MTECH3A / FINALS

Instructor: Maria Theda Ann B. Mondejar

Enterobacteriaceae 3.0
Lesson

 Pleseomonas Classification of Enterics (Lactose fermentation)

➢ New member of Enterobacteriaceae

➢ only oxidase positive member

General Characteristics

Key characteristics of the family Enterobacteriaceae

• Facultative anaerobes, non-sporeforming

• All motile species have peritrichous flagella except Tatumella

ptyseos which is monotrichous.

• All are catalase (+) except for Shigella dysenteriae.

The Enterics
• Some strains of Salmonella, Shigella, Escherichia,
Enterobacter and Proteus possess fimbriae or pili.  part of the normal intestinal flora
• Grow on most media at 35°C within 24 hours  cause gastrointestinal disease
• Some may grow at 1 - 5°C (psychrophiles) especially Colony Morphology
Yersinia enterocolitica and some strains of Serratia

Enterobacteriaceae divided into two broad categories

1. Opportunistic pathogens - part of the usual intestinal

microbiota

- may produce infection outside their normal body sites

(extraintestinal)

- produces significant virulent factors

2. Primary pathogens - not present as commensal biota in the GI

tract of humans

- produce infections resulting from ingestion of contaminated

food and drink

Salmonella spp. Yersinia spp.

Shigella spp. Plesiomonas shigellosis Classification

Classification of Enterics (Habitat)  Subcategorized into Tribes

 Use of tribes in classifying the members – proposed by

Ewing

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 The enterics have three major surface antigens

O antigen – the most external component of the

lipopolysaccharide (LPS) of gram negative bacteria

▪ Heat stable, Somatic antigen

▪ Antibodies to O antigen are predominantly IgM

▪ used for E. coli & Shigella spp. serotyping

H antigen – flagellar antigen

▪ only bacteria that are motile will possess this antigen

▪ Heat-labile

▪ Protein in nature

▪ Used for Salmonella spp. serotyping

Enterics reaction to TSI
K antigen – capsular antigen or envelope antigen

▪ Heat-labile polysaccharide found only in encapsulated

species

✓ Klebsiella spp.

✓ K1 antigen in Escherichia coli

✓ Vi antigen of Salmonella enterica subsp.

enterica serotype Typhi

Opportunistic Members of Enterobacteriaceae and Associated

Virulence and Antigenic factor
Infections
 Virulence of Enterobacteriaceae genera is affected by
their ability to:

• Adhere

• Colonize

• Produce toxin

• Invade tissues
Escherichia coli (colon bacillus)
• Harbor plasmid that can provide antimicrobial resistance
Major facultative inhabitant of the large intestine

❖ Indicator organism for fecal contamination of water

and foods
❖ normally resides in the colon without causing disease
❖ may acquire virulence factors and cause disease

Colonial morphology:

➢ MAC

✓ Lactose-positive pink colonies

with surrounding area of
precipitated bile salts

➢ EMB

✓ Green metallic sheen

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 Enteroadherent Escherichia coli

2 kinds of disease

a. UTIs
b. diarrheal syndromes
2 types of enteroadherent Escherichia coli (EAEC)

a. DAEC – diffusely adherent Escherichia coli

b. EAEC – enteroaggregative Escherichia coli
DAEC

➢ pediatric UTIs and diarrheal disease

Uropathogenic Escherichia coli ➢ cystitis in children
➢ acute pyelonephritis in pregnant women
EAEC

➢ cause diarrhea by adhering to the surface of the

intestinal mucosa
➢ persistent watery diarrhea
➢ WBC and RBC are absent in stool

Enterohemorrhagic Escherichia coli (EHEC) serotype

0157:H7

• Hemolytic Uremic Syndrome (HUS) – most severe

Gastrointestinal Pathogens manifestation of EHEC

Strains of Escherichia coli that cause diarrhea in man: • Hemorrhagic colitis

Enteropathogenic Escherichia coli (EPEC)
• does not produce enterotoxin
• associated with infantile diarrhea
• causes nursery outbreak diarrhea
• watery diarrhea with mucus but no blood
• serologic typing – to identify EPEC serotypes for
epidemiologic studies
Enterotoxigenic Escherichia coli (ETEC)
• produce enterotoxins
• major causes of traveller’s diarrhea
• heat - labile toxin (LT) similar to cholera exotoxin
(rice water stool)
• heat-stable toxin (ST)
• Infective dose: 106 - 1010
Enteroinvasive Escherichia coli (EIEC)
• bacillary dysentery in all age groups similar to
shigellosis
• Main virulence factor is encoded in a plasmid
shared by Shigella spp. and Escherichia coli
• HUS - characterized by low platelet, hemolytic anemia
and kidney failure
• Bloody diarrhea w/o WBC which distinguishes it from
dysentery caused by Shigella spp. or EIEC
• Appears colorless in MacConkey agar
Cytotoxins: Verotoxin I and II
1. Verotoxin I
✓ identical to the Shiga toxin (Stx) of Shigella
dysenteriae
✓ damages Vero cells (African green monkey
kidneys cells)
✓ neutralize by antibody against Stx
2. Verotoxin II
✓ Not neutralize by antibody against Stx
Uropathogenic Escherichia coli (UPEC) serotype

• Watery diarrhea is bloody with WBC, RBC and mucus o Most common cause of UTI in human

• detect invasiveness using monolayer cell cultures with Extraintestinal infection

Hep-2 (Human epithelial-2) cells
Neonates:
• Invasive E. coli is identified by Sereny Test (+)  septicemia
 meningitis
Virulence factor:
➢ K1 antigen
✓ Immunochemically identical to the capsular antigen of
Neisseria meningitidis group B

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 Klebsiella pneumoniae subsp. pneumoniae

 Bacillus mucosus capsulatum, Friedlander’s bacillus

 Possess large polysacharride capsule

➢ Protects organism against:

✓ Phagocytosis

✓ Antimicrobial
absorption

✓ Responsible for
colonial
characteristics:

✓ moist

✓ mucoid

Common characteristics of Tribe Klebsielleae

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 Enterobacter Genus Serratia

Serratia

General Characteristics

Enterobacter, Cronobacter and Pantoea  Motile GN rods, NLF but sucrose fermenters

Enterobacter spp.  Differentiated from other members of the tribe by their

ability to produce Dnase
 Colony morphology of many of the species resembles
Klebsiella when growing on MAC agar (LF)  produce a non-water soluble red to pink pigment
(prodigiosin)
 Common characteristics
➢ Serratia rubidaea
• Growth in potassium cyanide broth
➢ Serratia marcescens
• IMViC - - + +
➢ Serratia plymuthica
• In contrast to Klebsiella, Enterobacter spp. usually
produce ornithine decarboxylase ➢ Pigment production is
typically a characteristic
• Lysine decarboxylase is produced by most species in strains of
but not by E. gergoviae or E. cloacae environmental origin
Pantoea spp. Serratia marcescens
 Pantoea (Enterobacter) agglomerans gained notoriety  species most significant clinically
with a nationwide outbreak of septicemia resulting
from contaminated intravenous fluids  Found frequently in nosocomial infections of the:

 Designated early on as E. agglomerans complex, it o urinary or respiratory tract

includes members that are:
• lysine-, ornithine-, and arginine-negative
or “triple decarboxylases-negative”
Cronobacter spp.
 Cronobacter (Enterobacter) sakazakii typically
produces a yellow pigment
o bacteremic outbreaks in nurseries and
cardiac surgery and burn units
 Contamination of antiseptic solution used for joint
injections has resulted in an epidemic of septic arthritis
Serratia odorifera
contains two biogroups

 IMViC - - + + TSI – A/A + gas ➢ Emits a dirty, musty odor resembling that of rotten potatoes

 documented as a pathogen in neonates causing ➢ S. odorifera biogroup 1 is isolated predominantly from the
meningitis and bacteremia, often coming from respiratory tract and is positive for sucrose, raffinose, and
powdered infant formula ornithine

 It has also been isolated from cultures taken from • biogroup 1 may be indole-positive (60%)
brain abscesses and respiratory and wound infections
➢ S. odorifera biogroup 2 is negative for sucrose, raffinose,
and ornithine and has been isolated from blood and CSF

• biogroup 2 may also be indole-positive (50%)

S. liquefaciens, S. rubidaea, and S. fonticola have also been

isolated from human sources

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 Hafnia sp.
 Genus Hafnia is composed of one species, H. alvei
 However, two distinct biotypes are recognized:
• H. alvei
• H. alvei biotype 1 – grows in the beer wort of
breweries and has not been isolated clinically
• Hafnia has been isolated from many anatomic
sites in humans and in the environment
 Hafnia has been linked to gastroenteritis and is
occasionally isolated from stool cultures
 A delayed positive citrate reaction is a major
characteristic of Hafnia
Proteus
General Characteristics
 Widely disseminated in the environment
 Part of normal intestinal microbiota
 Opportunistic pathogens
 Proteus mirabilis is the most common clinical isolate
 phenylalanine deaminase (+)
➢ only member of Enterobacteriaceae that synthesize
this enzyme
➢ all produce a bluish gray confluent surface growth
or translucent sheet of growth on moist blood agar
that gives off a “burnt gun powder” or “ burnt
chocolate” odor
 P. mirabilis and P. vulgaris are widely recognized
human pathogens
 Both species hydrolyze urea and produce H2S,
although some strains of P. vulgaris are negative for
H 2S
 P. mirabilis is differentiated from P. vulgaris by the
indole and ornithine decarboxylase tests
 P. mirabilis is indole-negative but ornithine-positive
 P. vulgaris is indole-positive but ornithine-negative
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 Both species, particularly P. mirabilis, can produce
“swarming” colonies on non-selective media, such as
SBA and CHOC
 Swarming is a result of a tightly regulated cycle of
differentiation from standard vegetative cells
(swimmers) to hyperflagellated, elongated, polyploid
cells (swarmers) capable of coordinated surface
movement
 P. vulgaris ferments sucrose and gives an acid/acid
reaction in TSI agar
 Diene’s phenomenon
• Different strains of Proteus when inoculated on a
culture media swarm towards each other but do
not merge and are separated by a narrow
demarcation line between them
 Swarming of Proteus is inhibited by:
• 5 % agar
• Salt –free culture media
• PEA
• CLED (cystine lactose electrolyte difficient)
Diene’s phenomenon
Antigenic Structure
 All possess O, H and K antigens
 Proteus vulgaris
➢ same antigenic structure as Rickettsia
➢ O antigens (OX-19, OX-2) of some strains
✓ detect the rickettsial antibodies in the Weil-
Felix test
 Proteus mirabilis
➢ Source of O antigen (OX-K) that detect the
rickettsial antibodies in the Weil-Felix test
Clinical Infection
 Majority of human infections are caused by Proteus
 leading cause of community-acquired UTI
 major cause of nosocomial infections
 urease activity of Proteus mirabilis
➢ leads to struvite kidney stones (calculi)
 Providencia

Edwardsiella tarda is the only recognized human pathogen

Edwardsiella

Genus Morganella  Members of this genus are:

• Negative for urea

• IMViC + + - -
• Lysine decarboxylase-positive
• TSI K/A + gas + H2S

Edwardsiella tarda

• isolated from humans and associated with

✓ diarrhea
✓ wound infections
✓ bacteremia
• isolated from the environment and many cold
Morganella morganii
blooded and warm blooded animals

• NLF and H2S (+) – differentiates it from Escherichia

coli

Citrobacteriaceae

✓ Morganella spp. is motile but does not swarm  Earlier classifications of the family Enterobacteriaceae
included Citrobacter within the tribe Salmonelleae,
which formerly consisted of the genera Salmonella,
Citrobacter, and Arizona

 However, changes in the classification and

nomenclature have caused the reclassification of genus
Citrobacter into its own tribe, Citrobacteriaceae, and
of Arizona as a subspecies of Salmonella

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 Citrobacter
 Genus Citrobacter consists of at least 11 species that
all have been isolated from clinical specimens
3 species most often isolated:
• Citrobacter freundii
• Citrobacter koseri
• Citrobacter braakii
 Most hydrolyze urea slowly and ferment lactose
slowly, producing colonies on MAC agar that
resemble those of E. coli
 All species grow on Simmons citrate medium and
give positive reactions in the methyl red test
 inhabitants of the GI tract and are associated with
hospital-acquired infections, most frequently UTI
 C. freundii can be isolated in diarrheal stool cultures,
and although it is a known extra-intestinal pathogen,
its pathogenic role in intestinal disease is not
established
 C. freundii has been associated with infectious
diseases acquired in hospital settings; UTI,
pneumonias, and intraabdominal abscesses have been
reported
 C. freundii has been associated with endocarditis in IV
drug abusers
Citrobacter freundii
 80% produces H2S
 Some strains (50%) fail to ferment lactose
 Colonies on primary selective media - mistaken for
Salmonella when isolated from stool cultures
Primary Intestinal Pathogens of the Family Enterobacteriaceae
2 species designation of Salmonella
Salmonella enterica
Salmonella bongori
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Salmonella
 Members of the genus Salmonella produce significant
infections in humans and in certain animals
 Salmonella serotypes are typically found in cold-
blooded animals as well as in rodents and birds, which
serve as their natural hosts
 Salmonellae are gram-negative, facultatively
anaerobic bacilli that morphologically resemble other
enteric bacteria
 On selective and differential media used primarily to
isolate enteric pathogens (e.g., MAC), salmonellae
produce clear, colorless, non-lactose-fermenting
colonies
 Colonies with black centers are seen if the media
(e.g., HE or XLD) contain indicators for H2S
production
 The biochemical features for the genus include the
following:
• In almost every case, they do not ferment
lactose
• Negative for indole, Voges-Proskauer,
phenylalanine deaminase, and urease
• Most produce H2S – a major exception is
Salmonella Paratyphi A, which does not
produce H2S
• Do not grow in medium containing
potassium cyanide
Classification of Salmonella
➢ type species of genus Salmonella
➢ Rarely isolated from human
Within the species S. enterica are six subspecies:
➢ S. enterica subsp. enterica (also called subspecies I)
➢ S. enterica subsp. salamae (subspecies II)
➢ S. enterica subsp. arizonae (subspecies IIIa)
➢ S. enterica subsp. diarizonae (subspecies IIIb)
➢ S. enterica subsp. houtenae (subspecies IV)
➢ S. enterica subsp. indica (subspecies VI)
❖ All former species are now serotypes of Salmonella
enterica subsp. enterica
➢ e.g. Salmonella enterica subsp. enterica serotype
Typhi or simply written as Salmonella Typhi
(serotype is capitalized and not italized
❖ Members of the former genus Arizona, now subspecies IIIa
of S. enterica, are found in infections with symptoms
identical to those of Salmonella infections and may be
transmitted to humans from pet turtles, snakes, and fish
Antigenic structures
O antigen – the most external component of the
lipopolysaccharide (LPS) of gram negative bacteria
▪ somatic antigen
▪ Antibodies to O antigen are predominantly IgM
▪ Heat stable
H antigen – flagellar antigen
▪ only bacteria that are motile will possess this
antigen
▪ Heat-labile
2 phases:
Phase 1 (specific phase)
• shared by a few organisms
• determine immunologic identity
• agglutinate only with homologous antisera
Phase 2 (nonspecific phase)
• shared by many organisms
• react heterologous antisera
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Vi antigen – capsular antigen
▪ Heat-labile polysaccharide found only in
encapsulated species
▪ Prevents phagocytosis
✓ Salmonella serotype Typhi
✓ Salmonella serotype
Choleraesuis
Clinical Infections
 In humans, salmonellosis may occur in several forms,
as follows:
o Acute gastroenteritis or food poisoning
characterized by vomiting and diarrhea
o Typhoid fever, the most severe form of enteric
fever, caused by Salmonella serotype Typhi, and
enteric fevers caused by other Salmonella
serotypes (e.g., Salmonella Paratyphi and
Choleraesuis)
o Non-typhoidal bacteremia
o Carrier state following Salmonella infection
 Gastroenteritis
o One of the most common forms of “food
poisoning”
o Results from the ingestion of the organisms
through contaminated food
o Salmonella strains associated with gastroenteritis
are usually strains found in animals; most such
strains are members of S. enterica subsp. enterica
o Source of the infection has been attributed
primarily to poultry, milk, eggs, and egg products
as well as to handling pets
o Insufficiently cooked eggs and domestic fowl,
such as chicken, turkey, and duck, are common
sources of infection
o Salmonellosis is a common cause of GI tract
infections
o Cooking utensils, such as knives, pans, and
cutting boards used in preparing the contaminated
meat, can spread the bacteria to other food
o Direct transmission from person to person has
been reported in institutions
o Salmonella gastroenteritis occurs when a
sufficient number of organisms contaminate food
that is maintained under inadequate refrigeration,
allowing growth and multiplication of the
organisms
o The infective dose necessary to initiate the
disease, 106 bacteria
 Enteric Fevers
o The clinical features of enteric fevers include:
o Prolonged fever
o Bacteremia
o Involvement of the reticuloendothelial system,
particularly the liver, spleen, intestines, and mesentery
o Dissemination to multiple organs
o Enteric fever caused by Salmonella Typhi is known as
typhoid fever
o Salmonella Typhi does not have a known animal
reservoir; humans are the only known source of
infection
o Other enteric fevers include paratyphoid fevers, which
may be due to Salmonella serotypes Paratyphi A, B,
and C and Salmonella serotype Choleraesuis
o Clinical manifestations of paratyphoid fevers are
similar to typhoid fever but are less severe, and the
fatality rate is lower
o Typhoid fever develops approximately 9 to 14 days
after ingestion of the organisms
o The onset of symptoms depends on the number of
organisms ingested; the larger the inoculum, the shorter
the incubation period
o Organisms seem to be resistant to gastric acids
o On reaching the proximal end of the small intestine,
they subsequently invade and penetrate the intestinal
mucosa
 Bacteremia
o Salmonella bacteremia, caused by non-typhoidal
Salmonella, is characterized primarily by prolonged
fever and intermittent bacteremia
o Serotypes most commonly associated with bacteremia
are Typhimurium, Paratyphi, and Choleraesuis
o Salmonella infection has been observed among two
different groups:
✓ Young children – experience fever and gastroenteritis
with brief episodes of bacteremia
✓ Adults – experience transient bacteremia during
episodes of gastroenteritis or develop symptoms of
septicemia without gastroenteritis
o Clinical manifestations
Mode of transmission
• Typhoid fever
- human carrier is the sole source of the organism ,
Salmonella serotype Typhi
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• Nontyphoidal salmonellosis
- is transmitted by ingestion of contaminated food (usually
eggs, poultry and beef products) and water; fecal-oral
route
- aside from the human source, animals and animal
products are the major sources
 Humans acquire the infection by ingesting the
organisms in food, water, and milk contaminated with
human or animal excreta
Carrier state
➢ Individuals who recover from infection may harbor the
organisms in the gall bladder
➢ Excrete the organisms in feces
➢ May be terminated by
✓ antimicrobial therapy
✓ Cholecystectomy – only solution to
the carrier state
Shigella
General characteristics
 slender, aerobic, non- motile, non-encapsulated, gram-
negative rods
 generally non-lactose fermenters and do not produce
H2S (distinguished from Escherichia coli as lactose
fermenters)
 do not produce gas from glucose (anaerogenic) except
for certain types of Shigella flexneri
 all species can cause bacillary dysentery and are the
major causes of it
 Never considered as part of the normal intestinal flora
Factors contributing to virulence
➢ smooth lipopolysaccharide (LPS) structure – may be
the one responsible for the organism’s ability to resist
gastric acidity
➢ Invasiveness
➢ Shiga toxin – interferes with protein synthesis
and is neurotoxic, cytotoxic and enterotoxic
Clinical infection
➢ bacillary dysentery or Shigellosis
❖ 1. acute toxigenic gastroenteritis
- diarrhea with profuse, watery stools and fever of a
self -limited course
 ❖ 2. acute tissue-invasive gastroenteritis Disease Produced: Plague

- diarrhea with tenesmus, bloody or blood streaked and  Yersinia pestis is primarily pathogenic to rodents
mucoid stools, vomiting, fever, abdominal cramps and
abdominal tenderness  humans are accidental hosts only

Laboratory diagnosis a. Mode of transmission (to humans)

 Specimen: • Via bites of infected rat fleas (Xenopsylla

cheopis)
 rectal swab of ulcer taken by sigmoidoscopy (best
specimen) • By handling the carcass of an infected animal

 feces – usual specimen • Via infected aerosol droplets from person to

person
*specimens must be placed quickly on isolation media
or transport medium because they are susceptible to the Clinical forms
acid pH of stool Bubonic plague
a. stool examination  Characterized by infected and swollen lymph
 if toxigenic, no abnormal findings nodes (called buboes)

 if invasive: pus cells, red blood cells and  occur most commonly in the groin and less
macrophages are seen frequently in the axillary and cervical nodes

Classification of Shigella according to serogrouping Septicemic plague

 Prominent finding is disseminated

intravascular coagulation (DIC)

 Schwartzman phenomenon (presence of black

areas of hemorrhages all over the body)

 Highly fatal

Pneumonic plague (the Black Death; Pandemic plague)

 Usually arises from septic embolization to the

lungs
Yersinia
 With bloody, bacteria-rich, highly infectious
Only 3 species are pathogenic for humans: sputum
 Yersinia pestis  Transmitted by inhalation and is highly
 Yersinia enterocolitica contagious

 Yersinia pseudotuberculosis Biochemical Identification of Gram negative Bacteria

- facultatively intracellular 1. IMViC

- primarily animal pathogens 2. ONPG (Ortho – Nitrophenyl – β – D –

galactopyranoside test
Yersinia pestis
3. TSI agar
Morphology
4. Oxidation – Fermentation test
 Facultative anaerobic, nonmotile, gram-negative
coccobacillus , short, encapsulated 5. Gelatin liquefaction

 Shows marked 6. Motility

bipolar staining with 7. Nitrate and nitrite reduction
Wayson’s stain,
staining the polar 8. Urease
bodies blue and
giving the cells a 9. Dnase
“safety pin” 10. Malonate utilization test
appearance

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