Enterobacteriaceae lecture noteCopy.pptx

Enterobacteriaceae
BY
Dr SB Zailani
CLINICAL MICROBIOLOGIST
UNIVERSITY OF MAIDUGURI
November 17, 2024 1

OUTLINE
• Introduction
• Classification
• Morphology
• Properties
• Epidemiology
• Diseases
Pathogenesis & treatment
• Laboratory Identification/Biochemical Tests
• Prevention & control
• Conclusion
November 17, 2024 2

Introduction
• The Enterobacteriaceae are a large family of
bacteria that are Gram negative, rod shaped
(1-5μm in length), oxidase negative, catalase
positive, nitrate-reducing, facultative
anaerobes, fermenting sugars to produce
lactic acid and various other end products,
that live in mammalian Gastrointestinal tracts
November 17, 2024 3

Scientific Classification
• Kingdom: Bacteria
• Phylum: Protobacteria
• Class: Gamma Proteobacteria
• Order: Enterobacteriales
• Family: Enterobacteriaceae
Rahn, 1937
• Over 30 genera and 170 species with more species being named
every year.
• >95% of the clinically significant strains fall into 10 genera and
about 25 species
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Family Enterobacteriaceae
• Escherichia coli
Opportunistic Escherichia coli
ETEC = enterotoxigenic E. coli
EIEC = enteroinvasive E. coli
EPEC = enteropathogenic E. coli
EHEC = enterohemorrhagic E. coli
EaggEC = enteroaggregative E. coli
UPEC = uropathogenic E. coli
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Citrobacter
Citrobacter freundii
Citrobacter diversus
Enterobacter spp
Enterobacter aerogenes
Enterobacter agglomerans
Enterobacter cloacae
Morganella morganii
Klebsiella Spp
Klebsiella pneumoniae
k.ozaena
k.rhinoscleromatis
Klebsiella oxytoca
klebsiella granulomatis
Proteus spp
Proteus mirabilis
Proteus vulgaris
Providencia spp
Providencia alcalifaciens
Providencia rettgeri
Providencia stuartii
Serratia spp
Serratia marcesans
Serratia liquifaciens
Shigella spp
Shigella dysenteriae
Shigella flexneri
Shigella boydii
Shigella sonnei
Yersinia spp
Yersinia enterocolitica
Yersinia pestis
Yersinia pseudotuberculosis
Salmonella spp
Salmonella
Salmonella enterica
Salmonella typhi
Salmonella paratyphi
Salmonella enteritidis
Salmonella cholerae-suis
Salmonella typhimurium
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Others
• Edwardsiella
• Hafnia
• Erwinia
• Pectinobacterium
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MORPHOLOGY
• (A)Flagella
Enterobacteriaceae are motile via peritrichous
flagella except Shigella and Klebsiella which are non-
motile.
• Constitute the H antigens (H = Hauch,German for breath)
-used for typing.
-Non motile organisms lack flagellar H antigens.
• The flagella are maximal in young cultures
November 17, 2024 8

(B)Capsule
• The capsule is a thin layer of surface polysaccharide.
• Constitutes the K antigen (K= Kapseal, German for
capsule).
• Formation enhanced by sugar-containing media.
• Capsules are particularly heavy in Klebsiella, it forms
mucoid colonies.
• Capsule -interfere with O agglutination
-inhibits phagocytosis
-may cross react with capsular
antigens of other bacteria.
November 17, 2024 9

(C) Fimbriae (Pili)
• Hair-like projections of protein on cell surface
• Promote adhesion and are developed in old (24-48
hours) broth cultures.
• The ability to agglutinate red cells, inhibition by
mannose and the width differentiate the types.
• Fimbriae may interfere with H agglutination.
• They are preserved by formalin and destroyed by
heat
November 17, 2024 10

(D) Cell Wall
• Consists of an outer layer and an inner layers
Outer layer
• Is a lipopolysaccharide (LPS) complex
• The side chains of repeating sugar units project from the
outer LPS layer constituting the O (somatic) antigen (O= Ohne,
German for without).
-used for typing
-Smooth colonies
-Resistance to killing by complement.
-Absence, is associated with rough colonies,
autoagglutination, non-virulence and killing by complement

Cont’d
• Core glycolipids form the basal layer to
which side chains are attached (The
enterobacterial common antigen)
• Phospholipid membrane
• Proteins (outer membrane proteins, OMPs)
are present in the phospholipids membrane.
They include those responsible for solute
transport (‘porins’) and structural
lipoproteins.

Cont’d
• Lipid A forms a layer between the
lipopolysaccharide and phospholipids.
Is the toxic moiety of ‘endotoxin’.
Biologic effects;
• induction of host febrile response by production
of IL-1 and prostaglandins,
• activation of complement,
• induction of interferon production,
• production of tumour necrosis factor,
• production of colony stimulating factor
• activity as a B-cell mitogen.

Cont’d
Inner layer of cell wall
• Consists of peptidoglycan that maintains
the cell wall rigidity

Antigens
• Somatic O antigens- these are heat stable polysaccharide
part of LPS
Variation from smooth to rough
colonial forms is accompanied by
progressive loss of smooth O Antigen
• Flagellar H antigens-heat labile
• Envelope /capsule K antigen-overlay the surface O antigen
and may block agglutination by O specific antisera
 Boiling for 15min destroy K antigen and unmask
O antigen
 K antigen is called Vi antigen in salmonella

Properties1
– Gram-negative, non-sporing, rod shaped bacteria
– Oxidase –ve, facultative anaerobes
– Ferment glucose aerogenically or anaerogenically
– Reduce nitrate to nitrite
– Many are normal inhabitants of the intestinal tract of man
and other animals
– Some are enteric pathogens and others are urinary or
respiratory tract pathogens
– Differentiation is based on biochemical reactions and
differences in antigenic structure

Properties2
– Most grow well on a variety of lab media including
a lot of selective and differential media originally
developed for the the selective isolation of enteric
pathogens.
• Most of this media is selective by incorporation of dyes
and bile salts that inhibit G+ organisms and may
suppress the growth of nonpathogenic species of
Enterobacteriaceae.
• Many are differential on the basis of whether or not the
organisms ferment lactose and/or produce H2S.

Properties3 - colonial morphology
– They all produce similar colonies usually moist, dull grey (except
Serratia marcescens which appears red) or mucoid colonies on
blood agar (Klebsiella) and smooth colonies on non-selective
media.
– They may or may not be hemolytic
– Colonies appearing as thin films or as waves (swarming) suggest
motility (Proteus)
– Colonies that appear red on McConkey agar or have a green
sheen on eosin methylene blue (EMB) agar indicate acid
production from lactose
– The three most useful media for screening stool cultures for
potential pathogens are TSI, kIA, and urea or phenylalanine agar.

Epidemiology
Habitat
• Normal intestinal microbial flora from birth,
present in the intestines of humans and other
animals, fish, insects, as well as in plants, soil and
water, decaying matter, or parasites on a variety
of different animals and plants
Prevalence
• Enterics are responsible for a majority of
nosocomial infections

Escherichia
Escherichia coli
• Normal inhabitant of the
G.I.T tract
• Some strains cause various
forms of gastroenteritis
• Is a major cause of urinary
tract infection and neonatal
meningitis and septicemia
• May have a capsule
• Most are motile

E. coli
– Antigenic structure - has O, H, and K antigens; K1 has a strong
association with virulence, particularly meningitis in neonates
– Virulence factors
(A)Toxins
-Enterotoxin (ETEC) LT&ST
-Enteroaggregative toxin ST- like toxin
-Haemolysin ;cell bound or secreted (lyse
RBC& leucocyte ,inhibits phagocytosis when
cell bound)
-Endotoxin
-EHEC (Shiga-like toxin);verotoxin: cytotoxic, enterotoxic
&neurotoxic inhibits protein synthesis

E. coli toxins

LT vs ST activity

(B)Type III secretion system to deliver effector
molecules directly into host cells
-Involved in uptake of EIEC into intestinal cells
-Involved in development of attachment &
effacing lesions in EPEC xterised by villi
destruction & pedastal formation
(C)Adhesion; colonization factors includes pilli or
fimbrae & non-fimbraefactors e.g. intimin

(D) Virulence factors that prevent host from
defence
-Capsule
-Iron capturing ability(enterochellin)
(E) Outer membrane proteins; Help the
organism to invade by helping in attachment &
initiatng endocytosis

E. coli toxins
» Both enterotoxins are composed of five beta
» subunits (for binding) and 1 alpha subunit
» (has the toxic enzymatic activity).

Type III secretion system

Pedestal formation

Types of adhesions
(intimin)

E. Coli
 Urinary Tract Infections
Commonly caused by E.coli uropathogenic strain
Host factors & organism factors allow seemingly
‘normal’ individuals to develop UTIs
 New evidence in women who suffer from
recurrent UTIs suggests that this is due to the
formation of pod-like E. coli biofilms inside
bladder epithelial cells

Ascending urinary tract infection

Pod-like biofilm

E. coli
Neonatal meningitis
E.coli strains with the K1 capsular antigen is
the leading cause it is associated with
septicaemia and has high mortality
Gastroenteritis
Several distinct types are involved and result
in different patterns of gastroenteritis

Various types of E. coli

– ETEC – is a common cause of traveler’s diarrhea and diarrhea in children
in developing countries.
– The organism attaches to the intestinal mucosa via colonization factors
and then liberates enterotoxin.
– The disease is characterized by a watery diarrhea, nausea, abdominal
cramps and low-grade fever for 1-5 days.
– Transmission is via contaminated food or water

• EPEC – Bundle forming pili are involved in attachment to the
intestinal mucosa.
• The type III secretion system inserts the tir (translocated
intimin receptor) into target cells, and intimate attachment
of the non-fimbrial adhesion called intimin to tir occurs.
• Host cell kinases activated to phosphorylate tir which then
causes a reorganization of host cytoskeletal elements
resulting in pedestal formation and development of an
attaching and effacing lesion
• The exact mode of pathogenesis is unclear, but it is probably
due to the attachment and effacement.
• Diarrhea with copius mucous without blood or pus along
with vomiting, malaise and low grade fever occurs

BFP
EPEC
EPEC
EPEC

Pedestal formation

EPEC
Tir injected

• EIEC – The organism attaches to the intestinal mucosa via pili and
outer membrane proteins are involved in direct penetration,
invasion and destruction of the intestinal mucosa.
• There is lateral movement of the organism from one cell to
adjacent cells.
• Symptoms include fever, severe abdominal cramps, malaise, and
watery diarrhea followed by scanty stools containing blood,
mucous, and pus.
• EAEC – Mucous associated autoagglutinins cause aggregation of
the bacteria at the cell surface and result in the formation of a
mucous biofilm.
• The organisms attach via pili and liberate a cytotoxin distinct
from, but similar to the ST and LT enterotoxins liberated by ETEC.
• Symptoms include watery diarrhea,vomiting, dehydration and
occasional abdominal pain.

• EHEC – The organism attaches via pili to the intestinal mucosa and
liberates the shiga-like toxin.
• The symptoms start with a watery diarrhea,progresses to bloody
diarrhea without pus and crampy abdominal pain with a low-grade
fever.
• This may progress to hemolytic-uremic syndrome that
• Its most often caused by serotypes O157:H7.
• This strain of E. coli can be differentiated from other strains of E. coli
by the fact that it does not ferment sorbitol in 48 hours (other
strains do).
• A sorbitol-Mac (SMAC) plate (contains sorbitol instead of lactose) is
used to selectively isolate this organism.
• One must confirm the isolate as E. coli O157:H7 using serological
testing, also confirm production of the shiga-like toxin
• Serotypes of E. coli other than O157H7 have now been found to
cause this disease

Summary of E.coli strains that cause
gastroenteritis.

E.coli
– Antimicrobic therapy- E. coli is usually susceptible to a
variety of chemotherapeutic agents, though drug resistant
strains are increasingly prevalent.
– It is essential to do susceptibility testing.
– Treatment of patients with EHEC infections is not
recommended because it can increase the release of shiga-
like toxins and actually trigger HUS

Shigella
(dysentery bacterium )
Shigella
Contains four species that
differ antigenically and, to a
lesser extent, biochemically.
S. dysenteriae (Group A)
12 serotypes
S. flexneri (Group B)
6 serotypes
S. boydii (Group C)
18 serotypes
S. sonnei (Group D)
1 serotype

Shigella species
Antigenic structure
• Differentiation into groups (A, B, C, and D) is based on O
antigen serotyping; K antigens may interfere with serotyping,
but are heat labile.
• O antigen is similar to E. coli, so it is important to ID as
Shigella before doing serotyping.
• Virulence factors
-Shiga toxin – is produced by S. dysenteriae and in smaller
amounts by S. flexneri and S. sonnei.
-Acts to inhibit protein synthesis by inactivating the 60S
ribosomal subunit by cleaving a glycosidic bond in the 28S
rRNA constituents.
-This plays a role in the ulceration of the intestinal mucosa.

Shigella
• Outer membrane and secreted proteins
• These proteins are expressed at body temperature and upon
contact with M cells in the intestinal mucosa they induce
phagocytosis of the bacteria into vacuoles.
• Shigella destroy the vacuoles to escape into the cytoplasm.
• From there they spread laterally (Polymerization of actin
filaments propels them through the cytoplasm.) to epithelial
cells where they multiply but do not usually disseminate
beyond the epithelium

Shigella attachment and penetration

Shigella attachment

Shigella penetration

Shigella invasion continued

Clinical significance
• Causes shigellosis or bacillary dysentery.
• Transmission is via the fecal-oral route.
• The infective dose required to cause infection is very low (10-200
organisms).
• There is an incubation of 1-7 days followed by fever, cramping,
abdominal pain, and watery diarrhea (due to the toxin)for 1-3
days.
• This may be followed by frequent, scant stools with blood,
mucous, and pus (due to invasion of intestinal mucosa).
• It is rare for the organism to disseminate.
• The severity of the disease depends upon the species one is
infected with.
• S. dysenteria is the most pathogenic followed by S. flexneri, S.
sonnei and S. boydii.

Shigella
– Antimicrobial therapy
• Sulfonamides are commonly used as are streptomycin,
tetracycline, ampicillin, and chloramphenicol.
• Resistant strains are becoming increasingly common, so
sensitivity testing is required.

Salmonella

Salmonella
• Classification has been changing in the last few years.
• There is now 1 species: S. enteritica, and 7 subspecies: 1,
2 ,3a ,3b ,4 ,5, and 6.
• Subgroup 1 causes most human infections
• Clinically Salmonella isolates are often still reported out as
serogroups or serotypes based on the Kauffman-White scheme
of classification.
• Based on O and H (flagella) antigens
• The H antigens occur in two phases; 1 and 2 and only 1 phase is
expressed at a given time.
• Polyvalent antisera is used followed by group specific antisera (A,
B, C1, C2, D, and E)
• Salmonella typhi also has a Vi antigen which is a capsular antigen

Salmonella virulence factors
• Capsule (for S. typhi and some strains of S. paratyphi)
• Adhesions – both fimbrial and non-fimbrial
• Type III secretion systems and effector molecules – 2 different
systems ;One promotes entry into intestinal epithelial cells
&The other type aids in survival of Salmonella inside
macrophages
• Outer membrane proteins - involved in the ability of
Salmonella to survive inside macrophages
• Flagella – help bacteria to move through intestinal mucous
• Enterotoxin - may be involved in gastroenteritis
• Iron capturing ability

Salmonella
– Clinical Significance – causes two different kinds of
disease: enteric fevers and gastroenteritis.
– Both types of disease begin in the same way, but
with the gastroenteritis the bacteria remains
restricted to the intestine and with the enteric fevers,
the organism spreads
– Transmission is via a fecal-oral route, i.e., via
ingestion of contaminated food or water

Salmonella
• The organism moves through the intestinal mucosa and adheres
to intestinal epithelium.
• Effector proteins of the type III secretion system mediate
invasion of enterocytes and M cells via an induced endocytic
mechanism.
• Salmonella multiplies within the endosome which moves to the
basal side of the cell and is released for phagocytoses by
macrophages
• In gastroenteritis inflammatory response mounted prevents the
spread beyond the GI tract and eventually kills the bacteria
• In enteric fevers the Salmonella disseminate before the
inflammatory response

Salmonella invasion of epithelial cells

Hemorrhage;
perforation
Cholecystitis;
Carrier state
Fever;
Relatively slow
pulse;
Enlarged liver
And spleen;
Rose spots;
Normal or low
WBC count
Inflammation and
ulceration of
Peyer’s patches
gallbladder
Small
intestine
Mesenteric
Lymph nodes
Transient
(primary)
bacteremia
Multiplication
in macrophages
in
liver,
spleen,
kidney,and
bone marrow
Septicemia
(second)
Ingestion of S. typhi
Bile
Bile
Thoracic duct
Lymph
nodes
Signs and
symptoms
Incubation
period
Fever,
malaise

Salmonella
• Diagnosis
• Blood cultures positive during 1st(75-90%)
& 3rd(30%)
week
• Stool culture positive during the 3rd
(80%) & 2nd
(40-50%)
week
• Urine culture positive during the 2nd
(25%) week
• Widal test, 4-fold rise in titer between acute and convalescent
stages
• 10% become short-term carriers & few % long-term carriers
due to persistence of bacteria in the Gall/Urinary bladder

Salmonella
– Antimicrobial therapy
• Enteric fevers – use chloramphenicol usually. Resistant
strains have emerged making antimicrobial
susceptibility testing essential.
• Gastroenteritis – usually doesn’t require antimicrobic
therapy.
– Replace lost fluids and electrolytes.

Comparison of Shigella versus
Salmonella invasion
Shigella Salmonella

Enterobacteriacea
• KLEBSIELLA
– NF of the GIT and respiratory tract, but potential pathogen
elsewhere
– Non- motile ,capsulated
– Has both O and K antigens
– Virulence factors: -capsule
-Adhesions
- Fe capturing ability
– Causes pneumonia, mostly in immunocompromised
– A major cause of nosocomial infections; UTI,
pneumonia ,septicaemia & meningitis
– Rhinoscleroma (granuloma of nose & pharynx), ozena
(progressive atrophy of nasal mucosa) &chronic genital
ulcerative disease are other manifestations

Enterobacteriaceae
• Citrobacter
• Are opportunistic pathogens causing urinary
tract, respiratory tract infections and
occasionally wound infections, osteomyelitis,
endocarditis, and meningitis
• Edwardsiella tarda
–Causes GI disease in tropical and subtropical
countries

Enterobacteriaceae
• Enterobacter
-NF of GI tract
-Motile ,capsulated
-Produces mucoid colonies
-Lactose fermenting
-Incriminated in Nosocomial infections; UTI, wound
& device
-Bacteremia in burns patients
-Possess ampC a xsomal ß-lactamase
-Most are resistant to ampicillin, 1st
& 2nd
generation
cephalosporins

Enterobacteriaceae
• Proteus, Providencia, and Morganella
• All are NF of the GIT
• Motile, with proteus swarming
• Virulence factors
-Urease; the ammonia produced may
damage the epithelial cells of the UT
• UT infections, as well as pneumonia,
septicemia, and wound infections

Enterobacteriaceae
Biochemical Reactions

IMViC Test
• Indole, Methyl Red, Voges-Prosakauer, Citrate
(IMViC) Tests:
– The following four tests comprise a series of
important determinations that are collectively
called the IMViC series of reactions
– The IMViC series of reactions allows for the
differentiation of the various members of
Enterobacteriaceae.

IMViC: Indole test
 Principle
 Certain microorganisms can metabolize
tryptophan by tryptophanase
 The enzymatic degradation leads to the
formation of pyruvic acid, indole and ammonia
 The presence of indole is detected by addition
of Kovac's reagent.
Tryptophan
amino acids
Tryptophanase
Indole + Pyurvic acid + NH3
Kovac’s Reagent
Red color in upper organic layer`

IMViC: Indole test
 Method:
 Inoculate tryptone water with the tested
microorganism
 Incubate at 37°C for 24 hours
 After incubation interval, add 1 ml
Kovacs reagent, shake the tube gently and read
immediately

IMViC: Indole test
 Result:
 A bright pink color in the top layer
indicates the presence of indole
 The absence of color means that
indole was not produced i.e.
indole is negative
 Special Features:
 Used in the differentiation of genera
and species. e.g. E. coli (+) from
Klebsiella (-).
Positive test
e.g. E. coli
Negative test
e.g. Klebsiella

IMViC test: MR/VP test
Results
Methyl Red test Voges-Proskauer test
Red: Positive MR (E. coli)
Yellow or orange: Negative MR (Klebsiella)
Pink: Positive VP (Klebsiella)
No pink: Negative VP (E. coli)

Citrate Utilization Test
 Incubate at 37°C for 24 hours.
Method
 Streak a Simmon's Citrate agar slant with
the organism

Citrate Utilization Test
Examine for growth (+)
Growth on the medium is
accompanied by a rise in
pH to change the
medium from its initial
green color to deep blue
Result
Positive
Klebsiella, Enterobacter
Negative
E. coli

Urease Test
 Urea agar contains urea and phenol red
 Urease is an enzyme that catalyzes the conversion of urea
to CO2 and NH3
 Ammonia combines with water to produce ammonium
hydroxide, a strong base which ↑ pH of the medium.
 ↑ in the pH causes phenol red to turn to deep pink. This is
indicative of a positive reaction for urease
Urea
Urease
CO2 + NH3
H2O
NH4 OH ↑ in pH
Phenol Red
Pink
Positive test
 Streak a urea agar tube with the organism
 incubate at 37°C for 24 h
Method
Principle

Urease Test
• If color of medium turns
from yellow to pink
indicates positive test.
• Proteus give positive
reaction after 4 h while
Klebsiella and Enterobacter
gave positive results after
24 h
Result
Positive test Negative test

Reaction on Triple Sugar Iron (TSI) Agar
• TSI contains
– Three different types of sugars
• Glucose (1 part)
• Lactose (10 part)
• Sucrose (10 part)
– Phenol red (acidic: Yellow)
• TSI dispensed in tubes with equal butt & slant
• Principle
– To determine the ability of an organism to attack a specific carbohydrate
incorporated into a basal growth medium, with or without the production
of gas, along with the determination of possible hydrogen sulphide
production.

Reaction on TSI
• Method:
– Inoculate TSI medium with an organism by
inoculating needle by stabbing the butt and
streaking the slant
– Incubate at 37°C for 24 hours

Typical reactions are as follows:
• Alkaline slant/Alkaline butt
• No carbohydrate fermentation
• Non fermenters
• E.g. Pseudomonas aeruginosa
• Alkaline slant/ Acid butt
• Glucose fermented
• Lactose (or sucrose for TSIA) not fermented
• Non-lactose-fermenting bacteria
• E.g. Shigella spp

• Alkaline slant/Acid (Black) butt
Glucose fermented
Lactose not fermented
H2S produced
Non-lactose fermenting- H2S producing bacteria
E.g. Salmonella spp, Citrobacter spp and Proteus spp
• Acid slant/ Acid butt
Glucose and Lactose (Sucrose for TSIA) fermented
Lactose fermenting coliforms
E.g. E. coli and the Klebsiella-Enterobacter spp.

Example
Result
Reaction on TSI
H2
S Slant
color
Butt
color
Non fermenter
e.g.
Pseudomonas
Alk/Alk/-
(No action on sugars)
Negative Red Red
LNF
e.g. Shigella
A/Alk/-
(Glucose fermented
without H2
S)
Negative
Red Yellow
LNF
e.g. Salmonella &
Proteus
A/Alk/+
(Glucose fermented
with H2
S)
Positive
black in
butt Red Yellow
LF
e.g. E. coli,
Klebsiella,
Enterobacter
A/A/-
(three sugars are
fermented)
Negative
Yellow Yellow
Result

EMB SS MacCon
key
O/F Nitrate
reductase
Oxidase Gram
stain
Metallic
sheen
LF LF O+/F+ +ve -ve -ve rod E. coli
Dark LF LF O+/F+ +ve -ve -ve rods Citrobacter
Dark LF LF O+/F+ +ve -ve -ve rods Klebsiella
Dark LF LF O+/F+ +ve -ve -ve rods Enterobacter
Colorless NLF/
H2S
NLF O+/F+ +ve -ve -ve rods Salmonella
Colorless NLF NLF O+/F+ +ve -ve -ve rods Shigella
Colorless NLF/
H2S
NLF O+/F+ +ve -ve -ve rods Proteus
Summary of morphology, cultural characteristics,
and biochemical reactions of Enterobacteriaceae

Motility Urease Citrate VP MR Indole TSI
Motile -ve -ve -ve +ve +ve A/A/- E. coli
Motile -ve +ve -ve +ve +ve A/A/- Citrobacter
freundii
Non
motile
+ve +ve +ve -ve -ve A/A/- Klebsiella
pneumoniae
Motile +ve +ve +ve -ve -ve A/A/- Enterobacter
cloacae
Motile -ve +ve -ve +ve -ve A/Alk/+ Salmonella
typhi
Non
motile
-ve -ve -ve +ve -ve A/Alk/- Shigella
boydii
Motile
Swarwing
+ve +ve -ve +ve -ve A/Alk/+ Proteus
mirabilis
Summary of morphology, cultural characteristics,
and biochemical reactions of Enterobacteriaceae

Oxidase Test
Positive
Negative
Pseudomonas
Enterobacteriaceae
 Nitrate test: +ve further
reduction to N2
 Growth on cetrimide agar
Pale colonies with green
pigmentation
MacConkey’s agar
& TSI
Lactose fermenter
Pink colonies on MacConkey
& acidic butt and slant on TSI
colorless colonies on MacConkey
& acidic butt alkaline slant onTSI
Lactose non-fermenter
IMViC test
& EMB
IMViC
++ - -
& black
colonies with
metalic shines
on EMB
E.coli
IMViC
- - ++
No H2S production
(no blacking in TSI)
H2S production
(blacking in TSI)
Shigella
Urease production
+ve
Proteus
-ve
SS agar
colorless colonies with black centers
Salmonella
 O/F test: O+
/F-
Motility
Not motile Motile

Prevention and control
There control depends on;
• Hand washing,
• Asepsis, sterilisation and disinfection,
• Minimal IV therapy
• Strict precautions in keeping the urinary tract
sterile (closed drainage)
• Proper handling and processing of foods and
animal products e.t.c

You have been a great
audience

Campylobacter &
Helicobacter
rRNA Superfamily VI of Class Proteobacteria

 Gram-negative
 Helical (spiral or curved) morphology; Tend to be
pleomorphic
 Characteristics that facilitate penetration and
colonization of mucosal environments (e.g.,
motile by polar flagella; corkscrew shape)
 Microaerophilic atmospheric requirements
 Become coccoid when exposed to oxygen or upon
prolonged culture
 Neither ferment nor oxidize carbohydrates
General Characteristics Common
to Superfamily

 First isolated as Vibrio fetus in 1909 from
spontaneous abortions in livestock
 Campylobacter enteritis was not recognized until
the mid-1970s when selective isolation media
were developed for culturing campylobacters
from human feces
 Most common form of acute infectious diarrhea in
developed countries; Higher incidence than
Salmonella & Shigella combined
 In the U.S., >2 million cases annually, an annual
incidence close to the 1.1% observed in the
United Kingdom; Estimated 200-700 deaths
History of Campylobacter

 Small, thin (0.2 - 0.5 um X 0.5 - 5.0 um), helical
(spiral or curved) cells with typical gram-negative
cell wall; “Gull-winged” appearance
• Tendency to form coccoid & elongated forms
on prolonged culture or when exposed to O2
 Distinctive rapid darting motility
• Long sheathed polar flagellum at one (polar) or
both (bipolar) ends of the cell
• Motility slows quickly in wet mount preparation
 Microaerophilic & capnophilic 5%O2,10%CO2,85%N2
 Thermophilic (42-43C) (except C. fetus)
• Body temperature of natural avian reservoir
 May become nonculturable in nature
Morphology & Physiology of Campylobacter

Campylobacter Species Associated
with Human Disease

 Low incidence potential sequela
 Reactive, self-limited, autoimmune disease
 Campylobacter jejuni most frequent antecedent
pathogen
 Immune response to specific O-antigens cross-
reacts with ganglioside surface components of
peripheral nerves (molecular or antigenic mimicry)
• Acute inflammatory demyelinating neuropathy
(85% of cases) from cross reaction with
Schwann-cells or myelin
• Acute axonal forms of GBS (15% of cases) from
molecular mimicry of axonal membrane
Guillain-Barre Syndrome (GBS)

 Zoonotic infections in many animals particularly
avian (bird) reservoirs
 Spontaneous abortions in cattle, sheep, and
swine, but generally asymptomatic carriage in
animal reservoir
 Humans acquire via ingestion of contaminated
food (particularly poultry), unpasteurized milk, or
improperly treated water
 Infectious dose is reduced by foods that neutralize
gastric acidity, e.g., milk. Fecal-oral transmission
also occurs
Epidemiology of Campylobacteriosis

 Contaminated poultry accounts for more than half
of the camylobacteriosis cases in developed
countries but different epidemiological picture in
developing countries
 In U.S. and developed countries: Peak incidence
in children below one year of age and young
adults (15-24 years old)
 In developing countries where campylobacters
are hyperendemic: Symptomatic disease occurs
in young children and persistent, asymptomatic
carriage in adults
Epidemiology of Campylobacteriosis(cont.)

 Sporadic infections in humans far outnumber
those affected in point-source outbreaks
 Sporadic cases peak in the summer in temperate
climates with a secondary peak in the late fall
seen in the U.S.
 Globally, C. jejuni subsp. jejuni accounts for more
than 80% of all Campylobacter enteriti
 C. coli accounts for only 2-5% of the total cases
in the U.S.; C. coli accounts for a higher
percentage of cases in developing countries

 Infectious dose and host immunity determine
whether gastroenteric disease develops
• Some people infected with as few as 500 organisms
while others need >106
CFU
 Pathogenesis not fully characterized
• No good animal model
• Damage (ulcerated, edematous and bloody) to the
mucosal surfaces of the jejunum, ileum, colon
• Inflammatory process consistent with invasion of the
organisms into the intestinal tissue; M-cell (Peyer’s
patches) uptake and presentation of antigen to
underlying lymphatic system
 Non-motile & adhesin-lacking strains are avirulent
Pathogenesis & Immunity

Cellular components:
Endotoxin
Flagellum: Motility
Adhesins: Mediate attachment to mucosa
Invasins
GBS is associated with C. jejuni serogroup O19
S-layer protein “microcapsule” in C. fetus:
Extracellular components:
Enterotoxins
Cytopathic toxins
Putative Virulence Factors

Specimen Collection and Processing:
 Feces refrigerated & examined within few hours
 Rectal swabs in semisolid transport medium
 Blood drawn for C. fetus
 Care to avoid oxygen exposure
 Selective isolation by filtration of stool specimen
 Enrichment broth & selective media
 Filtration: pass through 0.45 μm filters
Microscopy:
 Gull-wing appearance in gram stain
 Darting motility in fresh stool (rarely done in clinical lab)
 Fecal leukocytes are commonly present
Identification:
 Growth at 25o
, 37o
, or 42-43o
C
 Hippurate hydrolysis (C. jejuni is positive)
 Susceptibility to nalidixic acid & cephalothin
Laboratory Identification

Laboratory Identification (cont.)

 Gastroenteritis:
•Self-limiting; Replace fluids and electrolytes
•Antibiotic treatment can shorten the excretion period;
Erythromycin is drug of choice for severe or complicated
enteritis & bacteremia; Fluroquinolones are highly active
(e.g., ciprofloxacin was becoming drug of choice) but
fluoroquinolone resistance has developed rapidly since the
mid-1980s apparently related to unrestricted use and the
use of enrofloxacin in poultry
•Azithromycin was effective in recent human clinical trials
•Control should be directed at domestic animal reservoirs
and interrupting transmission to humans
 Guillain-Barre Syndrome (GBS)
•Favorable prognosis with optimal supportive care
•Intensive-care unit for 33% of cases
Treatment, Prevention & Control

History & Taxonomy of Helicobacter
 Family not yet named (17 species by rRNA sequencing)
 First observed in 1983 as Campylobacter-like
organisms (formerly Campylobacter pyloridis) in the
stomachs of patients with type B gastritis
 Nomenclature of Helicobacter was first established
in 1989, but only three species are currently
considered to be human pathogens
Important Human Pathogens:
 Helicobacter pylori (human; no animal reservoir)
 H. cinaedi (male homosexuals; rodents)
 H. fenneliae (male homosexuals; rodents)

 Helicobacter pylori is major human pathogen
associated with gastric antral epithelium in patients
with active chronic gastritis
 Stomach of many animal species also colonized
 Urease (gastric strains only), mucinase, and
catalase positive highly motile microorganisms
 Other Helicobacters: H. cinnaedi and H. fenneliae
• Colonize human intestinal tract
• Isolated from homosexual men with proctitis,
proctocolitis, enteritis, and bacteremia and are
often transmitted through sexual practices
General Characteristics of Helicobacter

 Gram-negative; Helical (spiral or curved) (0.5-1.0
um X 2.5-5.0 um); Blunted/rounded ends in gastric
biopsy specimens; Cells become rod-like and
coccoid on prolonged culture
 Produce urease, mucinase, and catalase
 H. pylori tuft (lophotrichous) of 4-6 sheathed flagella
(30um X 2.5nm) attached at one pole
 Single polar flagellum on H. fennellae & H. cinaedi
 Smooth cell wall with unusual fatty acids
Morphology & Physiology of
Helicobacter

Helicobacter on Paramagnetic Beads

Helicobacter Species Associated with
Human Disease

 Family Clusters
 Orally transmitted person-to-person (?)
Worldwide:
 ~ 20% below the age of 40 years are infected
 50% above the age of 60 years are infected
 H. pylori is uncommon in young children
Epidemiology of Helicobacter Infections

Developed Countries:
 United States: 30% of total population infected
• Of those, ~1% per year develop duodenal ulcer
• ~1/3 eventually have peptic ulcer disease(PUD)
 70% gastric ulcer cases colonized with H. pylori
 Low socioeconomic status predicts H. pylori infection
Developing Countries:
 Hyperendemic
 About 10% acquisition rate per year for children
between 2 and 8 years of age
 Most adults infected but no disease
• Protective immunity from multiple childhood infections
Epidemiology of Helicobacter Infections (cont.)

 Colonize mucosal lining of stomach & duodenum in
man & animals
• Adherent to gastric surface epithelium or pit epithelial
cells deep within the mucosal crypts adjacent to gastric
mucosal cells
• Mucosa protects the stomach wall from its own gastric
milleu of digestive enzymes and hydrochloric acid
• Mucosa also protects Helicobacter from immune
response
 Most gastric adenocarcinomas and lymphomas are
concurrent with or preceded by an infection with H.
pylori
Pathogenesis of Helicobacter Infections

Virulence Factors of Helicobacter

 Multiple polar, sheathed flagella
• Corkscrew motility enables penetration into viscous
environment (mucus)
 Adhesins: Hemagglutinins; Sialic acid binding
adhesin; Lewis blood group adhesin
 Mucinase: Degrades gastric mucus; Localized
tissue damage
 Urease converts urea (abundant in saliva and
gastric juices) into bicarbonate (to CO2) and
ammonia
• Neutralize the local acid environment
• Localized tissue damage
 Acid-inhibitory protein

Urease
C=O(NH2)2 + H+
+ 2H2O  HCO3
-
+ 2 (NH4
+
)
Urea Bicarbonate Ammonium
ions
And then… HCO3
-

CO2 + OH-
Urea Hydrolysis

Tissue damage:
 Vacuolating cytotoxin: Epithelial cell damage
 Invasin(s)(??): Poorly defined (e.g., hemolysins;
phospholipases; alcohol dehydrogenase)
Protection from phagocytosis & intracellular killing:
 Superoxide dismutase
 Catalase
Virulence Factors of Helicobacter (cont.)

Laboratory Identification
Recovered from or detected in endoscopic antral
gastric biopsy material; Multiple biopsies are taken
Many different transport media
Culture media containing whole or lysed blood
Microaerophilic
Grow well at 37oC, but not at 25 nor 42oC
Like Campylobacter, does not use
carbohydrates, neither fermentatively nor oxidatively

Triple Chemotherapy (synergism):
Proton pump inhibitor (e.g., omeprazole =
Prilosec(R))
One or more antibiotics (e.g., clarithromycin;
amoxicillin; metronidazole)
Bismuth compound
Inadequate treatment results in recurrence of symptoms

REVIEW
Campylobacter & Helicobacter
Superfamily

 Gram-negative
 Helical (spiral or curved) morphology; Tend to be
pleomorphic
 Characteristics that facilitate penetration and
colonization of mucosal environments (e.g.,
motile by polar flagella; corkscrew shape)
 Microaerophilic atmospheric requirements
 Become coccoid when exposed to oxygen or upon
prolonged culture
 Neither ferment nor oxidize carbohydrates
General Characteristics Common
to Superfamily
REVIEW

 First isolated as Vibrio fetus in 1909 from
spontaneous abortions in livestock
 Campylobacter enteritis was not recognized until
the mid-1970s when selective isolation media
were developed for culturing campylobacters from
human feces
 Most common form of acute infectious diarrhea in
developed countries; Higher incidence than
Salmonella & Shigella combined
 In the U.S., >2 million cases annually, an annual
incidence close to the 1.1% observed in the
United Kingdom; Estimated 200-700 deaths
History of Campylobacter
REVIEW

 Small, thin (0.2 - 0.5 um X 0.5 - 5.0 um), helical
(spiral or curved) cells with typical gram-negative
cell wall; “Gull-winged” appearance
• Tendency to form coccoid & elongated forms
on prolonged culture or when exposed to O2
 Distinctive rapid darting motility
• Long sheathed polar flagellum at one (polar) or
both (bipolar) ends of the cell
• Motility slows quickly in wet mount preparation
 Microaerophilic & capnophilic 5%O2,10%CO2,85%N2
 Thermophilic (42-43C) (except C. fetus)
• Body temperature of natural avian reservoir
 May become nonculturable in nature
Morphology & Physiology of Campylobacter
REVIEW

Campylobacter Species Associated
with Human Disease
REVIEW

 Low incidence potential sequela
 Reactive, self-limited, autoimmune disease
 Campylobacter jejuni most frequent antecedent
pathogen
 Immune response to specific O-antigens cross-
reacts with ganglioside surface components of
peripheral nerves (molecular or antigenic mimicry)
• Acute inflammatory demyelinating neuropathy
(85% of cases) from cross reaction with
Schwann-cells or myelin
• Acute axonal forms of GBS (15% of cases) from
molecular mimicry of axonal membrane
Guillain-Barre Syndrome (GBS)
REVIEW

 Zoonotic infections in many animals particularly
avian (bird) reservoirs
 Spontaneous abortions in cattle, sheep, and
swine, but generally asymptomatic carriage in
animal reservoir
 Humans acquire via ingestion of contaminated
food (particularly poultry), unpasteurized milk, or
improperly treated water
 Infectious dose is reduced by foods that neutralize
gastric acidity, e.g., milk. Fecal-oral transmission
also occurs
Epidemiology of Campylobacteriosis
REVIEW

 Contaminated poultry accounts for more than half
of the camylobacteriosis cases in developed
countries but different epidemiological picture in
developing countries
 In U.S. and developed countries: Peak incidence
in children below one year of age and young
adults (15-24 years old)
 In developing countries where campylobacters
are hyperendemic: Symptomatic disease occurs
in young children and persistent, asymptomatic
carriage in adults
REVIEW

 Sporadic infections in humans far outnumber
those affected in point-source outbreaks
 Sporadic cases peak in the summer in temperate
climates with a secondary peak in the late fall
seen in the U.S.
 Globally, C. jejuni subsp. jejuni accounts for more
than 80% of all Campylobacter enteriti
 C. coli accounts for only 2-5% of the total cases
in the U.S.; C. coli accounts for a higher
percentage of cases in developing countries
REVIEW

History & Taxonomy of Helicobacter
 Family not yet named (17 species by rRNA sequencing)
 First observed in 1983 as Campylobacter-like
organisms (formerly Campylobacter pyloridis) in the
stomachs of patients with type B gastritis
 Nomenclature of Helicobacter was first established
in 1989, but only three species are currently
considered to be human pathogens
Important Human Pathogens:
 Helicobacter pylori (human; no animal reservoir)
 H. cinaedi (male homosexuals; rodents)
 H. fenneliae (male homosexuals; rodents)
REVIEW

 Helicobacter pylori is major human pathogen
associated with gastric antral epithelium in patients
with active chronic gastritis
 Stomach of many animal species also colonized
 Urease (gastric strains only), mucinase, and
catalase positive highly motile microorganisms
 Other Helicobacters: H. cinnaedi and H. fenneliae
• Colonize human intestinal tract
• Isolated from homosexual men with proctitis,
proctocolitis, enteritis, and bacteremia and are
often transmitted through sexual practices
General Characteristics of Helicobacter
REVIEW

 Gram-negative; Helical (spiral or curved) (0.5-1.0
um X 2.5-5.0 um); Blunted/rounded ends in gastric
biopsy specimens; Cells become rod-like and
coccoid on prolonged culture
 Produce urease, mucinase, and catalase
 H. pylori tuft (lophotrichous) of 4-6 sheathed flagella
(30um X 2.5nm) attached at one pole
 Single polar flagellum on H. fennellae & H. cinaedi
 Smooth cell wall with unusual fatty acids
Morphology & Physiology of
Helicobacter
REVIEW

Helicobacter Species Associated with
Human Disease
REVIEW

 Family Clusters
 Orally transmitted person-to-person
 ~ 20% below the age of 40 years are infected
 50% above the age of 60 years are infected
 H. pylori is uncommon in young children
Epidemiology of Helicobacter Infections
REVIEW

Developed Countries:
 United States: 30% of total population infected
• Of those, ~1% per year develop duodenal ulcer
• ~1/3 eventually have peptic ulcer disease(PUD)
 70% gastric ulcer cases colonized with H. pylori
 Low socioeconomic status predicts H. pylori infection
Developing Countries:
 Hyperendemic
 About 10% acquisition rate per year for children
between 2 and 8 years of age
 Most adults infected but no disease
• Protective immunity from multiple childhood infections
Epidemiology of Helicobacter Infections (cont.)
REVIEW

 Colonize mucosal lining of stomach & duodenum in
man & animals
• Adherent to gastric surface epithelium or pit epithelial
cells deep within the mucosal crypts adjacent to gastric
mucosal cells
• Mucosa protects the stomach wall from its own gastric
milleu of digestive enzymes and hydrochloric acid
• Mucosa also protects Helicobacter from immune
response
 Most gastric adenocarcinomas and lymphomas are
concurrent with or preceded by an infection with H.
pylori
Pathogenesis of Helicobacter Infections
REVIEW

REVIEW

Triple Chemotherapy (synergism):
Proton pump inhibitor (e.g., omeprazole =
Prilosec(R))
One or more antibiotics (e.g., clarithromycin;
amoxicillin; metronidazole)
Bismuth compound
Inadequate treatment results in recurrence of symptoms
REVIEW

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