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Enterobacteriaceae
Enterobacteriaceae
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Classification – more than15 different genera
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Escherichia
Shigella
Edwardsiella
Salmonella
Citrobacter
Klebsiella
Enterobacter
Hafnia
Serratia
Enterobacteriaceae
Proteus
 Providencia
 Morganella
 Yersinia
 Erwinia
 Pectinobacterium
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Enterobacteriaceae
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Morphology and General Characteristics
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Gram-negative, non-sporing, rod shaped bacteria
Oxidase –
Ferment glucose and may or may not produce gas
in the process (aerogenic vs anaerogenic)
Reduce nitrate to nitrite (there are a few
exceptions)
Enterobacteriaceae
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Are facultative anaerobes
If motile, motility is by peritrichous flagella
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
Enterobacteriaceae
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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.
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Enterobacteriaceae
On CBA they all produce similar colonies
that are relatively large and dull gray. They
may or may not be hemolytic.
 The three most useful media for screening
stool cultures for potential pathogens are
TSI, LIA, and urea or phenylalanine agar.
 The antigenic structure is used to
differentiate organisms within a genus or
species.
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Three major classes of antigens are found:
Enterobacteriaceae
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Somatic O antigens – these are the heat stable
polysaccharide part of the LPS.
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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 the surface O
antigen and may block agglutination by O specific
antisera.
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Boiling for 15 minutes will destroy the K antigen and unmask
O antigens.
The K antigen is called the Vi (virulence) antigen in
Salmonella typhi.
Antigenic Structure of
Enterobacteriaceae
Enterobacteriaceae
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Escherichia coli
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Normal inhabitant of the G.I. 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.
Biochemistry
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Most are motile.
E. coli
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May be hemolytic on CBA – more common in pathogenic
strains
KEY tests for the normal strain:
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TSI is A/A + gas
LIA K/K
Urea –
Indole +
Citrate –
Motility +
There is an inactive biotype that is anaerogenic, lactose –,
and nonmotile.
E. coli
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Antigenic structure - has O, H, and K antigens. K1
has a strong association with virulence, particularly
meningitis in neonates.
Virulence factors
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Toxins
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Enterotoxins – produced by enterotoxigenic strains of E. coli
(ETEC). Causes a movement of water and ions from the
tissues to the bowel resulting in watery diarrhea. There are
two types of enterotoxin:
LT – is heat labile and binds to specific Gm1
gangliosides on the epithelial cells of the small intestine
where it ADP-ribosylates Gs which stimulates adenylate
cyclase to increase 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.
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E. coli toxins
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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
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Both enterotoxins are composed of five beta
subunits (for binding) and 1 alpha subunit (has the
toxic enzymatic activity).
Composition of subunits of
enterotoxins
E. coli toxins
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Shiga-type toxin – also called the verotoxin -produced
by enterohemorrhagic strains of E. coli (EHEC) – is
cytotoxic, enterotoxic, neurotoxic, and may cause
diarrhea and ulceration of the G.I. tract.
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There are two types shiga-like toxin 1 and shigalike toxin 2.
 Inhibit protein synthesis by cleaving a 28S rRNA
that’s part of the 60S subunit
E. coli toxins
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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
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Type III secretion system to deliver effector molecules
directly into the host cells.
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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
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Adhesions – are also called colonization factors and
include both pili or fimbriae and non-fimbrial factors
involved in attachment (e.g. intimin).
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Virulence factors that protect the bacteria from host
defenses
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There are at least 21 different types of adhesions.
Antibodies to these may protect one from colonization.
Capsule
Iron capturing ability (enterochelin)
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
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Clinical significance
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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)
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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.
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Bacteria living on the edges of the biofilms nay
break off leading to a round of infection.
Pod-like biofilm
E. coli infections
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Neonatal meningitis – is the leading cause of neonatal
meningitis and septicemia with a high mortality rate.
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Usually caused by strains with the K1 capsular antigen.
Gastroenteritis – there are several distinct types of E. coli
that are involved in different types of gastroenteritis:
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enterotoxigenic E. coli (ETEC),
enteroinvasive E. coli (EIEC),
enteropathogenic E. coli (EPEC) ,
enteroaggregative E. coli (EAEC), and
enterohemorrhagic E. coli (EHEC).
Various types of E. coli
E. coli gastroenteritis
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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 large amounts of mucous without blood or pus
occurs along with vomiting, malaise and low grade fever.
This is a problem mainly in hospitalized infants and in day care
centers.
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BFP
EPEC
EPEC
EPEC
Pedestal formation
EPEC
Tir injected
E. coli gastroenteritis
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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.
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.
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E. coli gastroenteritis
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EHEC – The organism attaches via pili to the intestinal mucosa and
liberates the shiga-like toxin.
The symptoms 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.
This is 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 that the isolate is E. coli O1547:H7 using
serological testing and confirm production of the shiga-like
toxin before reporting out results.
Serotypes of E. coli other than O157H7 have now been found
to cause this disease
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Summary of E.coli strains that
cause gastroenteritis.
E.coli
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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 species
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Shigella
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Contains four species that differ antigenically and, to a lesser
extent, biochemically.
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S. dysenteriae (Group A)
S. flexneri (Group B)
S. boydii (Group C)
S. sonnei (Group D)
Biochemistry
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TSI K/A with NO gas
LIA K/A
Urea –
Motility All ferment mannitol except S. dysenteriae
S. sonnei may show delayed lactose fermentation
Shigella species
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Antigenic structure
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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
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Shiga toxin – is produced by S. dysenteriae and in smaller
amounts by S. flexneri and S. sonnei.
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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 species
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Outer membrane and secreted proteins
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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
Shigella
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Clinical significance
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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.
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S. dysenteria is the most pathogenic followed by S. flexneri, S.
sonnei and S. boydii.
Shigella
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Antimicrobial therapy
Sulfonamides are commonly used as are
streptomycin, tetracycline, ampicillin, and
chloramphenicol.
 Resistant strains are becoming increasingly
common, so sensitivity testing is required.
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Salmonella
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Salmonella
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Classification has been changing in the last few years.
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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.
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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.
Phase variation of Salmonella
Salmonella
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Biochemistry
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TSI K/A + gas and H2S: S. typhi produces only a small
amount of H2S and no gas , and S. paratyphi A produces
no H2S
LIA K/K with H2S with S. paratyphi A giving K/A results
Urea –
Motility +
Citrate +/Indole -
Virulence factors
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Endotoxin – may play a role in intracellular survival
Capsule (for S. typhi and some strains of S. paratyphi)
Adhesions – both fimbrial and non-fimbrial
Salmonella virulence factors
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Type III secretion systems and effector molecules – 2
different systems may be found:
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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
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
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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.
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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.
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Salmonella invasion of epithelial
cells
Salmonella
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The endosome moves to the basal side of the cell and
Salmonella are released and may be phagocytosed by
macrophages.
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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.
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.
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Salmonella
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The bacteria move via the lymphatics and bloodstream
to the liver and spleen where phagocytosis and
multiplication occurs.
The bacteria 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)
Salmonella
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Diagnosis of typhoid fever
Blood cultures are positive during the first week and
after the second week
Stool cultures and sometimes urine cultures are positive
after the second 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.
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Salmonella
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Antimicrobial therapy
Enteric fevers – use chloramphenicol usually.
Resistant strains have emerged making
antimicrobial susceptibility testing essential.
 Gastroenteritis – usually doesn’t require
antimicrobic therapy.
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Replace lost fluids and electrolytes.
Comparison of Shigella versus
Salmonella invasion
Shigella
Salmonella
Enterobacteriaceae
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Citrobacter
TSI K/A or A/A both + gas and H2S
 LIA K/A + H2S
 Urea usually +
 Motility +
 Are opportunistic pathogens causing urinary
tract or respiratory tract infections and
occasionally wound infections,
osteomyelitis, endocarditis, and meningitis.
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Enterobacteriaceae
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Edwardsiella tarda
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TSI K/A + gas and H2S
LIA K/K +H2S
Urea –
Citrate –
Indole +
Clinical significance – causes GI disease in tropical
and subtropical countries
Enterobacteriaceae
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Klebsiella
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NF of GI tract, but potential pathogen in other areas
TSI A/A + gas
LIA K/K
Urea +
Citrate +
MR-, VP+
Motility Has both O and K antigens
Klebsiella
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Virulence factors
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Capsule
Adhesions
Iron capturing ability
Clinical significance
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Causes pneumonia, mostly in immunocompromised hosts.
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Permanent lung damage is a frequent occurrence (rare in
other types of bacterial pneumonia)
A major cause of nosocomial infections such as
septicemia and meningitis
Enterobacteriaceae
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Enterobacter
NF of GI tract
 TSI, LIA, and urea give variable results
depending upon species
 Citrate +
 Clinical significance
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Nosocomial infections
 Bacteremia in burn patients
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Enterobacteriaceae
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Serratia
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A free-living saprophyte
TSI A/A or K/A; +/- gas (does not ferment lactose)
LIA usually K/K
Citrate +
Motility +
Urea +/Has been found in RT and UT infections
Is resistant to many antimicrobics
Enterobacteriaceae
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Proteus, Providencia, and Morganella
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Are all part of the NF of the GI tract (except
Providencia).
All motile, with Proteus swarming
PA +
Lysine deamination + (LIA R/A)
Urea + for most, strongly + for Proteus
TSI variable (know the reactions for each in the
lab!)
Indole – only P. mirabilis is -
Proteus, Providencia, and
Morganella
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Virulence factors
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Clinical Significance
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Urease – the ammonia produced may damage the
epithelial cells of the UT
UT infections, as well as pneumonia, septicemia, and
wound infections
Yersinia
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Three species are important pathogens in man
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Yersinia pestis – causes plague
Yersinis enterocolitica – enteropathogenic
Yersinia pseudotuberculosis – enteropathogenic
Yersinia species
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Identification
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Y. pestis can be separated from Y. enterocolitica and Y.
pseudotuberculosis by the fact that it is non-motile. Y.
enterocolitica and Y. pseudotuberculosis are both nonmotile at 370 C, and motile at 220 C.
Y. pestis is identified based on the following:
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Non-motile
Bipolar staining
Slow growth of small colonies on ordinary culture media – it
grows better at lower temperature (25-300 C)
Yersinia pestis bipolar staining
Yersinia species
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TSI K/A no gas
LIA K/A
Urea –
Guinea pig or mouse pathogenicity studies: LD50<10
Direct fluorescent antibody test
New DNA probe test
Yersinia pestis – virulence characteristics
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Endotoxin – is responsible for many of the symptoms
Murine toxin – causes edema and necrosis in mice and rats,
but has not been shown to play a role in human disease
Y. pestis
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Fraction 1 – a protein component of the antiphagocytic
protein capsule. Also blocks flea digestion.
V antigen – a secreted protein that controls expression
of many of the virulence genes plus it appears to have
another unknown function that is essential for
virulence
Pla – a protease that activates plasminogen activator
(acts as a fibrinolysin) and degrades C3b (prevents
formation of complement membrane attack complex)
and C5a (prevents attraction of phagocytes)
Psa – a pilus adhesion for attachment
Iron acquisition and sequestering system
Type III secretion system
 YopB and YopD – disrupt actin cytoskeleton in
phagocytic cells to evade phagocytosis
Y. pestis
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Y. pestis – clinical significance
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In man plague occurs in two forms; bubonic and pneumonic
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Bubonic plague – transmitted by fleas from an infected rodent
(is endemic in our local mountains).
The bacteria travel in the blood to the nearest lymph node
where they are engulfed by fixed macrophages.
A high fever develops and the lymph nodes in the groin and
armpit become enlarged (buboes) as the bacteria proliferate
and stimulate an inflammatory response.
The bacteria growing in the lymph node leak into the
bloodstream.
Lysis of the bacteria releases LPS, causing septic shock.
Subcutaneous hemorrhages, probably due to LPS causing DIC
gave the disease the name, the black death, in the middle
ages.
The untreated mortality rate is quite high.
Buboes and pneumonia
Y. pestis
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Eventually bacteria reach the lungs where they are
ingested by lung macrophages to cause pneumonic
plague.
 Pneumonic plague – this can be transmitted
directly to others via aerosol. Direct inhalation of
aerosols containing the organism produces a form
of the disease that progresses much more rapidly
and the mortality rate is close to 100%.
Treatment for plague
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Streptomycin or tetracycline are effective
Yersinia species
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Yersinia enterocolitica and Yersinia
pseudotuberculosis identification –
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Both are motile at 22-250 C, but non-motile at 370 C
Both exhibit bipolar staining
Both grow better at lower temperatures and produce
small colonies at 370 C
TSI A/A (sucrose, not lactose fermentation) for Y.
enterocolitica; K/A for Y. pseudotuberculosis
LIA K/A for both
Urea + for both
ODC + for Y. enterocolitica only
Yersinia species
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Cefsulodin-irgasan-novobiocin (CIN) agar is a selective
media developed specifically for the isolation of Y.
enterocolitica from gastrointestinal specimens.
The media also contains mannitol and phenol red to
differentiate mannitol from non-mannitol fermenting
organisms.
The media is incubated at room temperature and
Yersinia are the only Enterobacteriaceae that will grow
on the media.
Aeromonas and Pleisiomonas, both members of the
Vibrionaceae will also grow.
After 48 hours at RT, Y. enterocolitica and Y.
pseudotuberculosis both produce typical pink (from
mannitol fermentation) colonies with a bulls-eye
appearance.
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Y. enterocolitica growth on CIN
Yersinia species
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Y. enterocolotica – virulence factors
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Enterotoxin similar to E. coli ST (increases cGMP leading to
watery diarrhea)
Adhesions – include both fimbrial and non-fimbrial
adhesions.
At least four different adhesions have been identified
thus far.
Antiphagocytic proteins – include both outer membrane and
secreted proteins.
Some are actually injected directly into the host via a
type III secretion mechanism.
Some interfere with signal transduction in host cells,
thus interfering with the ability of PMNs to respond to
signals leading them to the invading bacteria.
Others disrupt the actin cytoskeleton and lead to death
of the PMNs.
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Yersinia species
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V antigen - a secreted protein that controls expression of
many of the virulence genes plus it appears to have another
unknown function that is essential for virulence
Iron capturing ability
Yad A – an outer membrane protein that interferes with C3b
binding to bacteria thus preventing the formation of a
membrane attack complex.
Endotoxin
Y. pseudotuberculosis – virulence factors
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Has all of the same virulence factors as Y. enterocolitica
except the enterotoxin.
Yersinia species
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Yersinia enterocolitica and Y. pseudotuberculosis – clinical
significance
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Both are acquired by ingestion of contaminated food or
water.
Y. enterocolitica is a common cause of human disease,
whereas, Y. pseudotuberculosis is mainly a disease of other
animals.
Both cause a disease involving fever and abdominal pain. Y.
enterocolitica also causes a watery diarrhea.
After ingestion, the bacteria invade the intestinal epithelium
by invasion of M cells.
They are transcytosed through the M cells and released
at the basal surface.
Once through the intestional epithelium, the bacteria
penetrate into the underlying lymphoid tissue, where
they multiply both inside and outside host cells.
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Yersinia species
Multiplication of the bacteria produces an
inflammatory response that is responsible for the
extreme pain associated with the infections
(resembles acute appendicitis)
 Fever is due to the activity of the LPS endotoxin.
 Sometimes they drain into adjacent mesenteric
lymph nodes, causing mesenteric lymphadenitis.
Reactive arthritis may occur in some people following
Y. enterocolitica infection.
 It is thought to be due to cross reacting T cells or
antibodies that attack the joints.
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Summary of Yersinia infections
Yersinia species
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Antimicrobic susceptibility - must do
antimicrobial susceptibility testing.