Enterobacteriaceae

Enterobacteriaceae
 Classification – 44 genera, more than 170
species
Enterobacteriaceae
 Escherichia
 Extraintestinal infection, diarrhea

 Shigella
 Typhoid fever, sepsis, gastroenteritis (food poison)

 Salmonella
 Dysentery
Enterobacteriaceae

 Morphology and General Characteristics

 Gram–, non-sporing, rod shape
 Oxidase–, differentiate from other G- rod
 Ferment glucose and may or may not produce gas
(aerogenic vs anaerogenic)
 Reduce nitrate to nitrite (a few exceptions)
Enterobacteriaceae
 Are facultative anaerobes
 If motile, motility is by peritrichous flagella
 Many are normal inhabitants of the intestinal tract
 Some are enteric pathogens and others are urinary
or respiratory tract pathogens
 Differentiation is based on biochemical reactions
and differences in antigenic structure
Enterobacteriaceae
 Most grow well on a variety of selective and
differential media originally developed for the
selective isolation of enteric pathogens.
 Most of these media are selective by adding dyes
and bile salts that inhibit G+ and suppress non-
pathogenic species of Enterobacteriaceae grow.
 Many are differential on the basis of whether or not
the organisms ferment lactose and/or produce H2S.
Enterobacteriaceae

 On CBA (China blue agar) they all produce

similar colonies: large and dull gray. May or
may not be hemolytic.
 The antigenic structure is used to differentiate
organisms within a genus or species.
 Three major classes of antigens are found:
Enterobacteriaceae
 Somatic O antigens –are the heat stable polysaccharide
part of the LPS.
 Variation from smooth to rough colonial forms is
accompanied by progressive loss of smooth O Antigen.
 Flagellar H antigens – are heat labile
 Envelope or capsule K antigens – overlay O antigen and
may block agglutination by O specific antisera.
 The K antigen is called the Vi (virulence) antigen in
Salmonella typhi.
Antigenic Structure of
Enterobacteriaceae
Escherichia
 6 species
 Escherichia coli
 Some are normal flora of the G.I. tract, cause
opportunistic infections or extraintestinal infection
 Some strains cause various forms of gastroenteritis.
 Is a major cause of urinary tract infection and neonatal
meningitis and septicemia.
 May have a capsule.
 Biochemistry: Most are motile.
E. coli

 May be hemolytic on CBA – more common in pathogenic

strains
 KEY tests for the normal strain:
 IMVIC (Indole, Methyl Red, Voges-Prosakaur, Citrate) Tests: ++--
 Motility +

 More than 170 kinds of O serotypes

E. coli

A B C
 A. E. coli on Eosin Methylene Blue agar plate.
 B. E. coli on blood agar plate.
 C. E. coli on MacC agar plate.
E. coli
 Antigenic structure - has O, H, and K antigens.
 Pathogenic factors:
 Invasiveness:
 Invasive outer membrane protein
 K antigen

 Pili/adhesin

 Endotoxin
 Enterotoxin
 Heat labile enterotoxin, LT
 Heat stable enterotoxin, ST

 Shiga-type toxin

 Hemolysin
E. coli toxins
 Virulence factors
Toxins

Enterotoxins –produced by ETEC. Causes a movement of

water and ions from tissues to the bowel resulting in watery
diarrhea.
 Two types of enterotoxin:

LT – is heat labile and binds to specific Gm1 gangliosides on
the epithelial cells of the small intestine which increases
production of cAMP. Increased cAMP alters the activity of
sodium and chloride transporters producing an ion
imbalance that results in fluid transport into the bowel.
E. coli toxins

ST– is heat stable and binds to specific receptors to
stimulate the production of cGMP with the same
results as with LT.
LT vs ST activity
E. coli toxins

Both enterotoxins are composed of five beta
subunits (for binding) and 1 alpha subunit (has
the toxic enzymatic activity).
Composition of enterotoxins
E. coli toxins
 Shiga-type toxin – also called the
verotoxin- produced by EHEC – is
cytotoxic, enterotoxic, neurotoxic, and
may cause diarrhea and ulceration of
the G.I. tract.

Two types: shiga-like toxin 1 and
shiga-like toxin 2.

Inhibit protein synthesis by
cleaving a 28S rRNA that’s part of
the 60S subunit
E. coli toxins
 Enteroaggregative ST-like toxin – produced by
enteroaggregative strains of E. coli (EAEC) – causes
watery diarrhea.
 Hemolysins – two different types may be found: cell
bound and secreted.

They lyse RBCs and leukocytes and may help to
inhibit phagocytosis when cell bound.
 Endotoxin
E. coli toxins
 Type III secretion system to deliver effector
molecules directly into the host cells.
 Involved in inducing uptake of EIEC into intestinal
cells.
 Involved in development of an attachment and
effacing lesion in EPEC characterized by microvilli
destruction and pedestal formation.
Type III secretion system
Pedestal formation
E. coli
 Adhesions – also called colonization factors, include pili,
fimbriae and non-fimbrial factors involved in attachment
 At least 21 different types.
 Antibodies to these may protect one from colonization.
 Virulence factors that protect bacteria from host defenses
 Capsule
 Iron capturing ability
 Outer membrane proteins - are involved in helping the
organism to invade by helping in attachment (acting as
adhesion) and in initiating endocytosis.
Types of adhesions

(intimin)
E. coli
 Clinical significance
 Is the leading cause of urinary tract infections
which can lead to acute cystitis (bladder
infection) and pyelonephritis (kidney infection).
Ascending urinary tract infection
Urinary tract infections (UTI)
 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.
 Bacteria living on the edges of the biofilms nay break
off leading to a round of infection.
Pod-like biofilm
E. coli infections
 Neonatal meningitis – is the leading cause of neonatal
meningitis and septicemia with a high mortality rate.
 Usually caused by strains with the K1 capsular antigen.
 Gastroenteritis –several distinct types of E. coli are
involved in different types of gastroenteritis:
 enterotoxigenic E. coli (ETEC),
 enteroinvasive E. coli (EIEC),
 enteropathogenic E. coli (EPEC) ,
 enteroaggregative E. coli (EAEC),
 enterohemorrhagic E. coli (EHEC).
ETEC

 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 (LT, ST).

Cause watery diarrhea, nausea, abdominal
cramps and low-grade fever for 1-5 days.

Transmission is via contaminated food or water.
EPEC
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 cause intimate
attachment of the non-fimbrial adhesion.

Diarrhea with large amounts of mucous without blood or
pus occurs along with vomiting, malaise and low grade
fever.

Mainly in hospitalized infants and in day care centers.
EIEC
 EIEC – The organism attaches to the intestinal mucosa via
pili and outer membrane proteins are involved in direct
penetration, invasion of the intestinal cells, and destruction of
the intestinal mucosa.

There is lateral movement of the organism from one cell
to adjacent cells.

Cause fever, severe abdominal cramps, malaise, and
watery diarrhea followed by scanty stools containing
blood, mucous, and pus.
EAEC
 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.

Cause watery diarrhea, vomiting, dehydration and
occasional abdominal pain.
EHEC
 EHEC – The organism attaches via pili to the intestinal
mucosa and liberates the shiga-like toxin.

Start with a watery diarrhea that progresses to bloody
diarrhea without pus and crampy abdominal pain with
no fever or a low-grade fever.

This may progress to hemolytic-uremic syndrome that
is characterized by low platlet count, hemolytic anemia,
and kidney failure.
EHEC

Most often caused by serotypes O157:H7.This strain
can be differentiated from others by it does not
ferment sorbitol in 48 hours.

A sorbitol-Mac (SMAC) plate (contains sorbitol instead
of lactose) is used to selectively isolate this organism.

One must confirm that the isolate is E. coli O1547:H7
by serological testing and confirm production of shiga-
like toxin before reporting out results.
Summary
Transmission in Humans
 Undercooked or unpasteurized animal
products
 Ground beef**
 Other meats
 Milk, cheese
 Foods contaminated with
feces
 Fruits
 Vegetables

Center for Food Security and Public

Health, Iowa State University, 2012
Diagnosis
 Culture
 Feces, food, environmental samples
 Differential and selective media

 Suspected colonies confirmed with:

 Biochemical tests
 Immunoassays

 Phage typing

 PFGE

 Serology
Center for Food Security and Public
Health, Iowa State University, 2012
Treatment
 Mainly supportive
 Antibiotics
 Usually avoided
 Do not reduce symptoms, prevent complications, or
reduce shedding
 May increase risk of HUS
 Antimotility drugs
 May increase risk of HUS

Center for Food Security and Public

Health, Iowa State University, 2012
Pathogenic E. coli
Site of
Full name action Disease

ETEC Enterotoxigenic Small Traveler’s diarrhea, infant diarrhea;

E. coli intestine watery diarrhea, vomiting, cramps,
nausea, low-grade fever.
EIEC Enteroinvasive Large Fever, abdominal pain, watery diarrhea
E. coli intestine followed by bloody stool.
EPEC Enteropathogenic Small Infant diarrhea with fever, nausea,
E. coli intestine vomiting, non-bloody stools.
EHEC Enterohemorrhagic Large Hemorrhagic colitis with severe
E. coli intestine abdominal cramps, watery diarrhea
followed by grossly bloody diarrhea;
almost no fever; hemolytic uremic
syndrome.
EAEC Enteroaggregative Small Infant diarrhea, persistent watery
E. coli intestine diarrhea with vomiting, dehydration,
and low grade fever
Shigella species
 Contains four species that differ antigenically and, to a
lesser extent, biochemically
 S. dysenteriae (Group A)
 S. flexneri (Group B)
 S. boydii (Group C)
 S. sonnei (Group D)
 Biochemistry
 IMVIC test: -+--
 Motility -
 All ferment mannitol except S. dysenteriae
 S. sonnei may show delayed lactose fermentation
Shigella species

Species Types Subtypes

S. dysenteriae A 1-10 8a, 8b, 8c

1a, 1b, 2a, 2b, 3a,
S. flexncri B 1-6, X, Y variety
3b, 3c, 4a, 4b
S. boydii C 1-18

S. sonnei D1
Shigella

A B

A. On MacConkey agar plate. E. coli colonies show pink and ferment lactose.
S. flexncri colonies are small, colorless and transparent, non-lactose
fermanting (18h, 37°C).
B. On SS agar plate. E. coli colonies are larger and red color, while S. flexncri
colonies are small, colorless and transpatent (18-24h)
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.
Shigella species

 Virulence factors
 Shiga toxin （ Exotoxin ） – 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 28S rRNA.
 Plays a role in the ulceration of the intestinal mucosa.
Shigella species

 Outer membrane and secreted proteins

 They 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.
Pathogenisis
Shigella attachment
Shigella penetration
Clinical Syndrome
 Causes shigellosis or bacillary dysentery.
 Transmit 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.
Clinical Syndrome

 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.
Immune and Diagnosis

 The immunity to Shigella cannot last long.

 Diagnosis of Shigella infection:
 Specimens: fresh stool, mucus flecks, rectal swabs
 Isolation and culture:
 Streaked on SS agar or EMB agar. Colorless colonies.
Biochemical test and serotype test are followed.
Treatment
 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
Salmonella
 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
 Biochemistry
 Urea –, Motility +, Citrate +/-, Indole -
Species Glucose Lactose Mannitol H2S VP Mehtylred Citrate
S. paratyphi A  -  -/+ - + +
S. schottmuelleri  -  +++ - + +
S. typhimurium  -  +++ - ++ +
S.hirschfeldii  -  + - + +
S. cholerae-suis  -  +/- - + +
S. typhi + - + -/+ - + +
S. enteritidis  -  +++ - + +

+: positive/produce acid; : produce both acid and gas; -: negative

Salmonella

 Virulence factors
 Endotoxin – may play a role in intracellular survival
 Capsule (for S. typhi and some strains of S.
paratyphi)
 Adhesions – both fimbrial and non-fimbrial
Virulence factors
 Type III secretion systems and effector molecules – 2
different systems may be found:
 One type is involved in promoting entry into intestinal
epithelial cells
 The other type is involved in the ability of Salmonella
to survive inside macrophages
Virulence factors
 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
Clinical Syndrome
 Causes two different kinds of disease: enteric
fevers and gastroenteritis.
 Both diseases begin in the same way, but for
gastroenteritis the bacteria remains restricted to the
intestine, and for 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.
Salmonella invasion of epithelial
cells
Clinical Syndrome
 The endosome moves to the basal side of the cell and
Salmonella are released and may be phagocytosed by
macrophages.
 For gastroenteritis the Salmonella multiply and their presence
induces a strong inflammatory response which causes most of
the symptoms seen in gastroenteritis (mild to moderate fever
with diarrhea and abdominal cramps).

The inflammatory response prevents the spread beyond
the GI tract and eventually kills the bacteria.
Clinical Syndrome
 In enteric fevers (typhoid and paratyphoid) the
Salmonella disseminate before they multiply to high
enough levels to stimulate a strong inflammatory
response, so the initial symptoms are only a low-
grade fever and constipation.
Salmonella
The bacteria move via the lymphatics and bloodstream to the liver
and spleen where phagocytosis and multiplication occurs.

They re-enter the bloodstream to disseminate throughout the body
to all organs causing fever, headaches, myalgia, and GI problems.

Rose spots (erythematous, muculopapular lesions) are seen on the
abdomen. Osteomyelitis, cystitis, and gall bladder infections may
occur.

Symptoms of paratyphoid fevers (due to S. paratyphi A, B, or C) are
similar to but less severe than those that occur with typhoid fever
(due to S. typhi)
Diagnosis of typhoid fever


Blood cultures are + during 1st week and after 2nd week.

Stool cultures and sometimes urine cultures are + after 2nd week.

The Widal test is a serological test for antibodies against Salmonella
typhi. One looks for a 4-fold rise in titer between acute and
convalescent stages.

10% of those infected become short term carriers and a smaller %
become long-term carriers due to persistence of the bacteria in the
gallbladder or 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