Enterobacteriaceae family

General characteristics of Enterobacteriaceae

• Gram negative non-spore forming bacilli

• Facultative anaerobes and motile by peritrichous flagella (except

klebsiella and shigella)

• Ferment glucose with acid production ( with or without gas production)

• Catalase positive and oxidase negative

• Reduce nitrates to nitrites

• Many are normal inhabitants of large intestine of man and animals

• Grow readily and rapidly on simple media.

 Gram negative bacilli

Aerobes that do not ferment Facultative anaerobes that

Anaerobes
carbohydrates - Ferment glucose
- Oxidase negative
- Reduce nitrates to nitrites
Pseudomonas, Brucella,
Burkholderia
Enterobacteriaceae family

Lactose fermentation on MacConkey agar

Positive (LF) Negative (NLF)

Escherichia coli Salmonella Edwardisella
Klebsiella Shigella Hafnia
Citrobacter Proteus Serratia
Enterobacter Morgagnella Erwinia
Providentia Yersinia
Further differentiation is based on biochemical reactions and differences in antigenic structure
 Common biochemical tests for identification of enteric bacteria
• Growth on MacConkey agar
• Oxidase test
• Nitrate reduction test
• IMViC test (Indole, Methyl red, Voges Prausker, and Citrate)
• Fermentation of sugars
• Utilization of amino acids
• H2S production, Urease test

API (Analytical profile index) strip test for species identification

BACTERIAL DIARRHEA
 Definitions
 Watery Diarrhea (secretory diarrhea/ Non-inflammatory):
 Passage of abnormally liquid or unformed feces at an increased frequency ( 3 or more
times in 24 hours)
 Small bowel enteritis that disrupts the normal absorption & secretory process of the
small bowel

 Inflammatory diarrhea (Dysentery/ bloody diarrhea):

 Passage of loose, frequent feces containing blood , pus, and mucus

 This indicates inflammation or colonic tissue damage caused by invasion of bacteria

or toxin
 General features
 Associated with ingestion of contaminated food and water

 Similar manifestations despite different causative agents

 Symptoms include nausea, vomiting, diarrhea, abdominal pain, and cramps

 Common etiology of diarrhea
Watery diarrhea (non-inflammatory) Inflammatory diarrhea (dysentery)
 Bacterial  Bacterial
• Preformed enterotoxin • Cytotoxin production
 Staphylococcus aureus  E coli (Enterohemorrhagic)
 Bacillus cereus  Clostridium difficile
 Clostridium perfringens

• Enterotoxin production • Mucosal Invasion

 Enterotoxigenic E coli  Shigella
 Enteroaggregative E coli  Enteroinvasive E coli
 Vibrio cholerae  Campylobacter jejuni
 Viral  Salmonella spp.
 Norwalk virus  Yersinia enterocolitica
 Rotavirus
 Protozoal
 Protozoal  Entamoeba histolytica
 Giardia lamblia
 Cryptosporidium
 Isospora belli
 Escherichia coli
• Named after Escherich, first to describe colon bacillus

• Commensal enteric bacterium normally found in human and animal

intestine, but some strains are pathogenic

• Over 700 antigenic types or serotypes of E. coli have been

recognized based on O, H, and K antigens.

• Only few serotypes actually cause disease & each pathogenic serotype has
specific virulence factors (plasmid or bacteriophage encoded)

• Major cause of gastroenteritis, urinary tract infection ,neonatal

meningitis, respiratory tract infection, wound infection and
septicemia.
 General characteristics

• Family: Enterobacteriaceae

• Gram negative, motile rods

• Catalase positive and oxidase negative

• Lactose fermenting colonies on MacConkey medium

• IMViC test (+ + - -)
 Diarrhea causing E.coli
• Diarrhea-causing E coli are conveniently classified according to their
virulence properties

Each group causes disease by a different mechanism, and the resulting syndromes usually
differ clinically and epidemiologically.
 Virulence Factors
 Virulence factors: Surface Antigens & Toxins
• Surface Antigens
 Lipopolysaccharide (LPS) :
– Lipid A – endotoxic shock (septic shock)
– O polysaccharide - antigenic
 Pili / Fimbriae : colonization factors ( specific types in different strains)
 Capsule (in some strains) : protects against phagocytosis and
antibacterial factors in serum & capsular antigen is called “K” antigen

• Toxins
 LT (heat labile) & ST (heat stable) toxins ( ETEC)
 VT (verocytotoxin or shiga- like toxin) – EHEC
 Enteroaggregative heat stable (EAST) – EAEC
 Enterotoxigenic E.coli (ETEC)
 Common cause of “Traveller's diarrhea” in people visiting developing
countries & diarrhea in children residing in developing countries

 Transmission: consumption of contaminated food and water

 Noninvasive, but toxigenic

 Causes diarrhea resembling cholera
 Virulence factors:
 Specialized fimbriae, Colonization factor antigens ( CFAs)
 Binding of ETEC to intestinal epithelial cells
 Enterotoxins (plasmid coded toxins (LT/ ST)
 Mediates hyper secretion of water and electrolytes by the intestinal epithelial
cells by ADP ribosylation and activation of adenyl cyclase
 Mechanism of action of LT of enterotoxigenic E. coli

Heat labile toxin (LT)

 Complex of polypeptide subunits
 One subunit of A (enzymic action )
 Five subunits of B (binding)

LT resembles cholera toxin

• Heat labile toxin causes activation of adenyl cyclase and increased production cAMP
• Heat stable (ST) toxin causes activation of guanyl cyclase and increased production of cGMP
 Pathogenesis of ETEC diarrhea
Enters through Fecal-oral route transmission

Attaches to intestinal epithelium through CFAs

Enterotoxins (LT and ST) mediate hyper secretion of water and

electrolytes (Na+ , Cl-) into the bowel lumen. resulting in watery
diarrhea

Self limiting diarrhea and resolves in 1-2 days

 Enteropathogenic E.coli (EPEC)
 Causes diarrhea in infants (infantile diarrhea) and
young children
 Noninvasive, Nontoxigenic
 Pathogenesis:
 Adhesion via bundle forming pilus (Bfp)

 Type III secretion system injects Esps (E.coli

secretion proteins) including Intimin, Tir

 Cytoskeleton reorganization & disruption of

micrvilli and formation of pedestal like lesions
(Attachment and Effacement lesion)
Typical lesion “attachment-effacement”
 Malabsorption and diarrhea phenomenon at the brush border (tight
binding & disruption of brush border causes
malabsorption and diarrhea)
 Enteroinvasive E.coli (EIEC)
• Non-toxigenic but invasive strain
• Invade into the tissue and cause inflammatory mediated damage
resulting in bloody diarrhea (resembles shigella dysentery)
• Humans are the only known reservoir and transmits by
consumption of contaminated food and water
• Common in children < 5 years of age in developing countries
• Pathogenesis:
 Invasion of enterocytes in large intestine
 Inhibits protein synthesis and lysis of host cells
 Lateral spread to adjacent cells
 Inflammation, tissue destruction, necrosis and ulceration.
 Passage of blood, mucus & leucocytes (pus cells) in stool
 Pathogenesis of Enteroinvasive E coli
• Entry through specialized M cells
• Transcytosed through M cells into underlying
macrophages
• Inside the macrophage they escape phagosome
and cause apoptosis & also inflammation
causes seperation of tight columnar
junctions and permit entry of large number
of organisms to lamina propria
• Bacteria released from the lysed macrophages
then attach to basolateral surface of the
epithelial cell and invade into the cell mediated
by invasion plasmid antigens (Ipa)
• Multiplies inside and spreads laterally to
adjacent cells resulting cell death and
necrosis of the cell creating ulcer
• All these events leads to passage of loose
stools consisting exudates of blood, mucus,
WBCs (dysentery). Minute mucosal ulcers
are also seen.
 Enterohemorrhagic E.coli (EHEC))

• Most common serotype identified is O157:H7.

• RESERVOIR: Intestine of many animals (cattle)

• Source of infections: Consumption of contaminated animal products

(Meat products such as improperly cooked hamburger, unpasteurized
milk, fresh vegetables)
• More common in developed countries

• Pathogenesis:
• Combination of attachment/effacement (A/E) lesion + cytolytic action of toxin
(shiga like toxin (Stx1& Stx2)
Pathogenesis of EHEC
 Complications of EHEC
• Complications (esp. in young children and elderly)
• Hemorrhagic colitis

• Hemolytic uremic syndrome

• Microangiopathic hemolytic anemia

• Thrombocytopenia

• Acute renal injury & Renal failure

• Receptors for Stx are found on Renal micro vascular endothelial cells

• Binding to vascular endothelium leads to activation of coagulation cascades, formation

of micro thrombi, intravascular hemolysis, and ischemia.

• Results in decrease in renal perfusion, oliguria, hematuria ( hemolysis) and renal failure
 Enteroaggregative E.coli
• Persistent mucus diarrhea in children in
developing countries
Pathogenesis:
• Adherence to intestinal mucosa

• Biofilm formation on enterocyte surface

• Release of toxin

• Induction of proinflammatory response

• Mucosal toxicity and increased intestinal

secretion resulting in mucus diarrhea
 Laboratory diagnosis
• Tests for genus identification:
– Microscopy of the stool sample

– Culture on MacConkey ( Lactose fermenting colonies)

– Biochemical tests including IMViC test ( + + - -)

• Tests for identification of diarrhogenic strains

– Growth on Sorbitol MacConkey agar

• Only EHEC do not ferment sorbitol

– Immunoassays for detection of toxins by using monoclonal antibodies ( ETEC,

EHEC)

– DNA probes and PCR for detection of toxic genes

– Other tests: Serotyping , Sereny’s test for EIEC, Rabbit ileal loop test for ETEC
 Summary of Diarrheogenic E. Coli
group Site Disease Virulence factors Pathogenesis identification
• Colonization factor antigens
Travelers’ (CFAs) encoded on plasmid. • LT  ligated
diarrhea, Hypersecretion of fluids rabbit ileal loop,
Enterotoxigenic E. Coli Small
diarrhea in • two toxins: and electrolytes into gut
(ETEC) intestine LT (heat labile) • DNA probe
malnourished lumen  watery diarrhea.
infant ST (heat stable) • ELISA

Destruction of microvilli
Infant diarrhea
Enteropathogenic E. Coli Small Bundle forming pili   malabsorption, watery Serotyping with
in developing
(EPEC) intestine localized adherence diarrhea (mucoid, no specific antisera.
countries
blood).

• Cytopathic effect
Intracellular multiplication. on HEP-2 cell
Diarrhea in cultures
Extension to adjacent
Enteroinvasive E. Coli Large children (like Invasion of epithelial cell of
epithelial cells  diarrhea • Detection of
(EIEC) intestine bacillary colon
(blood, mucus, pus in virulence marker
dysentery)
stool). antigen (VMA) by
ELISA

Inhibition of protein • Serotyping with

Hemorrhagic
Enterohaemorrhagic E. synthesis, destruction of specific antisera.
colitis,
Coli (EHEC) or intestinal microvilli  • No sorbitol
Large hemolytic Verotoxin (cytotoxic on vero
decrease absorption of fermentation.
Verotoxigenic E. coli intestine uremic cell line tissue culture)
water and electrolytes (like
(VTEC) syndrome
shiga toxin)  bloody • DNA probe
(HUS) • ELISA
diarrhea.

• Bundle forming pili. Adherence to intestinal • adherence to HEP-2

Enteroagreggative E. coli Small
Infant diarrhea • heat stable enteroaggregative mucosa  watery, mucoid tissue culture.
(EAEC) intestine
enterotoxin. diarrhea. • DNA probe.
 Vibrios
• Gram negative curved (comma shaped) rods
• Primarily found in water sources ( surface water & marine water)
• Possess polar flagella
 Vibrio cholerae
• Causative agent of major epidemic and pandemic disease “cholera”

• Gram negative, curved (comma) shaped bacteria

• Polar flagellum , Darting motility

• Catalase + ve & Oxidase +ve

• It grows well in alkaline environment (pH 7.4 -9.6)

• Readily cultivated in varieties of medium

– TCBS (thiosulphate citrate bile salt sucrose) is the selective medium
Classification Scheme of V.cholerae
Mechanism of action of cholera toxin
 Pathogenesis of cholera
• Vibrio cholerae ingested in large numbers (fecal-oral route)

• Sensitive to gastric pH , large dose needed to cause disease unless

patient is achlorhydric or taking antacids

• Colonization of small intestine through Toxin co regulated pilus A

• Produces mucinase (destroys mucosal lining of the intestinal

epithelium) & powerful enterotoxin ( cholera toxin)

• Cholera toxin mediates massive loss of fluid and electrolytes similar to

heat labile toxin (LT) of ETEC ( no damage of enterocytes, no blood or
leukocytes in stool)
 Clinical features of cholera
 Incubation period: 2-3 days

 High infectious dose: >108 CFU

 103 -105 CFU with achlorhydria or hypochlorhydria (lack of or

reduced stomach acid)

 Abrupt onset of vomiting and life-threatening watery diarrhea (15-

20 liters/day)

 Produces characteristic rice-water stools (colorless, odorless, contain

mucus flakes)
 If not treated, death may occur due to hypovolemic shock &
metabolic acidosis
 Laboratory diagnosis of cholera
• Specimens -
- From patients - watery stools and mucous flakes
- From contacts and carriers - rectal swabs
TCBS medium
• Direct Microscopy:
 Gram’s stain: Gram negative curved rods
 Hanging drop: Darting motility
• Transport or Holding media
- Venkataraman Ramakrishnan (VR) medium
• Enrichment media : Alkaline peptone water
• Selective media- TCBS ( Thiosulphate Citrate Bile Sucrose)
- yellow colonies (indicates sucrose fermentation)
• Biochemical reactions
 Oxidase, Utilization of amino acids, Fermentation of sugars
  Slide agglutination test for identification of serogroups and serotypes
 Serogroups or Serovars identification ( O1 & other antiserum)
 Serotype identification(Ogawa, Inaba, Hikojima)

 Differentiation of Classical and El Tor biotypes by biochemical tests

Differences between Classical vibrio Vs El Tor Vibrio

Test Classical vibrio El Tor vibrio
Hemolysis on Blood agar - +
Voges Proskauer(VP) - +
Chick erythrocyte agglutination - +
Polymixin B sensitivity + -
Group IV phage susceptibility + -

El Tor phage V susceptibility - +

 Treatment and prophylaxis
• Treatment
– Replace Fluid and Electrolytes – MOST IMPORTANT
– Antibiotic treatment – will reduce the severity
– Tetracycline is the drug of choice

• Vaccines:
– Whole cell killed vaccine + recombinant B subunit (WC/rBS)
– Live attenuated vaccine
– Efficacy is 50-70% & protection is only for a short period of 3-6 months

• Prevention:
– Good hygiene, Clean & Safe drinking water, and food supply
– Proper sewage treatment
 Vibrio parahemolyticus
• Halophilic vibrios (high requirement of salt )
• Natural habitat : Sea water, marine life

• Morphology: resembles vibrio except

• Do not ferment sucrose
• Tolerates high conc. of salt ( 8-10% Nacl)

• Food poisoning from improperly cooked sea foods( Shell fish, Raw
oysters, Crabs etc)
• Pathogenesis:
 Little is known about its pathogenesis, except that an enterotoxin similar to cholera
toxin is secreted and limited invasion sometimes occurs.
 Self limiting watery diarrhea, fecal leucocytes often present
 Disease can be prevented by proper refrigeration and cooking of seafood.