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Enterobacteriaceae
General Features of Enterobacteria
Present in large intestine
Gram negative bacteria
Aerobic or facultative anaerobic
Motile by peritrichate flagella or non motile
Grow on ordinary media (non fastidious)
Ferments glucose with acid & gas or only acid
Reduce nitrates to nitrites
Catalase + ve & oxidase -ve
Classification of Enterobacteriaceae

Based on lactose fermentation – oldest method :
1.
Lactose fermenters e.g. Escherichia, Klebsiella.
2.
Late lactose fermenters e.g. Shigella sonnei
3.
Non lactose fermenters e.g Salmonella, Shigella
- Commensal intestinal bacteria: Coliform bacilli LF
- Intestinal pathogens: NLF
- Paracolon bacilli
Classification of Enterobacteriaceae

Modern taxonomy – group together bacteria
that possess:
1. Common morphological and biochemical
properties
2. Similar DNA base compositions

Family – Tribe / Group - Genera
Enterobacteriaceae (Tribes & Genera)
CDC 1989
Tribe 1 Eshcherichieae Tribe 2 Klebsielleae Tribe 3 Proteeae
Klebsiella
Escherichia
Proteus
Enterobacter
Shigella
Providentia
Serratia
Edwardsiella
Morganella
Hafnia
Salmonella
Citrobacter
Tribe 4 Erwinieae
Erwinia
Tribe 5 Yersinieae
Yersinia
Escherichia coli

Named after Escherich, first to describe colon
bacillus

Normal flora of the human & animal intestine.

Remains viable in the feces for few days.

Detection of E.coli in the drinking water –
indicates recent pollution with human or animal
feces.
Morphology & Culture
Morphology
- Gram negative straight rod, occasinally capsulated
- Motile by peritrichate flagella
Cultural characters:
- Facultative anaerobe, non fastidious
- Colonies are smooth, translucent,
- BA: haemolytic colonies
- MA: lactose fermenting pink colonies.
Antigenic Structure of Gram –ve Bacteria

Three antigens –
serotyping of E.coli
1. H – flagellar antigen
2. O – somatic antigen
3. K – capsular antigen
Majority do not possess K Ag.
Virulence Factors
- Two types of virulence factors: Surface Ags &
Toxins
1. Surface Antigens
- LPS surface O Ag – endotoxic activity, protects
from phagocytosis and bactericidal effects of
complement
- Envelope or K Ag – protects against
phagocytosis and antibacterial factors inserum
- Fimbriae – colonisation factors, found in strains
causing diarrhoea and urinary tract infections
Virulence Factors
2. Toxins (Exotoxins) – two types
- Enterotoxins – pathogenesis of diarrhoea
3 types :
- LT (heat labile toxin),
- ST (heat stable toxin) &
-
VT (verocytotoxin or shiga- like toxin)
- Hemolysins – may be nephrotoxic
Heat Labile Toxin (LT)
Resembles cholera toxin in its
structure, function and mode of
action
Complex of polypeptide subunits.
one subunit of A (action- enzymic),
five subunits of B (binding)
Heat Labile Toxin (LT)
Escherichia coli /
Vibrio cholerae
Gut lumen
Intestinal
epithelial cell
E.coli toxins
Heat Labile Toxin (LT)
Heat Stable Toxin (ST)
Activates Adenyl cyclase
Activates guanyl cyclase
increased production of
cAMP
Increased secretion of Na,
Cl and water from the
cell
Increased production of
cGMP
Inhibition of ionic uptake
in intestinal cells
Osmotic loss of water
from cells
Verocytotoxin (VT)
So named because of cytotoxic effect on Vero
cell lines
Similar to Shigella dysenteriae type 1 toxin in
its physical, antigenic and biological properties
VT gene is phage coded.
Pathogenicity/ Clinical Infection
1.
Urinary tract infection
2.
Diarrhoea
3.
Pyogenic infections
-
Wound infection, especially after surgery of lower
intestinal tract.
-
Peritonitis.
-
Biliary tract infection.
-
Neonatal meningitis.
Septicemia – can lead to fatal conditions like
4.
-
Septic shock
-
Systemic Inflammatory Response Syndrome
Lab Diagnosis of UTI
Specimens
Urine Mid stream urine (MSU)
Catheter specimen urine (CSU)
Supra pubic aspiration (SPA)
Microscopy
Wet mount
Pus cells / hpf
Bacteria / crystals/ casts
Gram stain
Gram negative bacteria
(1bacteria / oil field is significant)
Urine Culture
Kass semi-qauntative method
Standard loop technique
To know significant bacteriuria
Lab Diagnosis of E. coli UTI
Significant bacteriuria > 105 organism / ml of MSU
Culture
BA / MAC : LF (flat)
Identification tests
I M Vi C test: + + - TSI agar
AST
Acid, no gas
Diarrheagenic E.coli
 Enteropathogenic E.coli (EPEC)
 Enterotoxigenic E.coli (ETEC)
 Enteroinvasive E.coli (EIEC)
 Enterohemorrhagic E.coli (EHEC) or Verotoxigenic
E.coli (VTEC)
 Enteroaggregative E.coli (EAEC) : “stacked brick”
appearance.
 Diffusely adherent E.coli (DAEC)
Enteropathogenic E.coli (EPEC)
Infantile diarrhea
Institutional outbreaks
Noninvasive, nontoxigenic
Pathogenesis – adhesion via fimbriae, to the
enterocyte membrane & disruption of brush border
microvilli
Clinical features – fever, diarrhea, vomiting, nausea,
non bloody stools
Lab Diagnosis – testing colonies grown on BA/ MA
with EPEC O antisera
Enterotoxigenic E.coli (ETEC)
Traveller’s diarrhea: From developed to endemic areas
Resembles cholera
Noninvasive, toxigenic
Pathogenesis – production of plasmid coded toxins
(LT/ ST) along with colonisation factor Ags (CFA)
Clinical features - Diarrhea, vomiting and abdominal
pain
Lab Diagnosis – demonstration of enterotoxin by in
vitro or in vivo methods, detection of LT/ St by gene
probes
Demonstration of ETEC toxins
Assay
In vivo tests
LT
ST
Ligated rabbit illeal loop
Infant rabbit bowel
+
+
+
Infant mouse intragastric
-
+
Adult rabbit skin (Vascular permeability
factor)
In vitro tests
Tissue culture tests
Rounding of Y1 mouse adrenal cells
ELISA
Passive agglutination tests
+
-
Enteroinvasive E.coli (EIEC)

Bloody diarrhea (dysentery), resembles Shigella
dysentery

Passage of blood, mucus & leucocytes in stool

Pathogenesis - Invades epithelial cells by endocytosis
and can spread laterally to adjacent cells, causes
tissue destruction, necrosis and ulceration.

Lab Diagnosis:
1. Sereny test- instillation of suspension of freshly
isolated EIEC or Shigella in the eyes of guinea pig –
mucopurulent conjunctivitis and severe keratitis
2. Penetration of HeLa or Hep2 cells in tissue culture
Enterohemorrhagic E.coli (EHEC)

Produces verocytotoxin (VT), a shiga-like toxin (SLT);
hence also known as Verocytotoxigenic E.coli (VTEC)

Pathogenesis – EHEC attaches to the colonic mucosa
and releases VT. VT targets vascular endothelial
cells, inhibits protein synthesis - cytotoxicity

Clinical features - Mild diarrhea (bloody) to fatal
complications (esp. in young children and elderly):
1. Hemorrhagic colitis – destruction of mucosa
followed by hemorrhage.
Enterohemorrhagic E.coli (EHEC)
2. Hemolytic Uremic syndrome – triad of acute renal
failure, hemolytic anemia and thrombocytopenia.

Serotype O157: H7 is most commonly involved.

Outbreaks of food poisonings (fast foods, contaminated
hamburgers)
Enterohemorrhagic E.coli (EHEC)

Lab Diagnosis:
1. Demonstration of bacilli or VT in feces or in
culture
2. Sorbitol MacConkey agar for O157:H7 – does
not ferment sorbitol unlike other E.coli
3. Cytotoxic effects on Vero or HeLa cells
4. DNA probes to detect toxins
Enteroaggregative E.coli (EAEC)
Persistent diarrhea in children in developing
countries.
Aggregate to give a “Stacked brick appearance” on
Hep2 cells or glass (due to fimbria)
Pathogenesis – shortening of villi, mucus biofilm,
heat stable cytotoxin (hemorrhagic necrosis and
edema)
Klebsiella
Normal gut flora in the intestine
Gram negative coccobacilli (short & plump)
Capsulated, non-motile, Mucoid LF colonies on MAC
Species
K. pneumoniae
K. oxytoca
K. ozaenae
Pneumonia, Urinary tract infections
Atrophic rhinits
K. rhinoscleromatis Rhinoscleroma
Pathogenicity of Klebsiella pneumoniae

Pulmonary infections - Pneumonia (lobar):
1. High fatality
2. In middle aged or older persons with medical problems
like DM, alcoholism, chronic bronchopulmonary disease
3. Extensive necrosis & hemorrhage resulting in thick,
mucoid, brick red sputum “currant jelly like”

Extrapulmonary infections –
1. Meningitis & enteritis in infants
2. UTI
3. Septicemia

An important cause of nosocomial infections.
Lab Diagnosis - Klebsiella
Specimens
Urine, sputum, nasal secretions /
swab, blood
Culture
BA / MAC : LF (mucoid)
Identification tests
I M Vi C test: - - + +
TSI agar
Urease
Acid with gas
Positive
Proteus
Normal gut flora in the intestine
Gram negative bacilli, pleomorphic
Motile, Non lactose fermenter NLF on MAC
Swarms on BA, Urease +, H2S +
Species
P mirabilis, P vulgaris
UTI
Pneumonia Wound infections
Urease converts urea to NH4 & CO2 causing alkalinization of
urine leading to renal calculi (stones)
Proteus antigens are used in the Weil - Felix test to diagnose
Rickettsial diseases
Lab Diagnosis - Proteus
Specimens
Urine, sputum, wound swab
Culture
BA: swarming
MAC : NLF, fishy/ seminal smell
Identification tests
Indole: PM - / PV +
TSI agar
K / A (H2S)
Urease +
Enterobacter, Serratia, Citrobacter
Moist environments in hospitals – common
reservoirs.
Pathogenicity –
- UTI,
- Wound & respiratory infections in hospitalized
patients,
- Outbreaks in ICUs, burn units & other special units