Chapter 16 Enterobacteriaceae
MLAB 2434 – Clinical Microbiology
Cecile Sanders & Keri Brophy-Martinez
Chapter 16 - Enterics
Family Enterobacteriaceae often
referred to as “enterics”
 Four major features:

All ferment glucose (dextrose)
 All reduce nitrates to nitrites
 All are oxidase negative
 All except Klebsiella, Shigella and
Yersinia are motile

Microscopic and Colony
Morphology
Gram negative bacilli or coccobacilli
 Non-spore forming
 Colony morphology on BAP or CA of
little value, as they look the same,
except for Klebsiella
 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 1611), species are grouped into Tribes,
which have similar characteristics
(Table 16-1, page 466)
 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
Many enterics possess antigens that can
be used to identify groups



O antigen – somatic, heat-stable antigen
located in the cell wall
H antigen – flagellar, heat labile antigen
K antigen – capsular, heat-labile antigen
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 –
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)

Characteristics

Dry, pink (lactose positive) colony
with surrounding pink area on
MacConkey
Escherichia coli (cont’d)
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

Escherichia coli (cont’d)

Infections
Wide range including meningitis,
gastrointestinal, urinary tract,
wound, and bacteremia
 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)
• Enterohemorrhagic (EHEC serotype
0157:H7) – associated with
hemorrhagic diarrhea and hemolyticuremic syndrome (HUS), which includes
low platelet count, hemolytic anemia,
and kidney failure; potentially fatal,
especially in young children;
undercooked hamburger, unpasteurized
milk and apple cider have spread the
infection; does NOT ferment sucrose;
identified by serotyping
Escherichia coli (cont’d)
• Enteroaggregative (EaggEC) – cause
diarrhea by adhering to the mucosal
surface of the intestine; watery
diarrhea; symptoms may persist for
over two weeks

Urinary Tract Infections
• E. coli is most common cause of UTI
and kidney infection in humans
• Usually originate in the large instestine
• 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 hermannii – yellow pigmented;
isolated from CSF, wounds and blood
Escherichia vulneris - wounds
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
Four major species
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

Colonies resemble Klebsiella
 Motile
 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 bacteremic outbreaks
in nurseries, cardiac surgery, and
burn units
 Fairly resistant to antibiotics

Serratia species (cont’d)

Major characteristics
Ferments lactose slowly
 Produce characteristic pink 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 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
 Providenicia 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
 Resembles Salmonella sp.

Salmonella
Produce significant infections in
humans and certain animals
 On differential selective agar,
produces clear, colorless, nonlactose fermenting colonies with
black centers (if media contains
indicator for hydrogen sulfide)

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
 Large and complex group of
organisms; grouped by O, H, and Vi
(for virulence) antigens

Salmonella (cont’d)

Clinical Infections

Acute gastroenteritis or food poisoning
• Source = handling pets, insufficiently cooked
eggs and chicken, and contaminated cooking
utensils
• Occurs 8 to 36 hours after ingestion
• Requires a high microbial load for infection
• Self-limiting in health 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)
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
 All species cause bacillary
dysentery
 S. dysenteriae (Group A)
 S. flexneri (Group B)
 S. boydii (Group C)
 S. sonnei (Group D)

Shigella (cont’d)

Characteristics








Non-motile
Do not produce gas from glucose
Do not hydrolyze urea
Do not produce H2S on TSI
Lysine decarboxylase negative
ONPG positive (delayed lactose +)
Fragile organisms
Possess O and some have K antigens
Shigella (cont’d)

Clinical Infections





Cause dysentery (bloody stools, mucous,
and numerous WBC)
S. sonnei is most common, followed by S.
flexneri (“gay bowel syndrome”)
Humans are only known reservoir
Oral-fecal transmission
Fewer than 200 bacilli are needed for
infection in health individuals
Shigella (cont’d)
Yersinia species


Consists of 11 named species
Yersinia pestis



Causes plague, which is a disease primarily
of rodents; transmitted by fleas
Two forms of plague, bubonic and
pneumonic
Gram-negative, short, plump bacillus,
exhibiting “safety-pin” or “bipolar”
staining
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
Yersinia species

Yersinia pseudotuberculosis
Pathogen of rodents, particularly
guinea pigs
 Septicemia with mesenteric
lymphadenitis, similar to
appendicitis
 Motile at 18 to 22 degrees C

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 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
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
Screening Stools for
Pathogens
Because stools have numerous
microbial flora, efficient screening
methods must be used to recover
any pathogens
 Enteric pathogens include
Salmonella, Shigella, Aeromonas,
Campylobacter, Yersinia, Vibrio, and
E. coli 0157:H7

Screening Stools for
Pathogens (cont’d)
Most labs screen for Salmonella,
Shigella, and Campylobacter; many
screen for E. coli 0157:H7
 Fecal pathogens are generally
lactose-negative (although Proteus,
Providencia, Serratia, Citrobacter
and Pseudomonas are also lactosenegative)
