Enterobacteriaceae

Dr. Ng Woei Kean

Ph.D. (Molecular Medicine)
 General Characteristics
• Gram negative rods
• Facultative anaerobes
• Ferment glucose to acid with or without gas
• Reduce nitrate to nitrites
• Oxidase negative
• Growth not enhanced by NaCl
• All motile except a few - Klebsiella and Shigella
 Classifications
• 31 genera, over 100 species.
• Only a few are pathogenic to human being.
• Different metabolic profiles namely sugar
fermentation and H2S production.
• They are differentiated into “lactose fermenter”
and “non-lactose fermenter” groups.
 MacConkey medium

Lactose Non-lactose
fermenter fermenter
- E. coli, -Salmonella,
Klebsiella Shigella
spp.
Genera of Enterobacteriaceae known as human pathogen
Pathogenic potential of Enterobacteriaceae
 Virulence Factors
1. Antigens
- K (capsular antigen) - >100
- O (lipopolysaccharide antigen) - >150
- H (flagellar antigen) - >50
2. Pili or fimbriae
3. Toxins
Antigenic structure of Escherichia coli. The O antigen is contained in
the repeating polysaccharide units of the lipopolysaccharide (LPS) in
the outer membrane of the cell wall. The H antigen is flagellar
protein. The K antigen is the polysaccharide capsule present in
Pili
• It mediate attachment. There are type 1-4 pili.
• Type I pili: Mannose sensitive - attaches to
enterocytes.
• Type 2 Pili: Mannose resistant - mediate
attachment to various tissue outside normal
habitat.
Type 2 Pili:
• P pili - present in E. coli causing
urinary tract infection (UTI).
• Bundle forming Pili (BFP) - present in E.
coli
causing gasterointestinal tract infection
(GIT).
• X adhesin present in E.coli causing UTI.
Toxins
• Protein exotoxins - damaging membranes,
inhibiting protein synthesis, or altering metabolic
pathways, leading to cell death / physiological
change
Escherichia coli
 Escherichia coli
• Ubiquitous in nature and is present as normal
flora in the body, but it is also a pathogen.
• Diseases produced by E. coli
- Diarrhea
- Urinary tract infection (UTI)
- Meningitis
- Other: septicemia, wound
infection, abscess, infection of
various tissue
Gram stain
– E. coli
 Classification of diarrhea causing E. coli
• Enterotoxigenic (ETEC) 3 types-based on
• Enteropathogenic (EPEC) bacterial attachment
pattern to cell
• Enteroaggregative (EAEC) • localized
• Enteroinvasive (EIEC) • diffuse
• Enterohaemorrhagic (EHEC) • stack brick
 Enterotoxigenic (ETEC)
Virulence factors
• Colonization factor Antigen I & II (CFA I & II).
• Produce LT (heat labile) and or ST (heat stable)
toxin. Plasmid mediated.
Pathogenesis
• Transmission: oral-fecal
• Site of infection - small intestine
• Secretion of fluid and electrolytes by LT & ST
• LT - activates adenylyl cyclase.
• ST - activates guanylyl cyclase.
 Enteropathogenic (EPEC)

Virulence factors
• BFP - forms clustered micro colonies on
enterocytes surface.
• Pathogenicity Island (PAI) genes for attachment
and effacement of microvilli.
Pathogenesis
• Transmission: oral-fecal.
• Site of infection - small intestine.
• Produce “pedestals” and effacement of microvilli.
• Perturbs enterocyte cytoskeleton proteins.
 Enteroaggregative (EAEC)
Virulence factors
• Aggregative adhrence fimbriae (AAF/I).
• Enteroaggregative ST1 and LT toxins.

Pathogenesis (exact mechanism not known)

• Transmission: oral-fecal.
• Site of infection - small intestine.
• Adheres tightly to enterocytes.
• Perturbs mucosal absorption by forming mucus-
bacteria biofilm on the intestinal surface.
 Enteroinvasive (EIEC)
• Typical serotype O29:H

Virulence factors
• Contains large plasmid encoding Ipa gene

Pathogenesis
• Similar to Shigella
 Enterohaemorrhagic (EHEC)
• Typical serotype O157:H7

Virulence factors
• Contains attaching and effacing factor (eae
gene).
• Produce shiga like toxin I and II
(verotoxin).
Pathogenesis
• Site of infection - colon
• Damages epithelial cells
• Shiga like toxin causes capillary thrombosis &
inflammation of colonic mucosa and hemorrhage
 Diagnosis of Diarrhoeagenic
E. coli
• Culture of stool - MacConkey media.
• E.coli grows as lactose fermenting pink colony.
• Problem: non-pathogenic E.coli can not be
differentiated from diarrhoegenic E. coli
by culture characteristics - they look
same.
Special Tests
• ETEC - Detection of LT and ST toxin by ELISA
in culture supernatant or by in vitro cell assay
and in vivo assay in mice
• EPEC - Detection of A/F gene by molecular
technique or detection by EPEC antisera
• EAEC - Cell adherence assay (Hep2, HeLa)
• EIEC - By Sereny’s test in rabbit eye
• EHEC -Using anti-sera.
 Treatment
• Gasterointestinal infection: oral rehydration.
• More severe infections e.g. hemolytic uremiuc
syndrome (HUS) caused y EHEC - transfusion
and hemodialysis.
• May develop resistance: antimicrobial drug
therapy may be contraindicated for non
life threatening infections.
Salmonella
 Characteristic of Salmonella
• Causes spectrum of illness from local to
systemic infection.
• Motile and non-lactose fermenting
enterobacteriaceae.
 Classification of Salmonella
• Previously: biochemical reaction, O, H and Vi
antigen
• Recently: all are classified into a single
species -
Salmonella enterica.
• All serotypes are now placed under S.
enterica
• Salmonella typhi = Salmonella enterica serotype
Typhi
 Diseases
• Typhoid fever
• Enterocolitis
• Bacterimia
 Pathogenesis of Enterocolitis
• Oral transmission
• Infective dose > 1000 organisms
• Incubation period: 8-48 hours
• M cells ==> sub-mucosa (multiply)
• Macrophage apoptosis
• cyclic AMP, toxin and inflammatory mediators
• Fluid secretion, inflammation
• Toxins to inhibit protein synthesis
• Cleared by phagocytosis
 Pathogenesis of Typhoid
Fever
• The ingested salmonellae reach the small
intestine, from which they enter the lymphatics
and then the bloodstream.
• They are carried by the blood to many organs,
including the intestine.
• The organisms multiply in intestinal lymphoid
tissue and are excreted in stools.
• After an incubation period of 10–14 days, fever,
malaise, headache, constipation, bradycardia,
and myalgia occur.
• The fever rises to a high plateau, and the spleen
and liver become enlarged.
• In the preantibiotic era, the chief complications
of enteric fever were intestinal hemorrhage and
perforation, and the mortality rate was 10–
15%.
• Treatment with antibiotics has reduced the
mortality rate to less than 1%.
 Laboratory Diagnosis
• Detection of antibody or antigen in blood
• Culture
- Urine, stool, blood, duodenal fluids
- Special sample: Bone marrow
Culture of Urine and Stool
• Transport media - Cary-Blair
• Media - MacConkey,
• Identification by biochemical tests
• Confirmation by specific antisera
Blood Culture
• Blood culture is positive alone in 50-70% cases.
• Multiple blood culture is 75-97% sensitive.
• Blood culture is positive in about 40% cases if
the patient is on antibiotic.
• Bone marrow culture is 90% sensitive. It is not
affected by antibiotics.
Isolation of organism from various sample in
relation to timing of the illness
100

90 Blood
80
Stool
70

60
Series1
50 Series
2
40
Series
3
30 Urine
20

10

0
1 2 3 4 5 6

Weeks after onset of fever

Serological Diagnosis – Widal Test
• Widal test - tube and slide agglutination tests.
- Detects antibody to O and H antigens.
- Detects antibody to Vi antigen for carrier.
• Factors affecting the tests
- The duration of the disease
- Antibody
- Antimicrobial therapy
- Test procedure - Tube vs slide test
 Vaccination
• Parenteral
- Heat killed phenol treated vaccine
- Acetone inactivated vaccine
*Three dose schedule
• Live vaccine: Oral Ty21a
• Protection is variable.
• Recommended for travelers.
Shigella
 Shigella spp.
• Caused dysentery.
• Self limiting disease.
• Non-motile and non-lactose fermenting.
 Classification and characteristics
Species Serogroups Key Features
S. dysenteriae Gr A Serotype 1 produce shiga
12 Serotypes toxin. World wide distribution.

S. flexneri Gr B Does not produce shiga

14 Serotypes toxin.

S. boyedii Gr C
18 Serotypes -

S. sonnei Gr D
1 Serotype Most common in USA
 Virulence Factors
1. Ipa proteins (Ipa A,B,C and D).
2. Ics (IcsA and IcsB)
3. Shiga toxin
4. ShET1 and ShET2
5. LPS
Action of Shiga Toxin (A-B Toxin)
• Being released
• Stimulates fluid secretion
• Acts like a neurotoxin
• Causes cell death
• Causes apoptosis of epithelial cells
 Pathogenesis
• Shigella infections are almost always limited to
the gastrointestinal tract; bloodstream invasion is
quite rare.
• Shigellae are highly communicable; the infective
dose is on the order of 103 organisms.
• The essential pathologic process is invasion of
the mucosal epithelial cells (eg, M cells) by
induced phagocytosis, escape from the
phagocytic vacuole, multiplication and spread
within the epithelial cell cytoplasm, and passage
to adjacent cells.
• Microabscesses in the wall of the large intestine
and terminal ileum lead to necrosis of the
mucous membrane, superficial ulceration,
bleeding, and formation of a “pseudomembrane”
on the ulcerated area.
• This consists of fibrin, leukocytes, cell debris, a
necrotic mucous membrane, and bacteria.
• As the process subsides, granulation tissue fills
the ulcers, and scar tissue forms.
 Laboratory Diagnosis
1. Examination of stool
2. Microscopy of stool
3. Culture of stool
- Transport media: Cary-Blair transport
media
- Culture in: MacConkey media (identification
by biochemical tests - sugar fermentation)
4. Confirmation by specific antisera
 Treatment and Prevention
Treatment
• Fluid replacement
• Antibiotics

Control and Prevention

• Hygiene