Lecture 5

Enterobacteriaceae: General
introduction, Escherichia coli &
Shigella, Salmonella, other Gram- rods
 Morphology and General Characteristics
• Gram-negative, no-spore forming, rod shaped bacteria, facultatively
anaerobic
• Catalase positive, oxidase negative (except for Plesiomonas) and reduce
nitrate to nitrite(there are a few exceptions)
• Ferment glucose and may or may not produce gas in the process
(aerogenic vs anaerogenic)
• If motile, motility is peritrichous flagella (except Shigella and Klebsiella)
 Enterobacteriaceae Classification
• The Enterobacteriaceae are a large, heterogeneous
group of Gram-negative rods.
• Due to nucleic acid hybridization and nucleic acid
sequencing technologies 63 genera have been defined;
however, the clinically significant Enterobacteriaceae
comprise 20–25 species, and other species are
encountered infrequent:
 Escherichia
 Shigella
 Edwardsiella
• Resident microflora:
 Salmonella  Escherichia – is a part of
 Citrobacter
 Klebsiella the normal microbiota and
 Enterobacter incidentally cause disease
 Hafnia
 Serratia  Klebsiella
 Proteus  Proteus
 Providencia
 Morganella  Enterobacter
 Yersinia
 Erwinia
 Pectinobacterium Exist in carrier state:
 Growth of Enterobacteriaceae on selective
media
• On Chocolate Blood Agar (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:
• Triple sugar iron agar (TSI),
• Lysine Iron Agar (LIA), and
• urea or phenylalanine agar.
• The antigenic structure is used to differentiate organisms
within a genus or species.
Three major classes of antigens are found:
 Escherichia coli
• 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 – lactose fermenting
• Most are motile.

E.Coli on ENDO agar

 Antigenic structure
• Has O, H, and K antigens.
• K1 has a strong association with virulence,
particularly meningitis in neonates.
Virulence factors
Toxins:
• 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.
 E. coli Enterotoxins
• There are two types of
enterotoxins:
• 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 (cyclic adenine mono
phosphate). Increased cAMP alters
the activity of sodium and chloride
transporters producing an ion
imbalance that results in fluid
transport into the bowel.
 E.coli Enterotoxins
• ST – is heat stable and
binds to specific
receptors to stimulate
the production of cGMP
(cyclic guanine mono
phosphate) with the
same results as with LT.
 E. coli toxins
• E. coli Shiga-like 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.
• There are two types:
 shiga-like toxin 1
 shiga-like toxin 2.
• Enteroaggregative ST-like toxin – produced by enteroaggregative
strains of E. coli (EAEC) – causes watery diarrhea.
• Hemolysins – two different types may be found:
 cell bound
 secreted.
• They lyse RBCs and leukocytes and may help to inhibit phagocytosis
when cell bound.
• Endotoxin Type III secretion system to deliver effector molecules
directly into the host cells. Involved in inducing uptake of
enteroinvasive E.coli (EIEC) into intestinal cells.
 Enteropathogenic E.coli Enterohemmoragic E.coli Eneterotoxigenic E.coli

Enteroaggregative E.coli Enteroinvasive E.coli Diffusely adherent E.coli

 E. coli: Adhesins
• Adhesins are also called colonization factors and include both
pili or fimbriae and non-fimbrial factors involved in
attachment.
• There are at least 21 different types of adhesions. Antibodies
to these may protect one from colonization.
• Virulence factors that protect the bacteria from host defenses:
 Capsule
 Iron capturing ability (enterochelin)
• Outer membrane proteins - are involved in helping the
organism to invade by helping in attachment and in initiating
endocytosis.
 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 – there are several distinct types of E.
coli that are involved in different types of
gastroenteritis:
 enterotoxigenic E. coli (ETEC),
 enteroinvasive E. coli (EIEC),
 enteropathogenic E. coli (EPEC) ,
 enteroaggregative E. coli (EAEC), and
 enterohemorrhagic E. coli (EHEC).
 E. coli gastroenteritis EPEC
• EPEC –Enteropathogenic E.coli bundle forming pili are
involved in attachment to the intestinal mucosa. This leads
to changes in signal transduction in the cells, effacement
of the microvilli, and to intimate attachment via a non-
fimbrial adhesion called intimin.
• EPEC adhere to the mucosal cells of the small bowel.
• After attachment, there is loss of microvilli (effacement);
formation of filamentous actin pedestals or cuplike
structures; and, occasionally, entry of the EPEC into the
mucosal cells.
• The exact mode of pathogenesis is unclear, but 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.
Adhesion of E.coli to jejunal mucosa

Electron micrograph of enteropathogenic Escherichia coli (EPEC) adherent to

the jejunal mucosa and demonstrating the attaching and effacing lesion, also
called pedestal formation. Tightly adherent E. coli are obliterating the brush
border.
Pedestal formation
 E. Coli gastroenteritis ETEC:
• ETEC –Eneterotoxigenic E.coli 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
• Some strains of ETEC produce a heat-labile enterotoxin (LT) that
is under the genetic control of a plasmid and is closely related to
cholera toxin.
• Some strains of ETEC produce the heat-stable enterotoxin STa,
which is under the genetic control of a heterogeneous group of
plasmids.

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.
 E. coli gastroenteritis EIEC
• EIEC –Enteroinvasive E.coli- 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.
• EIEC produce a disease very similar to shigellosis.
• The disease occurs most commonly in children in
developing countries and in traveler’s to these
countries.
• Similar to Shigella, EIEC strains are nonlactose or
late lactose fermenters and are nonmotile.
 E. coli gastroenteritis EAEC:
• EAEC – Enteroaggregative E.coli - 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.
• EAEC causes acute and chronic diarrhea (>14 days in
duration) in persons in developing countries. These
organisms also are the cause of foodborne illnesses in
industrialized countries and have been associated with
traveler’s diarrhea and persistent diarrhea in patients with
HIV.
• Some strains of EAEC produce ST-like toxin (see earlier
discussion on E. coli O104:H11); others a plasmid-encoded
enterotoxin that produces cellular damage; hemolysin
• Symptoms include watery diarrhea, vomiting, dehydration
and occasional abdominal pain.
 E. coli gastroenteritis :
• EHEC pr STEC – Enterohaemorrhagic E.coli - 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.
• STEC has been associated with mild non-bloody diarrhea, hemorrhagic
colitis, a severe form of diarrhea, and with hemolytic uremic syndrome, a
disease resulting in acute renal failure, microangiopathic hemolytic
anemia, and thrombocytopenia.
• A low infectious dose (< 200 CFU) is associated with infection. More than
150 E. coli serotypes that produce Shiga toxin, O157:H7 is the most
common and is the one that can be identified most readily in clinical
specimens.
• This may progress to hemolytic-uremic syndrome (HUS) that is
characterized by low platelet count, hemolytic anemia, and kidney
failure. This is most often caused by serotypes O157:H7.
 Urinary tract infection
• E. coli is the most common cause of urinary tract
infection and accounts for approximately 90% of
first urinary tract infections in young women.
• The symptoms and signs include urinary
frequency, dysuria, hematuria, and pyuria. Flank
pain is associated with upper tract infection.
• Urinary tract infection can result in bacteremia
with clinical signs of sepsis.
Ascending urinary tract infection
• Most of the urinary tract infections
that involve the bladder or kidney in an
otherwise healthy host are caused by a
small number of O antigen types that
have specifically elaborated virulence
factors that facilitate colonization and
subsequent clinical infections.
• These organisms are designated as
uropathogenic E. coli.
• Typically, these organisms produce
hemolysin, which is cytotoxic and
facilitates tissue invasion.
• Strains that cause pyelonephritis
express K antigen and elaborate a
specific type of pilus, P fimbriae, which
binds to the P blood group antigen.
 E.coli: Antimicrobial therapy

• E. coli is usually susceptible to a variety of

chemotherapeutic agents, though drug
resistant strains are increasingly prevalent. In
general, the sulfonamides, ampicillin,
cephalosporins, fluoroquinolones, and
aminoglycosides have good to excellent
antimicrobial efficacy.
• It is essential to do susceptibility testing.
 Shigella species :
Shigella 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:
• Urea –
• Motility –
• All ferment mannitol except S. dysenteriae and
S. sonnei may show delayed lactose fermentation
 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 one
of the rRNA constituents. This plays a role in the
ulceration of the intestinal mucosa.
Shigella attachment and invasion
 Type three secretion system
• Type three secretion system (often
written Type III secretion system and
abbreviated TTSS or T3SS, also
called Injectisome) is a protein
appendage found in several Gram-
negative bacteria.
• This is a needle-like structure is used
as a sensory probe to detect the
presence of eukaryotic organisms
and secrete proteins that help the
bacteria infect them. The
secreted effector proteins are
secreted directly from the
bacterial cell into the eukaryotic
(host) cell, where they exert a
number of effects that help the
pathogen to survive and to escape an
immune response.
 Shigella species : 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: 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. dysenteriae 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 : Classification
• 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.
 Salmonella :
• Virulence factors:
 Endotoxin – may play a
role in intracellular
survival
 Capsule (for S. typhi
and some strains of S.
paratyphi)
S. typhimurium and S. typhi possess partly
 Adhesions – both overlapping and a partly distinct repertoire of
virulence factors. Both serovars express the
fimbrial and non- type III secretion system, lipopolysaccharide,
fimbrial and other surface polysaccharides, fimbrae,
flagellin.
 Selective media for Salmonella
• The most commonly used media selective for Salmonella are SS agar,
bismuth sulfite agar, Hektoen enteric (HE) medium, brilliant green agar
and xylose-lisine-deoxycholate (XLD) agar. All these media contain both
selective and differential ingredients and they are commercially available.

Growth of Salmonella (white non lactose Growth of Salmonella Growth of Salmonella on a Hektoen enteric (HE)
agar. S. Typhimurium colonies grown on HE agar
fermenting) and E.coli (pink- lactose on a XLD agar. Black are blue-green in color indcating that the
fermenting) on a McConkey agar. centers indicate on bacterium does not ferment lactose However it
does produce hydrogen sulfide, (H2S), as indicated
production of H2S by black deposits in the centers of the colonies
Salmonella and Shigella on SS agar
 Salmonella virulence factors :
• Salmonella 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 - outer membrane proteins - is 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

• Salmonella causes two different kinds of disease:

 enteric fevers
 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.
Salmonella invasion of epithelial cells
a, Salmonella invasion. Entry
into host cells is mediated by
the Salmonella pathogenicity
island-1 (SPI-1) type III
secretion system (TTSS) and its
effectors.
 Salmonella Typhi manifestation
• 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)
 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
Salmonella: Antimicrobial therapy
• Enteric fevers – use cephalosporins and
azithromycin usually.
• Resistant strains have emerged making
antimicrobial susceptibility testing essential.
• Gastroenteritis – usually doesn’t require
antimicrobic therapy.
• Replace lost fluids and electrolytes.
 Enterobacteriaceae: Citrobacter
• Are opportunistic pathogens causing urinary
tract or respiratory tract infections and
occasionally wound infections, osteomyelitis,
endocarditis, and meningitis.
 Enterobacteriaceae: Klebsiella
• May cause Neurofibromatosis (NF -
neurocutaneous syndrome) of GI tract, but
potential pathogen in other areas
• Motility –
• Has both O and K antigens
 Klebsiella: Virulence factors

• Capsule
• Adhesions
• Iron capturing ability
Clinical significance
• Causes pneumonia, mostly in immunocompromised
hosts. 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: Enterobacter

• Normal flora of Gi tract

• Clinical significance:
 Nosocomial infections
 Bacteremia in burn
patients
 Enterobacteriaceae: Serratia
• A free-living saprophyte
• Has been found in
respiratory tract (RT)
and urinary tract (UT)
infections.
• Is resistant to many
antimicrobials
 Enterobacteriaceae: Proteus, Providencia,
and Morganella
• Are all part of the NF of the GI tract (except
Providencia).
• All motile

Proteus mirabilis
 Proteus, Providencia, and Morganella
Virulence factors
• Urease – the ammonia produced may damage
the epithelial cells of the UT
Clinical Significance
• UT infections, as well as pneumonia,
septicemia, and wound infections
Read:
CHAPTER 15 Enteric Gram-Negative
Rods (Enterobacteriaceae)

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