-fermenting

shigellae from Philippines.
 S. flexneri, based on type-specific (I–VI) and group-

specific (1–8) antigens, have been classified into six

serotypes (1–6) and several subtypes (1a, 1b; 2a,
2b; 3a,3b, 3c; 4a, 4b; 5a, 5b).
Shigella boydii (group C):
 This group is named after Boyd, who first described

these strains from India (1931). A total of 19

serotypes have been identified in this group.
 Members of S. boydii group resemble biochemically,

but not antigenically,with those of S. flexneri

Shigella sonnei (group D):
▪ This group is named after Sonnei, who first described

these strains from Denmark (1915).

▪ S. sonnei have been divided into 26 colicin types by

using 33 indicator strains.

Culture
Nutrient agar:
▪ They grow on ordinary media, such as nutrient agar

or Mueller–Hinton agar. Shigella colonies on nutrient

agar, after overnight incubation, are small, circular,
convex, smooth, and translucent.
▪ Occasionally on primary isolation and frequently in

subcultures, a proportion of the colonies may be of

the rough type.
MacConkey agar:
▪ Shigella spp. on MacConkey agar produce non

lactose-fermenting pale, colorless colonies.

▪ S. sonnei (which ferments lactose late) forms pale

pink colonies on prolonged incubation.

Selective media:
▪ Deoxycholate citrate agar (DCA), xylose lysine
deoxycholate (XLD) agar, Salmonella–Shigella (SS)
agar, and Hektoen enteric (HE) agar are frequently
used selective media for isolation of Shigella
species.
▪ DCA is a useful selective medium for isolation of
Shigella spp. from feces. On this medium, Shigella
spp. produce small colonies, which on prolonged
incubation produce lactose-fermenting pink colonies.
Shigella on XLD
Shigella boydii on HEA
 XLD agar is another selective medium, which is less
inhibitory to S. dysenteriae and S. flexneri. Shigella
spp. forms red colonies on this medium.
 Shigella spp. on SS agar form colorless colonies.
Shigella spp. on HE agar forms green colonies.
Liquid media:
 Selenite F and Gram-negative (GN) broth are

commonly used enrichment media.

 Enrichment of feces in GN broth for 4–6 hours

followed by subculture on XLD or HE medium is

useful for isolation of Shigella from clinical
specimens.
Biochemical reactions
▪ Shigella ferments mannitol, forming acid but no gas.
 Mannitol fermentation test is an important
biochemical test, which is used to classify shigellae
into mannitol-fermenting and-non fermenting
species.
 S. flexneri, S. boydii, and S. sonnei are mannitol-
fermenting species, while S. dysenteriae is mannitol
non fermenting species.
 XLD agar is another selective medium, which is less
inhibitory to S. dysenteriae and S. flexneri. Shigella
spp. forms red colonies on this medium.
 Shigella spp. on SS agar form colorless
colonies.Shigella spp. on HE agar forms green
colonies,
characteristics Shigella Shigella flexneri Shigella boydii Shigella sonnei
dysenteriae

Number of serotypes 12 6+2 18 2 phases;26 colicin

variant types

Lactose - - - -

sucrose - - - -

Mannitol - + + +

Dulcitol - - v -

Xylose - - v v

indole v v v -

Lysine decaboxylase - - - -

Ornithine - - - +
decarboxylase
Cell Wall Components
 The cell wall of shigellae, like other Gram-negative
bacilli, contains a lipopolysaccharide (LPS) structure.
 The LPS is liberated during lysis of the cell and, to
some extent, during culture. The LPS moiety functions
as an endotoxin and is an important component of
the virulence of the bacteria.
Antigenic structure
 The antigenic structure of shigellae is simple, unlike
the complex antigenic structure of salmonellae.
 Shigellae possess somatic O antigens and certain
strains possess K antigens.Shigella K antigens, when
present, may sometimes interfere with agglutination
by O antisera.
 Shigellae strains also possess fimbrial antigens.
Common fimbrial antigens are also found
Virulence factors
Endotoxin
▪ Endotoxin increases the cytotoxic activity of Shiga

toxin on human vascular endothelial cells.

▪ The endotoxin is expressed by chromosomal genes

of the bacteria.
Shiga toxin
▪ Shiga toxin is an exotoxin produced by S.
dysenteriae. It is a heat-labile protein and acts as
enterotoxin and neurotoxin.
▪ Shiga toxin (Stx) is a group of cytotoxins that
contain two major immunologically non–cross-
reactive groups called Stx1 and Stx2.
▪ Both Stx1 and Stx2 groups are encoded by a
bacteriophage inserted into the chromosome of the
bacteria. Shiga toxins have one A subunit and five B
subunits:
❑ The main function of B subunit is to bind toxins to
host cell glycolipid (Gb3) surface receptor, present
on the brush border of epithelial cell of the
intestines. It also mediates transfer of the A subunit
into the cell.
❑ Subunit A is a 32-kDa polypeptide. It cleaves the
28S rRNA in the 60S ribosomal subunit, thereby
preventing the binding of aminoacyl -transfer RNA
and disrupting protein synthesis.
Shiga toxin
1. Neurotoxic activity: This activity is demonstrable by
paralysis and death of experimental animal
following injection with the toxin. Although called
neurotoxin, the primary site of its action is not the
neural tissue but is the blood vessels, neurological
manifestations being secondary.
2. Enterotoxic activity: These toxins are enterotoxic for
ligated rabbit intestinal segments with induction of
fluid accumulation in ligated rabbit ileal loop. Two
new Shigella enterotoxins, designated as S. ET-l
and S. ET-2—the former confined to S. flexneri 2a
and the latter more widespread—have been
identified.
3. Cytotoxic activity: This is demonstrated by
cytotoxicity of toxin for Vero, HeLa, and some
selected endothelial cells, such as human renal
vascular endothelial cells. This appears to be the
same as vero toxin 1 (or Shiga-like toxin)
produced by certain strains of Escherichia coli
(VTEe).
Pathogenecity
 The infectivity dose (ID) is extremely low. As few as 10
S. dysenteriae bacilli can cause clinical disease, whereas
100–200 bacilli are needed for S. sonnei or S. flexneri
infection.Shigella spp.cause disease by invading and
replicating in cells lining the intestinal mucosa of the
colon.
 Structural proteins, such as intestinal adhesion factor,
endotoxin, and exotoxin mediate the adherence of the
bacteria to the cells as well as their invasion,
intracellular replication, and cell-to-cell spread.
 The bacilli infect the epithelial cells of the villi in the
large intestine and multiply inside them.
Subsequently, bacteria spread laterally to involve
adjacent cells and penetrate into the lamina
propria. Shigellae lyse the phagocytic vacuole and
multiply in the host cell cytoplasm. Shigellae survive
phagocytosis by inducing programed cell death or
apoptosis.
 This mechanism also leads to the release of
interleukin-1 resulting in the attraction of
polymorpho nuclear leukocytes into the infected
tissues. This in turn alters the integrity of the
intestinal wall and allows the bacteria to reach the
deeper epithelial cells
 Shiga toxin produced by the bacteria also plays an
important role in progression of mucosal lesions
after invasion of the colonic cells. The toxin also
induces vascular damage in the colonic mucosa.
Clinical syndrome
 Arthritis, toxic neuritis, conjunctivitis, and, in children ,
intussusception.
 Hemolytic uremic syndrome may also occur
following infection with S. dysenteriae because of
vasculo pathymediated by Shiga toxin.
 Reiter syndrome (arthritis, urethritis, conjunctivitis) is
usually observed in adults with HLA-B27 histo
compatibility antigen.
 Shigella septicemia is rare, except in malnourished
children with S. dysenteriae infection.
 Disseminated intravascular coagulation,
bronchopneumonia,and multiple organ failure may
occur in lethal cases of Shigella septicemia.
Typing of Shigella
For epidemiological purposes, Shigella species have
been classified into many colicin types, depending
upon the biochemical characteristics of the
microorganisms
Identification of bacteria
 Pale non–lactose-fermenting colonies on MacConkey
agar are identified by carrying out motility test,
biochemical tests, and slide agglutination test with
specific Shigella antisera (polyvalent and monovalent
sera)
Serodiagnosis
 Serological tests are not useful in the diagnosis of
shigellosis.
Thank You…!!