Shigella and Salmonella Lecture

SHIGELLA AND SALMONELLA
Ms. Clemencia Tjazuko
MSc. Medical Microbiology

 Morphology
 Short gram-negative rods
 0.5 by 1-3 µm in size
 Non-motile
 Non-sporing
 Non-capsulated
 Fimbriae may be present
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Shigella
 Agent of bacillary dysentery
 Dysentery- infection of the intestines which causes
the passage of bloody stools mixed with mucus.
 Named after Japanese microbiologist Kiyoshi
Shiga, who isolated the first member 
S.dysenteriae in 1886 from epidemic dysentery
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 Antigenic Structure
 Simple
 ‘Major’ antigens and ‘Minor’ somatic antigens
 Some have K antigens
 Fimbrial antigens
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Classification
 Classified into four species based on combination
of biochemical and serological characteristics
 Serotypes distinguished within the species
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1. S.dysenteriae (Group A)
 Does not ferment mannitol
 15 serotypes
 Serotype 1- S.shigae
 Only shigella producing shiga toxin (exotoxin)
 Only member of enterobacteriaceae that is catalase
negative
 Indole negative
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 Serotype 2- S.schmitzi
 Forms indole
 Ferments sorbitol and rhamnose
 Serotype 3-7
 Formerly Large-Sachs group
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2. S.flexneri (Group B)
 Based on specific type antigen
 6 serotypes
 Complex antigenically
 Serotype 6
 Indole negative and has three biotypes
 Manchester, Newcastle and Boyd 88
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3. S. Boydii (Group C)
 19 serotypes
 Isolated least frequently
 Resemble S.flexneri biochemically but not
antigenically
 Described by Boyd (1931) from India
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4. S. Sonnei (Group C)
 Sonne (1915), Denmark
 Ferments lactose and sucrose late
 Indole negative
 Antigenically homogenous
 2 forms: Phase I and Phase II
 Phase II colonies are larger, flatter and irregular
 Mildest form of bacillary dysentry
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 Toxins
1. Endotoxin
 May be due to the LPS of Gram-negative cell wall
 Has irritating effect on intestinal wall which causes
diarrhoea and subsequently intestinal ulcers
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2. Exotoxin
 Produced by S.dysenteriae type 1
 Also called Shiga toxin or verotoxin
 Acts as enterotoxin and neurotoxin
 Inhibit protein synthesis
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3. Verocytotoxin (VT)
 Cytotoxin produced by S.dysenteriae type 1
 Acts on vero cells
 VT1 and VT2
 VT1 two subunits A and B.
A binds cytotoxin to cells
B inhibit protein synthesis
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Pathogenesis of Shigella
 Mode of transmission:
infection through contaminated fingers
contaminated food/water
flies (rare)
transmitted sexually (homosexuals), gay bowel
syndrome
 Minimum Infective Dose:
10-100 bacilli
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 Entry via M cell:
Bacilli enter mucosa via M cell
Cross the basolateral side of M cells to reach submucosa where
they are engulfed by macrophages
Bacilli released by macrophages
Cytokines released which attract inflammatory cells to infected
site inflammation acute collitis
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 Invasion:
Inside submucosa, shigellae induce own uptake
into adjacent epithelial
Determined by large virulence plasmid, codes
for ipa proteins and Type III secretion system
 Direct cell-to-cell spread:
from one host cell to the other by inducing actin
polymerisation cells of host
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Bacillary dysentery
 Short incubation period (1-7 days)
 Clinical features:
 Frequent passage of loose, scanty feces containing
blood and mucus, with abdominal cramps and
tenesmus
 Complications:
 Arthritis, toxic neuritis, conjunctivitis, parotitis,
intussusception in children
 Hemolytic uremic syndrome in severe cases
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Laboratory Diagnosis
 Specimen
 Isolating bacillus from feces
 Fresh feces inoculated or transported in Sach’s
buffered glycerol saline or Gram-negative broth @
pH7.0-7.4
 Rectal swabs not satisfactory
 Gram-negative broth sodium deoxycholate to
inhibit gram-positive bacteria, subcultured 6-8hrs
after incubation
 Selenite F Broth/Salmonella Shigella (SS) broth
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 Microscopy- plenty of pus cells, erythrocytes and
macrophages
 Culture- best to use mucus flakes
 MacConkey, DCA/ XLD
 Incubation @ 37 ºC, plates inspected for pale or pink-
coloured colonies, identified by biochemical reactions
 Non-motile bacillus that is : urease, citrate, H2S and
KCN negative further investigated by biochemical tests
 Confirmed by slide agglutination with sera
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 Salmonella Shigella Agar - colonies colourless
 Hektoen enteric (HE) Agar – contains bile salts as
inhibitory agents and some dyes, colonies appear
green with fading colour to the periphery
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 Treatment
 Acute cases infants and young children,
dehydration corrected propmptly
 Antibiotics reserved for severe toxic cases
 Many strains still sensitive to Nalidixic acid and
Norfloxacin
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Salmonella
 Morphology
 Gram-negative rods,1-3 by 0.5 µm in size
 Fimbriae – present
 Motile with petrichate flagella
 But S.Gallinarum and S.Pullorum are non-motile
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Clinical Classification
1. Typhoidal Salmonella
 Serotypes S.Typhi and S.Paratyphi restricted to
human hosts causing enteric fever
(typhoid/paratyphoid fever)
2. Non-typhoidal Salmonella
 Remaining serotypes are the food poisoning
group, infect human beings causing
gastroenteritis, septicemia or localised infections
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Antigenic Classification
 Kauffmann-White scheme
 Based on the presence of different somatic (O) and
flagellar (H) which is detected by agglutination with
respective antisera
 Based on O antigen:
 Serogroup 2
 Serogroup 4
 Serogroup 9
 Within each group further diffrentiation by Phase 1
and Phase 2 flagellar antigens
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Antigenic Structure
Flagellar antigen H
Somatic antigen O
Surface antigen Vi
H antigen
Present on flagella, heat labile
Destroyed by boiling or alcohol
H suspensions agglutinate rapidly to form large, loose,
fluffy clumps
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 Strongly immunogenic, induce antibody formation
 Occurs in two phases
O antigen
 Phosholipid-protein-polysaccharide complex
 Identical to endotoxin
 Heat and alcohol stable
 67 O antigens
 Less immunogenic
 O suspensions form compact, chalky, granular clumps
with antisera
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Vi antigen
 Surface polysachharide envelope or capsular antigen
covering the O antigen
 Inhibits phagocytosis, resists complement
activation, bacterial lysis
 Heat labile, Poorly immunogenic
 Present bacilli is inagglutinable with O antisera.
Becomes agglutinable after boiling or heating at 100
ºC for 1hr
 S.Paratyphi C
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Antigenic Variation
 H-O Variation
 Associated with the loss of flagella. Induced by:
Phenol agar: inhibits flagella
Mutation: Non-motile mutant of S.Typhi 901-O
strain subcultured in Craigie’s tube
 Phase variation:
Phase 1: serotype specific (a, b,c etc)
Phase 2: nospecific/group antigens (1,2, etc)
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 V-W Variation
 V forms of S.Typhi isolates with Vi antigens are
agglutinated by Vi antisera but not O antisera
 After serial subcultures, Vi antigen is lost W
forms agglutinable by O antisera but not Vi
antisera
 Also VW forms agglutinable with both
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 S-R Variations
 Smooth to rough variation, due to loss of of O
antigen side chain from LPS leading to exposure of
core polysaccharide portion
 Smooth colonies produced by virulent strains
 Rough strains form large, round and irregular
colonies, avirulent due to loss of O antigen
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Pathogenesis of Salmonella
 S.Typhi and S.Paratyphi A, B, C  Typhoidal
salmonellae
 Cause Enteric fever also known as Typhoid fever
 Infective Dose:103 – 106 bacilli
 Risk factors: stomach acidity, intestinal integrity
 Entry through epithelial cells (M cells): lines the
mucosa. Salmonellae cause formation of
membrane ruffles. Ruffles enclose adherent
bacteria bacteria mediated endocytosis (BME)
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 Mechanism of BME: Type III secretion system
delivery of bacterial proteins into cytoplasm of
epithelial cells alters actin cytoskeleton required for
bacterial uptake
 Entry into macrophages: Salmonellae containing
vacuoles cross epithelial layersubmucosa
phagocytosed
 Survival inside macrophages:
 S.Typhi alters its surface so that it is no longer
suspectible to lysosomal enzymes of macrrophages
 Encode Type III secretion system that delivers bacterial
proteins into macrophage cytoplasm
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 Primary bacteremia: Salmonellae inside
macrophages spread via lymphatics to enter blood
stream
 Spread: disseminate throughout liver, spleen,
lymph nodes and bone marrow; and gallbladder,
kidneys and lungs further multiplication
 Secondary bacteremia: from clinical organs
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Clinical Manifestations
 Gradual onset with headac he,
malaise,anorexia, coated tongue and abdominal
discomfort with constipation or diarrhea
 Soft, palpable spleen. Hepatomegally
 Rashes/Rose spots that fade on pressure
 Complications: gastrointestinal bleeding and
intestinal perforation in third or fourth week of
illness
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 Carriage
 10% of untreated patients become carriers and excrete
S.Typhi in urine or feces
 Two types of carriers:
 Fecal carriers: bacilli multiply in gall bladder and
excreted in feces. Common
 Urinary carriers: multiplication in kidneys and bacilli
excreted in urine
 Tyhoid Mary-chronic carrier, a New York cook who
caused 1300 cases outbreaks
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Laboratory Diagnosis
 Specimen and Culture Isolation
 Blood and Bone marrow culture:
 In first week of illness in blood culture bottle
(BHI broth, automated BACTEC)
 Stool Culture (3-4 weeks of illness)
 Enrichment broth such as Selenite F Broth,
tetrathionate broth and gram-negative broth
 MacConkey Agar NLF
 DCA, XLD agar, SS agar and Wilson Blair’s
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 Urine Culture
 In 3-4 weeks of illness
 On MacConkey agar
 Culture Smear and Motility
 Motile, gram-neg bacilli
 Biochemical Identification
 Catalase positive and oxidase negative
 Nitrate reduced to nitrite
 Indole and Urease negative
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 Slide Agglutination
 Confirm the serotype
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 Serum Ab detection (Widal Test)
 2-3 weeks of illness, Abs detected against TO, TH,
AH, BH antigens
 S.Typhi infection: high TO and TH Abs
 S.Paratyphi A infection: high TO and AH Abs
 S.Paratyphi B infection: high To and BH Abs
 Antigen detection (serum and urine)
 ELISA, CIEP
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 Molecular Methods
 PCR detecting flagellin gene, iroB, and fliC
gene
 Antimicrobial testing
 Done on Mueller-Hinton Agar
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Assignment due 22 Feb 2017
1. To which family does Shigella and Salmonella
belong to? What are the general properties of
that family?
2. Name the Non-Typhoidal Salmonellae
species? Write short notes about what
diseases they cause
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Shigella and Salmonella Lecture

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