Enterobacteriaceae

1
Enterobacteriaceae

• Biochemical Reactions

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IMViC Test

• Indole, Methyl Red, Voges-Prosakaur, Citrate
  (IMViC) Tests
• The following four tests comprise a series of
  important determinations that are collectively
  called the IMViC series of reactions
• The IMViC series of reactions allows for the
  differentiation of the various members of
  Enterobacteriaceae.

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IMViC Indole test

• Principle
• Certain microorganisms can metabolize tryptophan
  by tryptophanase
• The enzymatic degradation leads to the formation
  of pyruvic acid, indole and ammonia
• The presence of indole is detected by addition of
  Kovac's reagent.

Tryptophanase
Tryptophane amino acids
Indole Pyurvic acid NH3
Kovacs Reagent
Red color in upper organic layer
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IMViC Indole test

• Method
• Inoculate tryptone water with the tested
  microorganism
• Incubate at 37C for 24 hours
• After incubation interval, add 1 ml Kovacs
  reagent, shake the tube gently and read
  immediately

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IMViC Indole test
Positive test e.g. E. coli
Negative test e.g. Klebsiella

• Result
• A bright pink color in the top layer indicates
  the presence of indole
• The absence of color means that indole was not
  produced i.e. indole is negative
• Special Features
• Used in the differentiation of genera and
  species. e.g. E. coli () from Klebsiella (-).

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IMViC testMethyl Red-Voges Proskauer (MR-VP)
Tests

• Different bacteria convert dextrose and glucose
  to pyruvate using different metabolic pathways.
• Some of these pathways produce unstable acidic
  products which quickly convert to neutral
  compounds. Some organisms use the butylene glycol
  pathway, which produces neutral end products,
  including acetoin and 2,3-butanediol.
• Other organisms use the mixed acid pathway, which
  produces acidic end products such as lactic,
  acetic, and formic acid. These acidic end
  products are stable and will remain acidic.

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IMViC testMethyl Red-Voges Proskauer (MR-VP)
Tests
Principle
Glucose
Acidic pathway
Or
Neutral pathway
Acety methyl carbinol (ACETOIN)
Mixed acids ? pH less than 4.4
Barrits A (?-naphthol) Barrits B (40 KOH)
Methyl Red indicator
VP positive Klebsiella
MR positive E. coli
Pink color
Red color
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IMViC test MRVP test

• Method

• Inoculate the tested organism into One tube of
  MRVP broth
• Incubate the tubes at 37C for 24 hours
• AFTER INCUBATION  Pour 1/3 of the suspension
  into a clean nonsterile tube 
• Run the MR test in the tube with 2/3, and the VP
  test in the open tube with 1/3.
• For methyl red  Add 6-8 drops of methyl red
  reagent.
• For Voges-Proskauer  Add 12 drops of Barritt's A
  (?-naphthol), mix, 4 drops of Barritt's B (40
  KOH), mix
• Let sit, undisturbed, for at least 1hour

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IMViC test MR/VP test

• Results

Voges-Proskauer test
Methyl Red test

• Red Positive MR (E. coli)

• Pink Positive VP (Klebsiella)

• No pink Negative VP (E. coli)

• Yellow or orange Negative MR (Klebsiella)

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IMViC test Citrate Utilization Test
Principle
Na2CO3
CO2 Na H2O
Pyruvate
Citrate
Alkaline,?pH
Simmones Citrate media
Contains Citrate as a sole of C source
Bromothymol blue
Blue colour
Positive test

• Positive test Klebsiella, Enterobacter,
  Citrobacter

• Negative test E. coli

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IMViC test Citrate Utilization Test
Method
Streak a Simmon's Citrate agar slant with the
organism

• Incubate at 37C for 24 hours.

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IMViC test Citrate Utilization Test
Result

• Examine for growth ()
• Growth on the medium is accompanied by a rise in
  pH to change the medium from its initial green
  color to deep blue

Positive Klebsiella, Enterobacter
Negative E. coli
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Urease Test
Principle

• Urea agar contains urea and phenol red
• Urease is an enzyme that catalyzes the conversion
  of urea to CO2 and NH3
• Ammonia combines with water to produce ammonium
  hydroxide, a strong base which ? pH of the
  medium.
• ? in the pH causes phenol red r to turn a deep
  pink. This is indicative of a positive reaction
  for urease

H2O
Urease
Urea
NH4 OH
CO2 NH3
? in pH
Phenol Red
Method
Pink Positive test

• Streak a urea agar tube with the organism
• incubate at 37C for 24 h

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Urease Test
Result

• If color of medium turns from yellow to pink
  indicates positive test.
• Proteus give positive reaction after 4 h while
  Kelebsiella and Enterobacter gave positive
  results after 24 h

Positive test
Negative test
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Reaction on Triple Sugar Iron (TSI) Agar

• TSI contains
• Three different types of sugars
• Glucose (1 part)
• Lactose (10 part)
• Sucrose (10 part)
• Phenol red (acidic Yellow)
• TSI dispensed in tubes with equal butt slant
• Principle
• To determine the ability of an organism to attack
  a specific carbohydrate incorporated into a basal
  growth medium, with or without the production of
  gas, along with the determination of possible
  hydrogen sulphide production.

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Reaction on TSI

• Method
• Inoculate TSI medium with an organism by
  inoculating needle by stabbing the butt and
  streaking the slant
• Incubate at 37C for 24 hours

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Summary of morphology, cultural characteristics,
and biochemical reactions of Enterobacteriaceae
EMB SS MacConkey Nitrate reductase Oxidase Gram stain
Metallic sheen LF LF ve -ve -ve rod E. coli
Dark LF LF ve -ve -ve rods Citrobacter
Dark LF LF ve -ve -ve rods Klebsiella
Dark LF LF ve -ve -ve rods Enterobacter
Colorless NLF/H2S NLF ve -ve -ve rods Salmonella
Colorless NLF NLF ve -ve -ve rods Shigella
Colorless NLF/H2S NLF ve -ve -ve rods Proteus
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Summary of morphology, cultural characteristics,
and biochemical reactions of Enterobacteriaceae
Motility Urease Citrate VP MR Indole TSI
Motile -ve -ve -ve ve ve A/A/- E. coli
Motile -ve ve -ve ve ve A/A/- Citrobacter freundii
Non motile ve ve ve -ve -ve A/A/- Klebsiella pneumoniae
Motile ve ve ve -ve -ve A/A/- Enterobacter cloacae
Motile -ve ve -ve ve -ve A/Alk/ Salmonella typhi
Non motile -ve -ve -ve ve -ve A/Alk/- Shigella boydii
Motile Swarwing ve ve -ve ve -ve A/Alk/ Proteus mirabilis
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Practical Work

• Reaction on TSI
• Indole Test
• MR test
• VP test
• Citrate Utilization test
• Urease test

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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

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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

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Klebsiella species (contd)

• Significant biochemical reactions
• Lactose positive
• Most are urease positive
• Non-motile

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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

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Enterobacter species (contd)
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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

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Serratia species (contd)

• Major characteristics
• Ferments lactose slowly
• Produce characteristic pink pigment, especially
  when cultures are left at room temperature
• S. marscens on
• nutrient agar ?

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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

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Proteus, Morganella Providencia species

• All are normal intestinal flora
• Opportunistic pathogens
• Deaminate phenylalanine
• All are lactose negative

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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 non-selective
  media and have a distinctive burned chocolate
  odor
• Both are strongly urease positive
• Both are phenylalanine deaminase positive

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Proteus species (contd)

• A exhibits characteristic swarming
• B shows urease positive on right

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Morganella species

• Morganella morganii is only species
• Documented cause of UTI
• Isolated from other anatomical sites
• Urease positive
• Phenylalanine deaminase positive

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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

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Citrobacter species

• Citrobacter freundii associated with nosocomial
  infections (UTI, pneumonias, and intraabdominal
  abscesses)
• Ferments lactose and hydrolyzes urea slowly
• Resembles Salmonella sp.

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Treatment Klebsiella b-lactamse ve Ampicillin
and amoxicillin with b-lactamase inhibitor such
as clavulanic acid Cephalosporins (Cefuroxime,
Cefotaxime and fluoroquinolones) Cephalosporine
resistance often sensitive to gentamicin Multi
resistance serious hospital infection
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Treatment Klebsiella UTI trimethoprim,
nitrofurantion, or oral cephalosporin Pneumonia
Vigorous treatment with aminoglycosides or
Cephalosporins Vaccine - LPS
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Treatment Enterobacter b-lactamse ve and
Cephalosporinase ve Some resistance to
tetracyclines. Most strains are sensetive to
fluoroquinolones, co-trimoxazole and
carbapenems Differ from Serratia sensitive to
polymyxins
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Treatment Hafnia Sensitive to Aminoglycosides,
fluroquinolones and carbapenems
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Treatment Serratia Resistant to Cephalosporins
Resistance to ampicillin and gentamicin is
variable Gentamicin is the first-line In
recalcitrant cases- Fluoroquinolones or
carbapenems
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Treatment Proteus P. mirabilis b-lactamase ve
in general P. vulgaris usually resistant to
penicillins and cephalosporines and may sensitive
to b-lactamase stable derivatives
(cefotaxime) Treatment in infection associated
with renal stones usually unsyccessful Treatment
usually by lab findings
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Pseudomonas aeruginosa
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Characteristics

• Gram negative
• Motile
• Aerobic
• Nonfermentive
• Two forms
• Planktonic
• Biofilm
• Optimum growth
• 37C, can grow in 42 C
• Minimal nutritional requirements
• Three colony types
• Rough
• Smooth
• Mucoid

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Colony types
Small rough colonies
Large smooth colonies
Mucoid colonies
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Virulence

• Elastase
• Elastase has several activities that relate to
  virulence. The enzyme cleaves collagen, IgG, IgA,
  and complement. It also lyses fibronectin to
  expose receptors for bacterial attachment on the
  mucosa of the lung. Elastase disrupts the
  respiratory epithelium and interferes with
  ciliary function.
• Protease
• Alkaline protease interferes with fibrin
  formation and will lyse fibrin.
• Exotoxin A
• Exotoxin A prevents elongation in eukaryotic
  protein synthesis

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Virulence

• P. aeruginosa produces three other soluble
  proteins involved in invasion
• a cytotoxin (mw 25 kDa)
• The cytotoxin is a pore-forming protein.
• Two hemolysins.
• a phospholipase
• a lecithinase.
• One Pseudomonas pigment is probably a determinant
  of virulence for the pathogen.
• Pyocyanin
• The blue pigment impairs the normal function of
  human nasal cilia, disrupts the respiratory
  epithelium, and exerts a proinflammatory effect
  on phagocytes.

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Pathogenesis

• Opportunistic pathogen that can infect almost any
  body site given the right predisposing
  conditions.

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Diseases

• Endocarditis
• Respiratory infections
• Bacteremia and Septicemia
• Central Nervous System infections
• Ear infections including external otitis
• Eye infections
• Bone and joint infections
• Urinary tract infections
• Gastrointestinal infections
• Skin and soft tissue infections, including wound
  infections, pyoderma and dermatitis

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Bacterial Endocarditis

• Pseudomonas aeruginosa infects heart valves.
• IV drug users
• prosthetic heart valves.
• The organism establishes itself on the
  endocardium by direct invasion from the blood
  stream.

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Repiratory Infections

• Pneumonia
• Bacteremic pneumonia commonly occurs in
  neutropenic cancer patients undergoing
  chemotherapy.
• Lower respiratory tract colonization of cystic
  fibrosis patients

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Cystic Fibrosis

• The most common lethal inherited disorder among
  Caucasians, with an incidence of approximately 1
  in 2500 live births.
• Characteristics
• pancreatic insufficiency
• abnormal sweat electrolyte concentrations
• production of very viscid bronchial secretions
• tend to lead to stasis in the lungs and this
  predisposes to infection.

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Cystic Fibrosis
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Bacteremia and Septicemia

• Primarily in immunocompromised patients.
• Predisposing conditions
• hematologic malignancies,
• immunodeficiency relating to AIDS
• Neutropenia
• diabetes mellitus
• severe burns.

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Bacteremia and Septicemia

• Ecthyma gangrenosum
• Think Pseudomonas aeruginosa in neutropenic
  patients

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Central Nervous System Infections

• Pseudomonas aeruginosa causes meningitis and
  brain abscesses.
• Portal of Entry
• Inner ear or paranasal sinus
• Inoculated directly
• Surgery
• Invasive diagnostic procedures
• Spreads from a another site of infection like the
  urinary tract

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Ear infections

• External otitis
• "swimmer's ear"

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Eye infections

• It is one of the most common causes of bacterial
  keratitis, and has been isolated as the etiologic
  agent of neonatal ophthalmia, which occurs in
  1-12 of newborn infants.

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Bone and Joint Infections

• Direct inoculation of the bacteria or the
  hematogenous spread of the bacteria from other
  primary sites of infection.
• Blood-borne infections are most often seen in IV
  drug users, and in conjunction with urinary tract
  or pelvic infections.
• Chronic contiguous osteomyelitis
• The most common sites of involvement are the
  vertebral column, the pelvis, and the
  sternoclavicular joint
• Osteochondritis
• puncture wounds of the foot

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Urinary tract infections

• Usually hospital-acquired and related to urinary
  tract catheterization, instrumentation or
  surgery.
• 3rd leading cause of hospital-acquired UTIs
• about 12 percent of all infections of this type.
• The bacterium is among the most adherent of
  common urinary pathogens to the bladder
  uroepithelium.
• Pseudomonas can invade the bloodstream from the
  urinary tract.
• source of nearly 40 percent of Pseudomonas
  bacteremias.

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Gastrointestinal infections

• It can produce disease in any part of the
  gastrointestinal tract.
• Perirectal infections
• Pediatric diarrhea
• Gastroenteritis
• Necrotizing enterocolitis.
• The GI tract is also an important portal of entry
  in Pseudomonas septicemia.

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Skin and Soft tissue infections

• wound infections, pyoderma and dermatitis
• Pseudomonas aeruginosa can cause a variety of
  skin infections, both localized and diffuse. It
  has also been implicated in folliculitis and
  unmanageable forms of acne vulgaris.
• The common predisposing factors
• are breakdown of the integument
• Burns, trauma or dermatitis
• high moisture conditions
• ear of swimmers and the toe webs of athletes and
  combat troops, in the perineal region and under
  diapers of infants, and on the skin of whirlpool
  and hot tub users
• AIDS

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Treatment

• Pseudomonas aeruginosa is frequently resistant to
  many commonly used antibiotics.
• Although many strains are susceptible to
  gentamicin, tobramycin, colistin, and amikacin,
  resistant forms have developed. 
• The combination of gentamicin and carbenicillin
  is frequently used to treat severe Pseudomonas
  infections.

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Immune Defenses

• Phagocytosis by polymorphonuclear leukocytes is
  important in resistance to Pseudomonas
  infections. Antibodies to somatic antigens and
  exotoxins also contribute to recovery.
• Once P. aeruginosa infection is established,
  other antibodies, such as antitoxin, may be
  important in controlling disease.
• Cell-mediated immunity does not seem to play a
  major role in resistance or defense against
  Pseudomonas infections.