Enterobacteriaceae

• Enterobacteriaceae


  • Escherichia
  • Klebsiella
  • Enterobacter
  • Serratia
  • Pantoea
  • Cronobacter
  • Haffnia
  • Citrobacter
  • Shigella
  • Salmonella
  • Edwardsiella
  • Proteus
  • Providencia
  • Morganella
  • Yersinia
  • Plesiomonas

  
• Enterobacteriaceae

  • Members are called "enterobacteria" which are predominant facultative flora of the human bowels
  • Most species are not intestinal pathogens but opportunistic organisms which are responsible for the majority of Nosocomial infections
  • Small, straight sided, facultative anaerobic, gram-negative, non-sporeforming rods
  • All members grow luxuriantly grow on BAP as moist, smooth, gray, shiny, entire, convex and opaque colonies
  • Beta hemolysis is seen in some strains

  
• Enterobacteriaceae


  • All members are motile at 35°C with peritrichous flagella, except Shigella, Klebsiella and some species of Yersinia
  • All are catalase-positive, except for one group of Shigella species
  • All are oxidase negative, except Plesiomonas
  • All members are non-encapsulated, except Klebsiella and Enterobacter
  • Almost all are able to reduce nitrate to nitrite, except Photorhabdus and Xenorhabdus

  
• Enterobacteriaceae


  • Some organisms may grow at 1°-5°C (psychrophiles) especially Yersinia enterocolitica and some strains of Serratia
  • Some are Rapid Urease producers: Proteus, Providencia, Morganella
  • Some are Late Urease Producers: Citrobacter, Klebsiella, Enterobacter, Yersinia and Serratia
  • All members ferment glucose
  • Some are lactose fermenters: RLF - lactose permease & B-galactosidase, LLF - B-galactosidase, NLF - no enzyme present

  
• Culture Media

  • EMB - Gram (+) inhibitor, Methylene blue, Lactose, Eosin Y
  • MAC - Gram (+) inhibitor, Bile salt, Crystal violet, Lactose, Neutral red
  • SSA - Gram (+) inhibitor, Bile salt, Lactose, Neutral red
  • XLD - Gram (+) inhibitor, Bile salt, Lactose, Xylose, Sucrose, Phenol red
  • HEA - Gram (+) inhibitor, Bile salt, Lactose, Salicin, Sucrose, BTB
  • BSA - Gram (+) inhibitor, Brilliant green, Glucose, Bismuth sulfite
  • TCBS - Gram (+) inhibitor, Bile salt, Citrate, Sucrose, BTB
• Antigenic Determinants

  • O Antigen - Somatic Antigen (first described by Ohne; heat stable)
  • H antigen - Flagellar Antigen (first described by Haunch, a german bacteriologist; heat labile)
  • K Antigen - also Vi Antigen (heat labile)
  • Fimbrial Antigens - most members of Enterobacteriaceae are piliated specifically common pili; these are readily agglutinated by specific antibodies
• Antimicrobial Resistance

  • Carbapenem-Resistant Enterobacteriaceae (CRE) - Resistance to imipenem, meropenem, doripenem, or entapenem due to carbapenemase
  • Extended Spectrum Beta-lactamases (ESBL) - Plasmid-mediated antibiotic resistance to extended spectrum of cephalosporins due to carbapenemase, cephalosporinase, metallo-B-lactamase, in E.coli and Klebsiella
  • Klebsiella pneumoniae Carbapenemase (KPC) - Resistance to carbapenems by Klebsiella, Enterobacter, Serratia

  
• Biochemical Tests

  • TSI
  • LIA
  • SCA
  • SIM
  • Urea
  • IMViC
  • ONPG
• Triple Sugar Iron Agar (TSI)

  • A butt-slant medium considered to be a modification of Kligler's iron agar, with the only difference being the sucrose not included in KIA
  • CHO content: TSI - glucose (0.1%), lactose (1.0%), sucrose (1.0 %)
  • pH indicator - phenol red (acid - yellow; alk - red)
  • H2S indicator - ferrous ammonium sulfate
  • Source of sulfur - sodium thiosulfate

  
• TSI Principle

  1. Glucose utilization by certain microorganisms occurs both aerobically on the slant where oxygen is available and anaerobically in the butt

  2. Once glucose is fermented, acids will be produced. These acids in the medium will cause the phenol red to assume a yellow color. Thus, the butt and slant will appear yellow after 6 hours of incubation

  3. After depletion of the limited glucose (0.1%), some organisms utilize lactose and continue making acid end products. The slant and butt will remain yellow after 18-24 hours of incubation. The reaction is called acid over acid (A/A) fermenting 2 or all sugars

  4. Some organisms are not able to utilize lactose, but instead breakdown the peptone in the medium. The by-product of these peptone metabolism, which occur in the surface of the slant are alkaline and causes the phenol red to revert back to its original red color. After 18-24 hours of incubation, the TSI will thus, show a red slant and retained yellow butt. The reaction is called alkaline over acid (K/A), signifying that only glucose was fermented

  5. Glucose non-fermenters may also produce alkaline products from peptone utilization. Reaction seen will be alkaline over alkaline (K/K). This signifies that no sugar was fermented

  6. Some organisms have the ability to produce gas from fermentation of sugars. While others produce large amounts of hydrogen sulfide gas (with H2S)
• TSI Reactions

  • A/A - Serratia, Escherichia, Enterobacter, Klebsiella
  • A/A, H2S - Citrobacter, Arizona
  • K/A, H2S - Salmonella, Proteus, Arizona, Citrobacter, Edwardsiella
  • K/A - Shigella, Citrobacter, Providencia, Yersinia
  • K/K - Pseudomonas

  
• Lysine Iron Agar (LIA)


  • Medium used to determine whether a gram-negative rod decarboxylates or deaminates lysine and forms H2S
  • LIA contains lysine, peptones, small amount of glucose, ferric ammonium citrate and sodium thiosulfate
  • When glucose is fermented, the butt of the medium becomes acidic (yellow). If organism produces lysine decarboxylase, cadaverine is formed. Cadaverine neutralizes the organic acids formed by glucose fermentation, and the butt of the medium reverts to alkaline state (purple)
  • If decarboxylase is not produced, the butt remains acidic (yellow)
  • If oxidative deamination of lysine occurs, a compound is formed that, in the presence of ferric ammonium citrate and coenzyme, flavin mononucleotide, forms a burgundy color on the slant
  • If deamination does not occur, LIA slant remains purple

  
• LIA Reactions

  • Lysine deaminase (slant): (+) red, (-) purple
  • Lysine Decarboxylase (butt): (+) purple, (-) yellow
  • Cadaverine - end product of L. decarboxylase
  • Blackening of the medium - indicates H2S production but it cannot detect Proteus (SACEd)
  • Only Proteus, Providencia and Morganella are positive for deamination but negative for Lysine decarboxylation of all the members of Enterobacteriaceae

  
• Simmon Citrate Agar (SCA)


  Medium used to determine if organisms can utilize citrate as their sole source of carbon producing acetate and other alkaline carbonate end products in the process, which change the color of the indicator from green to blue
• Indole Test

  Principle: Organisms that produce the enzyme tryptophanase are able to degrade tryptophan into indole, which can be detected by its ability to combine with certain aldehydes to form a colored compound
• Motility Test


  Principle: Performed on SIM (Sulfide Indole Motility) or any semi-solid motility test medium to determine if an organism is motile or non-motile
• Hydrogen Sulfide Production

  Test used for the detection of hydrogen sulfide (H2S) gas produced by an organism, which reacts with the iron compound to form the black precipitate of ferric sulfide. Possible Organisms: Salmonella, Proteus, Arizona, Citrobacter, Edwardsiella
• Urea Hydrolysis Test

  Principle: Based upon the ability of some bacteria to hydrolyze urea into ammonia water and CO2 by means of the enzyme urease. If urea is split to form ammonia, the medium becomes alkaline and the indicator turns from yellow to red.
• Methyl Red (MR)

  Principle: Some organisms produce large amount of acids from dextrose while others produce less. This test is based upon the final hydrogen ion concentration (acidity) reached by the culture. The test will be positive if a pH is 4.5 or lower, and negative above pH 4.5
• Voges Proskauer (VP)


  Principle: Some bacteria have the ability to produce acetoine (acetylmethylcarbinol), which reacts with guanidine compounds present in broth to give the red colored compound.
• ONPG Test


  Principle: Used to detect the presence of the enzyme beta-galactosidase, which hydrolyzes the colourless ONPG substrate to produce a yellow coloured product.

  
• Voges Proskauer (VP) test

  Tests for the production of acetoine (acetylmethylcarbinol) which reacts with guanidine compounds in the broth to give a red color
• ONPG

  1. nitrophenyl-β-D-galactoside, a rapid test to detect slow lactose fermenters due to the enzyme beta-galactosidase
• Escherichia coli is the most significant species of the genus Escherichia
• Escherichia coli


  • Short rods, motile by peritrichous flagella, non-encapsulated, non-spore forming
  • Part of the normal colon flora of both man and animals

  
• Escherichia coli


  • S-type fimbriae/pili (adhesins)
  • Enterotoxins - heat stable non antigenic toxin (ST toxin); heat labile antigenic toxin (LT toxin)
  • Verotoxin - human-derived cytotoxin which causes an irreversible cytotoxic effect on Vero tissue culture cells (similar to shiga toxin, thus referred to as Shiga-like toxins)
  • Invasiveness
  • Hemolysin

  
• Escherichia coli strains

  • Commensal
  • Pathogenic
  • Intestinal (EPEC, ETEC, EIEC, EAEC, EHEC/VTEC)
  • Extraintestinal (UPEC, SPEC, MNEC)

  
• Escherichia coli is the leading cause of UTI, and can also cause pneumonia, neonatal meningitis, wound infections, septicemia, and diarrheal diseases
• EPEC

  Does not produce enterotoxins, but associated with infantile diarrhea
• ETEC

  Produces enterotoxins, major cause of Traveller's diarrhea (Turista), may resemble cholera
• EIEC

  Causes Bacillary dysentery in all age groups similar to Shigellosis
• EAEC


  Produce non-fimbrial antigens that attach the organisms to their target cells (also produced by EPEC and VTEC)

  
• EHEC


  Associated with diarrhea, hemorrhagic colitis and Hemolytic Uremic Syndrome (EHEC O157:H7 is the most virulent strain)
• SERENY TEST is a classic test for the invasiveness of Escherichia coli, in which a suspension of the organism is instilled in the conjunctival sac of the guinea pig or rabbit causing purulent and exudative conjunctivitis

  
• Escherichia coli identification on culture media

  • MacConkey - dry pink colonies due to lactose fermentation
  • EMB - colonies with greenish metallic sheen
  • XLD - dry yellow colonies
  • SMAC - a medium that substitutes sorbitol for lactose in MacConkey, used as screening test for EHEC O157:H7

  
• Escherichia coli biochemical tests

  • TSI - A/A with gas
  • IMViC (++--)
  • L. deaminase (-)
  • Urease (-)
  • Utilizes acetate as a source of carbon
  • MUG (+), except EHEC O157:H7

  
• MUG test

  Escherichia coli produces the enzyme beta-D-glucoronidase, which hydrolyzes beta-D-glucopyranosid-uronic derivatives to a glycons and D-glucoronic acid. The substrate 4-methylumbelliferyl-beta-D-glucoronide is impregnated in the disk and is hydrolyzed by the enzyme to yield 4-methylumbelliferyl moiety, which fluoresces blue under long wavelength UV light.

  
• Other species of Escherichia include Escherichia albertii, Escherichia blattae, Escherichia fergosonii, and Escherichia vulneris
• Treatment for Escherichia coli infections includes fluid and electrolyte replacement in diarrheal cases, and antibiotics like sulfonamide, Ampicillin and Chloramphenicol
• Klebsiella


  • Gram-negative, encapsulated and non-motile
  • Has polysaccharide capsule
  • All are lactose fermenters except Klebsiella ozaenae and Klebsiella rhinoscleromatis
  • Colonies are large, moist and mucoid
  • Possess "O" and "K" antigens
  • Urease (+)
  • IMViC: --++
  • TSI A/A with gas