Gram Negative

Bacteria:

INTRODUCTION
TO

ENTEROBACTERI
ACEAE

Study Outline /
Review
Quick Assessment For Residual Knowledge

GNB Types & Short overview, Position of

Enterobacteriaceae among them, Gut picture.
Enterobacteriaceae History, Definition,
Characteristics,
Classification,
Importance,
Diseases, Antigenic structures & Virulence
factors.
GI
diseases

Terminologies,
Brief
mechanisms of pathogenesis.
Lab Dx Approach for the GI & other disorders
[with emphasis on Enterobacteriaceae].

Categories of GNB
1. Related to Enteric tract:

Enterobacteriaceae Esch coli, Salmonella

Vibrionaceae Vibrio cholerae
Pseudomonadaceae Pseudomonas aeroginosa
Bacteroidaceae Bacteroides fragillis
Helicobactor / Campylobacter

2. Related to Respiratory tract:

Haemophilus, Legionella, Bordetella,

Acinetobacter
Enterobacteriaceae Klebsiella

3. Related to Urogenital tract:

Gardnerella, Haemophilus ducreyi, Acinetobacter

Enterobacteriaceae Proteus, UPEC, C.
granulomatis

4. Related to Nervous System:

Haemophilus, Pseudomonas
Enterobacteriaceae NMEC, Klebsiella, Serratia

Categories of GNB (contd.)

5. Related to Musculoskeletal system:

Pseudomonas, Acinetobacter
Enterobacteriaceae E. coli, Klebsiella, Serratia

6. Related to CVS:

HACEK group (Haemophilus, Actinobacillus

actinomycetemcomitans,
Cardiobacterium
hominis, Eikenella corrodens and Kingella spp.)
Non-HACEK group Pseudomonas,
Enterobacteriaceae (Enterobacter, Citrobacter,
Providentia, Serratia, Proteus, Salmonella, E.
coli)

7. Related to Zoonosis (Animal sources):

Brucella, Francisella, pasteurella,

Enterobacteriaceae Yersinia

Enterobacteriaceae
Scientific
classification

Domain: Bacteria
Phylum: Proteobacteria
Class: Gammaproteobacteria
Order: Enterobacteriales
Family: Enterobacteriaceae
Rahn, 1937

HISTORY
In the 1800s, the first member Serratia
marcescens was discovered by Bizio in 1823 on
a dish of Italian barley (Polenta).
Klebsiella
1880s.

and

Proteus

were

discovered

in

In 1893, Theobald Smith distinguished Lactose

Fermenter (Benign Organisms/Coliforms) and
Non lactose fermenters (Dangerous pathogens).
The third group, Paracolon bacilli (Delayed
lactose fermentation) were defined in 1897.

 In 1937, Otto Rahn first proposed the

name Enterobacteriaceae family with a
single genus, Enterobacter and 112
species.
In 1944, Borman, Stuart & Wheeler
defined the family characteristics.
In 1972, there were 11 genera and 26
species.
In 1985, 22 genera, 69 species.
In 2004, 40 genera, and 200 species.
At present, 47 genera and >200
species.

DEFINITION
Enterobacteriaceae is a complex and
heterogeneous
bacterial
family
consisting of large number of related
genera that are found in the small &
mostly large intestines (colons) of
human, animals & insects, and also in
soil, plants, water, foods & decaying
matters
having
the
following
distinguished [] characteristics:-

CHARACTERISTIC
1. GNB = Gram Negative Bacilli/Rods
S
2. Facultative anaerobes ---- [Cf. Pseudomonas,
Helicobacter pylori]

3. Either Motile (peritrichous flagella) OR nonmotile ------- [Cf. Vibrio, Pseudomonas, H. pylori]
4. Ferment D-glucose [Dextrose] (with production
of Lactic acid gas & others)
5. Oxidase Negative, which means, Lack of
cytochrome C oxidase ------ [Cf. Vibrio,
Pseudomonas]

ONLY Exception: Plesiomonas (formerly

Aeromonas) shigelloides

6.

Characteristics
Reduce Nitrate
[NO ] to Nitrite (NO )
(contd.)
3

(Very
spp.)

few

exceptions,

e.g.

Photorhabdus

--- [Mycobacteria also reduce Nitrate to Nitrite].

7.

Some have capsule (Klebsiella) or slime

layer.
8.
Some ferment Lactose along with or
without other substances.
9. Non spore-forming.
10. Have pili or fimbriae.
11.
Sharing
and
definitive
antigenic
characteristics.

Members of
Enterobacteriaceae
There are 47 genera and >200
species
Numbers are increasing from time to time
1.
2.
3.
4.
5.
6.
7.
8.

Escherichia Esch. coli

Salmonella S. typhi, S. paratyphi.
Shigella
S. dysentery, S. sonni,
Klebsiella
K. pneumonae
Proteus P. mirabilis
Yersinia Y. pestis, Y. enterocolitica
Enterobacter E. cloacae
Citrobacter
C. freundi

Other members:
1.
2.
3.
4.
5.
6.
7.
8.

Serratia S. marcescens
Hafnia H. alvei
Morganella
M. morganii
Calymmatobacterium
C. granulomatis
Erwinia
Pectinobacterium
Edward siella
Plesiomonas etc.

10 species cause 90% of the human

diseases, whereas 23-25 species consist
99% of all Enterobacteriaceae isolates
from human

Shigella with no Flagella (Atrichous)

Escherichia coli with Peritrichous Flagella

Vibrio cholerae with Polar Monotrichous Flagella

Campylobacter jejuni
with Amphitrichous flagella

Helicobacter curved rod with Lophotrichous Flagella

CLASSIFICATION
Traditional Classification:
Based on disease production,
Based on site of the disease
(Clinical classification),
Based on Lactose fermentation,
Coliforms (LF),
Non-coliforms (NLF)

Based of Motility.

NEW (MODERN)
Classification.

On The Basis of Disease

Production
Normal flora OR Opportunistic (potential)
pathogen :
Most of them, e.g. E.coli, Enterobacter,
Citrobacter, Proteus

Primary pathogen:
Salmonella, Shigella, Yersinia,

Clinical Classification
Primarily Intestinal Pathogen
Shigella , Yersinia enterocolitica

Intestinal & Extra-intestinal Pathogen

Salmonella, Esch. coli,

Extra-intestinal PathogenKlebsiella , Proteus, Yersinia pestis

On The Basis of
Lactose Fermentation
Lactose fermenters (LF):
Esch coli, Klebsiella (except K.
rhinoscleromatis), Enterobacter,
Citrobacter,

Late Lactose ferments (LLF) :

Shigella sonni, Providencia, Serratia,

Lactose non-fermenters (NLF):

Salmonella, Shigella (except S. sonni),
Proteus, Klebsiella rhinoscleromatis,
Yersinia

On The Basis of Motility

(Hanging drop, Soft agar)
Motile:
Salmonella, Esch. coli, Proteus

Non-motile:
Shigella, Klebsiella etc.

Motility in Soft (Semi-solid) Agar Media

Left & Right: Motile.

Center: Non-motile

Modern Classification
TRIBES
No
I
II
III
IV
V
VI

NAME
Escherichie
ae
Edwardsiele
ae
Salmonellac
eae
Citrobacter
eae
Klebsiellace
ae
Proteeae

Examples of
Enterobacteriaceae
Escherichia, Shigella
Edwardsiella
Salmonella
Citrobacter
Klebsiella, Enterobacter,
Hafnia, Serratia
Proteus, Morganella,
Providencia

Antigenic
Structures
antigenic structures are

The
used to
differentiate organisms within a genus or
species.
Three major classes of antigens are found:
O (Ohne Hauch) Somatic or cell wall
antigens,
H (Hauch) Flagellar antigens and
K (Kapsel) Capsular (or Virulence)
antigens.

Plays important role in epidemiology,

classification as well as serotyping.

Somatic or Cell Wall [O]

Antigens
Heat stable
Outer component (polysaccharide part or
side chain) of the LPS core of cell wall.
Repeating oligosaccharide (3-5 sugars)
subunits.
One of the major antigenic determinants of
the GN cell wall.
Antibody against O polysaccharide IgM.
Variation causes many antigenic types of
Salmonella & E. coli and perhaps other GNB.
In E. coli & Salmonella, loss of the O Ag
results in partial loss of virulence.

Flageller [H] Antigens

Heat labile.
Constructed from proteins.
Antigenic variation is due to the difference in
Amino Acid sequences.
Antibody against H proteins IgG.
The flagellar filament is composed of a single
protein, Flagellin.
Molecular weights of Flagellin range from 33,000
to 60,000 among different bacterial species.
In some bacterial genera, such as Salmonella,
many varieties of flagellar antigen have been
identified.

Capsular [K or Vi] Antigens

Heat labile or relatively heat stable (??).
Polysaccharides in nature (anti-phagocytic).
Found on the surface of many bacterial
species:

Klebseilla
Certain strain of Esch. Coli
Salmonella typhi (K-like Ag),
certain Citrobacter (K-like Ag) etc

Often constitutes the outermost layer of the

cell.
Boiling for 15 minutes to 1 hour would destroy
the K antigen and unmask O antigens.

K or Vi Antigens (contd.)
Linked to the bacterial
covalent
attachment
phospholipid or lipid-A.

surface via
to
either

Overlay the surface O Ag and may block

agglutination by O specific antisera (antiphagocytic).
May mediate direct interactions between
the
bacterium
and
its
immediate
environment.

Called the Vi (Virulence) antigen in

Salmonella (as well as certain Citrobacter
etc.)
Important virulence factor.

Determinants of
Pathogenicity

Ability to colonize, some colonization factors are:

Adhesins,
Pili or Fimbria
Enzymes: IgA protease, Urease etc.

Invasiveness
Toxins:
Endotoxin (LPS)
Exotoxins (e.g. Enterotoxin)

Motility (Peritrichous flagella) H Ag

Capsule (K or Vi Ag) Resists phagocytosis
Outer polysaccharide O Ag
Sequestration of Nutritional factors especially
Iron,
e.g., by siderophores.

Virulence Factors (contd.)

Antigenic phase variation:
Ability to alternately express or not express
either capsule or flagella
Thus avoid host immune response.

Susceptibility of the host (NOT a virulence

factor):
Gastric acid suppression
Anomalies, circumcision etc.

Other factors:
Enzymes (e.g. Urease, -lactamases)
Resistance plasmid (Drug resistance)
Bile solubility (Bile salt tolerance)
Ability to resist killing inside the cells etc.

Mechanisms inovlved
INVASION
Salmonella, Shigella, Campylobacter,
Escherichia coli, Entamoeba histolytica.

NON-INVASIVE MECHANISM
Toxin production
Vibrio cholerae, ETEC

Decreased absorption
Rotavirus, norwalk agent, giardia lamblia

ENZYMES
Proteus

Diseases Produced
1. GIT: Salmonella, Shigella, Esch. Coli,
Yersinia
2. UTI: Esch. Coli, Proteus, Klebsiella,
Citrobacter, Providentia.
3. Wound infection: Esch. Coli, Proteus,
Klebsiella, Enterobacter.
4. Pneumonia: Esch. Coli, Klebsiella, Proteus.
5. Bacteraemia: Salmonella, Klebsiella,
Esch.
Coli, Proteus.
6. Meningitis: Esch. Coli, Klebsiella,
Salmonella,
Proteus (rare)

Spectrum of GIT
diseases

Diarrhea

- Frequent &/or loose/fluid stool (small

intestine).

Dysentery

- Stool with blood/pus ( fever)

accompanied by pain, abdominal cramps
(inflammatory disorder of large intestine). Or, any
bloody diarrhea.

Gastroenteritis

- Syndrome with GIT symptoms

nausea/vomiting, diarrhea, abdominal discomfort.

Gastritis

- Syndrome with stomach symptoms i.e.

nausea, vomiting, upper abdominal discomfort.

Spectrum of GIT diseases

Enteritis

- Syndrome associated with

inflammation of small intestine.

Enterocolitis

- Syndrome associated
with inflammation of both intestines.

Colitis

- Syndrome associated
inflammation of large intestine.

Food

with

poisoning Syndrome of infection

associated
with
consumption
of
contaminated food - Food associated
infection.

Lab.
Diagnosis
DIARRHEAL
DISEASES
Emphasizing on
ENTEROBACTERIACEA
E

PRINCIPLE
It is based on demonstration of the
causative agent by M/E and isolation
&
identification
by
standard
microbiological techniques and also
by immunological tests and some
special tests. Molecular techniques
are also employed.

STEPS-1:
1. Specimen:
the lesion:

According to the site of

Freshly passed stool or rectal swab, Urine,

Sputum, Blood, Pus, CSF & other body fluids
etc.

2. Microscopic
CRUCIAL

Examination

(M/E):

Saline, Iodine, and Z-N preparation:

For E. histolytica, Giardia, Cryptosporidium.
- Giardia & Cryptosporidium both are acid
fast in
modified Z-N staining.
Pus Cells, Macrophage, RBC:
Helps to detect type of diarrhea

STEPS (contd.) 2:
3. Isolation & Identification (Culture):
Specimen should either be immediately
cultured
OR should be placed in transport
media
(Stuart,
Amies, Cary-Blair etc.) in
case of stool.
OR in case of urine, should be stored at 40C,
if
not inoculated in the media within 30
minutes.
Incubation at 370C aerobically for usual
pathogens & at 420C microaerophilically for
Campylobacter.
Selection of culture media depends on
provisional diagnosis &/or information provided by
the
referring physician. Stool culture is of

STEPS (contd.) 3:
Culture Media:
Mac ConKeys Agar (MA),
Shigella-Salmonella (SS) Agar,
Triple Sugar Iron (TSI) etc.
[]
Thiosulfate Citrate Bile-salts Sucrose (TCBS),
Campy-BA etc.
Additionally For
URINE:
Cystine Lactose Electrolyte Deficient (CLED),
Chromogenic UTI agar (HiCrome) etc.

Depends on Provisional diagnosis

Information provided by the Referring
physician.

STEPS (contd.) 4:
Isolated colonies are identified by:
- Morphology & Growth on the Type of Media
- Staining & M/E,
- Biochemical tests (Fermentation, IMViC etc),
- Motility test,
- Serologic grouping by specific antisera,
- Toxigenicty test,
- Animal inoculation test etc.
Biochemical Test: Accordingly.
Urease production: Urease producing
(Urease positive) bacteria are:
Proteus, Klebsiella, Yersinia
pseudotuberculosis, Helicobacter pylori,
Ureaplasma urealyticum etc.

STEPS (contd.) 5:
Immunological test: Serogrouping by antisera,
Detection of rota viral Ag, C. diffcile toxin from
stool by LAT or other tests.
Special tests:
For ETEC, EPEC, VTEC etc.:
Done in reference Labs.
NAAT (Neucleic Acid Amplification Test):
By PCR (Preferentially Multiplex)

ANY
QUESTION
?????