Enterobacteriaceae
MLAB 2434 – Microbiology
Keri Brophy-Martinez
Overview- Enterics
Family Enterobacteriaceae often
referred to as “enterics”
 Four major features:

All ferment glucose (dextrose)
 All reduce nitrates to nitrites
 All are oxidase negative, except
Plesiomonas
 All except Klebsiella, Shigella and
Yersinia are motile

Microscopic and Colony
Morphology




Gram negative bacilli or coccobacilli
Non-spore forming
Facultative anaerobe
Colony morphology on BAP or CA of little
value, as they look the same


large, moist and grey on SBA
Selective and differential media are used
for initial colony evaluation

ex. MacConkey, HE, XLD agars
Classification of Enterics
Due to the very large number of
organisms in the Family
Enterobacteriaceae (see Table 19-1,
page 429), species are grouped into
Tribes, which have similar
characteristics
 Within each Tribe, species are
further subgrouped under genera

Virulence and Antigenic
Factors of Enterics


Ability to colonize, adhere, produce various
toxins and invade tissues
Some possess plasmids that may mediate
resistance to antibiotics
 ESBL’s: Extended-Spectrum beta
lactamases
• Inactivate extended spectrum
antibiotics like penicillins,
cephalosporins and aztreonam
Virulence and Antigenic
Factors of Enterics

Many enterics possess antigens that can be used to
identify groups
 O antigen – somatic, heat-stable antigen located in
the cell wall, made by all bacteria, stimulates
EARLY antibody production
 H antigen – flagellar, made by bacteria with
flagella, heat labile antigen,stimulates LATE
antibody production
 K antigen – capsular, heat-labile antigen, made by
some bacteria
• Vi- capsular antigen of S. typhii
Clinical Significance of
Enterics
Enterics are ubiquitous in nature
 Except for few, most are present in
the intestinal tract of animals and
humans as commensal flora;
therefore, they are sometimes call
“fecal coliforms”
 Some live in water, soil and sewage

Clinical Significance of
Enterics (cont’d)

Based on clinical infections produced,
enterics are divided into two categories:


Opportunistic pathogens – normally part
of the usual intestinal flora that may
produce infection outside the intestine
Primary intestinal pathogens – not
commensal flora, they produce infection
from ingestion of contaminated food or
water (Salmonella, Shigella, and Yersinia
sp.)
Escherichia coli
Most significant species in the
genus
 Important potential pathogen in
humans
 Common isolate from colon flora

Escherichia coli (cont’d)

Infections


Wide range including meningitis,
gastrointestinal, urinary tract,
wound, and bacteremia
Urinary Tract Infections
• E. coli is most common cause of UTI and kidney
infection in humans
• Usually originate in the large intestine
• Able to adhere to epithelial cells in the urinary
tract
Escherichia coli (cont’d)

Septicemia & Meningitis
• E. coli is one of the most common causes of
septicemia and meningitis among neonates;
acquired in the birth canal before or during
delivery
• E. coli also causes bacteremia in adults,
primarily from a genitourinary tract infection
or a gastrointestinal source
Escherichia coli (cont’d)

Gastrointestinal Infections
• Enteropathogenic (EPEC) – primarily in
infants and children; outbreaks in hospital
nurseries and day care centers; stool has
mucous but not blood; identified by
serotyping
Escherichia coli (cont’d)
• Enterotoxigenic (ETEC) – “traveler’s
diarrhea”; watery diarrhea without blood;
self-limiting; usually not identified, other
than patient history and lactose-positive
organisms cultured on differential media
• Enteroinvasive (EIEC) – produce dysentery
with bowel penetration, invasion and
destruction of intestinal mucosa; watery
diarrhea with blood; do NOT ferment
lactose; identified via DNA probes
Escherichia coli (cont’d)
• Enteroadherent E. coli
• Enteroaggregative (EAEC)
• Cause diarrhea by adhering to the
mucosal surface of the intestine;
watery diarrhea; symptoms may
persist for over two weeks
• Diffusely adherent(DAEC)
• Associated with UTI’s and diarrheal
disease, esp. in children and pregnant
women
Escherichia coli (cont’d)
•
Enterohemorrhagic (EHEC serotype 0157:H7)
• Origin
• Isolated in 1970
• Undercooked hamburger, unpasteurized milk and
apple cider have spread the infection
• Pathogenesis
• Circulating Shiga toxin binds to kidney
endothelium causing an inflammatory response
• Macrophages and neutrophils damage the
endothelium and glomerular basement membrane
• Associated with hemorrhagic colitis, TTP and
hemolytic-uremic syndrome (HUS), which includes
low platelet count, hemolytic anemia, and kidney
failure; potentially fatal, especially in young
children
Escherichia coli (cont’d)

Enterohemorrhagic (EHEC serotype 0157:H7)


Clinical Symptoms
• Starts with a watery diarrhea then progresses to
bloody diarrhea. No WBC’s are found in stool
•
Laboratory Diagnosis
• Does NOT ferment sorbitol
• Identified by serotyping, latex tests
Escherichia coli (cont’d)

General Characteristics

Dry, pink (lactose positive) colony
with surrounding pink area on
MacConkey
Other Escherichia species
Escherichia hermannii – yellow
pigmented; isolated from CSF,
wounds and blood
 Escherichia vulneris - yellow
pigmented; wounds

Escherichia coli (cont’d)

Characteristics
• Ferments glucose, lactose, trehalose, & xylose
• Positive indole and methyl red tests
• Does NOT produce H2S or phenylalanine
deaminase
• Simmons citrate negative
• Usually motile
• Voges-Proskauer test negative
• Fimbriae
• O, H, K antigens
Klebsiella, Enterobacter,
Serratia & Hafnia sp.



Usually found in intestinal tract
Wide variety of infections, primarily
pneumonia, wound, and UTI
General characteristics:






Some species are non-motile
Simmons citrate positive
H2S negative
Phenylalanine deaminase negative
Some weakly urease positive
MR negative; VP positive
Klebsiella species


Usually found in GI tract
K. pneumoniae is mostly commonly isolated
species



Possesses a polysaccharide capsule, which
protects against phagocytosis and
antibiotics AND makes the colonies moist
and mucoid
Has a distinctive “yeasty” odor
Frequent cause of nosocomial pneumonia
Klebsiella species (cont’d)

Significant biochemical reactions
• Lactose positive
• Most are urease positive
• Non-motile
Enterobacter species



Comprised of 12 species; E. cloacae and E.
aerogenes are most common
Isolated from wounds, urine, blood and
CSF
Major characteristics



Motile
Simmons citrate positive
MR negative; VP positive
Enterobacter species
(cont’d)
Serratia species
Seven species, but S. marcescens is
the only one clinically important
 Frequently found in nosocomial
infections of urinary or respiratory
tracts
 Implicated in bacteremia,
septicemia, cardiac surgery, and
burn units
 Fairly resistant to antibiotics

Serratia species (cont’d)

Major characteristics
Ferments lactose slowly
 Produce characteristic pink or red
pigment, especially when cultures
are left at room temperature

S. marscens on
nutrient agar →
Hafnia species
Hafnia alvei is only species
 Has been isolated from many
anatomical sites in humans and the
environment
 Occasionally isolated from stools
 Delayed citrate reaction is major
characteristic

Proteus, Morganella &
Providencia species
All are normal intestinal flora
 Opportunistic pathogens
 Deaminate phenylalanine
 All are lactose negative

Proteus species





P. mirabilis and P. vulgaris are widely
recognized human pathogens
Isolated from urine, wounds, and ear and
bacteremic infections
Both produce swarming colonies on nonselective media and have a distinctive
“burned chocolate” odor
Both are strongly urease positive
Both are phenylalanine deaminase positive
Proteus

Lactose negative
Proteus species (cont’d)


A: exhibits characteristic “swarming”
B: shows urease positive on right
Morganella species
Morganella morganii is only species
 Documented cause of UTI
 Isolated from other anatomical
sites
 Urease positive
 Phenylalanine deaminase positive

Providencia species
Providencia rettgeri is pathogen of
urinary tract and has caused
nosocomial outbreaks
 Providencia stuartii can cause
nosocomial outbreaks in burn units
and has been isolated from urine
 Both are phenylalanine deaminase
positive

Citrobacter species




Citrobacter freundii
associated with
nosocomial infections
(UTI, pneumonias, and
intraabdominal
abscesses)
Ferments lactose and
hydrolyzes urea slowly
Methyl red positive,
Simmons citrate positive
Resembles Salmonella sp.
Salmonella
Produce significant infections in
humans and certain animals
 Infections caused by ingestion of
food or water contaminated with
either human or animal feces

Salmonella (cont’d)

Salmonella on MacConkey
Salmonella (cont’d)
Lactose negative
 Negative for indole, VP,
phenylalanine deaminase, and urease
 Most produce H2S
 Do not grow in potassium cyanide

Salmonella

Virulence Factors
 Fimbriae- help in attachment
 Enterotoxin
 Anitgenic Structures
• O and H structures are used for serologic
grouping
• Vi- helpful in ID of Salmonella typhi
Salmonella (cont’d)

Clinical Infections

Acute gastroenteritis or food poisoning
• Source = handling pets, insufficiently cooked
eggs and chicken,milk, and contaminated
cooking utensils
• Occurs 8 to 36 hours after ingestion
• Symptoms include vomiting, chills, watery
diarrhea and abdominal pain
• Requires a high microbial load for infection
• Self-limiting in healthy individuals (antibiotics
and antidiarrheal agents may prolong
symptoms)
Salmonella (cont’d)

Typhoid and Other Enteric Fevers
• Prolonged fever
• Bacteremia
• Involvement of the RE system,
particularly liver, spleen, intestines, and
mesentery
• Dissemination to multiple organs
• Occurs more often in tropical and
subtropical countries
Salmonella (cont’d)

Typhoid fever





S. Typhii causative organism
• Invades intestinal mucosa causing constipation
• Gains entrance into lymphatic system and bloodstream then
into the liver, spleen and bone marrow where they are
phagocytized by PMN’s. They multiply in the PMN’s and are
eventually released into the blood stream.
• Finally, they invade the gall bladder and other parts of the
intestinal tract to initiate GI symptoms.
Found in contaminated food originating from infected
individuals
Other causes, improper disposal of sewage, poor
sanitation, lack of modern H2O systems
Develops 9-14 days after ingestion with symptoms of
fever, malaise, anorexia, myalgia, continuous dull
headache, rash
Severe
Salmonella (cont’d)
Salmonella Bacteremia
 Carrier State

• Organisms shed in feces
• Gallbladder is the site of organisms
(removal of gallbladder may be the only
solution to carrier state)
Shigella species

Closely related to the Escherichia

Fragile organisms

All species cause bacillary
dysentery, not normal GI flora
S.
 S.
 S.
 S.

dysenteriae (Group A)
flexneri (Group B)
boydii (Group C)
sonnei (Group D)
Shigella (cont’d)

Characteristics






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Non-motile
Do not produce gas from glucose
Do not hydrolyze urea
Do not produce H2S on TSI
On differential, selective media appear as
clear, NLF
Lysine decarboxylase negative
Citrate negative
Possess O and some have K antigens
lack the H antigen
Shigella (cont’d)

Clinical Infections

Cause dysentery (bloody stools, mucous,
and numerous WBC)
• Endotoxin- LPS
• Exotoxin- Shiga toxin




Confined to GI tract,septicemia rare
Humans are only known reservoir
Oral-fecal transmission, person-person
Fewer than 200 bacilli are needed for
infection in healthy individuals= takes a
low infective dose
Shigella (cont’d)
Yersinia species


Consists of 11 named species
Yersinia pestis


Causes plague, which is a disease primarily
of rodents; transmitted by rat fleas
Two forms of plague
• bubonic- flea bite, buboes
• Pneumonic- secondary to bubonic, organisms
multiply in bloodstream and resp. tract
Characteristic bubo
Yersinia gram stain
Yersinia species

Yersinia enterocolitica
 Most common form of Yersinia
 Found worldwide
 Found in pigs, cats and dogs
 Human also infected by ingestion of contaminated
food or water
 Some infections result from eating contaminated
market meat and vacuum-packed beef
 Is able to survive refrigerator temperatures (can
use “cold enrichment” to isolate)
 Mainly causes acute gastroenteritis with fever,
headaches
 Stool may be bloody
 Can mimic appendicitis
Laboratory Diagnosis of
Enterics

Collection and Handling


If not processed quickly, should be
collected and transported in CaryBlair, Amies, or Stuart media
Isolation and Identification
Site of origin must be considered
 Enterics from sterile body sites are
highly significant
 Routinely cultured from stool

Laboratory Diagnosis of
Enterics (cont’d)

Media for Isolation and Identification of
Enterics



Most labs use BAP, CA and a
selective/differential medium such as
MacConkey
On BA, produce large, greyish, smooth
colonies; can be β-hemolytic or
nonhemolytic
On MacConkey, lactose positive are pink;
lactose negative are clear and colorless
Laboratory Diagnosis of
Enterics (cont’d)



For stools, highly selective media, such as
Hektoen Enteric (HE), XLD, or SS is used
along with MacConkey agar
CIN- selects for Yersenia, hold at room
temp.
Identification

Most labs use a miniaturized or automated
commercial identification system, rather
than multiple tubes inoculated manually
Laboratory Diagnosis of
Enterics (cont’d)

Identification (cont’d)

All enterics are
• Oxidase negative
• Ferment glucose
• Reduce nitrates to nitrites
Laboratory Diagnosis of
Enterics (cont’d)

Common Biochemical Tests
Lactose fermentation and utilization
of carbohydrates
 Triple Sugar Iron (TSI)
 ONPG
 Glucose metabolism

• Methyl red
• Voges-Proskauer
Laboratory Diagnosis of
Enterics (cont’d)

Common Biochemical Tests (cont’d)

Miscellaneous Reactions
•
•
•
•
•
•
Indole
Citrate utilization
Urease production
Motility
Phenylalanine deaminase
Decarboxylase tests
Identification Clues
•
•
Lactose Fermenter
• Think: E. coli, Klebsiella, Enterobacter, possibly
Citrobacter
Lactose Fermenter with mucoid colony
Think: Klebsiella, Enterobacter
H2S +
• Think: Proteus, Salmonella, Citrobacter
Nonmotile
• Think: Klebsiella, Shigella
•
•
•
•
Voges- Proskauer +
•
Think: Klebsiella, Enterobacter, Serratia
Screening Stools for
Pathogens




Because stools have numerous
microbial flora, efficient screening
methods must be used to recover any
pathogens
Fecal WBC
Fecal pathogens are generally lactosenegative
Enteric pathogens include Salmonella,
Shigella, Aeromonas, Campylobacter,
Yersinia, Vibrio, and E. coli 0157:H7
References


Engelkirk, P., & Duben-Engelkirk, J. (2008). Laboratory Diagnosis of
Infectious Diseases: Essentials of Diagnostic Microbiology .
Baltimore, MD: Lippincott Williams and Wilkins.
Mahon, C. R., Lehman, D. C., & Manuselis, G. (2011). Textbook of
Diagnostic Microbiology (4th ed.). Maryland Heights, MO: Saunders.